THE UNIQUE SIGNALLING REQUIREMENTS OF C-KIT' CELLS

STIMULATED BY MEMBRANE-BOUND STEEL FACTOR

Nadia 2. Klebasz

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Depertment of Immunology

University of Toronto

O Copyright by Nadia Z. Klebasz ZOO1

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THE UNIQUE SIGNALLINC REQUIREMENTS OF C-KIT' CELLS STIMULATED BY MEMBRANE-BOUND STEEL FACTOR

Nadia 2. Klebasz Master of Science. Department of Immunology University of Toronto. 200 1

ABSTRACT

Signalling requirements for PI3-kinase and PLC-y following stimulation of c-Kit- cells by

either soluble Steel Factor or membrane-bound Stcrl Factor were cxamined. By studying

33D-KitYF728 and YF719 cells. it was found that PLC-y activation is required for

stimulation by membrane-bound Steel Factor. Stimulation by membrane-bound Steel Factor

of 32D cells expressing a wild-type c-Kit receptor was inhibitrd by neomycin sulfate.

Stimulation of bone rnarrow-derivrd mast cells by mSLF was inhibited by neomycin sulfate

and olric acid. A study of both antagonists revealed that nrithrr inhibits c-Kit or PLC-y

phosphorylation. Pbkinase is recruited to the c-Kit receptor following mSLF stimulation of

murine bone marrowderived mast cells. To test the etlïcacy of the P1.C antagonists in vivo.

the rffects of neomycin sulfate and oleic acid were studied in micr. Oleic acid. but not

neomycin sulfate. has some potential for reducing ovenll skin mast ceil numbers without

visibly affecting surrounding tissue.

ACKNOWLEDGEMENTS

There are so many people to whom 1 am grateful not only for helping me through my Master's and the writing of this thesis but also for fnendship and support long before 1 even decided to attempt a graduate degree.

1 owe al1 of my Fnends an enormous "Thank you" for the times that they al1 made an effort to get in touch and find out how things were going with me. That has meant so much to me. Heather. Susannah, Jen, Grytjse, Julie. Karen and everyone else who cared, you guys are the best.

Daily life in the lab, with its ups and doms. was made more bearable by interesting lab- mates. I'd like to thank Jon for dl of his help over the past two years. Dim sum and noodle lunches were always a welcome break. I'm also grateful to Dino and Rowena for the Fnendships and the talks. They always helped me keep my perspective.

1 would not be writing this had it not been for my supe~isor. Dr. Stuart A. Berger. 1 cannot imagine that there could ever be a graduate supervisor more enthusiastic than he. For this. and for his patience and guidance, 1 will always be extremely grateful. 1 am convinced that it is because of him that 1 had the best possible graduate student expenence. 1 am also grateful to my cornmittee members. Dr. Tania Watts and Dr. Eleanor Fish. who both took the time out of their always-busy schedules to help out.

Above al1 else. 1 am grateful that 1 have had the love and encouragement of my family. In my sister 1 have something that very few people have: a life-long best fiend. Thank-you for always being there for me. If I have been successfid in my scholastic endeavors then 1 have managed to do so largely because 1 have always received the support and love of my Mother and Faiher. They reminded me to always believe in myself. Thank-you both for giving me the chance ro l e m and grow. This achievement is not mine alone: it is ours.

To my fiancé Bruce: Thank-you. Sweetheart. for your love and understanding. You have been the perfect partner. mentor, and fiiend. 1 made it through this programme a stronger person because of you. A lifetime will not be long enough .....

iii

TABLE OF CONTENTS . .

ABSTRACT ................................................................................................................ 11

... ....................................................................................... ACKNO WLEDGEMENTS i i i

........................................................................................... TABLE OF CONTENTS iv

LIST OF FIGURES .................................................................................................... vi

... LIST OF TABLES .................................................................................................... viii

Mast Cells ................................................................................................................ 1 3 Mast Ce11 role in the Defence against Bacteria ...........................................................

Mast Cells in Allergy .................................................................................................. 3 Mast cells in inflammation and tissue repair .............................................................. 4 Mast Cell Role in the Specific Immune System ......................................................... 5 Mast cell-fibroblast interaction ................................................................................... 6 c-Kit c-Kit c-Kit c-Kit Steel

............................................................................................................ Receptor 6 ........................................................................... ...................... structure .... 6

expression ....................................................................................................... 9 Mutations ......................................................................................................... 10 Factor ............................................................................................................ Il

Identification of the Kit Ligand .............................................................................. 11 SLF Structure .......................................................................................................... 11 Signal Transduction by the SLF-KIT Complex ........................................................ 15

........................................................................................................ Mechanism 1 5 Physiological roles of SLF-induced Signal Transduction through Kit ..................... 16

................................................................................................................. PI3-kinase 18 17 Phospholipase C-y ......................................................................................................

....................................................................................................... Thesis Rationale 23

MATERIALS AND METHODS ............................................................................. 26

Cell Culture .............................................................................................................. 26 Reagents and Antibodies .......................................................................................... 27 Production of Recombinant SLF .............................................................................. 27

............................................................. Immunoprecipitation and Western Blotting 28 ...................................................................... Stimulation assays ................... ..... -30

Viability assays ...................................................................................................... 1 Flow cytometry ......................................................................................................... 31

........................................................................................................... In vivo studies 32

III . ............................................................................................................... RE S ULTS 3 3

Kit YF728 recepton do not induce Steel Factor-stimulated PLC-y recruitment ...... 34 KitWT . KitW719 and KitYF728 but not KitYF719lYF728 32D celis respond to

.......................................................................................................................... sSLF 37 Response of KitYF728 receptors to membrane-bound Steel Factor is impaired ..... 30 KitWT and KitYF7 19 receptor but not KitYF728 receptors respond to plate-bound

................................................................................................ anti-c-Kit antibodies -43 Neornycin sulfate inhibits stimulation of KitYF719- but not KitWT- or KitYF728- expressing cells by soluble Steel Factor ................................................................... 46 Neomycin sulfate inhibits stimulation by membrane-bound Steel Factor or . . immobilized anti-c-Kit antibodies ......... ,. .............................................................. 46 Bone marrow-derived mast cells are stimulated by sSLF and X9/D3 cells but not SVSI' ......................................................................................................................... 49 Neornycin sulfate inhibits BMMC stimulation by mSLF but not sSLF ................... 54 Ionomycin revend of neomycin sulfate inhibition of BMMCs stimulated by mSLF .................................................................................................................................. 54 Oleic Acid cm inhibit BMMCs stimulated by mSLF but not sSLF ......................... 57 BMMC stimulation by mSLF-expressing X91D3 stroma1 cells phosphotylates c-Kit

Neomycin sulfate and oleic acid do not affect mSLF-mediated c-Kit tyrosine phosphorylation ........................................................................................................ 63 Neomycin sulfate and oleic acid do not affect mSLF-mediated PLC-y tyrosine phosphorylation ........................................................................................................ 66 PI3-kinase is recruited to the c-Kit receptor following BMMC stimulation with mSLF and Akt may be phosphorylated .................................................................... 71 OIeic acid decreases murine dermai mast ce11 numbers in vivo ............................... 74

32D myelomonocytic ce11 mode1 and c-Kit receptor mutants .................................. 78 Neomycin sulfate as a PLC antagonist ..................................................................... 78 PLC-y requirement for signalling through c-Kit by mSLF ....................................... 80 c-Kit sipalling in bone marrow-derived mast cells ................................................. 80 Oleic acid as a PLC-y inhibitor .............................................................................. 82 Biochemical studies of the mode of action of neomycin sulfate and oleic acid ....... 85 PU-kinase recruitment following stimulation by mSLF .......................................... 86 In vivo efTects of oleic acid and neomycin sulfate .................................................... 87

FUTURE DIRECTIONS .......................................................................................... 88

REFERENCES ......................................................................................................... 9 I

LIST OF FIGURES

Figure 1 .

Figure 2 .

Figure 3 .

Figure 4 .

Figure 5 .

Figure 6 .

Figure 7 .

Figure 8 .

Figure 9 .

Figure 10 .

Figure 1 1 .

Figure 12 .

Figure 13 .

Figure 14 .

Figure 15 .

Structural domains of the c-Kit receptor ............................................................ 7

Generation of membrane-bound and soluble Steel Factor ............................... 13

Akt activation by P13-kinase ............................................................................ 20

Basic mechanisms of cellular calcium signalling ............................................ 24

SLF-stimulated tyrosine phosphorylation of PLC-y in BMMCs and 32D

KitWT cells but not 32D KitYF728 cells ....................................................... 35

Stimulation of 32D infectants with sSLF ......................................................... 38

Stimulation of 32D KitWT and KitYF719 but not KitYF728 ce11 lines with

1 mSLF on X9/D3 stroma1 cells ......................................................................... 4,

Stimulation of 32D KitWT and KitYF719 ce11 lines but not KitYF728 cells

with plate-bound anti-c-Kit antibody .............................................................. -44

Neomycin sulfate inhibits stimulation of KitYF719- but not KitWT- or

KitYF728-expressing cells ............................................................................... 47

Neomycin sulfate inhibits stimulation of 32D KitWT cells by mSLF and

irnmobilized anti-c-Kit antibodies ................................................................... 50

Stimulation of bone-mmw denved mast cells by sSLF and mSLF .............. 52

- - Neomycin sulfate inhibits BMMC stimulation by mSLF but not sSLF .......... 33

Ionomycin restores neomycin sulfate-induced inhibition of BMMCs

stimulated by mSLF ...................................................................................... -33

Oleic acid inhibits BMMC stimulation by mSLF but not sSLF ....................Al

BMMC stimulation by X9/D3 stroma1 cells phosphorylates c-Kit .................. 64

Figure 16 . PLC antagonists do not affect c-Kit phosphorylation ...................................... 67

. Figure 17 PLC antagonists do not affect PLC-y2 phosphorylation .................................. 69

Figure 18 . PI3-kinase recruitrnent and possible Akt activation following stimulation of

BMMC with mSLF .......................................................................................... 72

vii

LIST OF TABLES

Table 1. Ef%ect of neomycin sulfate and oleic acid topicai treatment on dermal mast ceil

. . densities ................................................... . ........................................................ 75

viii

ATP

BH

BMMC

CTL

DAG

EGF

ER

FBS

k

IL

MF

IP3

ITAM

kDa

LPS

MHC

mRNA

mSLF

PDGF

PH

PI3 K

adenosine triphosphate

breakpoint-cluster-region homology

bone marrow-derived mast cells

cytotoxic T lymphocyte

diac y lglycerol

epidermal growth factor

endoplasmic reticulum

fetal bovine senun

irnmunoglobulin

interieukin

interferon

inositol 1 -4.5-triphosphate

immunoreceptor tyrosine-based activation motif

kiloDalton

lipopoly saccaride

major histocompatibility complex

messenger ribonucleic acid

membrane-bound Steel Factor

plateletderived growth factor

pleckstrin homology

phosphatidylinositol3 ' kinase

PIP2

PIP~

PLC-y

RTK

SH2

SLF

sSLF

TNF

phosphatidylinositol4.5-bisphosphate

phosphatidylinositol3,4,5-triphosphate

phospholipase C gamma

receptor tyrosine kinase

Src-homology 2 domain

Steel Factor

soluble Steel Factor

tumor necrosis factor

1. INTRODUCTION

A. Mast Cells

Mast cells originate fiom ~ ~ 3 4 ' pluripotent progenitor cells in the bone marrow. They

circulate in the penphery as undifferentiated CD34'. FceRL* and c-Kt' mononuclear cells

and. following migration into tissue. mature under local influences (reviewed by Reischl et

al.. 1999). Mature mast cells are widely distributrd throughout the body in comective

tissues. beneath epithelial surfaces. and in close proximity to blood vessels in vinually al1

vascularized tissues (reviewed by Gdli et al.. 1999). They are classically known to be the

primary effectors of allergic ruid inflamrnatory responses. but recent advances have s h o w

that mast cells likely have many diverse roles. They have been implicated in tissue

remodelling and wound repair. arthritis. pathological fibrosis. angiogenesis. and host

reactions to neoplasia (reviewed by Bradding and Holgate. 1999). Their diverse biological

roles may in part be due to the vast nurnber of lipid mediators. proteases and cytokines that

they are capable of synthesizing and releasing. There is also evidence that heterogeneity

exists among human mast cells with respect to their cytokine production. perhaps further

contributing to their divenity.

1. Mast Ce11 role in the Defence against Bacteria

Mast cells are a crucial component of the innate immune system in the defence against

bacterial infection. This was clearly demonstrated by Malaviya and colleagues who observed

that FVlW' mat-ce11 deficient mice are unable to clear virulent strains of Klebsiella

pneumoniae applied intranasally or intrapentoneally (Malaviya et al.. 1996). Normal mice.

or CY/V mice in which the mast cells were reconstituted, were able to clear the bacterial

infections. They showed that neutrophil influx was required for bacterial clearance and that

turnor necrosis factor (RIF)a was the mast ce11 cytokine that mediated this process. Mast

ce11 activation and subsequent TNFa production was demonstrated at lest in part to be due

to contact with the type I tirnbrial subunit. FimH (Malaviya et al.. 1996). However. other

bacterial components such as lipopolysaccharide have also been demonstrated to induce mast

crll cytokine release ( Leal-Benunen et al.. 1994).

Unlike FimH and lipopolysaccharide that directly cause mast cells to secrete certain

mediaton. mast cells can also be stimulated indirectly via the activation of the complement

system by a pathogen. Mice that are deficient in the complement component C3 (C3-/-) have

an increased mortality rate following caecal ligation and puncture. a mode1 of acute baterial

pentonitis. as compared to wild type mice (Prodeus et ai., 1997). C3-/- mice that were

subjected to the CLP procedure also demonstrated Iowa levels of intrapentoneal TNFa

production, degranulation of peritoneal mast ceils. and neutrophil recruitrnent. These defects

were improved when purified C3 was used to treat C3-/- mice.

2. Mast Cells in Allergy

Perhaps one of the most studied areas of mast ce11 function is the role they play in

irnmunoglobulin (Ig) E-mediated and IgE-dependent allergic responses. After migration of

the undifferentiated CD34'. FcaRI' and c-Kit' cells into tissue. Steel Factor (SLF) and other

cytokines. such as interleukin (U)-3. IL4 and IL-6, can induce the expression of the hi&-

affinity IgE receptor (FcaRI) (Metcalfe et al.. 1995; Tom et al., 1996). This receptor is

expressed as an a-P-y-y heterotetramer. but does not have intrinsic kinase activity (Wofsy et

al.. 1997). Cross-linking of FcsRI receptorj is mediated by multivalent antigen-bound.

allergen-specific IgE antibodies. This initiates a complex sipalling cascade. The fint step.

cornmon to al1 subsequent downstream signals. is the recruitment of the protein kinase lyn

(reviewed by Reischl et al.. 1999). This results in the phosphorylation of immunoreceptor

tyrosine-based activation motifs (ITAMs) on the FcsRI P- and y-chains. Syk kinase is

recruited to the y-chah ITAM and activated syk is capable of mediating phospholipase C-y1

(PLC-y) phosphorylation ( reviewed by Ortega et al .. 1 999). The downstream consequences

of phospholipase C activation are PKC activation and intracellular calcium mobilization.

Activated PKC results in the activation of nuclear protein c-Fos and cJun. It also activates

myosin and promotes actin polymenzation.

While the PLC-y casade is so far the best-understood pathway. other pathways are

also involved in signalling through FcsRi. The activation of the Ras pathway results in the

activation of transcription factors such as NFicB. NF-AT, Elk- 1 and Jun (Graves et al.. 1996).

Mast cells activated by multivalent antigen bound to IgE rapidly release histamine.

proteoglycans. proteases and other pre-made inflammatory mediators. FcsRI activation also

leads to the rapid synthesis and secretion of cytokines such as IL-2, iL-3. IL-4, IL-5, IL-6,

interferon (iNF)II, TNFa, GM-CSF and macrophage intlammatoty protein a (Burd et al.,

1989; Plaut et al., 1989; Wodnar-Filipowicz et al., 1989).

3. Mast cells in inflammation and tissue repair

The positioning of mast cells close to blood vessels implies that the vast number of mediaton

that they release likely contribute to the recruitment of cells required for repair of damaged

tissue. Acute tissue injury is typically followed by vascular changes resulting in edema and

later by the emigration of leukocytes ftom the blood vessels into the damaged tissue. Upon

histamine release by mast cells. endothelial cells which line blood vessels can be induced to

express the adhesion molecule P-selectin on their ceIl surface (Bonfanti et al.. 1989). P-

selectin mediates the rolling of leucocytes along the endotheliurn. Furthemore. mast cell

produced TNFa increases the expression of ICAM-1. VCAM-1 and E-selectin. al1 of which

are involved in adhesion of leucocytes firmly to endothelial cells (Carlos and Harlan. 1994).

It has been demonstrated in vitro that mast ceIl cytokines IL-4 and IL-13 promote

transendothelial migration of leucocytes (Ebisawa et al.. 1992: Moser et al.. 1993).

Specifically. chernotaxis of eosinophils is observed in the presence of mast ceIl cytokines

GM-CSF and I L 4 (reviewed by Bradding and Holgate, 1999). Neutrophils are

chemotactically attmted when mast cells secrete [L-8. while IL-16 and MCP-1 are both

potent chemoartractants of T cells (Rumsaeng et al.. 1997). The recruitmrnt of leucocytes

allows for the removal of cellular and tissue debris and the remodelling of new exiracellular

matrix components.

4. Mast Cell Role in the Specific Immune System

Mast cells are generally considered to be part of the innate immune system. They are also

important cells in numerous interactions that are critical in the development of the specific

immune system.

One role that mast cells play in the specific immune system is that of antigen

presentation. Both human and murine mast cells express Class 11 MHC (HLA) antigens and

studies have shown that murine bone marrow-denved mast celIs (BMMCs) are able to

present soluble exogenous antigens to T cells resulting in T ceil proliferation (Frandji et al..

1993). This antigen presentation is enhanced by IL-4 and GM-CSF but inhibited by MFy

(Fnndji et al.. 1993). This observation by Frandji and CO-workers suggests that m a t cells

can not only ampli@ their antigen presenting activity. but they can also down-regulate it

whrn a Th 1 cell-mrdiated response is occurring. consistent with the importance of mast cell-

derived cytokines in Th2-like responses such as allergy and infection. In fact. mast ceIl

secretion of I L 4 following binding of the BMMC MHC Class II molecule to the T ce11

receptor complex has been demonstrated. although not essential. to be one way in which T

ce11 differentiation down the Th2 pathway is induced (Huels et al.. 1995). Gauchat and

colleagues showed that the production of I L 4 and IL43 by mast cells is also important in

intluencing IgE production by B cells via the CD40 ligand expressed on their ce11 surface

(Gauchat et ai.. 1993).

5. Mast cell-fibroblast interaction

Not only are mast cells supported by Steel Factor-expressing fibroblasts but fibroblast

activity also may depend on proteins. cytokines and other mediaton secreted by mast cells.

Tissue fibrosis, the hallmark of chronic inflammation associated with increased mast

cell numbers, is largely the result of the production and deposition of matnx proteins by

fibroblasts. A number of mast ceil mediators have k e n demonstrated to af3ect fibroblasts.

IL-4 has been shown to stimulate murine fibroblast proliferation (Monroe et al.. 1988). In

human fibroblasts, IL-4 has been demonstrated to induce a proliferative signal (Trautmann et

al.. 1998) and is capable of inducing fibroblasts to secrete collagen (Postlethwaite et ai..

1992). Histamine has also been show to promote human fibroblast growth (Boucek and

Noble. 1973). as has I N F a (Sugarman et ai.. 1985).

B. c-Kit Receptor

1. c-Kit structure

c-Kit is a msrnembranr receptor tyrosine kinase. It belongs to the class III.

irnrnunoglobulin superfmily of receptors. which includes the platelet-derived growth factor

(PDGF) receptor and the receptor for colony-stimulating factor 1 (Qiu et al.. 1988). In total.

the c-Kit protein is cornprised of 976 amino acids. 497 of which form the extracellular

region, 23 are in the transmembrane domain and the remaining form a cytoplasmic region

(Yarden et al., 1987). Figure I is a diagammatic representation of the structure of the c-Kit

Figure 1. Stmctural domains of the c-Kit receptor. The extracellular domain of the c-Kit

receptor is composed of five Ig-like domains. The three domains farthest away from the cell

membrane are involved in ligand binding. The intracellular region contains the catalytic

domain that is separated into two parts by a kinase insert. About 30 amino acids fom the

juxtmrmbrane region of c-Kit and anchor the receptor in the plasma membrane (reviewed

by L w et al.. 1994).

Extracellular Domain

Transmembrane 1

Domain Domain

lntracellular Domain 1 + Kinase lnsert

receptor. The extracellular domain consists of five Ig-like domains, with the second and the

fifth domains each potentiaily containing two cysteine bridges. There are up to nine N-

glycosylation sites, mostly concentrated in the carboxy terminal half of the extracellular

domain. Heterogeneous N-glycosylation atrects the molecular mass of c-Kit which varies

behveen 145 kDa to 160 kDa (Qiu et al.. 1988: Yarden et al.. 1987). The intracçllular

domain of c-Kit is comprised of a consensus ATP-binding site and a tyrosine kinase domain

that is bisected by a small. hydrophillic kinase insert ( Yarden et al.. 1987). This domain

underpes ligand-dependant tyrosine autophosphorylation. Post-transcriptionai modification.

specifically alternative splicing events. can produce variants of the c-Kit receptor that are

biologically functional (Hayashi et al.. 199 1 ; Reith et al.. 199 1 : Rossi et al.. 1992).

2. c-Kit expression

The c-kir mRNA transcript is widely expressed during development in the embryonic gem

layers (Qiu et al.. 1988: Yarden et al.. 1987) and in hematopoietic stem and progenitor cells

( Ashrnan et ai.. 1 99 1 ). In fact. embryonic expression of c-kir mRNA can be detected in skin.

brain. spinal cord. liver, bone, lung, kidney, gut. teeth and nasal tissue (reviewed by Ashman.

1999: Lev et al., 1994). By birth. the expression of c-kit is much more restricted. High levels

of c-kit expression are observed in hematopoietic tissue. during the maturation of germ cells

and neural-crest-denved melanocytes (reviewed by Galli et al.. 1994). Dermal mast cells are

one of the few mature ce11 types to express the c-Kit receptor (Mayrhofer et al.. 1987). High

levels of c-kit expression have also been detected in many types of hurnan rnalipancies.

including myeloid leukemia (Gadd and Ashrnan. 1985: Lemer et al.. 1991: Wang et al..

1989), melanoma (Funasaka et al.. 1992), breast and rend cancers (Natali et al., 1992; Turner

et al., 1992) and glioblastoma (Yarden et al., 1987).

3. +Kit Mutations

The c-Kit receptor is the product of the W locus. which in mice is located on chromosome 5

(Chabot et al., 1988: Geissler et al.. 1988). Genetic studies have dernonstrated that the

different severities in phenotype exhibited by mutant mice depend on the specific mutation of

the W alleles. For example, mice that are WBV. b ~ ~ ~ f i ~ ~ ~ and P2/W'' have xvere macrocytic

anemia and die perinatally or at the late fetal stage (Nocka et al.. 1990): (Geissler et al..

198 1). Heteroqgotes at these loci survive. but display a wide range of phrnotypcs. W/+

mice rxhibit only small areas without coat pigmentation. often referred to as "dominant white

spotting" (Nocka et al.. 1990). while W"/+ mice have severe macrocytic anemia lack al1 coat

pigmentation. and have small gonads (Geissler et al.. 198 1 ). Other homozygous mutations at

the W locus such as W'W are viable. but result in mice that have severe mast ce11

deficiencies. are depigmented and are sterile (Nocka et al.. 1990). Finally. some mutations at

the W locus. such as FV4' and ndv? result in only a mild anemia and slight coat

depigmentation (Nocka et al.. 1990: Reith et al.. 1990). The severity of the phenotype

displayed in homoygous mice is believed to be related to the arnount of impairment of

kinase activity in the mutant receptor (Lev et ai.. 1994). Thus. the w.'~ and W'-' alleles. which

are the result of missense mutations. almost completely abolish tyrosine kinase activity and.

even though the receptor is expressed at proper levels on the ce11 surface. the result is death in

utero (Nocka et ai.. 1990: Reith et al.. 1990). The less severe phenotypes. such as those

pmduced by W and W". are the result of consenative substitutions that only partially impair

tyrosine kinase activity.

C. Steel Factor

1. Identification of the Kit Ligand

The ligand for the c-Kit receptor is cdled Steel Factor. Some alternative names for this

molecule are mast-ceIl growth factor. stem-ce11 factor and Kit ligand. Mutations at the S~er l

(SI) locus result in phenotypes that are complementary to those observed in mice with

mutations at the W locus. Early studies showed that bone marrow transplanted from SI

donors into W mice resulted in animals that were hematologically nomal and these

observations were confirmed in vitro (Dexter and Moore. 1977). However. the converse (i.e.

manow From FV mice transplanted into SI mice) does not produce normal animals. These

observations led to the identification of c-Kit and Steel Factor as a receptor-ligand pair.

2. SLF Structure

The SI gene encodes a transmembrane protein 273 arnino acids in length. It is comprised of a

short 25 amino acid leader sequence. an extracellular domain of 185 amino acids. a

hydrophobie transmembrane sequence of 27 arnino acids and a 36 amino acid cytoplasmic

domain (Williams et al., 1992). Steel Factor undergoes heterogeneous glycosylation and as a

result its molecular weight c m range between 28 to 35 kDa (Zsebo et al., 1990). On the ce11

surface, it is believed to prirnarily exia as non-covalently linked dimers (Hsu et ai.. 1997).

hterestingly. a soluble, biologically active fom of SLF (sSLF) also exists, and is the

result of alternative splicing (Flanagan et al.. 1991; Huang et al.. 1992). Soluble Steel Factor

is produced if the SLF rnRNA ûmscnpt is spliced to include exon 6, which is not present in

the transcnpt encoding for the membrane-bound fonn of SLF (mSLF). This exon codes for

an extracellular region, proximal to the ce11 membrane. which can be recognized by chyrnases

and cleaved at a consensus site. thereby producing the soluble form of Steel Factor (Longley

et al.. 1997). Figure 2 compares the two biologically active foms of Steel Factor. The

regulation of expression of these two forms of SLF is believed to be tissue-specific. For

exarnple. in spleen and bone rnarrow it appears as though the expression of both forms is

relatively equal. while in the brain the expression of sSLF is about 100 tirnes greater (Huang

et al.. 1992). The reasons for the different expressions of the different forms are still unclear.

While both Forms are biologically active. it has been experimentally demonstrated that there

are some physiologically distinct fùnctions between the two forms of the ligand. Toksoz and

colleagues dernonstrated in vitro longer-term suppon of human hematopoetic cells on stroma1

cells expressing membrane-bound. than by soluble SLF (Toksoz et al.. 1992). Additionall y.

genetic evidence exists that strengthens the hypothesis that only membrane-bound Steel

Factor can support erythropoiesis (Kapur et al.. 1998). Steel-Dickie fi mice provide strong

in vivo support of the importance of mSLF. In these mice. stroma1 cells secrete only sSLF

but do not express mSLF. Such animals have significant hemopoietic and g e n ce11 defects.

lack coat pigmentation and almost completely lack dermal mast cells (Brannan et al.. 199 1 :

Flanagan et al.. 199 1). These observations suggest that. although the soluble form of Steel

Factor is sufficient for viability of mice. the membrane-bound form of SLF is crucial to the

Figure 2. Ceneration of membrane-bound and soluble Steel Factor. Alternative mRNA

splicing results in the formation of either soluble or membrane-bound Steel Factor. The

inclusion of exon 6 in the mRNA transcript results in the production of the SLF protein

containing a cleavage site in the extracellular domain that c m be recognised by proteolytic

enzymes. Cleavage at this point gives rise to the soluble form of SLF. Membrane-bound

Steel Factor is produced when exon 6 is spliced out thus deleting the proteolytic cleavage

site.

SLF without SLF with

proteolytic cleavag e site -*

Transmembrane - Dornain

Soluble SLF

development of hematopoietic progeniton. melanocytes and germ cells and is. as such. the

physiologically more relevant form.

D. Signal Transduction by the SLF-KIT Complex

1. Mechanism

Signal transduction through the c-Kit receptor occurs by a mechanism that is analogous to

other receptor tyrosine kinases (RTKs). The first step in this chain of signalling events is the

high affinity binding of SLF to the c-Kit receptor. It has been experimentally detennined that

the N-terminal part of the c-Kit ectodomain is the site at which the high aflinity binding

occurs (Blechrnan et al., 1993). Not only does the deletion of the third Ig domain result in an

obvious decrease in ligand affinity. but it was also demonstrated that monoclonal antibodies

(mAbs) which acted as ligand cornpetiton were in fact interacting with the three N-terminal

tg domains (Blechman et al.. 1993). SLF binding results in very rapid c-Kit receptor

homodimerization. Two possible models have been suggested to explain how this occun.

with some experimental support for each. One mode1 proposes that c-Kit dimerization

occurs oniy after the binding of dimerized ligand ( L w et al.. 1992; Philo et al.. 1996). while

the second mode1 proposes that monomeric SLF induces conformational changes to the

receptor which results in the interaction of the fourth Ig domain of c-Kit (Blechrnan et al..

1995). However. Lemmon and CO-worken have provided some evidence that the fourth Ig

domain rnay not be absolutely required for receptor dimerization (Lemmon et al.. 1997).

Upon dimerization. the c-Kit receptor undergoes extensive tyrosine autophosphoqdation.

thereby creating binding sites for numerous Src-homology 2 (SH2)-containing signal

transduction molecules (Heldin. 1995). Some of the proteins which become associated with

the phosphorylated receptor include phosphatidylinositol 3-kinase (P13K). PLCy, Stat 1. the

GTPase activating protein and members of the Src family kinases including Src. Lyn and Fyn

(reviewed by L i~ek in . 1 999).

2. Physiological roles of SLF-induced Signal Transduction through Kit

Steel Factor induced c-Kit signalling has been reported to be critical in mediating a wide

variety of biological outcomes in numerous ce11 types. This interaction has been

demonstrated to play a role in hematopoietic ce11 survival. proliferation and differentiation.

cell adhesion and migration, and meianogenesis and grnetogenesis (reviewed by Ashrnan.

1999). It is also possible that SLF and c-Kit influence other. as yet unknown. biological

outcomes in adult life but embryonically lethal W or SI alleles make these roles more difficult

to determine.

Steel Factor has k e n demonstrated to be a potent growth factor. albeit synergistically

with other factors and cytokines. not only for hematopoietic stem cells but also for nurnerous

differentiating hematopoietic lineages (reviewed by Broudy, 1997). Numerous studies have

supponed the hypothesis that SLF is critical for hematopoiesis in vivo. For exampie. when

added to an enriched population of cells containing hematopoeitic stem cells. SLF was

demonstrated to accelerate the entry of the ceils into ce11 cycle (Leary et al.. 1992). KitA cells

in this population display colony formation activity in the presence of SLF (ikuta and

Weissman. 1992). Funhermore. Steel Factor alone can transiently maintain the long-terni

survival ability of Lin'. Sca* populations of murine hematopoietic cells in vitro (Li and

Johnson, 1994). However, Steel Factor alone was unable to allow for the self-renewal of

stem ceils (Li and Johnson, 1994). Additionaily, hematopoirtic stem cells are capable of

surviving on stromal cells in viiro even in the presence of ACKZ. an antagonistic anti-c-kit

receptor antibody, suggesting that other cytokines are produced that are capable of promoting

stem ce11 survival in the absence of Steel Factor (Wineman et al., 1993). Synergy between

SLF and other various cytokines can have potent effects on stem cells. For example spergy

with IL-6 is crucial in promoting growth of mast cells (Saito et al.. 1996) and other factors.

like I L 4 are required for full maturation (Tom et al.. 1998).

There is evidence to suggest that Steel Factor c m also mediate adhesion of

hematopoietic cells to bone marrow stromal cells. Ce11 adhesion mediated by SLF is believed

to occur by two mechanisms. First. the binding of SLF by c-Kit' cells may mediate direct

attachent (Kaneko et al.. 1991). thereby helping to anchor the hematopoietic cells to the

fibroblasts. There is some evidence that this anchoring does not require c-Kit receptor kinase

activity. as mast cells which are derived frorn WIF" mice and thus lack c-Kit receptor kinase

activity. are still able to adhere normally to fibroblasts (Adachi et al.. 1992). Secondly.

signalling by SLF through c-Kit has been demonstrated to increase the avidity of

hematopoietic and mast ce11 integrins VLA-4 and VLA-5 for extracellular fibronectin or

VCAM- 1 (Dastych and Metcalfe. 1994; Kinashi and Springer, 1994; Kovach et al.. 1995).

Steel Factor has also k e n shown to be involved in hematopoietic stem ce11

mobilization fiom the bone marrow to peripheral blood and spleen. aithough the mechanisms

of this M c k i n g activity are not yet known (Fleming et al.. 1993: Yan et al.. 1994). A

similar result is seen when mice are injected with antibodies against VLA-4 or its receptor

VCAM-1 suggesting that VLA-4 on hematopoietic cells and VCAM-1 on supporting cells

play a role in ce11 traff~cking in vivo (Papayannopoulou and Nakamoto. 1993).

As discussed, W W and mice have severe mast ce11 deficiencies. suggesting that

SLF is critical for mast ce11 production. SLF has also been shown to promote mast ce11

survival. proliferation and maturation in vivo (lemura et al.. 1994; Tsai et al.. 1991).

Furthetmore. SLF promotes mast ce11 chernotavis (Meininger et al.. 1992). adhesion (Dastych

and Metcalfe. 1994) and secretory Function (Columbo et al.. 1992: Wenhil et al.. 1997).

One component of eukaryotic cell membranes is phosphatidylinositol. a phospholipid that

can be phosphorylated at a nurnber of its free hydroxyl groups. Derivatives of

phosphatidylinositol. or phosphoinositides. play an important role in a multitude of cellular

processes. The enzymes that are responsible for the phosphorylaiion of

phosphatidylinositides are called phosphoinositide kinases (PI3Ks) (reviewed by F m a n et

al.. 1998: Wymann and Pirola 1998). Specifically. PU-kinases catalyze the transfer of the y-

phosphate group of ATP to the D3 position of the phosphoinosides.

There are three classes of phosphoinositide kinases. but only class I PI3-kinases are

known to be activated by signalling through receptor tyrosine kinases. like c-Kit. These

kinases are heterodimers. composed of a 1 10-1 20 kDa catalytic subunit (typically p 1 10) and a

50-100 kDa regdatory subunit (typically p85) (Fruman et al.. 1998: Wymann and Pirola.

1998). The p85 subunit is composed of a Src homology 3 (SH3) domain. a breakpoint-

cluster-region homology (BH) domain flanked by two proline-rich regions and two SH2

domains separated by an inter-SHZ region (Escobedo et al., 199 1 : Otsu et al., 199 1 ; Skolnik

et al., 1991 ). None of these regions contain any catalytic activity. The SH3 domain is

involved in self-association (Kapeller et al., 1994). the proline rich regions are binding sites

for a number of SH3 domain-containing proteins that associate with P13K, like Fyn (Prasad et

al., 1993) and Lck (Kapeller et al.. 1994). and the BH domain specifically interacts with Rho

family proteins (Tolias et al.. 1995; Zheng et al.. 1994). The SH2 domains of p8S bind

specifically to phosphotyrosyl residues, as on the intracellular region of c-Kit. The inter-SHZ

domain is necessary and sufficient for interaction with the p l I O catalytic subunit (Hu et al..

1993). p85 cm not only be phosphorylated afier recruitment to phosphorylated tyrosine

residues on receptor tyrosine kinases. but it cm also be phosphorylated by the catalytic

subunit. which possesses intrinsic protein serine kinase activity (Carpenter et al.. 1993).

One of the best-snidied downstream consequences of PD-kinase activation is the

activation of the protein kinase AktPKB. A simplified representation of this pathway is

show in Figure 3. There are two PD-kinase dependent steps for the activation of Akt: the

pleckstrin homology (PH) domain of Akt promotes its translocation to the ce11 membrane and

binds to phosphatidylinositol 4.5-bisphosphate (P1P2) (a product of P13K) and the

phosphorylation of threonine 308 and serine 473. required for full activation of Ah. requires

phosphoinositide-dependent kinases (PDKI and PDK2) (Alessi et al.. 1996: Franke et al..

1997; Klippel et al., 1997). In tum. activated Akt can phosphorylate and inactivate

glycogen-synthase-kinase 3 (Cross et al.. 1995) and the pro-apoptotic protein BAD (Datta et

al.. 1997). Another target of activated Akt is integrin-linked kinase. It has k e n shoun to be

activated by phosphatidylinositol 3.4.5-triphosphate (PIP3) and to phosphorylate Akt on

Figure 3. Akt activation by PH-kinase. Two crucial events of Akt activation are

dependent on PI3-kinase: Akt transIocation to the plasma membrane occurs following PIP?

binding by the PH domain, and phosphorylation at theonine 308 and serine 173 requires

phosphoinositide-dependent kinases (PDK 1 and 2). Activated Akt cm inactivate the pro-

apoptotic factor BAD and glycogen-synthase-kinase 3 resulting in ce11 survival.

Phospholipid

phosphatase

1 \

. . . . . . . ....... ....... ....... . . . . . . . . . . . . . . 1 PDKl ,-* T308 . . . . . . . ....... ....... ....... Aktt PKB

.... . . . ....... I . . . . . . . . . . . . . .

Active Akt

Proliferation L Protein synthesis

senne 473 (Hannigan et al., 1996). Both PI3-kinase activity and calcium influx have been

demonstrated to be important for c-Kit receptor endocytosis and the inhibition of these two

signais disrupts the earliest stages of ligand-mediated receptor intemakation (Gommerman

et ai., 1997).

F. Phospholipase C-y

Another molecule that is recruited and activated upon ligand binding by the c-Kit receptor is

phospholipase C-y (Li et al.. 199 1 ).

PLC-y is a 145 kD single-peptide enzyme which is comprised of several distinct

domains. It contains two PH domains. One of these PH domains is split by two SH2

domains and a SH3 domain. whereas the second PH domain is intact (Mayer et al.. 1993).

Both PH domains are functional in binding phospholipids (Garcia et al.. 1995). The catalytic

domain of PLC. which is separated into two regions. is dependent on ca2+ for activity (Clark

et ai.. 199 1 ). PLC-y also contains a protein kinase C homology-2 domain that binds ca2*

(Essen et al.. 1 996).

Upon c-Kit autophosphorylation. receptor phosphotyrosines act as hi&-affinity

binding sites for PLC SH2 domains (Rottapel et al., 199 1). Interaction with phosphorylated

receptor domains increases phosphorylation of PLC-y senne and tyrosine residues. thereby

activating the enzyme (Wahl et al.. 1992). Activated PLC-y catalyzes the hydrolysis of PIPz

to generate the secondary signalling messengen inositol 1.4.5-triphosphate (IP,) and

diacylgiycerol (DAG). IP3 acts on receptors on the endoplasmic reticulum (ER) that causes

the release of ER-stored ca2' (reviewed by Bemdge et al.. 1998). Low ER calcium Ievels

induce extracellular ca2' entry into the ce11 via store-operated calcium channels (Berridge

and Irvine. 1989). hcreases in intracellular calcium levels is involved in cellular

proli feration and metabolism (reviewed by Bemdge et al .. 1 99 8). Diac ylgl ycerol activates

protein kinase C (PKC). Figure 4 is a simplified representation of some of the downstrearn

effects of PLC-y activation.

G. Thesis Rationale

The c-Kit receptor and its ligand Steel Factor are involved in initiating a complex series of

downstream signals. There is some redundancy in the cellular responses elicited by the

molecules recruited to the c-Kit receptor. While both the soluble and the membrane-bound

forms of Steel Factor are biologically active. research demonstrates that mSLF is the more

important form in vivo. Therefore. understanding the signalling that occurs when the

different ligand foms activate the receptor may be critical to the understanding of the

biological function and relevance of both forms of Steel Factor. Furthemore. knowledge

about the specific sipalling molecules involved in the activation of c-Kit* cells. like mast

cells. is critical if we hope to control mast cell pathologies. This thesis attempts to examine

the requirement of PD-kinase and PLC-y activation following stimulation by soluble Stcel

Factor or membrane-bound Steel Factor.

Figure 1. Basic mecbanisms of cellular calcium sipaliing. PLC-y activation results in the

production of IP3 and DAG. IP3 acts on recepton on the endoplasrnic reticulurn. causing the

relrase of calcium from the ER. Low calcium levels in the ER signal store-operated calcium

channels on the plasma membrane. allowing calcium to enter the ce11 from the extracellular

milieu. Higher cytoplasrnic calcium levels may trigger cellular metabolism and prolifention.

Excessive cytoplasmic calcium may lead to abnormally high levels of calcium k i n g taken up

by the mitochondria resulting in an activation of apoptosis.

Mitochondnon

II. MATERIALS AND METHODS

A. Ce11 Culture

32D cells (gift from Dr. Joel Greenberger. Pittsburg, PA) are an IL-3 dependent c-Kit

negative rnyelomonocytic ce11 line. The complementq DNAs (cDNAs) (obtained fiom Dr.

R. Rottapel) for the KitWT. YF719. and YF728 were transfected as previously described

(Gommemian et al.. 1997). Cells were routinely grown in RPMI (Gibco/Life Technologies.

Inc. Burlington, Ontario) supplemented with 10% fetal bovine serum (FBS) and 2%

supematant from WEHI-3 cells as a source of IL-3. Al1 ce11 cultures contained 55 pmol/L P-

mercaptoethanol and antibiotics (both Sigma Oakville. Ontario). l mg/mL of antibiotic

G418 (Gibco) was added to al1 cultures to select for transfectants.

Fibroblasts X9fD3 and SV SI^ (a gift fiom Dr. David Williams. Indianapolis. M) were

routinely grown in Du1 becco's modi fied Eagle's medium ( LiFe Technologies Inc. )

supplemented with 10% FBS and antibiotics (Sigma).

Bone marrow-derived mast cells were typically obtained tiom 4- to 6-week old

C57036 mice (Jackson Laboraties). Mice were sacrificed and marrow fiom the femur was

rinsed out with phosphate-buffered saline (PBS) containing 0.5% FBS. Cells were spun down

at 1250 rpm in a clinical centrifuge for 5 minutes and resuspended in 2 rnL ice-cold ACK. to

rupture red blood cells. and incubated on ice for 3 minutes. 5 rnL of cold PBS + 0.5% FBS

were added and cells were spun down. Cells were washed once more in PBS and then

resuspended in OPTI-MEM media containing 5% FBS. 55 pmoK P-mercaptoethanol.

antibiotics and 4% WEiii-3 supematant as a source of IL-3. WEHI-3 ce11 supematant was

added to the cells every second day and cells were passaged evex-y fourth day. Mast cells

were used d e r five weeks of culture up to a maximum of 10 weeks. During this time. the

ceils in culture stain positive for c-Kit and FceEU.

B. Reagents and Antibodies

Neomycin sulfate and oleic acid were obtained h m Calbiochem (La Jolla. CA). Bioactivity

was always assessed by 3~-thymidine incorporation. which was purchased from Mandel

(Guelph. ON). ITS (Insulin/Transferrin/Selenite) liquid media supplement was purchased

from Signa.

Antibodies used for immunoprecipitating and Western blottiny were used at

concentrations recomrnended by supplirrs. Thry include: rabbit anti-c-Kit and 4G 1 O anti-

phosphotyrosine (both Upstate Biotechnology. Lake Placid. NY). anti-PLC-yl. anti-PLC-y 1.

anti-phosphoAkt and anti-p85 (al1 Pharmingen. Mississauga. ON). Al1 secondary honeradish

peroxidase conjugated antibodies were used at 1:2500 and were obtained from Sigma.

C. Production of Recombinant SLF

Soluble SLF used in d l assays was produced as previously described by Gommerman et al.

(Gommerman et al.. 1997). Briefly. recombinant murine SLF was produced in soluble form

in Escherichia coli using the pFLAG.ATS isopropyl- l -thio-P-D-galactopyranoside (D'TG)-

inducible secretion expression vector (Invitrogen. Carlsbad. CA). This vector includes an

eight arnino acid N-terminal FLAG epitope (Interscience. Markham. Ontario). E. coli

containing the pFLAG.ATS plasmid were incubated ovemight at 37'C in LB broth with 100

@rnL ampicillin. This culture was then diluted 20-fold in fresh media and grown to an Aboo

of 0.4-0.5 before k ing induced with 0.033 g/L IPTG. The cultures were then incubated

ovemight at 37°C before they were centrifuged at 10.000 rpm for 20 min. The bacterial

supernatant was passed through a 0.22 micron filter and stored at -80°C with 1 mM CaC12

and 100 pM phenylmethylsylfonyl fluonde (PMSF). FLAG-SLF was purified by passing

supematants over a colurnn of Anti-FLAG Ml mouse monoclonal antibodies covaiently

attached to agarose gel. The column was first equilibrated with 30 mL PBS + 1 mM CaCl?.

Bacterial supematants were then passed over the M l column three times. The FLAG-SLF

fusion protein binding to the affinity column is ca2'-dependent: therefore. elution of FLAG-

SLF was achieved by adding EDTA. Six elutions with 1 mL of PBS + 2mM EDTA were

performed. These were collected. concentrated and assayed for bioactivity .

D. Immunoprecipitation and Western Blotting

For analysis of PLC-y recruitment in 32D infectants. 32D cells were starved for 6 hours in

RPMI + ITS Liquid Media Supplement + 0.05% BSA. Cells were spun down and

resuspended at a concentration of 1 x 10' cells/mL. A Zx solution of RPMI + ITS + SLF (2

pg/mL) preheated to 3PC was added to an -qua1 volume of cells and incubated for 5 minutes

in a 37°C water bath. RPMI + ITS alone was used as an unstimulated control. After

incubation. samples were removed and placed on ice. Cells were washed in ice-cold PBS

twice and resuspended in a bufTer containing 50 mmoVL Tris (pH 7.0). 1% NP-40. 50

mmoK EDTA. protease inhibitor cocktail (Roche. Laval. Quebec). phosphatase inhibitor

cocktail (Sigma). 200 p n o K sodium orthovanadate, 20 rnrnol/L NaF and 1 rnmollL PMSF.

Cells were lysed by 3 rounds of freezing in a dry ice/ethanol bath and thawing, and pelleted in

a microcentrifuge at 10,000 rpm for 20 minutes at 4°C. The supernatant was recovered and

50 pL of a 50% protein A slurry (AmcnhamPharmacia Biotech, Quebec). that was precoated

for one h o u with anti-PLCyl antisera (Pharmingen) was added. The samples were incubated

2 hours at 4°C with rotation and were then washed 3-5 times with lysis buffer. Afier the final

wash. the beads were resuspended in gel-loading buffer and boiled for 5 minutes. Samples

were resolved on a 6% polyacrylamide gel. The proteins were transferred to nitrocellulose

and blocked in Tris-buffered saline + 0.1% Tween-20 (TBST) containing 1% grlatin (Bio-

Rad. Mississauga. Ontario). The membrane was incubated wfth anti-phosphotyrosine

antibody at 0.5 pg/mL in a 1% gelatin-TBST solution ovemight at room temperature. The

membrane was washed 3 times and incubated with a secondary anti-mouse antibody

conjugated to horseradish peroxidase (Sigma) at a dilution of 12500 for 1 hour at room

temperature. The membrane was washed three times and then visualized using

chemiluminescence reagents (NEN Life Science Products. Guelph. ON). Blots were stripped

and repmbed with anti-PLC-y1 at 1:2000 to ven. even protein loading.

For experiments requiring stimulation of BMMCs by mSLF. X9/D3 fibroblasts were

plated in 6 well plates at 1 x 106 cells/well and allowed to adhere ovemight. SU SI^

fibroblasts were used as a control. BMMCs were added to the fibroblasts and the plates were

spun in a clinical centrifuge at 1250 rpm for 2 minutes and then incubated at 3PC for the

required tirne. Following stimulation. the plates were placed immediately on ice. Media was

gently removed and lysis buffer was added directly to the wells. tmmunoprecipitation was

canied out as described above.

E. Stimulation assays

For bioassays with soluble SLF, 32D infectants were washed 3 times in RPMI + 0.5% FBS.

BMMCs were washed and plated in RPMl + ITS + 0.1% BSA. Cells were plated with 150

ng/mL sSLF at a density of 2.5 x 104 cells/rnL. For irnmobilized anti-c-Kit assays. 96-well

tlat-bottom plates were coated with 10 pg/mL of a secondary mouse anti-rat monoclonal

antibody (Jackson, Mississauga ON) ovemight at 4°C. Excess antibody was then washed

from the plate and the plate was blocked with PBS + 1% FBS for 2 hours at 3TC. Varying

concentrations of ACK-2. an anti-c-Kit antibody, in PBS-FBS were added. Plates were

incubated for 2 hours at 4*C and washed several times. CeIls were then plated at 1.5 x 104

cells/mL. For CO-cultures with fibroblasts. X9/D3 cells and SIISI'' cells were treated with

Mitomycin C ( 5 pg/mL) for 2 hours at 37°C to arrest fibroblast division. washrd 3 tirnes with

PBS. trypsinized. counted and then plated at a concentration of 1 x lo4 celllwell in 96-well

plates that had been pre-coated with 0.1% gelatin (Sigma). Fibroblasts were allowed to

adhere 4 to 6 hours before plating 32D cells or BMMCs. In al1 cases. 32D infectants and

BMMCs were starved for 5-6 houn before plating. When used. neomycin sulfate was pre-

incubated with the cells for 20 minutes. whereas pre-incubation with oleic acid and vitamin E

was 1 hour. In ail cases. d e r 18 hous of stimulation. 1 pCi of 3~-thymidine was added to

each well for 6 hours. Cells were harvested and incorporated radioactivity was detemined by

scintillation counting. The degree of stimulation was determined by calculating the ratio of

radioactivity incorporated by the infectants in the presence of the SLF-expressing stroma1

cells to radioactivity incorporated by the infectants in the presence of stromal cells not

expressing SLF &er subtracting counts obtained with the stroma1 cells alone. according to

the formula:

This formula takes into account variations in background betweeen ce11 lines and also any

non-specific support of the 32D cells by stomal cells not related to SLF. Plate-coated ACK-2

results were plotted as a rneasure of incorporation with both secondary and ACK-2 over

incorporation with secondary alone.

F. Viability assays

32D iransfectants were washed and starved. as described above. and plated in 96-well tlat-

bonom plates with either 150 ng/mL sSLF. 2% WEHI-3 supernatant or with no growth factor

present. Cells were incubated ovemight at 37OC. Following incubation. the cells counted in

the presence of ûypan blue and scored as viable if they could exclude the dye.

G. Flow cytometry

1 x 106 BMMCs. 32D KitWT and KitYF728 cells were washed 2-3 times in PBS + 0.5%

FBS and resuspended in the same solution. An anti-c-Kit antibody. FITC IB8 (Pharmingen)

was added to the cells at 1: 100. Cells were also incubated with a FITC rat IgG2t,.rc isotypr

control. Al1 samples were incubated on ice for 60 minutes and then washed 3 times with cold

PBS + FBS. Cells were immediately andyzed for c-Kit expression levels by 80w cytometry.

H. In vivo studies

Groups of four 4- to 6-week old C57iB6 mice were fed an 11% Breeder's diet for at least one

week pior to the start of the experiment and hair from a dorsal patch of skin was removed

with the exfoliant 'NeetTM' one day pnor to the start of the experiment. Mice were treated 3

times daily for 4 days with cream composed of 80% polyethylene glycol (PEG 1000). 10%

water. and varying concentrations of oleic acid. vitamin E or neomycin sulfate. Jojoba oil

made up the rest of the oil component in oleic acid or vitamin E treatments alone. AHer 4

days of treatment. the rnice were sacrificed and the treated skin was removed. fixed in

formalin. embedded in parafin. sectioned. and stained with toluidine blue. a mast ce11

specific stain. The slides were blinded and randornized pnor to enumerating mast ceIl

drnsities by morphometry.

III. RESULTS

Some of the data presented in this chapter has been published in:

Gommerman. J.L., Sittaro. D., Klebasz. N.Z., Williams. D.A., and Berger. S.A. (2000)

Differential stimulation of c-Kit mutants by membrane-bound and soluble Steel

Factor correlates with leukemic potential. Blood, 96: 3 7 3 - 3 7 4 2

A. Kit YF728 receptors do not induee Steel Factor-stimulated PLC-y recruitment

Residue Y 728 in murine c-Kit is part of a short sequence of amino acids that closely matches

the consensus sequence identified for the PLC-y SH2 binding domain. It has been previously

demonstrated that PLC-y is recruited to the c-Kit receptor and becomes tyrosine

phosphorylated after stimulation by sSLF (Rottapel et al.. 199 1 ). Additionally. 32D YF728

crlls have bcen demonstrated to be unable to mobilize calcium in responsr to SLF

(Gommerman et al.. 1997). To confimi the involvement of Y728 in the activation of PLC-y.

the tyrosine phosphorylation of PLC-y1 in Kit YF728 mutants, as cornpared to KitWT cells

in response to sSLF. was rxarnined. To do this. 32D cells rxpressing either KitWT or

KitYF728 were stimulated with sSLF for 5 minutes. PLC-y1 was immunoprecipitated and

then analyzed by Western blotting with 4G 10. an anti-phosphotyosine antibody. PLC-y 1

from sSLF-stimulated BMMCs was also analyzed as an additional control. As s h o w in

Figure 5A. BMMCs express a higher c-Kit receptor level than do 32D cells. This difference

in c-Kit expression is likely the reason for the geater degee of PLC-y1 tyosine

phosphorylation in response to sSLF by BMMCs than by 32D KitWT cells, as shown in

Figure SB. No additional tyrosine phosphorylation was seen in lysates from 32D-KitF728

cells in response to sSLF. This result supports the hypothesis that the tyrosine at position 738

on the murine c-Kit receptor is required for PLC-y recniitment and activation. and thus

YF728 mutants are unable to activate PLC-y.

Figure 5. SLF-stirnulated tyrosine phosphorylation of PLC-y in BMMCs and 32D

KitWT cells but not 32D KitYF728 cells. (A) Sudàce expression of the c-Kit receptor on

3?D KitWT and KitYF728 cells was compared with BMMCs. Cells were incubated with

FITC 288. an anti-c-Kit mtibody and analyzed by flow cytometry. (B) PLC-y1 tiorn

BblMCs. 32D KitWT and 32D-KitYF728 cells stimulated with sSLF for 5 minutes was

imrnunoprecipitated. resolved on a 6% SDS-PAGE gel. transferred to nitrocellulose and

blotted with an anti-phosphotyrosine antibody. The blot was stripped and reprobed with an

anti-PLC-y 1 antibody.

IP: PLC-y

B BMMC KitWT YF728

SLF: + - + - + blot: 4Gl O

B. KitWT, KitYF719 and KitYF728 but not KitYF719NF728 32D cells respond to sSLF

Both PI3-kinase and PLC-y have been implicated in growth factor receptor-mediated

mitogenesis (Valius and Kazlauskas. 1993). Gommerman and CO-workers (Gommerman et

al.. 2000) have previously demonstrated that. in response to sSLF. the 3ID-KitWT and the

two single mutant cell lines are able to proliferate and incorporate thymidine equally wrll. In

contrast. the 32D KitYF719NF728 double-mutant cclk failcd to be stimulated by sSLF.

similar to untransfected c-Kit' 32D cells. This result could be interpreted as eithrr a loss of

ce11 viability or simply a growth arrest of the cells. In order to distinguish bebveen these two

possibilities. ce11 viability atter a 24 hour incubation in the presence or absence of soluble

SLF was e~arnined. All cell lines were also stimulated with 2% WEHI-3 ce11 supernatant.

which maintains excellent ce11 viability. as an additional positive control. The proportion of

viable cells was determined by a trypan blue exclusion viability assay. As shown in Figure 6.

sSLF maintains the viability of 31D cells expressing the WT receptor or recepton with either

the YF179 or YF728 mutations. In contrast. cells expressing the double mutant do not

remain viable in sSLF. Al1 ce11 lines demonstrated a viability of 93% or greater in the

presence of 2% WEHI-3 supernatant. These data are therefore consistent with the

requirement for either PI3-kinase or PLC-y activation for swival and mitogenic signals.

These results are also in agreement with those of Valius and Kazlauskas who demonstrated

that either the PI3-kinase or the PLC-y binding sites were sufficient to restore PDGF-

mediated mitogenesis. but that receptors bearing mutations at both of these sites were

mitogenically inert (Valius and Kazlauskas. 1993).

Figure 6. Stimulation of 32D infectants with sSLF. 32D infectants were incubated with

sSLF (empty bars) and percent viability ivas determinrd by sconng ability to exclude trypan

blue. Cells were aiso incubated with WEHI-3 cell supernatant (hatched bars) as a positive

control and without factor (dark bars) as a negative control.

C. Response of KitYF728 receptors to membrane-bound Steel Factor is impaired

Given that the KitWT and single-mutant receptor ce11 lines were capable of being

rnitogenically stimulated by sSLF. their stimulation by mSLF was next examined. Ln other

experiments. Gommerman and Berger (unpublished) have demonstrated mitogenic

stimulation of 32D KitWT and KitYF719 cells CO-cultured with NIH 3T3 tibroblasts

ex pressing mSLF. However. NIH 313 fibroblasts not only express mrm brane-bound S LF

but they also secrete the soluble form. To overcome this dificultly. X9/D3 stroma1 cells

were used as a source of mSLF. These cells were generated by transfecting SLF-negative

SI/SI'' cells with an expression vector encoding a form of murine SLF that produces only the

membrane bound and not the soluble form of the ligand. X9D3 and SVSI" cells were treated

with Mitornycin C to arrest ce11 division and cells were seeded on gelatin-coated plates.

which were found to aid fibroblast adhesion. Appmximately six hours later. KitWT.

KitYF719. KitYF728 or uninfected 32D cells were added. As shown in Figure 7. CO-culture

on X9/D3 cells of both K i t W and KitYF719 32D infectants resulted in 7- to 8-fold çreater

thymidine incorporation when compared to incorporation levels obtained with SLF-negative

SVS~" CO-cultures. after 24 hours. Thymidine incorporation levels of fibroblasts alone were

typically 9000 to 10000 cpm. whereas CO-cultures resulted in 12000 to 19000 cpm depending

on the ce11 type, and showed little variation fiom expenment to experiment. In contmst the

KitYF728 32D infectants were stimulated at most 3-fold by X91D3 cells above that seen

using SLF-negative SVSI" cells as the stimulus. Longer cotulture experiments of this nature

were not possible as the Mitomycin C-treated fibroblasts begin to Iose the ability to adhere to

Figure 7. Stimulation of 32D KitWT and KitYF719 but not KitYF728 cell lines with

mSLF on X9D3 stroma1 cetls. 32D infectants were CO-cultured with mSLF-expressing

X9/D3 stromal cells. KitWT. KitYF719, KitYF728 and 32D uninfected ce11 were incubated

with X9/D3 cells overnight followed by a 6 hour 'H-thpidine pulse. Cells were then

harvrsted and thymidine incorporation was determined by scintillation counting. Fold

stimulation represents stimulation of cells on X9/D3 cells compared with stimulation

observed on SLF-negative parental SIISI" cells. Error bars represent the standard error

determined fkom triplicate rneasurements. Similar results were obtained in three separate

experiments.

KiWT KitY F719 KitY F728

the plates after 36 to 40 hours. This expenment demonstrated that. although 32D KitYF728

cells are Fully stimulated by sSLF, these cells are poorly stimulated by mSLF. suggesting that

PLC-y activation may be critical for responding to the membrane-bound form of SLF.

D. KitWT and KitYF719 receptor but not KitYF728 receptors respond to plate-bound anti-c-Kit an tibodies

An altemate method that mimics stimulation by mSLF is the use of plate-bound ACK-2. a c-

Kit-specific antibody raised in rats (Kurosawa et al.. 1996). This fom of stimulation. which

is not complicated by the presence of other cellular factors. was therefore used to investigate

the response of cells bearing WT and mutant recepton. Tissue culture plate wells were pre-

coated with an anti-rat secondary antibody pnor to the addition of ACK-2 anti-c-Kit

antibody. Whrn both the secondary antibody and the anti-c-Kit antibody are plated togethrr.

32DKitWT and KitYF719 cells respond to the plate-bound antibodies in a concentration-

dependent fashion with maximal stimulation of 8- to 9-fold above background. as shown in

Figure 8. in contrast. KitYF728 cells exhibited stimulation no more than 2-fold above

background. Clearly. although 32D KitYF738 cells respond to sSLF. they fail to fully

respond to plate-bound anti-Kit antibodies. Given that this system mimics mSLF expressed

by fibroblasts and stornal rells. this result is consistent with a requirement for PLC-y

activation after stimulation with mSLF or immobilized ligand.

Figure 8. Stimulation of 32D KitWT and KitYF719 cell lines but not KitYF728 cells

with plate-bound anti-c-Kit antibody. Flat-boaom 96-well plates were tïrst coated with

mouse-anti-rat antibody followed by varying concentrations of anti-c-Kit antibody. KitWT

( filled), KitY F7 19 (hatched). KitY F E 8 (empty) and 32D uninfected cells (stippled) were

then added to the plates and incubated for 18 houn. Crlls were pulsed for 6 hours and

thymidine incorporation was determinrd by scintillation counting. Stimulation of 32D cells

with both anti-c-Kit antibody and secondary antibody was measured as fold stimulation over

counts obtained from stimulation with secondary antibody aione. Error bars represent the

standard emor detemined from tnplicate measurements. Similar results were obtained in

three separate experiments.

E. Neomycin sulfate inhibits stimulation of KitYF719- but not KitWT- or KitYF728-expressing cells by soluble Steel Factor

The observation that the YF719NF728 double c-Kit mutant does not mediate a suvival or

mitogenic signal in response to sSLF suggests that in the absence of the recniitment of PI3-

kinase, PLC-y activation is required for ce11 support. To fùrther confirm this requirement.

cells expressing either the KitWT or the single mutant c-Kit receptor were stimulated by

sSLF in the presence or absence of neomycin sulfate. an antagonist of PLC activity (Gabev et

al.. 1989). As shown in Figure 9. neomycin concentrations as high as 500 pmollL have linle

effect on stimulation by sSLF of 32D cells expressing the KitWT or KitYF728 receptor. In

contrat. 32D cells expressing KitYF719 are inhibited by neomycin sulfate in a dose

dependent manner. The ICso is approximately 100 poVL. which is a concentration in a

similar range as previously reported to be inhibitory for PLC-y in permeabilized rnast cells

(Cockcroft et al.. 1987). These data support the hypothesis that in the absence of PJ3-kinase

recruitmcnt. PLC activation is required for a full mitogcnic signal by sSLF.

F. Neomycin sulfate inhibits stimulation by mem brane-bound Steel Factor or imrnobilized anti-c-Kit antibodies

To Further test the hypothesis that PLC-y activation may be critical for responding to the

membrane-bond form of SLF. or immobilized ligand in the fom of anti-c-Kit antibodies.

the effect of the PLC antagonist neomycin sulfate on KitWT cells when stimulated by either

of these two forms of imrnobilized ligand was exarnined. As shown previously in Figure 9.

Figure 9. Neomycin sulfate inhibits stimulation of KitYF719- but not KitWT- o r

KitYF728-expressing cells. 32D Kit WT (squares). KitYF7 19 (circles) and KitY F718

(triangles) cells wrre incubated with sSLF and varyiny concentrations of nromycin sulfate

for 18 hours. Celis were then pulsed with 3~-thymidine for 6 hours and thymidine

incorporation was determined by scintillation counting. Percentage control refers to counts

measured in the absence of neomycin sulfate. Error bars represent the standard error

determined from triplicate measurements. Similar results were obtained in three separate

experiments.

10 100

neomycin sulfate (PM)

the addition of neornycin sulfate to KitWT cells stimulated by sSLF had only a slight effect

on ce11 proliferation and only at the highest concentrations of neomycin sulfate. in contrast.

neomycin sulfate specifically inhibits the ability of fibroblasts or immobilized anti-c-Kit

antibodies to support these cells (Figure 10A and 10B). Furthemore. the ICjo in both cases

is approximately 100 p o V L which was the concentration required to inhibit stimulation of

YF719 cells by sSLF. These results provide tùrther evidence that PLC-y activation is

important for cells stimulated by mSLF but not sSLF.

G. Bone marrow-derived mast cells are stimulated by sSLF and X9/D3 cells but not SVS~'

As shown previously in Figure 5. mature bone marrow-derived mast cells express hi& levels

of the c-Kit receptor and are able. therefore. to recruit both PI3-kinase and PLC-y to the

phosphorylated receptor following activation by Steel Factor (Rottapel et al.. 199 1 ). Similar

to the 33D KitWT cells. BMMCs were stimulated with both sSLF and mSLF on X9/D3

stroma1 cells and proliferation measured by thymidine incorporation after 24 houn. As

indicated by Figure 1 1. BMMCs stimulated by sSLF and by mSLF-expressing X9D3

fibroblasts were able to incorporate thymidine. This was not so with BMMCs coîultured on

S I /S I~ fibroblasts. There was very little th-midine incorporation above background and

visually these cells were clearly not being supported by the SIISI'' fibroblasts. These data are

consistent with data from a number of groups which demonstrated that. in the absence of

other factors. mast cells require either soluble or membrane-bound SLF for survival and

proliferation (lemura et al.. 1994: Tsai et al.. 199 1).

Figure 10. Neomycin sulfate inhibits stimulation of 32D KitWT cells by mSLF and

immo bilized anti-c-Kit antibodies. 32D Kit WT cells were incubated on X9/D3 t i broblasts

(A) and on anti-c-Kit antibodies (B) in the presence of varying concentrations of neomycin

sulfate for 18 hours. Cells were then pulsed with '~ - th~mid ine for 6 hours and thymidine

incorporation was determined by scintillation counting. Percent control refers to

incorporated counts measured in the absence of neomycin sulfate. Error bars represent the

standard error determined from triplicate measurements. Similar results were obtained in

three separate experiments.

10 100 lm neomycin sulfate (PM)

10 100 1000 neomycin sulfate (PM)

Figure 11. Stimulation of bone-marrow derived mast cells by sSLF and mSLF.

BMMCs were incubated either in the absence of factor. with sSLF. on X9/D3 fibroblasts. or

on sl/s14 fibroblasts for 18 hours. Celis were then pulsed with '~-th~rnidinr: for 6 houn and

thymidine incorporation was determined by scintillation counting. Similar results were

obtained in at least three separate experiments.

H. Neomycin sulfate inhibits BMMC stimulation by mSLF but not sSLF

The observations in 32D Kit transfectants supportrd the hypothesis that PLC-y activation is

required for stimulation by mSLF but not sSLF. Further independent support was obtained

by studying the effects of neomycin sulfate, a PLC-y antagonist. on crlls expressing the

KitWT receptor. By extension. these results should be reproducible in murine BMMCs that

endogenously express the c-Kit receptor and at levels much higher than the various 32D

transfectants. Murine BMMCs were stirnulated by either sSLF or mSLF in the presence or

absence of neornycin sulfate. As shown in Figure 12A. evrn the highest concentrations of

neomycin sulfate used have practically no effect on BMMC thymidine incorporation when

the cells are stimulated by sSLF. In contrat. Figure 12B shows the efect of neomycin

sulfate on BMMCs CO-cultured on mSLF-expressing fibroblasts. Support of the mast cells by

the fibroblasts is inhibited by neomycin sulfate in a dose-dependent manner. The ICro is

approximately 100 pmol/L. the same concentration that was inhibitory in the 32D

transfectant studies. These data support the observations obtained in 33D cell lines and the

conclusion that PLC activity is required for stimulation of Kit- cells by mSLF.

I. lonomycin reversai of neomycin sulfate inhibition of BMMCs stimulated by mSLF

Activation of PLC-y results in a number of downstream signalling effects. The hydrolysis of

P1(4.5)P2 by PLC-y produces IPs and diacylglycerol (DAG). IP3 binds to receptors on the

endoplasmic reticulurn thereby causing the release of ~ a " h m the ER store. Low ER

Figure 12. Neomycin sulfate inhibits BMMC stimulation by mSLF but not sSLF.

BMMCs were shuia ted by either sSLF (A) or rnSLF-expressing fibroblasts (B) in the

presence of varying concentrations of neomycin sulfate for 18 hours. Crlls were then pulsed

with 3~-thymidine for 6 houn and incorporated counts were detemined by scintillation

counting. Percentage control refers to incorponted counts observed in the absence of

neomycin sulfate. Error bars represent the standard m o r detemined from triplicate

rneasurements. Similar results were obtained in three separate exprriments.

10 100 lm

Neomycin Sulfate (PM)

Neomycin Sulfate (PM)

calcium levels trigger a calcium influx into the ce11 via the store operated calicum channels in

the plasma membrane. lncreased levels of cytoplasmic calcium have been associated with

cell proliferation and growth. secretion. and metabolism (reviewed by Bemdg et al.. 1998).

In the absence of PLC activation. a calcium ionophone such as ionomycin can increase

cytoplasmic calcium levels by creating channels in the plasma membrane. allowing calcium

to enter the cell. Thus. if neomycin sulfate is acting by preventing the activity of PLC-y

thereby blocking its downstrearn effect of elevating cytoplasrnic calcium levels. ionomycin

might be able to reverse this etfèct. This hypothesis was tested by CO-culturing BMMCs with

mSLF-expressing X9/D3 stroma1 cells in the presence of neomycin sulfate. Increasing

concentrations of ionomycin were added. As show in Figure 13. in the absence of

ionomycin. neomycin sulfate inhibits suppon of BMMCs by X91D3 fibroblasts. However.

0.1 nM ionomycin is sufficient to completely restore BMMC viability. even in the presence

of neomycin sulfate. At hi& doses of ionomycin BMMC viability decreased. Excessive

calcium levels in the cell can result in cell death and this may be the reason for the decreased

viability at hi& concentrations. This result supports the hypothesis that neomycin sulfate is

specifically acting by inhibiting PLC-y and that this inhibition cm be reversed by the calcium

ionophore ionomycin.

J. Oleic Acid can inhibit BMMCs stimulated by mSLF but not sSLF

Inhibition of cellular bc t ions by long chain cis-unsaturated free fatty acids (FFA) has been

extensively studied in cytotoxic T lymphocytes.' Free fatty acids have been used to inhibit T

ceil activation, degrandation (Richieri et ai., 1990) and signalling (Stulnig et al.. 2000). It

Figure 13. Ionomycin restores neomycin sulfate-induced inhibition of BMMCs

stimulated by mSLF. Bone marrow-derived mast cells were stimulated by X9/D3

fibroblasts in the prexnce of 250 pg/mL neomycin sulfate and increasing concentrations of

ionomycin for 18 hours. Cells were then pulsed with 3~-thymidine for 6 hours and

thymidine incorporation was determined by scintillation counting. Percent control refen to

incorporated counts observed as compared to counts incorporated by cells stimuaied on

X9/D3 in the absence of neomycin sulfate. Emor bars represent standard error determined

from triplicated measurements.

has been demonstrated that FFA inhibit receptor-mediated calcium influx although the

mechanism of this inhibition is not yet completely known. One possibility is that oleic acid

inhibits store-dependent ca2' influx by inhibiting the PLC-y pathway. To examine this

possibility, BMMCs were stimulated with either sSLF or mSLF in the presence of absence of

oleic acid. Vitamin E was included in sorne samples as an antioxidant. As s h o w in Figure

14A. oleic acid with or without Vitamin E. had very little inhibitory effect on BMMCs when

they were stimulated by sSLF. In contrast. BMMCs stimulated by mSLF-expressing X9/D3

stroma1 cells were inhibited by 5 pM oleic acid and 10 FM oleic acid in the presencr of

Vitamin E (Figure l l B and 14C). At higher concentrations of oleic acid inhibition is

reversed. This is consistent with results obtained by Gamberucci et al. who observed a dose-

dependent inhibition of thapsigargin-induced ca2' intlux by oleic acid up to 5 pM and a

decrease in inhibition at higher concentrations (Gamberucci et al.. 1997a). These data

suggest that in BMMCs oleic acid ma:y exen an inhibiting effect by intefiering with the

activity of PLC-y or one of its substrates and affecting the release of ca2& from interna1

stores.

K. BMMC stimulation by mSLF-expressing X91D3 stroma1 cells phosphorylates c-Kit

To confirm c-Kit receptor phosphorylation afier stimulation with mSLF in primary BMMCs.

murine BMMCs were stimulated with either sSLF or by CO-culture with mSLF-expressing

X9fD3 for given time points. immunoprecipitated with an anti-+Kit antibody and analyzed

by Western blotting with an antiphosphotyrosine antibody. BMMCs were also CO-cultured

Figure 14. Oleic acid inhibits BMMC stimulation by mSLF but not sSLF. (A) BMMCs

werr stimulated by sSLF in the presence (squares) or absence (circles) of Vitiunin E. (B)

BMMCs were stimulated by mSLF-expressing fibroblasts to which varying concentrations of

oleic acid were added in the presence of 1 O pM Vitamin E. Oleic acid inhibition of BMMCs

stimulated by mSLF fibroblasts in the absence of Vitamin E is s h o w in (C). Cells were thrn

pulsrd with 'H-thymidine for 6 hours and incorporated counts were determined by

scintillation counting. Percentage control refers to incorporated counts observed in the

absence of neomycin sulfate. Similar results were obtained in three separate experiments.

10 20 3 40

Oleic Acid (FM)

Oleic Acid (PM)

O 25 5 10 20 40

Oleic Acid (PM)

on SVSI" cells as a negative control and malyzed similady. As shown in Figure 15A.

tyrosine phosphorylation of the c-Kit receptor was observed in BMMCs stimulated with

sSLF. Furthenore. upon CO-culture of BMMCs with X9D3 stromal cells for 15 and 30

minutes tyrosine phosphorylation of the c-Kit receptor was easily detected. No c-Kit tyrosine

phosphorylation was seen in either unstimulated BMMCs or in the BMMCs CO-cultured uith

SVSI' stroma1 cetls. Furthermore. tyrosine phosphorylation of the c-Kit receptor persisted for

at least 60 minutes upon stimulation with either sSLF or mSLF. as sho\çn in Figure l5B.

Only afier 120 minutes was there a signifrcant decrease in the phosphorylation of the receptor

resulting from stimulation by rither fom of die SLF ligand. These results confirm that

stimulation of murine bone marrow-derived mast cells by either sSLF or mSLF-expressing

cells does indeed result in c-Kit tyrosine phosphorylation. and that receptor phosphorylation

penists For at least one hour afier receptor-l igand interaction.

L. Neomycin sulfate and oleic acid do not affect mSLF-mediated c-Kit tyrosine phosphorylation

As shown in Figures 12 and 14. neomycin sulfate and oleic acid were both able inhibit

BMMC stimulation by mSLF-expressing stromal cells but not stimulation by sSLF. One

possibility suggested by these results is that the PLC-y pathway may be inhibited at some

point by these antagonists. While it is knowm that neomycin sulfate acts by binding PIPz

(Gabev et al.. 1989; Schacht. 1978). the substrate for PLC-y. the exact mechanism of the

observed oleic acid inhibition is not yet known. To try to determine where the PLC-y

pathway is being interrupted. the effect of neomycin sulfate and oleic acid on c-Kit receptor

Figure 15. BMMC stimulation by X91D3 stroma1 cells phosphorylates c-Kit. (A)

BMMCs wrre incubated with either X9/D3 or SV SI^ tibroblasts for 15 or 30 minutes.

Lysates tvere immunoprecipitated with an mi-c-Kit antibody and blotted with an anti-

phosphotyrosine antibody. C~nstimulated cells and cells stimulated with sSLF were negative

and positive controls respectively. The blot was stripped and reprobed with an anti-c-Kit

antibody. ( B ) tmmunoprecipitation and western blotting were carried out as described in

(A) but CO-cultures with X9/D3 tibroblasts or stimulation with sSLF was carried out for 30,

60. or 130 minutes. The blot was also stnpped and re-probed with an mti-c-Kit antibody.

BMMC on BMMC X9/D3 +sSLF

IP: anti-c-Kit

blot: anti-P-Tyr

blot: anti-c-Kit

blot: anti-P-Tyr

blot: anti-c-Kit

tyrosine phosphorylation was Tust examined. Murine bone marrow-derived mast cells were

stimulated on X9D3 stroma1 cells in the presence or absence of neomycin sulfate and oleic

acid, c-Kit was immunoprecipitated and then visualized by Western blotting with an

antiphosphoiyrosine antibody. BMMCs stimulated with sSLF and on SVSI'. as positive and

negative controls respectively. were analyzed similarly . As s h o w in Figure 1 6. stimulation

with either sSLF or by X9D3 induces c-Kit receptor tyrosine phosphorylation. However.

neither 1 m M neomycin sulfate nor 5 pM oleic acid affects the phosphorylation of the c-Kit

receptor. Thesr results suggest that the inhibitors are not acting by atTecting the

phosphorylation of the c-Kit receptor but are most likely atiecting a stcp krther dowmstream.

M.Neomycin sulfate and oleic acid d o not affect mSLF-rnediated PLC-y tyrosine phosphorylation

The next strp was to determine if neomycin sulfate or oleic acid rnight be interferhg with

PLC-y recruitment and phospholylation. This would be one possible explmation given the in

vitro bioassay observations. In order to test this possibility. BMMCs were stimulated for 30

minutes on mSLF-expressing X 9 D 3 fibroblasts in the presence or absence of neomycin

sulfate and oleic acid. Cell lysates were immunoprecipitated with an anti-PLC-y? antibody

and then analyzed by Western blotting with an anti-phosphotyrosini: antibody. BMMCs

stimulated by sSLF and unstimulated cells were the positive and negative controls

respectively. Lystates fiom X9/D3 fibroblasts alone were aiso anal-d. As shoun in Figure

17. PLC-y is phosphorylated upon stimulation with sSLF and by mSLF. In the presence of

500 pM neomycin sulfate and 5 pM oleic acid. concentrations at which significant inhibition

Figure 16. PLC antagonisis do noi affect c-Kit phosphorylation. BMMCs were

incubated with X9/D3 fibroblasts in the presence or absence of momycin sulfate or olric

acid. Lysates were immunoprecipitated with an anti-c-Kit antibody and membranes were

blotted with an anti-phosphotyrosine antibody. Cells stimulated with sSLF served as a

positive control. The blot was stripped and re-probed with an anti-c-Kit antibody.

blot: 4G10

4-. P-c-Kit

blot: anti-c-Kit

Figure 17. PLC antagonists do not affect PLC-y2 phosphorylation. BMMCs were

incubated with X9/D3 tibroblasts in the presence or absence of neomycin sulfate or oleic

x i d . Cell I ysates wrre imrnunoprecipitated with an anti-PLC-y7 antibody and the membrane

was blotted with an anti-phospho~~osine antibody. Cells stimulated with sSLF were used as

n positive control. The blot was stripped and re-probed with an anti-phosphotyrosine

antibody.

IP: anti-PLC-y2

blot: anti-P-Tyr

biot: anti-PLC-y2

of BMMCs on X9/D3 fibroblasts was seen, PLC phosphorylation was clearly seen. No signal

was observable in unstimulated BMMCs or in X9D3 fibroblasts alone. These results supgest

that neither of the two PLC inhibiton studied acts by interfenng with PLC phosphorylation

and most likely have an effect elsewhere in the pathway.

N. PI3-kinase is recruited to the c-Kit receptor following BMMC stimulation with mSLF and Akt rnay be phosphorylated

Using the 32D mode1 system it was demonstrated that in the presence of sSLF activation of

either P13-kinase or PLC-y was necessary for mitogenic stimulation of the cells and that there

is clearly some redundancy in the two pathways (Gommerman et al.. 3000). Given the

observation that PLC activation is required for the stimulation of 32D crlls and BMMCs by

mSLF. the qucstion of whethcr the PI3-kinase pathway was being activated upon stimulation

by mSLF was rxamined. The activation of a main downstrearn target of PU-kinase. Akt. was

checked as well. The first possibility is that PI3-kinase is not k i n g recruited to the c-Kit

receptor. To address this question. BMMCs were stimulated by sSLF or mSLF. ce11 lysates

were made. c-Kit was immunoprecipitated and then analyzed by Western bloning with an

anti-p85 antibody. Figure 18A shows that receptor-binding subunit of PI3-kinase. p85. CO-

precipitates with c-Kit following stimulation with soluble Steel Factor. Immunoprecipitation

with an anti-c-Kit antibody of lysates made From BMMCs CO-cultured on mSLF-expressing

fibroblasts for 15. 30 or 60 minutes also showed p85 recruitment. These data suggest that

PI3-kinase is indeed k i n g recruited to the c-Kit receptor following stimulation by rnSLF. In

order to check if downstrearn targets of PU-kinase were k ing activated. lysates of BMMCs

Figure 18. Pi3-kinase recruitment and possible Akt activation following stimulation of

BMMCs with mSLF. (A) BMMCs were CO-cultured on X9/D3 fibroblasts for 15. 30. or 60

minutes. Crll lysates were immunoprecipitated with an anti-c-Kit antibody and the

membrane was bloned with anti-p85. Unstimulated cells were used as a negative control and

BMMCs stimulated with sSLF were used as a positive control. Stimulated human Jurkat cell

lysatr was provided with the p85 antibody as a control for the antibody The blot was

stripped and reprobed with anti-c-Ki t. (B) BMMCs were stimulated on X9/D3 fibroblasts

and lysates were run on an 8% SDS-PAGE gel. transferred to nitrocellulose and bloned with

an anti-Akt (P-Ser.173). BMMCs stimulated with sSLF were used as a positive control and

lysate from X9/D3 cells was used to assess background signalling from the fibroblasts. The

blot was stripped and re-probed with anti-c-Kit.

IP: anti-c-Kit

blot: anti-p85

P-AM (Ser 473)

stimulated either by sSLF or mSLF were made, proteins separated on an 8% polyacrylamide

gel and blotted with an anti-phosphoAkt antibody. Preliminary results. showm in Figure 18B,

suggest that there is an increase in phosphorylated Akt following CO-culture of BMMCs with

mSLF-expressing tibroblasts. Unfonunately, it is not clear firom this experiment if the

increase in Akt phosphorylation is solely in the BMMCs.

O. Ofeic acid decreases rnurine dermal mast cell numbers in vivo

Glucocorticostrroids are currently the most cornmonly used drugs for regulating allergic

inflammation. Local topical application of corticosteroids has brrn demonstrated to

significantly deplete murine dermal mast cells. This rffect is believed to be due to a

downregulation of SLF mRNA and a subsequent Iower SLF protrin production in fibroblasts

nther thm a direct effect on mast cells themselves (Finotto et al.. 1997). Conicosteroids

induce tissue atrophy. which is mostly a result of keratinocyte and tibroblast b t i o n

attenuation (Lavker and Schechter. 1985: Lehmann et al.. 1983: Wilson Jones. 1976). We

wanted to test the possibility that oleic acid and neomycin sulfate could specifically decrease

mast ce11 nurnben in vivo. To do this. each C57/B6 mouse was pre-treated with Weetm" to

remove hair from a small dond patch of skin one day pnor to the experiment. Three times a

day. for four days. oleic acid cream. neomycin cream or the control creams were applied

topically. Table 1 shows the results of two separate expenments. In both cases. there was a

decrease in mast cells in the group treated with oleic acid and vitamin E. either by 24% as in

the first expenment. or 20% as in the second experiment for the group treated with 0.5%

oleic acid with vitamin E. Vitamin E-treated mice and oIeic acid alone-treated mice did not

Table 1. Effect of neomycin sulfate and oleic acid topical treatment on dermal mast cell

densities. Mouse skin was treated 3 tirnrs daily for four days with cream composed of 80%

PEG. 10% water. and olric acid (OA). neomycin sulfate (NS) and vitamin E (VE) in

concentrations shotvn in the table. Jojoba oil (JJ) made up the rest of the oil component for

riiher olric acid or neoycin sulfate alone. Untreated (UNTR) mice were includrd as an

additional control. Mouse skin was tïxed in formalin. cmbedded in parafin. sectioned.

stained with toluidine blue and mast ce11 nurnbers per high power field was enumented by

morphometry. Al1 slides were blinded and randomized before counting.

have significantly fewer dermal mast cells than the control group. As s h o w in the second

experiment. neomycin sulfate did not decrease mast ce11 nurnbers. These results suggest that

oleic acid. applied topically in conjunction with an anti-oxidant. may be usehl for

specificaily decreasing mast ce11 numben in vivo in short-term experimrnts.

IV. DISCUSSION

A. 32D myelomonocytic cell model and c-Kit receptor mutants

The 32D ceil model is usehl for studying sipalling through the c-Kit receptor. 32D

cells do not normally express the c-Kit receptor and c m be transfected with cDNA for

different receptor mutants. The signalling through these mutant receptors is not confounded

by the presence of endogenous c-Kit receptor. Our group was able to demonstrate that 32D

KitWT. KitYF719 and KitYF778 cells were equally able to respond to sSLF. as measured by

thymidine incorporation assays and tryan blur exclusion assays (Gommerman et al.. 2000).

Howcver. this was not the case for the doublc mutant KitYF719flF728. which cannot recruit

rither PI3-kinase or PLC-y. These cells failrd to be mitogenically stimulütrd by sSLF. These

results suggest that either PU-kinase or PLC-y activation is required for stimulation by sSLF

and that there is some redundancy in fünction between these two molecules upon sSLF

stimulation. Bioassays in which neomycin sulfate. a PLC antagonist. inhibited the mitogenic

stimulation of the KitYF719 mutant but not the KitWT or KitYF728 cells supported Our

hypothesis.

B. Neomycin sulfate as a PLC antagonist

Neomycin sulfate is a polycationic antibiotic that is commonly used as a PLC-y

antagonist. Research canied out by Schacht and colleayes in the 1970s and 1980s revealed

that neomycin sulfate acts by binding strongly to PIP2 (Schacht. 1978: Wang et al.. 1984). It

was also demonstrated to bind PI and PIP. and at extremely high concentrations it can bind

IP3 and ATP. thus complicating its use as a specific tool as a PLC antagonist at high

concentrations (Prentki et al.. 1986). Arnong many other experimental techniques used to

demonstrate its specific ability to bind PIP2. Schacht and CO-workers were able to use

neomycin, coupled to glass beads. to purifj PtPz (Schacht. 1978). Neomycin has also been

demonstratcd to affect the polyphosphoinositides in cclls. although thcse cxperiments

revealed that different concentrations of neomycin sulfate are required to affect PIPI turnover

in different cells. For example, only 10-5 M neomycin was required to achieve a 50% drop in

activity of PLC in isolated platelet membranes (Rock and Jackowski. 1987). whereas

0 . 3 ~ 1 O" M neomycin was required to inhibit PLC in permeabilized mast cells (Cockcroft et

al.. 1987) and IO" M was required in sea urchin egg fragments (Whitaker and Aitchison.

1985). Although the exact reasons for such great différences in effective concentrations of

neomycin sulfate are unknown. one possible explanation for why hi& concentraions of

neomycin sulfate are required to block PIPz turnover in the plasma membrane of some cells is

that most PIPz in biological membranes is bound to positively charged regions of intrinsic

proteins that are close to the cytoplasmic surface of the cell membrane. making it inaccessible

to neomycin sulfate (Gabev et al.. 1989).

Neomycin sulfate has been successfuIly used in a number of different ce11 types to

demonstrate its PLC-y antagonist activity. In hamster fibroblasts. neornycin inhibits

thrombin-stimulated phosphoinositde turnover and ce11 proliferation at 2 mM without

affecthg thrombin binding. thymidine uptake or cellular protein synthesis (Carne. et al..

1985). In the murine embryonic fibroblast ce11 line C3H/lOT1/2. neomycin was used to

inhibit PDGF-induced IP3 formation and DNA synthesis but not the uptake of inorganic

phosphate (Vassbotn et al.. 1990).

C. PLC-y requirement for signalling through c-Kit by mSLF

The responsr of the c-Kit mutants stimulated by mSLF was also examincd. Both

KitWT and KitYF719 mutants were able to respond rnitogenically when CO-cultured with

fibroblasts expressing membrane-bound SLF. but this was not the case for the KitYF728

mutant. This ceIl line showed comparatively severe impairment of a rnitogrnic rrsponse to

mSLF. This was reproducibk when plate-bound anti-c-Kit antibodies werr used to stimulate

the cells. Once again. these results were supported by expcinments in which neomycin sulfate

was used as a PLC inhibitor. Stimulation and support by mSLF-expressing tïbroblasts or

anti-c-Kit antibodies of KirWT crlls was inhibited in a dose-dependent manner by neomycin

sulfate. These data suggest that PIC-y, although not absolutrly required for stimulation by

sSLF. is essential for stimulation by mSLF. This result is important because it has been

suggested that mSLF is the physiologically more relevant fonn (Flanagan a al.. 199 1: Kapur

et ai., 1998) and it is thus possible that PLC-y activation plays a critical role in supporting

Kit' cells in vivo.

D. c-Kit signalling in bone marrow-derived mast cells

One drawback of the 32D mode1 is that these cells express relatively low Ievels of the

c-Kit receptor. as compared to murine bone marrow-dcrîved mast cells. making biochemical

analyses of down-stream signalling difficutt. Furthermore. observations of primary BMMCs

are more likely to more closely approximate the behaviour of mast cells in vivo than

observations in transformed ce11 lines. For these reasons, signalling through the c-Ki t

receptor, and some of its downstream events. in prirnary murine bone marrow-derived mast

cells was examined.

BMMCs are factor-dependent cells. routinely cultured in media supplemented with

IL-3. In virro assays in which IL-3 is replaced with soluble Steel Factor show that sSLF is

capable of mitogenically stirnulating BMMCs. Furthermore. X9/D3 fibroblasts. which

express only mSLF and not sSLF. were able to support BMMCs. but SV SI^ fibroblasts. which

express neither fom of Steel Factor. were not. Stimulation of BMMC with plate-bound

monoclonal anti-c-Kit antibodies alone was not possible. most likely because of the lack of

any CO-stimulatory factors. As mentioned. IL-3 is one of the major mast ce11 growth factors.

but IL-4. IL-5 and IL-6 are also survival factors. Furthermore. autocrine production of CO-

stimulatory factors. like [LA. occurs upon extensive cross-linking of m a t ce11 surface

recepton. such as FcoRI. As discussed earlier. stroma1 cells express numerous adhesion

molecules that rnight also be critical in mast ceIl support and activity. It is possible that

subrnitogenic levels of I L 4 in addition to plate-bound anti-c-Kit antibodies. may have

supported BMMC in virro. These observations suggest that. similar to 32D KitWT cells.

either soluble- or membrane-bound Steel Factor can support of BMMCs in vitro. However.

prelirninary experiments were unsuccessful at rnimicking mSLF-expressing fibroblasts using

plate-bound monoclonal anti-c-Kit antibodies alone.

In order to examine the signalling requirements through the c-Kit receptor. BMMCs

were stimulated with either sSLF or mSLF in the presence of increasing concentrations of the

PLC inhibitor neomycin sulfate. It was obseived that BMMCs that were stimulated by sSLF

were unaftected by neomycin sulfate. even at the highest concentrations used. This suggests

that in BMMCs, like in 32D KitWT cells. either PLC-y or P13-kinase recuitment and

activation (but not both) is required for survival and mitogenesis. This was not the case when

the BMMCs were stimulated by mSLF-expressing fibroblasts. In this situation neornycin

sulfate clearly inhibited BMMC stimulation in a dose dependent manner. Additionally. this

inhibition was revenible upon the addition of ionomycin. a calcium ionophore. Calcium

mobilization t'rom the ER and through store-opented channels is one of the downstream

consequences of PLC activation. In the absence of PLC activity. rescue of the BMMCs was

possible by using ionomycin to mimic the downstrearn etTects of PLC. Taken together. thrse

data support the hypothesis that PLC-y recruitment and activation are required for stimulation

by mSLF.

E. Oleic acid as a PLC-y inhibitor

Fatty acids play a number of critical physiological roles. among thesct: they are the

building blocks of glycolipids and phospholipids. denvatives of fatty acids are important

intracellular messengers. and they are important energy-storing molecules hithin cells. Oleic

acid is an 18 carbon monounsaturated fatty acid with a single cis double bond bctween

carbon 9 and 10. The inhibitory role of oleic acid and other free fatty acids has been best

studied in T lymphocyte systems. although the exact mechanism of inhibition is as yet

unclear. Richieri and Kleinfeld examined the effects of oleic acid on tmsmembrane

signalling in cytotoxic T lymphocytes (CTLs) (Richieri and Kleinfeld. 1989: Richieri and

Kleinfeld. 1990; Richieri et al., 1990). They observed that treatrnent of CTLs with oleic acid

concentrations corresponding to less than 10% (moumol) bound to the ce11 completely

inhibits target ce11 or Concavalin (Con) A-mediated increases in intemal calcium ion

concentration (Richieri and Kleinfeld. 1989). These effects were not observed with stearic

acid, an 18-carbon saturatcd fatty acid. The inhibition of signalling by oleic acid could be

completely reversed by the addition of fatty acid free bovine senirn albumin (BSA). which

eficiently binds fatty acids. Richicri and Kleinfcld obscrvcd that ncither Con A binding nor

the production of inositol phosphate metabolites are afTected. implying that the inhibition

event occurs distal to T ce11 surface recognition events or receptor-PLC coupling (Richieri

and Kleinfeld. 1989). They suggest that the mechanism of inhibition is likely due to the

effkct of a physical perturbation of the T cell membrane lipids. Furthemore. Richien and

colleagues were able to show that cis unsaturated free fatty acids. including oleic acid.

inhibited CTL antigen-stimulated degranulation and that this was reversible on the addition

of fatty acid fiee BSA (Richien et al.. 1990). Garnberucci and coworkers studied the rffects

of oleic acid on Ehrlich ascites tumor ceils and demonstrated that oleic acid was able to

inhibit store-dependent capacitative ~ a " influ.. and that inhibition appeared to depend on the

ratio of fatty acid concentration to the concentration of cells rather than on the absolute

concentration of fatty acid alone (Gamberucci et al.. 1997a: Gamberucci et al.. 1997b).

Oleic acid has been demonstmted to inhibit PLC activation in response to epidermal

erowth factor (EGF) (Casabiell et al.. 1993). The effects of oleic acid on BMMCs that were C

stimulated by either sSLF or mSLF were studied in this project. Lnterestingly. it was found

that oleic acid had no effect on the mitognic stimulation of BMMCs by sSLF. In contrast.

the presence of oleic acid resulted in a significant decreasr in the BMMC proliferation. as

measured by thymidine incorporation by the cells. Ln the presence of the anti-oxidant vitamin

E, which on its own has no effect on the BMMCs in virro. significant decreases in BMMC

proliferation were again observed. In fact, it appears that slightl y lower concentrations of

oleic acid, than those used when vitamin E was not added, can achieve ma..imum BMMC

inhibition under these conditions. These data support previous results obtained with

neomycin and add strength to the hypothesis that PLC-y is required for stimulation by mSLF

but not sSLF. In both cases. oleic acid has an inhibitory effect up to a certain concentration

beyond which there appears to be a reversal of inhibition. Gambemcci and CO-workers

observed a similar reversal of effect of fatty acid inhibition in Ehrlich tumor cells

(Gamberucci et al.. 1997a). They suggest that the rffects of the fany acid appear to depend

on the ratio of [fatty acid] to [cells] rather than absolute fa@ acid concentration.

In order to clarify the reasons for the different responses of BMMCs upon stimulation

with soluble or membrane Steel Factor. 1 first investigated the effect on c-Kit receptor

tyrosine phosphorylation. Upon stimulation of BMMCs with sSLF or mSLF (on X 9 0 3

fibroblasts) c-Kit receptor tyrosine phosphorylation could be easily detected. This was not

observed for BMMCs CO-cultured on SI/SI' fibroblasts. Furthemore. stimulation with both

ligand Forms resulted in c-Kit tyrosine phosphorylation that could be detected for up to 60

minutes but this phosphorylation was greatly reduced b 120 minutes. These data concur

with results obtained by many other groups that observed c-Kit receptor phosphorylation in

response to Steel Factor (Rottapel et al.. 199 1 : Yarden et al.. 1987). They also confirm the

validity of the observations obtained using SVSI" fibroblasts as controls for in vitro assays.

F. Biochemical studies of the mode of action of neomycin sulfate and oleic acid

Of the two PLC-y inhibitors used in these studies. only the mode of action of

neornycin sulfate is completely known (Gabev et al.. 1989). There still remains some

arnbiguity as to the exact mode of inhibition by oleic acid. although a number of possibilities

have been tested. including the inhibition of store-dependent ca2' influv (Garnberucci et al..

1997a), and intercalation into the plasma membranc (Gamberucci et al.. 1997b). Given the in

virro observations presented earlier. the next step was to determine and confirm the stage at

which oleic acid and neomycin sulfate block c-Kit signalling in BMMCs CO-cultured on

fibroblasts expressing mSLF. It was demonstrated that neither neomycin sulfate nor oleic

acid act by interfering or inhibiting c-Kit receptor phosphorylation or mSLF-stirnulated PLC-

72 phosphorylation. Phosphorylation of PLC-y2 was investigated as this particular isozyme is

expressed only in hernatopoietic cells. The observations for neomycin sulfate are consistent

with its known ability to bind PIP2. the substrate of PLC-y. As such. neomycin sulfate would

not affect c-Kit receptor phosphorylation or the recruitment and activation of PLC-y. The

data also suggest that oleic acid inhibition of BMMC stimulation by mSLF does not occur at

the level of c-Kit receptor phosphorylation or PLC-y phosphorylation. It does not address the

question of whether oleic acid is somehow interfixing with the substrate of PLC-y. Richieri

and Kleinfeld studied the inhibiiory effects of oleic acid in cytotoxic T cell signalling

(Richieri and Kleinfeld. 1989). AAer 2 minutes. they obscrved an overall decrease in total

inositol phosphate metabolites after stimulating T cells with Con A in the presence of oleic

acid. However. the levels of total inositol phosphate metabolites rises d e r 15 minutes and

Richieri and Kleinîèld suggest that the inhibitory effect of Free fatty acids is independent of

phosphatidylinositol turnover (Richieri and Kleinfeld, 1989). However. preliminary work in

bone marrow-denved mast cells revealed that oleic acid c m inhibit SLF-dependent calcium

intluves as well as slightly decrease overall IP3 production (Jonathan Soboloff. University

Health Network, Toronto, persona1 communication).

G. PU-kinase recruitment following stimulation by mSLF

Observations in both 32D cells and BMMCs highlight the importance of PLC-y

activation in stimulation by rnSLF. Given these results. and the observation in 37D

KitYF728 mutants that PI3-kinase activation is suficient to mitogrnicaliy stimulate these

cells. it is possible that upon ce11 stimulation with mSLF the PI3-kinase pathway is not Iùlly.

if at all, activated. However. it was demonstrated that BMMCs CO-cultured on mSLF-

expressing fibroblnsts do recruit p85 to the c-Kit receptor even der 60 minutes.

Furthemore. Western blotting of lysates including BMMCs stimulated by X9/D3 tïbroblasts

shows Akt that is phosphorylated at senne 473. This rxpenment is inconclusive. as it is not

possible to determine if the Akt phosphoiylation signal is due solrly to mast ce11 stimulation.

Pre-incubation of the fibmblasts with wortmannin would help clariG this issue. Although

phosphoryiation of Akt at Ser473 does not completely address the question of whethrr Akt is

fully activated. these data do suggst that PI3-kinase is being recruited upon BMMC

stimulation by mSLF and that PI3-kinase may still be able to activate downstrearn targets.

H. In vivo effects of oleic acid and neomycin sulfate

Mast cells are important effectors of anti-bacteriül and anti-parasitic responsrs.

Udortunately. irnbalances in mast ce11 regulation cm result in and contribute to a large

varies of immunopathologies like intlammation. allergic reaction and autoirnmunity.

Currently, corticosteriods are commonly usrd to decrease mast ce11 nurnben in tissue. Such

treatment. however, cm tiike up to 3 weeks to be effective and results in massive atrophy of

tissue surrounding the mast cells (Lavker and Schcchter. 1985). The possibility of usine

neomycin sulfate and oleic acid to control dermal mast ce11 densities was examined.

Preliminary results suggest that neomycin sulfate is ineffective at reducing mast cell numben

in iViivo. This result is not too surprising. as neornycin sulfate is water- soluble and is most

likely being cleared very rapidly tiom the site of application. However. oleic acid (when

applied with vitamin E) appears to have some potential in reducing mast ce11 numbers.

[mportantly. this reduction is achirved quickly (within 4 days) and there are no visible signs

of tissue damage. Such a topical crearn could have potential use in the reduction of mast

cells in acute intlamrnatory processes.

V. FUTURE DIRECTIONS

Whrrr is rhr PD-kinuse ptrrh~vczy blockrd zipon mSLF srimulurion uj-the c-Kir recrpror?

The 32D mode1 providrd W e r support to the observations of Valius and Kazlauskas that.

following growth factor stimulation. PI3-kinase and PLC-y activity may have some overlap in

function (Valius and Kazlauskas. 1993). Both 32D YF719 and YF728 single mutants were

mitogenically stimulated by sSLF. but the double mutant was not. Furthermore. only

KitYF7 19 cells were supportcd by mSLF-expressing fibroblasts. This observation suggested

to us that perhaps the PI3-kinase pathway was not rven being activated in 32D cells and

BMMC stimulated by mSLF. But immunoprecipitations of the regulatory subunit of PD-

kinase. p85. with the c-Kit receptor following stimulation with mSLF suggested that PI3-

kinase is being recmited. PI3-kinase recruitment to the receptor does not necessarily mean

that the enzyme is fully activated. Preliminary studies attempted to address this question by

looking at the phosphorylation of senne 473 on Akt. As discussed earlier. it was impossible

to detemine definitively which cells were responsible for the increase in Akt phosphorylation

but conclusive results for this particular CO-culture rxperiment could be obtained after prr-

incubating fibroblasts in the presence of a PI3-kinase inhibitor like wortmannin. However.

even if an increased serine 473 phosphorylation was detectable. we could not conclude that

the Akt pathway is Mly activated. Phosphorylation of Akt at both critical sites. serine and

threonine. would have to be confinned and an in vitro Akt activity assay would be usehl in

assessing the actuai enzymatic activity of Akt. Furthemore. activation and activity of other

downstream targets of both PD-kinase and Akt. like phosphorylation of the pro-apoptotic

protein BAD. or the activity of caspase 9. could be tested.

Can oleic acid be used to stir<?v the in vivo rule of 'mczsi cells in immunopathologies?

Oleic acid. and other cis-unsaturated fa. acids. have been widely studied for the

immunosuppressive ctfccts they cxcrt on T cclls. Not only havc in vivo studics linkcd

increased free fatty acid concentrations to inhibition of T ceIl activation and cytolysis

(Richieri and Kleinfeld. 1990). but patient studies have also showm ihat elevated serum tiee

fatq acid concentrations inhibit T ce11 signalling (Stulnig et al.. 2000).

Our in vivo observations in rnice suggest that olric acid. when applied topically and in

conjunction with the anti-oxidant vitamin E. can reduce drrmal mast ceIl numben by

approximately 20% in four days. These studies were carried out in normal. wild-.pe C57/B6

mice that had no skin disorden. However. thrre are a number of immunopathologies. such as

allergic reactions. inflammations. fibrosis and scleroderma. in which mast ceIl numbers are

highly elevated and imbalances in mast ce11 function cm lead to inappropriate ce11

stimulation. tissue edema and even tissue darnage (reviewed by Reischl et al.. 1999). An

inhibitor which could specifically target mast cells. and inhibit their Function. could prove to

be extrernely useful in reducing the hamihl effects of these disorders by controlling mast ceil

nurnben and activity. A murine mode1 for scleroderma exists. Such animals. termed TSK

or Ti@-skin. have a genetically transmitted connective tissue disease characterized by skin

lesions similar to those seen in scleroderma patients. TSK-associated fibrosis is

characterized by an increase in mast ce11 numben overall and also increases in debmulated

mast cells. The rfficacy of topically applied oleic acid could br tested in TSK mice and

compared to untreated rnice. Furthemore. longer-term experiments in rnice would be useful

in revealing if surrounding tissue is atrected by oleic acid treatment. as compared to current

standard treatments.

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