Positive cross-talk between ecdysone and insulin signaling ...597/fulltext.pdf · 3 POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN INSECT GROWTH A dissertation presented

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POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN

INSECT GROWTH

A dissertation presented

by

Srikanth Subramanian

to

The Department of Biology

In partial fulfillment of requirements for the degree of

Doctor of Philosophy

in the field of

Biology

Northeastern University

Boston, Massachusetts

January 2012

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2012


ALL RIGHTS RESERVED

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POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN

INSECT GROWTH

A dissertation presented

by


ABSTRACT OF DISSERTATION

Submitted in partial fulfillment of the requirements

for the degree of Doctor of Philosophy in Biology

in the Graduate School of Arts and Sciences of

Northeastern University, January, 2012

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ABSTRACT

Wing imaginal discs are masses of undifferentiated cells that give rise to the

forewings and hindwings of adult insects. In this dissertation, I examined the roles of

insulin and ecdysone, a steroid molting hormone, in the regulation of imaginal wing disc

growth in the tobacco hornworm, Manduca sexta. Previous studies on a different

lepidopteran insect had suggested a requisite role for both hormones in disc growth

providing a means to coordinate nutritional regulation of growth with metamorphic

development. I studied the short-term and long-term stimulation of signaling events in

the insulin pathway, and in ecdysone receptor content, in order to determine loci at which

insulin and ecdysone might interact. My results clearly revealed a requirement for both

insulin and ecdysone in Manduca disc growth. This result countered earlier research on

growth in Drosophila, which is ecdysone independent, and was more closely in keeping

with steroid regulation of insulin response in vertebrates. The results raised questions

regarding the underlying mechanisms by which disc growth is stimulated. I showed that

ecdysone and insulin were required together to increase cell number, using an EdU click-

chemistry detection assay. I further examined whether specific signaling proteins were

synergistically enhanced by ecdysone and insulin and whether the effects of ecdysone on

growth were dependent upon insulin signaling. I found that RNAi-mediated knockdown

of the insulin receptor blocked hormone-stimulated growth, as well as blocking increases

in the ecdysone receptor, insulin receptor, and insulin receptor signaling pathway.

This research was the first demonstration of knockdown of the insulin receptor in M.

sexta. The results pinpointed critical cellular targets in the insulin pathway, specifically

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Akt, involved in cross-talk with ecdysone. Inhibitors of PI3-kinase and TOR, i.e.

upstream and downstream regulators of Akt, blocked growth and ecdysone receptor

content in a manner similar to insulin receptor knockdown. The results suggest that

downstream effects of Akt, such as reduced nuclear import of FOXO, may play crucial

roles in steroid-regulated growth. This work helps to improve our understanding of the

cellular mechanisms between insulin signaling and steroids that underlie post-embryonic

animal development.

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This thesis is dedicated to Pops, Mom and Eileen,

whose never-ending love and support

made this dissertation possible.

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TABLE OF CONTENTS

Abstract 4

Dedication 6

List of Figures 8

Abbreviations 9

Acknowledgements 12

Introduction 15

Chapter 1: The control of growth and differentiation of wing imaginal discs

in Manduca sexta. 24

Chapter 2: Effects of ecdysone and insulin on cell division 38

Chapter 3: Cellular interactions between ecdysone and insulin in the regulation

of disc growth 48

References 72

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LIST OF FIGURES

Figure 1. Ecdysteroid levels during the fifth instar of M. sexta.

Figure 2. A model of the insulin signaling pathway.

Figure 3. Growth and differentiation of the forewing imaginal disc.

Figure 4. Wing imaginal disc growth during first three days after entering the

wander stage.

Figure 5. Growth of wing imaginal discs in vitro.

Figure 6. Phosphoproteins response of cultured wing discs.

Figure 7. Phosphoprotein Akt and total protein response of cultured wing discs after

48 hour incubation.

Figure 8. Click reaction between EdU and azide modified dye.

Figure 9. Detection of cell proliferation with EdU in wing discs after 48 hour

incubation.

Figure 10. Number of proliferating cells (incorporating EdU) in wing discs after 48

hour incubation.

Figure 11. Effects of siRNA, LY294002, and rapamycin on growth in wing discs.

Figure 12. Detection of transcript in wing discs after 48 hour treatment.

Figure 13. Activation of phosphoproteins in wing discs after 48 hour incubation.

Figure 14. Total Akt in wing discs after 48 hour incubation.

Figure 15. Effects of rapamycin on p4EBP and EcR in wing discs after 48 hour

incubation.

Figure 16. Ecdysone receptor proteins in wing discs after 48 hour incubation.

Figure 17. Suggested mechanism through which ecdysone and insulin synergistically

interact to modulate EcR expression and activity.

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ABBREVIATIONS

4EBP initiation factor 4E binding protein

Akt2 human homolog of v-akt oncogene

BSA bovine serum albumin

Cdk cyclin-dependent kinase

cDNA complementary deoxyribonucleic acid DILP Drosophila insulin-like peptide

DIR Drosophila insulin receptor

DNA deoxyribonucleic acid

dsRNA double stranded ribonucleic acid E2 17- -estradiol Ec ecdysone EcR ecdysone receptor EcR-A ecdysone receptor isoform A EcR-B1 ecdysone receptor isoform B1 EdU 5-ethynyl-2’-deoxyuridine ERK extracellular signal-regulated kinase FOXO forkhead box-containing protein GFPsiRNA small-interfering RNA directed against green fluorescent protein Grb2 growth factor receptor-bound protein 2 GSK glycogen synthase kinase

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HRP horseradish peroxidase IDGF imaginal disc growth factor IGF insulin-like growth factor Ins insulin IR insulin receptor IRS insulin receptor substrate IRsiRNA small-interfering RNA directed against the

bombyxin/insulin receptor JH juvenile hormone MAPK mitogen-activated protein kinase MEK mitogen-activated ERK-activating kinase mRNA messenger ribonucleic acid mTOR mammalian target of rapamycin

p phosphorylated p70S6K 70 kDa S6 kinase PBS phosphate buffered saline PCR polymerase chain reaction PDK phosphoinositide-dependent protein kinases PI3K phosphatidylinositol-3-kinase

PIP3 phosphatidylinositol-(3,4,5)-triphosphate

PKB/Akt protein kinase B

PPAR- peroxisome-proliferator-activated-receptor PTEN phosphatidylinositol-3,4,5-triphosphate 3-phosphatase

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PTTH prothoracicotropic hormone qPCR quantitative real time PCR

Raf Raf serine/threonine kinase

Ras Ras GTP-binding protein

RNA ribonucleic acid

RNAi ribonucleic acid interference

ROS reactive oxygen species

Rp49 ribosomal protein 49 RT room temperature RXR retinoid X receptor S6 ribosomal protein S6 siRNA small-interfering RNA SOS son of sevenless STAT signal transducers and activators of transcription TOR target of rapamycin TSC tuberous sclerosis complex TZDs thiazolidinediones USP ultraspiracle V fifth instar

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ACKNOWLEDGEMENTS

I would like to take a moment, as my time at Northeastern University comes to an

end, to thank the people who have supported me in various ways and have helped make

my success possible. First and foremost, I thank my advisor, Dr. Wendy Smith. She has

been a great mentor and even greater friend to me. Her enthusiasm and sharp sense of

humor made the lab a wonderful place to work. She offered guidance when needed but

she also allowed me the independence to really take possession of my project. She

helped me grow as a person and as a scientist. I have learned so much from her and I am

grateful to be part of her lab. I hope to make her proud in the future. I also thank the

members of my doctoral committee Dr. Gail Begley, Dr. Erin Cram, Dr. Dick Deth and

Dr. Rebecca Rosengaus for their time and expert advice. I owe Dr. Rosengaus extra

thanks for helping me with statistics.

I thank the current and past members of the Smith lab, with whom I have had the

pleasure to work. First, I thank Dr. Amy (Walsh) D’Amico, a.k.a. “Coach” for

welcoming me into the lab, steering me clear of pitfalls, making me laugh constantly, and

for being a dear friend. I also thank Dr. Lou D’Amico for his constant encouragement

and sound advice. I thank Leon DeLalio for his willingness to listen and for sharing his

excellent taste in food, movies, and his lamp with me. There are also many

undergraduates that have been part of the lab who have helped me over the past few

years. I thank Abby Bootes and Erin Greguske for taking over management

responsibilities in the lab. I thank Jackie Olender for helping me troubleshoot real time

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PCR experiments and I thank Jade McPherson for her patience and hard work with the

EdU experiments. I also thank Bethany Kirpalani, Rhamy Zeid, Sauveur Jeanty, Dana

Galacchi, and Erin Vinoski for their friendship and dedication to the lab.

I thank the O’Malley lab for help with the confocal microscope. I thank Dr. Don

O’Malley for taking the time to teach me how to use the microscope properly. I also

thank Dr. Kristen Severi, Dr. Rebecca Westphal, and Sucharita Saha for being gracious

hosts during my lengthy visits to their lab.

The Davis lab has always had an open door policy with me and I thank them for

letting me take advantage of that. I thank Dr. Davis for his friendship and sense of

humor. I thank Dr. Andy Cary for sharing his technical expertise and offering

encouragement in his own unique way. Finally, I thank Dan Wreschnig for always

taking time to talk with me about science, teaching, literature, and life in general.

I thank the office staff in the Biology Department, especially Adrian Gilbert and

Aaron Roth, who have always found a way to make things work. I thank Frauke Argyros

and Patti Hampf for doing everything they could to make the teaching labs run smoothly.

I also thank Patti for letting me invade her office on long teaching days.

I thank the biology graduate students (including the Richards-Hall-Night-crew

and the Biohazards broomball team) for making this whole graduate experience as fun as

possible. I also have to thank my BHS friends (BA, Sarah, Larae, Karen, Jenn and Rene)

who have known me since we were kids and have been encouraging me ever since.

Finally, and most importantly, I need to thank my family. Their unconditional

love helped make this dissertation possible. Pops and Mom have been a constant source

of emotional support. I also thank Mani Mama, Bhagya Auntie, Karthik and Kavitha. I

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thank the Loughmans for welcoming me into their family. I’m saving the best for last; I

thank my wife, Eileen. There are no words that can fully encompass how much her love

and support (and patience) mean to me. I love you all.

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Introduction

Insect growth/development

The growth of a cell or tissue involves complex interactions between genes,

metabolism, nutrition, and extracellular signals. In order to understand these processes,

we need to use a model that can be studied physiologically and on a molecular level. The

tobacco hornworm, Manduca sexta has been a useful model to study the endocrine

regulation of growth, particularly post-embryonic growth that directs the transition from

juvenile to adult, because these holometablolous insects develop synchronously and have

clear markers for staging larval development (Nijhout and Williams, 1974; Gilbert et al.,

1980). The duration of growth in M.sexta, as in all insects, is controlled by changing

levels of hormones (Gilbert et al., 2002). The group of hormones that regulate insect

development are ecdysteroids, juvenile hormone (JH) and prothoracicotropic hormone

(PTTH) (Nijhout, 1994; Gilbert et al., 2002). During the fifth larval stage (instar), the

largest growth phase, PTTH secretion and ecdysone are inhibited by JH, a terpenoid

hormone with no vertebrate homolog (Nijhout and Williams, 1974; Roundtree and

Bollenbacher 1986; Gilbert, 2012). Larvae feed during the first part of the instar until

they reach a critical weight of about 6g (Nijhout and Williams, 1974; D’Amico et al.,

2001). JH levels then decrease, allowing the photoperiodically gated release of PTTH, a

neuropeptide, which causes the prothoracic glands to release ecdysone, a steroid (Figure

1) (Nijhout and Williams, 1974; Rountree and Bollenbacher, 1986). Markers for the

release of ecdysone, which is converted to the active ecdysteroid 20-hydroxyecdysone,

include cessation of feeding, purging of the gut, exposure of the dorsal vessel and

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wandering (increased motility) associated with searching for a suitable place to

pupate(Nijhout, 1994; Riddiford et al., 2003). Two days later, a second ecdysteroid peak

causes the animals to pupate (Nijhout, 1994, Riddiford et al., 2003). Body growth is

restricted to the larval instars, especially the fifth instar, so adult size is fixed at the larval-

to-pupal transition.

Figure 1. Ecdysteroid levels during the fifth instar of M. sexta. Abbreviations: wandering (W), ecdysis (E) (Bollenbacher et al., 1981; from Smith, 1995).

19 8 7 6 5 4 3 2 1 0 0

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Day of Fifth

Ecd

yste

roid

s

W E

0 1 2 3 4 5 6 7 8 9 0 1 2

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Insulin signaling pathway

In addition to the conventional hormones known to regulate insect growth, it is

becoming clear that insulin is also a key player in integrating metabolism and response to

nutrition. The insulin signaling pathway is conserved across species, and insulin-like

signaling molecules have been found in a variety of invertebrates including nematodes,

molluscs and insects (Claeys et al. 2002). Bombyxin, from the silkmoth Bombyx mori,

was the first insect insulin-like molecule to be discovered. A heterodimer of insulin-like

A- and B-chains, it is clearly a member of the insulin superfamily (Ishizaki and Suzuki,

1994; Claeys et al. 2002). Over 40 bombyxin genes have been identified, yet their

function(s) remain unclear (Adachi et al., 1989; Nagata et al., 1995; Kondo et al., 1996).

The insulin receptor (IR) is one of many growth factor receptors that regulate

cellular processes via the activation of receptor tyrosine kinases in the plasma membrane

(Myers et al. 2003; Claeys et al. 2002). The IR is comprised of - and -subunits joined

by disulfide bridges to form an -heterodimer (Myers et al. 2003). Ligand binding

specificity depends on cysteine-rich regions in -subunits located towards the

extracellular face of the plasma membrane. -subunits on the cytoplasmic face of the

membrane contain tyrosine kinase activity and undergo tyrosine phosphorylation by

adjacent subunits as a result of ligand binding (Claeys et al. 2002). These features are

also present in the insulin-like growth factor (IGF) receptor (Fernandez et al. 1995;

Myers et al. 2003).

The insulin receptor uses a group of scaffolding molecules called insulin receptor

substrates (IRS), to initiate cytosolic signaling (White, 1998; Lannigan, 2003; Claeys et

al. 2002). As shown in Figure 2, IRS binds to the phosphotyrosine in activated IR and

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itself becomes phosphorylated (White, 1998). IRS can then activate many growth related

pathways (Claeys et al. 2002; Lannigan, 2003). For example, the activation of Grb2/Sos

(son of sevenless) activates the mitogen-activated ERK-activating kinase/extracellular-

signal-regulated kinase (MEK/ERK) pathway via the proteins Ras and Raf (Lannigan,

2003). A second pathway leads to the activated IRS-phosphatidylinositol-3-OH kinase

(IRS-PI3K) complex, which generates phosphatidylinositol-(3,4,5)-triphosphate (PIP3)

and recruits phosphoinositide-dependent protein kinases (PDK), protein kinase B

(PKB/Akt), and other downstream kinases which affect cell growth and survival (Claeys

et al. 2002; Lannigan et al. 2003). Typically, Akt phosphorylation blocks the activity of

proteins such as glycogen synthase kinase (GSK), forkhead box-containing protein

(FOXO), and tuberous sclerosis complex (TSC) that stop growth in their non-

phosphorylated state (Figure 2) (Burgering and Medema, 2003; Krymskaya, 2003). For

example, non-phosphorylated TSC indirectly inhibits the growth regulating kinase, target

of rapamycin (TOR) (Krymskaya, 2003). TOR is a nutrition sensor that can regulate

translation by phosphorylating initiation factor 4E binding protein (4E-BP) and

p70S6kinase (Hay and Sonenberg, 2004).

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Figure 2. A model of the insulin signaling pathway. See text for details. Abbreviations: InR, insulin receptor; IRS, insulin receptor substrate; Grb2/SOS, adapter and guanine-nucleotide-exchange factor for Ras; Ras, Ras GTP-binding protein; Raf, Raf serine/threonine kinase; MEK, MAPK/ERK kinase; ERK, extracellular signal-regulated kinase; PI3K, phosphoinositide 3-kinase; PIP3, phosphatidylinositol-(3,4,5)-triphosphate; PDK, phosphoinositdie-dependent protein kinase; Akt protein kinase B; GSK, glycogen synthase kinase; FOXO, forkhead box-containing protein; TSC, tuberous sclerosis complex; TOR, target of rapamycin. 4EBP, binding protein for ribosomal initiation factor 4E; S6, ribosomal protein S6; p70S6K, 70 kDa S6 kinase.

Ecdysone and ecdysone receptor

Ecdysone is a steroid molting hormone which also stimulates metamorphosis-

related development. 20-hydroxyecdysone is the mature form of the hormone derived

from enzymatic modification of cholesterol. Ecdysone synthesis occurs in glands known

as the prothoracic glands, and is controlled by the brain via the neurosecretory hormone

prothoracicotropic hormone (PTTH). A PTTH-stimulated rise in ecdysone triggers each

larval molt. During the fifth instar, a rise in ecdysone induces the cessation of feeding

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and entry into the prepupal wandering stage (Bollenbacher et al., 1981; Baker et al.,

1987; Smith, 1995, and see Fig. 1).

The ecdysone receptor is part of the superfamily of nuclear receptors that are

ligand-dependent transcription factors (Riddiford et al., 2000; Billas and Moras, 2005).

This superfamily plays an important role in regulating the expression of an array of genes

during development and reproduction (Riddiford et al., 2000). Steroid receptors are

characterized by a C-terminal ligand-binding domain, a DNA-binding region, and

transcriptional activation domains (Jindra et al. 1996, 1997). The ecdysone receptor

(EcR) heterodimerizes with a nuclear receptor known as ultraspiracle (USP) (Yao et al.,

1993; Billas and Moras, 2005). USP is an ortholog of the vertebrate retinoid X receptor

(RXR) (Yao et al, 1993; Riddiford et al., 2001). There are two main ecdysone receptor

isoforms, EcR-A and EcR-B1, which differ in the N-terminal domain (Truman, 1996;

Jindra et al., 1996, 1997). EcR sub-types have been found to play specific roles in some

metamorphic events, particularly in the nervous system (Truman, 1996; Scauer et al.,

2011; Schwedes et al., 2011).

Insulin signaling and ecdysone in insects

A growing body of evidence indicates that metamorphosis involves an interplay

between ecdysteroid and insulin-stimulated developmental events. The regulation of

insect growth via the insulin signaling pathway is best understood in imaginal discs,

particularly in Drosophila. Imaginal discs are simple epithelia made up of a single layer

of cells that rapidly grow into complex structures in the adult (Johnston and Gallant,

2002). There is only one Drosophila insulin receptor (DIR) but it has been shown to be

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highly expressed in larval imaginal discs (Garofalo and Rosen, 1988). Overexpression or

loss-of-function of DIR increases or decreases body size, respectively (Brogiolo et al.,

2001). The DIR loss-of-function mimics the effects of starvation/nutrient-deficiency

(Brogiolo et al., 2001). Drosophila insulins or insulin-like peptides referred to as DILPs,

work together with a group of growth factors (secreted glycoproteins) called imaginal

disc growth factors (IDGFs) to control tissue and body growth primarily by regulating

cell size (Kawamura et al.,1999; Bryant, 2001; Varela et al., 2002; Goberdhan and

Wilson, 2003; Arquier et al., 2008; Zhu et al., 2008). At least part of this growth effect is

mediated by two small groups of neurons in the Drosophila brain which secrete DILPs

into the circulatory system at levels that are modulated by nutrition (Goberdhan and

Wilson, 2003; Arquier et al., 2008). Though DILPs and IDGFs are required for disc

growth in Drosophila, ecdysone does not seem to be required. In Drosophila fat body,

ecdysone appears to inhibit insulin/insulin-like growth factor signaling (Colombani et al.

2005, Francis et al., 2010).

In contrast, imaginal disc growth in Lepidoptera requires insulin and ecdysone.

Wing discs of Lepidoptera grow continuously and exponentially during the later larval

and prepupal stages (Kremen and Nijhout, 1998; Miner et al., 2000; Nijhout and Grunert,

2002; Nijhout et al., 2007). In feeding larvae, starvation and JH expression have been

shown to inhibit disc growth (Miner et al. 2000). However, in non-feeding larvae, i.e.,

once the animals have begun metamorphosis, discs continue to grow. In other words,

control of imaginal disc growth changes from being dependent on feeding to being

independent of feeding (Nardi et al., 1985; Nijhout et al., 2007). In the buckeye butterfly,

Precis coenia, imaginal discs from feeding animals stop growing (Nijhout and Grunert,

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2002). Disc growth in vitro is promoted by supplementing media with ecdysone and with

hemolymph containing the insulin-like growth factor, bombyxin (Nijhout and Grunert,

2002). Neither alone stimulates growth. Hence, unlike Drosophila, both ecdysone and

insulin-like hormones are required to stimulate growth. At the time I began my research,

little else was understood about the nature of the interaction between ecdysone and

insulin in regulating disc development. In the experiments described in Chapter 1, I

examined the roles of insulin and ecdysone in the regulation of wing imaginal disc

growth in Manduca sexta and explored the short-term and long-term stimulation of

insulin signaling events. Precedence for steroid regulation of insulin signaling can be

found in other animals as described in the introduction of Chapter 1.

Similar to Precis, I hypothesized that in Manduca, ecdysone was working synergistically

with insulin to enhance the insulin pathway and promote growth of imaginal discs. In

short-term (30 min) incubations, insulin activated growth factor signaling elements such

as Akt, while ecdysone did not. But after longer (2 day) incubations, ecdysone

stimulated identifiable members of the insulin pathway. Together, insulin and ecdysone

also stimulated disc growth. These observations were further explored in Chapters 2

and 3.

In experiments described in Chapter 2, I determined whether ecdysone was

responsible for cellular division, as is typical of steroid hormones (Revelli et al., 1998;

Fox et al., 2009). There was the possibility that insulin and ecdysone acted

synergistically, with neither having sole responsibility for an increase in cell number. By

using a novel DNA synthesis detection assay, I determined the effects of each hormone

specifically upon cell division (Chapter 2). Both insulin and ecdysone were seen to

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increase cell division, with ecdysone having a greater effect, in keeping with traditional

effects of steroid hormones.

In experiments described in Chapter 3, I further explored the interaction between

ecdysone and insulin in modulating growth. I hypothesized that insulin and ecdysone

would have mutually positive effects, particularly with regard to receptor expression.

Precedence in vertebrates is discussed in greater detail in Chapter 3. Using RNAi,

inhibitors, and antibodies directed against specific elements of the insulin pathway, I

further identified synergistic interactions between ecdysone and insulin, including but not

limited to receptor content, in the regulation of disc growth (Chapter 3).

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CHAPTER ONE

The control of growth and differentiation of wing imaginal discs

in Manduca sexta

(Published as Nijhout HF, Smith WA, Schachar I, Subramanian S, Tobler A, Grunert

LW. The control of growth and differentiation of the wing imaginal disks of Manduca

sexta. Dev Biol. 2007 Feb 15;302(2):569-76).

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INTRODUCTION

Initial studies with lepidopteran imaginal discs indicated that conditions required

for normal growth in Precis coenia differed from those in Drosophila (Nijhout and

Grunert, 2002). In Precis, during the feeding phase, growth of the discs depended on

nutrition (Nijhout and Grunert, 2002). When larvae were starved, disc growth ceased

within 4-6 hours (Miner et al., 2000; Nijhout and Grunert, 2002). Similarly, if larvae

were fed a low-nutrient diet, larval growth rate and growth rate of the discs were also

slow. In other words, wing disc growth was modulated to remain proportional to the

growth of the rest of the body.

When discs taken from feeding and growing larvae were placed in nutrient-rich

culture medium they failed to grow. However, discs were made to grow at a normal rate

by adding bombyxin and a steroid molting hormone 20-hydroxyecdysone to the culture

medium (Nijhout and Grunert, 2002). Precis disc growth thus required a classic insect

developmental trigger (ecdysone) as well as a hormone regulated by nutrition

(bombyxin), providing an appealing explanation for coordination of nutrition and

development in the regulation of body and organ size. In Precis, the bulk of disc growth

occurs during feeding. This may be an exception in developing insects rather than the

rule. For example, in most lepidopterans discs grow continuously and exponentially

throughout the late larval and prepupal periods (Miner et al., 2000).

In Manduca, imaginal discs continue to grow on their normal exponential trajectory, even

after larvae have stopped feeding (Williams, 1980; Nardi et al., 1985; Nijhout et al.,

2007). Given that growth continues in non-feeding in Manduca, the question arose as to

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whether insulin (and/or ecdysone) continued to play a positive role. To answer this

question, we studied the post-feeding wandering stage growth of the imaginal wing discs

of Manduca sexta. We showed that, as in Precis, disc growth in Manduca requires

ecdysone and insulin in vitro.

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MATERIALS AND METHODS

Animals and tissue culture

Manduca sexta larvae were reared on a standard artificial laboratory diet (Bell and

Joachim, 1976) at 25°C under a long-day 16:8 light:dark cycle. Day five, fifth instar

(wandering) animals were anesthetized for 10 min in water then surface-sterilized by

immersion for 2–3 min in a 1/750 solution of benzalkonium chloride (Matheson Coleman

and Bell). Discs were dissected in sterile saline in a sterile hood. Disks were cultured in

24-well plates (Costar 3524) in 300 l Grace's medium (Gibco, Invitrogen) supplemented

with 10% fetal calf serum (Gibco) and 10% antibiotic–antimicotic (Gibco), under a 95%

O2/5% CO2 atmosphere. Following incubations, disc length was measured under a

dissecting microscope using an ocular micrometer. Wing discs were then homogenized

in 2x SDS sample buffer, incubated for 3-5 minutes at 90°C, and stored at -20° C.

Western blots were subsequently performed (see below).

Hormones

Insulin (NovoRapid) was diluted in Grace’s medium to a final concentration of

30μM. 20-Hydroxyecdysone (Sigma) was dissolved in Grace’s medium to a final

concentration of 0.1 μg/ml. These concentrations were determined to provide maximal

growth based on preliminary tests of a range of doses for each hormone (0.1 μg/ml to 0.5

μg/ml for ecdysone, and 10 μM to 30μM for insulin, data not shown).

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Western blot analysis

Wing discs were dissected in pairs, placed in 40 μL of 2X SDS sample buffer, and

incubated at 90°C for three to five minutes. The samples were then run on a 10-20%

SDS-PAGE gradient gel (Bio-Rad) to separate the proteins. They were transferred from

gels to nitrocellulose membranes at 4°C. Membranes were then blocked in 3% BSA or

5% milk for one hour, rinsed quickly (15 minutes total) and placed on primary antibody

overnight. Primary antibodies included antiphospho-insulin receptor, antiphospho-Akt,

total Akt, and anti-Actin (Cell Signaling). Blots were rinsed and placed on secondary

anti-rabbit antibody with attached HRP (Cell Signaling) for 75 minutes. Blots were

treated with Western blotting chemiluminescence reagents (Pierce ECL Western blotting

substrate, Thermo Scientific) and were exposed on blue- sensitive autoradiographic film

(Marsh Bio Products) and developed (Kodak GBX fixer and developer). Blots were

scanned and analyzed using ImageJ (Abramoff et al., 2004). Protein bands were

normalized to actin as a loading control where appropriate.

Statistical Analysis

Protein expression in the wing imaginal discs was subjected to statistical analysis

using one way ANOVA, with a Dunnett’s post hoc test to compare specific treatments

against a reference. In all cases, p<0.05 was used to determine significance. All

statistical analyses were performed using SPSS v.19 (licensed to Northeastern University,

2011).

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RESULTS

Growth and differentiation

The morphology of the growing Manduca wing disc is shown in Fig. 3. During

the feeding stage (white box, Fig. 3) wing disc size stayed in proportion to body size. On

day 5 (V5) of the fifth instar, larvae enter the wandering stage and stop feeding. At this

point discs continued to grow and began to differentiate (Fig. 3). Disc differentiation

during the wandering stage involved development of the lacunae (spaces) that would

form the wing veins and the subsequent expansion of the tracheal system into those same

spaces. Wing vein differentiation or tracheal migration began 12 hours after the start of

wandering and the tracheal system developed fully at 60 hours (Fig. 3). In vivo, trachea

started to develop while the ecdysteroid levels were still low. In this regard, imaginal

discs of Manduca significantly differed from those of Precis. In Precis, tracheal

migration and wing vein differentiation occurred during the feeding stage of the last

larval instar (Miner et al., 2000). In Manduca, tracheal migration began before the peak

of ecdysone that initiates the pupal molt. At the same time growth rate increased

significantly in subsequent days after wandering (Fig. 4). It is clear that disc growth

increased as the larvae entered the non-feeding wandering stage. The size of the discs

increased from roughly 1 mm to several mm over a 3-day span (Fig. 3). The question

arose as to whether, as in Precis, ecdysone and insulin were involved in this change.

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Figure 3. Growth and differentiation of the forewing imaginal disc. The larval disc growth during the feeding stage is represented by the enclosed gray box. The developmental “age” of the feeding larva is indicated below each disc by the weight of the larva (in grams). During the wandering stage, developmental “age” is indicated as hours after the initiation of wandering. Numbers below wandering stage discs indicate scoring system used to quantify tracheal differentiation (from Nijhout et al., 2007).

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Figure 4. Wing imaginal disc growth during first three days after entering the wandering stage (from Nijhout et al., 2007).

Control of imaginal disc growth in vitro

We studied the direct effects of ecdysone and insulin on growth of wing imaginal

discs of Manduca sexta in tissue culture. Insulin was used rather than bombyxin as

Manduca bombyxin was not available in purified form. Insulin has been shown to affect

developing tissues in Manduca in a manner indistinguishable from bombyxin (Koyama

et al., 2008). As shown in Fig. 5, discs cultured with the addition of both ecdysone and

insulin doubled in size. Neither ecdysone alone nor insulin alone had a significant effect

on growth (Fig. 5).

32

Figure 5. Growth of wing imaginal discs in vitro. All discs were cultured for 48 hours alone in Grace’s medium (Control), with 20-hydroxyecdysone (Ec), with insulin (Ins) or with 20-hydroxyecdysone and insulin (Ec+Ins). Significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05) (disc pictures from Nijhout et al., 2007).

Short-term and long-term stimulation of insulin signaling events

Given a clear synergistic effect of ecdysone and insulin on growth, we next

investigated whether ecdysone and insulin had identifiable effects on growth-related

signaling pathways. Insulin-like hormones stimulate tyrosine phosphorylation of a highly

conserved amino acid sequence (YETDYY) of the insulin receptor (Nijhout et al., 2007).

Discs were exposed to ecdysone and/or insulin for 30 minutes, to capture short-lived

phosphorylation events. As seen in Figs. 6A and B, insulin alone stimulated the

a a a

b

33

phosphorylation of an 85kD protein, corresponding in size to the subunit of the

mammalian insulin receptor. A clone of the B. mori insulin receptor predicted a protein

of similar size (Swevers and Iatrou, 2003) which was also recently confirmed by the

sequencing of the M. sexta insulin receptor (Koyama et al., 2008). Anti-actin antibody,

which detected two actin fragments, one at 45kD and a smaller 30 kD fragment, was used

to control for loading of lanes on the Western blot (Fig. 6A). Samples were normalized

by dividing each respective band intensity by the intensity of actin.

An additional signaling kinase typically activated by insulin-like growth factors,

Akt/protein kinase B, was also studied using antibody directed against a highly conserved

phosphorylation domain (Fig. 6A and C). After 30-min incubation, insulin stimulated the

phosphorylation of Akt (65kD) in wing discs. Results from this experiment indicate that

mammalian insulin significantly stimulated (p<0.05) typical phosphoproteins in the

insulin signaling pathway. Conversely, ecdysone alone did not stimulate phosphorylation

of the insulin receptor or Akt within the 30 min incubation period (Fig. 6). When

combined, ecdysone and insulin significantly stimulated (p<0.05) phosphorylation of

both insulin receptor and Akt, though to no greater degree than insulin alone (Fig. 6B and

C).

34

Figure 6. Phosphoproteins response of cultured wing discs. A. 30-minute challenge in medium containing ecdysone (Ec), insulin (Ins), both (Ec+Ins), or no hormones (Control, C) (n=8). Molecular mass is shown in kilodaltons (kD) at right. B. Quantification of pIR. C. Quantification of pAkt Western blots were done using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).

b

b

b

b

a a

a a

B

C

Ec C Ins C Ec+Ins C

pIR

pAkt

Actin

- 85kD

- 62 kD

- 40 kD

- 30 kD

A

35

The same phosphoproteins were examined after long-term incubation. After 48

hours, insulin, or insulin combined with ecdysone (Ec+Ins), significantly increased pAkt

(p<0.05) (Fig. 7). 48 hour incubation in ecdysone alone also led to a significant increase

in pAkt (p<0.05) (Fig. 7B). The observed increases in pAkt after 48 hours were due, at

least in part, to increases in total Akt. As seen in Fig. 7C, there was a significant increase

in total Akt when discs were incubated with both ecdysone and insulin (Ec+Ins) (p<0.05)

(Fig. 7C).

Figure 7. Phosphoprotein Akt and total protein response of cultured wing discs after 48 hour incubation. Challenge in medium containing ecdysone (Ec), insulin (Ins), both (Ec+Ins). A. Representative Western blots. Molecular mass shown in kilodaltons (kD) at right. B. Quantification of pAkt. C. Quantification of total Akt. Western blots were done using Image J (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05)

a a a

a

36

DISCUSSION

The primary focus of the experiments described in this Chapter was to determine

whether growing discs in post-feeding insects, representing a typical period of

exponential disc growth, required both steroid hormones and insulin. Insulin-like growth

hormones had been implicated as the primary regulators of late larval insect growth,

using Drosophila as a model. Mutations in insulin signaling for genes such as IR, Chico

(an insulin receptor substrate), PI3K, TSC1/2, TOR, and FOXO, all caused changes in

Drosophila cell size or cell proliferation and affected overall body size (Brogiolo et al.,

2001; Britton et al., 2002; Kim et al., 2002; Garami et al., 2003; Hariharan and Bilder,

2006). In Drosophila, ecdysteroids reduced insulin signaling and inhibited growth

(Colombani et al., 2005). More recently, in lepidopteran insects such as Precis,

ecdysteroids were shown to work with insulin-like hormones to stimulate wing disc

growth. In Precis, disc growth occurs only in feeding larvae. A more typical pattern of

lepidopteran growth, however, is continuous through feeding and non-feeding (prepupal)

stages. We thus set out to determine if growth in the more typically developing tobacco

hornworm was driven by insulin alone, as in Drosophila, or by both insulin-like and

steroid hormones, as in Precis. Our results clearly implicate both hormones in Manduca

disc growth, despite the fact that growth occurs largely post feeding.

In the present studies, growth was shown to occur via the insulin signaling

pathway as indicated by phosphorylation of the Manduca insulin receptor and

downstream signaling kinase Akt (Fig. 6 and Fig. 7A). Short-term experiments indicated

that insulin was able to significantly activate the pathway by itself, but that ecdysone was

37

not (Fig. 6). After 48 hours, ecdysone significantly increased both total and

phosphorylated Akt on its own, as did insulin, although neither alone stimulated growth.

The maximal activation of Akt and significant growth in the wing discs was achieved by

incubating with both ecdysone and insulin (Fig. 7B and 7C). These results point strongly

to synergistic effects of ecdysone and insulin in regulating growth. Discs do not increase

in size when exposed to insulin alone despite clear demonstration that insulin can

enhance the phosphorylation of an endogenous insulin receptor as well as levels of

phospho- and total Akt. The dual-hormone requirement for Manduca disc growth means

that changes in somatic growth could come about by varying ecdysone levels, insulin

levels, or both.

The experiments described in this chapter delineated the parameters by which we

could reliably stimulate disc growth in Manduca sexta. The results raise questions

regarding the underlying mechanisms by which growth is stimulated. We chose to

investigate this in two ways. First we examined whether ecdysone and insulin were

together required to increase cell number. Cell division, a typical effect of steroids, may

necessitate additional input by insulin-like hormones in developing insects. Second, we

examined whether specific signaling proteins were enhanced by ecdysone and insulin and

whether the effects of ecdysone on growth were dependent upon insulin signaling. The

results of these approaches are developed in Chapters 2 and 3.

38

CHAPTER TWO

Effects of ecdysone and insulin on cell division

39

INTRODUCTION

During development, insulin signaling molecules function as growth factors

influencing size as well as differentiation of specialized cells. Insulin-like growth factors

(IGFs) have been known to affect cellular proliferation by stimulating cell cycle

progression (Dupont et al., 2000). Other growth factors such as fibroblast growth factor

and vascular endothelial growth factor, have been characterized as more potent mitogens,

but IGFs often synergize with these growth factors to produce an enhanced mitogenic

response (Kurenova et al., 2009; Shimotake et al., 2010; Guillemot et al., 2011; Wesche

Et al., 2011).

Steroids mediate their activity through binding to specific intranuclear receptor

proteins. These receptors may also be functionally linked to signaling pathways, at the

plasma membrane, for example activating tyrosine kinases or G protein coupled receptors

(Revelli et al., 1998). Steroids such as 17-beta-estradiol (E2), via binding to cytoplasmic

or membrane-associated receptors, have been seen to rapidly activate intracellular

signaling cascades such as ERK, PI3K and STATs (Fox et al., 2009).

Given this information, we wondered if ecdysone worked as a typical steroid

hormone and stimulated cellular division on its own, or if the requirement for insulin for

growth involved a co-requisite role in cell division. We used a novel detection assay,

EdU (5-ethynyl-2’-deoxyuridine), to directly measure de novo DNA synthesis (S-phase

of the cell cycle) using click chemistry (Salic and Mitchison, 2008; Click-iT EdU

imaging kit manual, Invitrogen, 2009). Click chemistry is a method of covalently

coupling an azide with an alkyne (Fig. 8). Detection of EdU employed the copper (I)

40

catalyzed click reaction with an azide modified fluorescent dye to form a stable triazole

ring. Because of the small size of the alkyne labeled nucleotide, no harsh denaturation

steps were needed to gain access to the DNA (Salic and Mitchison, 2008). In other

words, an alkyne modified nucleotide, EdU, once incorporated into DNA, could be easily

detected with an azide-modified fluorescent dye. These reagents were used to distinguish

the relative roles of insulin and ecdysone in cell division.

Figure 8. Click reaction between EdU and azide modified dye. EdU (5-ethynyl-2’-deoxyuridine, a thymidine analog, carries a terminal alkyne group. The terminal alkyne group reacts with azide in the presence of catalytic amounts of copper.

41


Animals, tissue culture, and hormones

Protocols for animal rearing, disc culture, and hormones employed for

experiments are as described in Chapter 1.

EdU labeling, fixing of and detection

EdU (Click-It EdU Imaging Kits-Invitrogen) was added to the Grace’s culture

medium at the start of incubation at a concentration of 10 M. After 48 hour incubation,

discs were washed two to three times in 3.7% formaldehyde in PBS and then incubated at

RT for 15 minutes. The discs were then washed twice in 3% BSA in PBS and

permeabilized and fixed using a standard formaldehyde fixation protocol (Invitrogen)

overnight. Discs were once again washed in 3% BSA in PBS and 0.5mL of Click-iT

reaction cocktail (43 μL 10X reaction buffer; 38 μL distilled water; 20 μL copper

sulphate; 1.2 μL Alexa Fluor 594 azide; 50 reaction buffer additive) was added to each

well. Samples were incubated for 30 minutes in the dark before being washed again with

3% BSA in PBS (Click-iT EdU imaging kit manual, Invitrogen). Discs were washed in

75% ethanol (x2), 95% ethanol (x2) and 100% ethanol (x2). Then discs were placed in

xylene for 60 seconds and moved to fresh xylene for 8 minutes. Discs were mounted on

slides (Fisherbrand Superfrost/Plus) with Permount and covered with cover slip. Slides

were stored at 4°C.

42

Imaging and analysis

Images were acquired using a BioRad MRC600 confocal imaging system

(Hercules, Ca, USA) with a Zeiss inverted microscope stand (wavelength 594 nm;

magnification of 40x, neutral density =5, F6 =1.0). Confocal microscopy equipment was

provided courtesy of the O’Malley Lab, Northeastern University. A grid was overlayed

on top of the slide to consistently map locations on the discs and subsequently removed

before pictures were taken.

Statistical analysis

Number of cells incorportated in EdU in the wing imaginal discs was subjected to

statistical analysis using one way ANOVA, with a Dunnett’s post hoc test to compare

specific treatments against a reference. In all cases, p<0.05 was used to determine

significance. All statistical analysis was performed using SPSS v.19 (licensed to

Northeastern University, 2011).

43

RESULTS

Cell proliferation

In conjunction with the stimulation of growth, ecdysone and insulin (Ec+Ins)

produced the greatest number of proliferating cells (Figs. 9 and 10). The center of the

discs were easy to distinguish under the microscope; but the edges were not. To solve this

problem, a grid was placed on top of the slides (at 40x magnification), and we

consistently chose the same 2-3 grids in the lower left area (roughly between the 6 and 8

o’clock positions) of the disc for the edge. Following incubation with ecdysone and

insulin, incorporation of EdU (DNA replication) increased significantly in both the center

and along the edge of the disc, although replication appeared more prevalent along the

outer edge of the disc (Fig. 9). Control samples showed little incorporation of EdU (Fig.

9). Discs treated with ecdysone alone showed significant cell division along the edges

(p<0.05), but not towards the center of the discs (Figs. 9 and 10). Conversely, discs

treated with insulin alone showed significant cell proliferation near the center of the disc

(p<0.05), but not along the edge. Overall, these results suggest that ecdysone and insulin

increase cell division, but ecdysone has a greater effect. Insulin does, however, augment

cell division when combined with ecdysone.

44

Figure 9. Detection of cell proliferation with EdU in wing discs after 48 hour incubation. Discs were treated with Grace’s medium (Control), ecdysone (Ec), insulin (Ins), or ecdysone and insulin (Ec+Ins) (n=8 for each treatment). Images are at 40x magnification taken at the center or at the outer edge of the disc using a confocal imaging system.

45

Figure 10. Number of proliferating cells (incorporating EdU) in wing discs after 48 hour incubation. Images were taken of discs treated with Grace’s medium (Control), ecdysone (Ec), insulin (Ins), or ecdysone and insulin (Ec+Ins) (n=8 for each treatment). A. The number of proliferating cells was counted in the center of each disc. B. The number of proliferating cells was counted at the outer edge of each disc. Significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).

b

b

b

b

a a

a a

A

B

46

DISCUSSION

In this chapter, we distinguished the influence of each hormone, ecdysone and

insulin, on cell division. As expected, the steroid hormone ecdysone successfully

stimulated cell division. Insulin by itself was also able to stimulate cell division in wing

discs. Potter et al. (2001) showed in Drosophila wing discs that insulin signaling was

responsible for increasing cell number as well as cell size. They overexpressed tuberous

sclerosis complex (TSC) homologues 1 and 2, which led to a decrease in cell number

(Potter et al., 2001). TSC is a downstream target that is normally inhibited in the insulin

pathway. Inactivating positive signaling components of the insulin pathway including

Drosophila insulin receptor (dinr), Drosophila PI3K (Dp110), and Drosophila Akt (dAkt)

all led to decreases in cell number and cell size (Leevers et al., 1996; Verdu et al., 1999;

Potter et al., 2001).

In Manduca, insulin significantly enhanced cell division by itself, but more

strongly in conjunction with ecdysone. This synergistic effect by a steroid and insulin on

cell division has also been shown in human breast cancer cells (Dupont et al., 2000;

Castoria et al., 2001; Hamelers and Steenbergh, 2003). Dupont et al. (2000) showed that

MCF-7 cells were stimulated to proliferate by both insulin-like growth factor I (IGF-I)

and the steroid estradiol (E2), individually. IGF induced proliferation by activating the

PI3K pathway. E2, via activation of the estrogen receptor, enhanced the expression of

growth related genes, including cyclin D1 as well as the PI3K pathway (Dupont et al.,

2000; Simoncini et al., 2000). It was further shown that a combination of these mitogens

resulted in a synergistic increase in cell division (Hamelers and Steenbergh, 2003).

47

Finally, if one of the receptors, either estrogen receptor or insulin receptor, was blocked,

then the proliferative response was suppressed (Dupont et al., 2000). This suppression

suggests that both hormones are needed to promote breast cancer cell proliferation.

In wing discs, ecdysone and insulin are both required for maximal cell division.

Ecdysone and insulin can also promote cell division on their own, suggesting that each

hormone can affect cyclins, proteins that control the progression of cells through the cell

cycle by activating cyclin-dependent kinase (Cdk) enzymes (see Uhlmann and Lopez-

Aviles, 2011). In MCF-7 breast cancer cells, insulin-like growth factor 1 receptor and

estrogen receptor were co-expressed and resulted in enhanced growth (Dupont and le

Roith, 2001). Estradiol induced expression of insulin receptor substrate (IRS-1) which

led to Akt activation. Estradiol also potentiated the effect of IGF-1 on the expression of

cyclin D1 and cyclin E, and phosphorylation of the retinoblastoma protein, a tumor

supressor (Dupont et al, 2000; Dupont and LeRoith, 2001). There are many cyclin

proteins, and they could have different sensitivities to ecdysone or insulin. These

hormones may also differentially regulate the expression of cyclin dependent kinase

inhibitors. This variability could explain why insulin promotes cell proliferation in the

center of the discs and ecdysone promotes cell proliferation towards the edge. When

ecdysone and insulin are both present, both sets of co-factors could be stimulated,

yielding greater overall proliferation and uniform tissue growth. Cell proliferation results

confirmed that the ecdysone and insulin signaling pathways were intertwined. Further

delineation of the relationships among signaling pathways involved in hormonally-

stimulated disc growth are described in Chapter 3.

48

CHAPTER THREE

Cellular interactions between ecdysone and insulin in the

regulation of disc growth

49

INTRODUCTION

As outlined in Chapter 1, lepidopteran growth occurs as an interplay between

insulin and the steroid hormone ecdysone. How is growth positively regulated by insulin

and steroids? Steroid hormones act through nuclear transcription factors to regulate gene

expression. In vertebrates, steroids such as progesterone, testosterone, and aldosterone

have all been shown to upregulate the insulin pathway (Berrie, 2001, Wu et al., 2010).

This relationship is best characterized for the interactions between estrogens and insulin-

like growth factors (IGFs). There is growing evidence to suggest that estrogen- and IGF-

mediated signaling pathways are linked. For example, in the brain, estrogen receptors

and IGF-I receptors are frequently expressed in the same cells. Estradiol and IGF-I

cooperate to regulate neuronal development, and both are activated in response to neural

tissue injury (Cardona-Gomez et al. 2003; Garcia-Segura et al., 2007). While the exact

molecular mechanisms involved in these interactions are still not well understood,

estrogen has been found to increase the expression of IGF-I receptors and IRS-I (Lee et

al. 2007). The estrogen receptor subtype known as estrogen receptor interacts with

PI3K, enhancing the activation of Akt and the phosphorylation of glycogen synthase

kinase-3 (GSK3) (Garcia-Segura et al. 2006). In addition, estradiol treatment results in

an increase in the phosphorylation of ERK (Garcia-Segura et al. 2006). These findings

suggest that estrogen effects in the brain may be mediated in part by the activation of the

signaling pathways of the IGF-I receptor.

A positive relationship also exists between estradiol and elements of the insulin

signaling cascade in cancer cell lines. The ligand-bound estrogen receptor was reported to

50

directly bind and activate the IGF type I receptor in human breast tumor cells (Hamelers

and Steenbergh, 2003). Castoria et al. (2001) reported that estradiol in MCF-7 breast

carcinoma cells activated the ERK pathway, PI3K, and Akt. They further showed that

inhibition of PI3K with the inhibitor LY294002 abolished the estradiol stimulation of Akt

and inhibited cell cycle progression (Castoria et al., 2001; Hamelers and Steenbergh,

2003).

Like the estrogen receptor, the peroxisome-proliferator-activated-receptor-

(PPAR- ) is a member of the nuclear hormone receptor family. Activation of PPAR-

enhances the regulation of glucose and lipid metabolism through regulation of insulin

signal transduction (Pandey et al., 2007). In some tissues, PPAR- activation leads to the

activation of insulin signaling pathways (Seto-Young et al., 2007). Thiazolidinediones

(TZDs), acting as PPAR- agonists, improve downstream insulin transduction in muscle

and stimulate PI3K and MAPK in blood vessels and liver cells (Belifiore et al., 2009),

and enhance insulin-signaling pathways and glucose uptake in type 2 diabetics (Pandey et

al., 2007; Choi et al., 2011). Seto-Young et al. (2007) have shown similar effects of

TZDs on insulin signaling in ovaries. This further supports the trend of positive cross-

talk between insulin signaling and the nuclear receptor family.

Testosterone, a steroid hormone, can also rapidly activate the insulin signaling

pathway. In cultured cardiomyocytes and in myoblasts, testosterone increased the

phosphorylation of ERK, Akt, TOR , and the downstream targets of TOR, 40S ribosomal

protein S6 kinase 1 (S6K1), and eukaryotic initiation factor 4E-binding protein 1 (4E-

BP1) (Altamirano et al., 2009; Wu et al., 2010). The S6K1 phosphorylation induced by

testosterone led to cardiac hypertrophy, and phosphorylation was blocked by the TOR

51

inhibitor, rapamycin. However, when Akt was inhibited, testosterone still activated TOR

and S6K1 (Altamirano et al., 2009). In a similar fashion, Serra et al. (2011) showed that

IGF-I was not essential for mediating testosterone’s effects on androgen-responsive

skeletal muscle. These findings suggest that although testosterone and insulin can work

together, testosterone does not necessarily require insulin; testosterone can stimulate

TOR by itself.

Just as steroids can enhance insulin signaling, insulin can alter steroid receptors.

For example, IGF-I and IGF-II are two of the most abundant growth factors in the

prostate (Pinches et al., 1991). IGF-I has been shown to activate androgen receptors and

thus stimulate cancer proliferation and growth in the absence of androgens

(Gnanapragasam et al., 2000; Pandini et al., 2005; Wu et al., 2006). Likewise, IRS-1

overexpression has been associated with tumor development in breast cancer.

Specifically, in estrogen sensitive breast cancer cell lines, IRS-1 was found to enhance

estrogen receptor activity, which led to an overgrowth of cells (Cesarone et al., 2006).

The effects of insulin on steroid receptors have received relatively little attention because

steroids are generally potent enough to stimulate growth on their own. A recent paper by

Morelli et al. (2010) provided underlying mechanisms for stimulatory effects of insulin

on steroid sensitivity in breast cancer cells. In estrogen-positive breast cancer cells, Akt2

modulated estrogen receptor transcriptional activity at multiple levels, including the

regulation of estrogen receptor expression, its nuclear retention, and the activation of

Forkhead transcription factor, FoxO3a (Morelli et al., 2010). FoxO3a co-localized and

co-precipitated with estrogen receptor in the nucleus, and further investigation suggested

a repressive effect of FoxO3a on estrogen receptor transcription (Morelli et al., 2010).

52

Hence, precedent exists for positive interactions between steroids and insulin in

vertebrates. Earlier chapters showed that, in insects, ecdysone and insulin stimulate

growth and cell division. In the present chapter, RNA interference (RNAi), kinase

inhibitors, and antibodies to receptors and signaling enzymes are employed to provide a

more detailed identification of interactions between ecdysone and insulin that stimulate

growth. In addition to further investigating the effects of ecdysone on insulin signaling,

the converse effects of insulin on ecdysone receptor content were explored. Together,

results confirm a mutually positive influence of steroids and insulin on growth-related

signaling pathways in growing wing discs.

53


Animals and tissue culture

Manduca sexta larvae were reared and discs were cultured as decribed in

Chapter 1.

Hormones

Insulin (NovoRapid) was diluted in Grace’s medium to a final concentration of

30μM. 20-Hydroxyecdysone (Sigma) was dissolved in Grace’s medium to a final

concentration of 0.1 μg/ml.

Knockdowns via RNAi

To determine if the observed disc growth was dependent upon the insulin receptor

(InR), RNA interference (RNAi) was used to reduce receptor content. The small-

interfering RNA (siRNA) was designed using the Whitehead siRNA Selection Web

Server (http://jura.wi.mit.edu/bioc/siRNA) (Yuan et al, 2004). Two separate siRNA

sequences,

IR-1 (5’-CCACCACGAACGGUUUAGUtt -3’) and

IR-2 (5’- CGCAGACGUUGUGAACAAUtt -3’), were directed against two different

regions of the insulin/bombyxin receptor gene. The Manduca insulin receptor sequence,

against which primers were designed, was obtained from the partial gene sequence of

Stefan Girgenrath (1999). A complete Manduca insulin receptor sequence was

subsequently published by Koyama et al. (2008) (GenBank accession no FJ169469) and

54

matched to the partial sequence used. A third siRNA, (5’-

GACACGUGCUGAAGUCAAGtt -3’), directed against green fluorescent protein (GFP),

was used as a non-specific sequence to test the effects of nonsense siRNA in culture.

Custom siRNA (40 nmol, standard purity, annealed) was synthesized by Ambion, Inc.

(www.ambion.com). siRNA was also generated using a siRNA synthesis PCR kit

(Invitrogen).

Quantitative real time PCR (qPCR) analysis of gene expression

Manduca bombyxin/insulin receptor (IR) and ribosomal protein 49 (RP49) were

analyzed by qPCR using a real-time sequence detection system (Applied Biosystems

7000). Individual wing discs were used from day five, fifth instar larvae. Total RNA

was collected from samples using SV Total RNA isolation kit (Promega). RNA was

tested spectrophometrically for purity and concentration, and run on a formaldehyde gel

to ensure quality. 100 ng of each RNA sample were reversed transcribed using random

hexamer primers in a Superscript III First Strand Synthesis System (Invitrogen)

according to the manufacturer’s protocol. Primers for qPCR analysis were designed

using Primer Express (Applied Biosystems). Primer sequences were as follows:

IR forward 5’- GGGATTTCGGCATGACCAGAGATATT-3’

IR reverse 5’-TCGTTCGACAGGCCCTGATATGG-3’

rp49forward 5’-GAGGAATTGGCGTAAACCTAGAG-3’

rp49reverse 5’-TGACGGGTCTTCTTGTTGGA-3’

PCR reactions were performed using SYBR green mastermix (Applied Biosystems). IR

55

PCR reactions contained 0.1 μM of each primer. RP49 PCR reactions contained 0.9 μM

of each primer. qPCR was performed in a final volume of 20 μl. All quantitative

reactions were subjected to 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of

95°C for 15s, and 60°C for 1 min. In addition, products were subjected to gel

electrophoresis to check product size for each primer pair. In order to account for

potential contamination, non-reverse-transcribed total RNA (with potential genomic

DNA contamination) and non-template controls were included. No products were

observed in these reactions. Dilution curves generated by serial dilutions of cDNA were

used to calculate amplification efficiencies. Transcript levels of the target genes were

normalized to the Manduca ribosomal gene rp49 after correcting for differences in

amplification efficiency (Pfaffl, 2001).

Western blot analysis

Wing discs were homogenized in 40 μL of 2X SDS sample buffer, and

incubated at 90°C for three to five minutes. The samples were then run on a 10-20%

SDS-PAGE gradient gel (Bio-Rad) to separate the proteins (Laemmli, 1970). They were

transferred from gels to nitrocellulose membranes at 4°C. Membranes were then blocked

in 3% BSA or 5% milk for one hour, rinsed quickly (15 minutes total) and placed on

primary antibody overnight. Primary antibodies included: anti-phospho-4EBP,

antiphospho-Akt, total Akt, anti-phospho-ERK, anti-actin, anti-phospho-GSK (Cell

Signaling), anti-USP (a gift from Prof. Fotis Kafatos, currently at Imperial College

London) and anti-EcR (Developmental Studies Hybridoma Bank, University of Iowa,

Department of Biology). Blots were rinsed and placed on secondary anti-rabbit antibody

56

with attached HRP (Cell Signaling) for 75 minutes. They were treated with Western

blotting chemiluminescence reagents (Pierce ECL Western blotting substrate, Thermo

Scientific). The blots were exposed on blue-sensitive autoradiographic film (Marsh Bio

Products) and developed (Kodak GBX fixer and developer). Blots were scanned and

analyzed using ImageJ (Abramoff et al., 2004). Protein bands were normalized to actin

as a loading control where appropriate.

Statistical analysis

Gene expression in the wing imaginal discs was subjected to statistical analysis

using one way ANOVA, with a Dunnett’s post hoc test to compare specific treatments

against a reference. Kaplan-Meir and Cox-regression tests were used to evaluate %

detection of real-time PCR data. In all cases, p<0.05 was used to determine significance.

All statistical analyses were performed using SPSS v.19 (licensed to Northeastern

University, 2011).

57

RESULTS

Inhibition of disc growth via siRNA against the bombyxin/insulin receptor (InR)

As previously observed in Chapter 1, discs incubated with ecdysone and insulin

(Ec+Ins) grew significantly (Dunnett Multiple Comparison test, p<0.05) (Fig. 11). The

relevance of InR to glandular growth was explored using RNAi-mediated knockdown of

the receptor. In the presence of siRNA directed against the bombyxin/insulin receptor,

ecdysone and insulin (Ec+Ins+IRsiRNA) no longer stimulated growth (Fig. 11).

Knockdown of the receptor was confirmed using real-time PCR (Fig. 12A, discussed in

greater detail on the following pages). Control RNAi, directed against green fluorescent

protein (GfpsiRNA), did not inhibit growth (Fig. 11). This indicated that the effects of

the RNAi were specific and that merely incubating discs with RNAi was not toxic. As

seen previously, discs treated with ecdysone alone (Ec) or insulin alone (Ins) did not

increase significantly in size relative to Grace’s medium alone (Control). The size of

discs incubated in ecdysone alone or insulin alone was not affected by IRsiRNA (Dunnett

Multiple Comparison test, p=0.123, p= 0.074, respectively) (Fig. 11).

58

Figure 11. Effects of siRNA, LY294002, and rapamycin on growth in wing discs. Discs were removed from day 5 fifth stage larvae and cultured for 24 hours in Grace’s medium/10% FBS/10% antibiotic-antimycotic, then transferred to the same medium containing Grace’s medium alone (control), 20-hydroxyecdysone and insulin (Ec+Ins), 20-hydroxyecdysone alone (Ec), or insulin alone (Ins) for 2 days. Discs were also treated with small interfering RNA directed against the bombyxin/insulin receptor (IRsiRNA), LY294002 (LY), an inhibitor of PI3K, or rapamycin, an inhibitor of TOR. Growth was assessed by measurement of disc width. Significant increase in growth is designated by “b” (Dunnett Multiple Comparison test, p<0.05).

Measurement of insulin receptor transcript

Levels of insulin receptor transcript (InR) were measured using real-time PCR

(Fig. 12A, C). Real-time PCR uses SYBR Green to detect the presence of double-

stranded DNA (dsDNA). The amount of fluorescence increases as the amount of double-

stranded DNA increases. As a control, the transcript levels of Rp49, a ribosomal gene,

were also measured (Fig. 12B, D). Fig. 12A and 12C indicate that there was a significant

and faster increase in InR in discs treated with both ecdysone and insulin (Ec+Ins)

(Kaplan-Meir/Mantel-

b b

a a a

a a

a

a

a

a a

a a a

a

a a

a a

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Figure 12. Detection of transcript in wing discs after 48 hour treatment. RNA was collected from wing discs (n=8) after treatment with ecdysone (Ec), insulin (Ins), ecdysone and insulin (Ec+Ins), or ecdysone and insulin and siRNA against the insulin receptor (Ec+Ins+IRsiRNA). 100ng of RNA from each sample was reverse transcribed with random hexomer primers and cDNA was analyzed using quantitative real time PCR A. The detection of insulin receptor transcript (InR) is indicated at various cycle times depending on treament. B. The detection of Rp49 transcript, a Manduca ribosomal gene was measured as a control. C. Fold changes in mRNA were calculated for InR and D. Rp49 using the Pffafl method. Significant differences are indicated by “b.”

Cox, p=0.00006; Pffafl method, p<0.05). InR dsDNA in the (Ec+Ins)-treated samples

was detected much earlier (cycle 5) than in controls (cycle 36), reflecting an increase in

InR transcript (Fig. 12A). InR dsRNA in (Ec+Ins)-treated samples were detected earlier

than ecdysone-treated (Ec) or insulin-treated (Ins) samples (Kaplan-Meir/Mantel-Cox,

p=0.00008). InR dsRNA in (Ec)- and (Ins)-treated samples were also detected

A B

b

a a a

C D

a a a a

60

significantly earlier than in untreated controls (Kaplan-Meir/Mantel-Cox, p=0.00048),

but differences were not observed between (Ec) and (Ins) treated groups (Kaplan-

Meir/Mantel-Cox, p=0.026) (Fig. 12A). The increase in insulin receptor transcript

stimulated by ecdysone and insulin independently was not enough to cause an increase in

overall disc size. Treatment with ecdysone, insulin and control siRNA

(Ec+Ins+GfpsiRNA) produced results similar to (Ec+Ins) (data not shown). As expected,

none of the treatments altered transcription of the ribosomal protein, Rp49 (Fig. 12B, D).

Treatment was a significant and independent predictor of detection (Wald 55.7, df

=4, p<0.00001). In this case “detection” refers to statistical distinction relative to control.

Relative to controls, ecdysone-treated samples were 27 times more likely to be detected

and insulin-treated samples were 23 times more likely to be detected relative to controls

(Wald 41.3, df=1, p<0.00001; Wald 40.6, df=1, p<0.00001). Together, ecdysone and

insulin were 52 times more likely to be detected than the controls (Wald 56.3, df= 1;

p<0.00001). Primer was an independent predictor (Wald 56, df=1, p<0.00001) after

controlling for the effect of treatment. Primer 1 (InR) was 27 times more likely to be

detected than primer 2 (Rp49) (Wald 56, df=1, p<0.00001).

Examination of insulin signaling pathways

RNAi against the bombyxin/insulin receptor blocked growth, so it was

hypothesized that it would also decrease the activation of downstream signaling proteins.

Western blots were used to measure changes in phosphorylated Akt (pAkt),

phosphorylated ERK (pERK), and phosphorylated 4EBP (p4EBP) (locations in signaling

61

Figure 13. Activation of phosphoproteins in wing discs after 48 hour incubation. Western blots of pAkt, pERK, and p4EBP were all quantified using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).

pathway shown in Fig. 2, results shown in Fig. 13). There was no significant increase in

pERK stimulated by any of the treatments (Fig. 13). Combined treatment with ecdysone

and insulin (Ec+Ins) significantly increased pAkt and p4EBP (Fig. 13, Fig. 16A). RNAi

against the bombyxin/insulin receptor (IRsiRNA) significantly decreased both pAkt and

p4EBP (Fig. 13). Insulin alone (Ins) stimulated a significant increase in pAkt. p4EBP

was not significantly increased by insulin alone in this particular set of experiments but

was in a later set of experiments (Fig. 16). Ecdysone (Ec) was able to stimulate pAkt, but

62

had almost no effect on p4EBP (Fig. 13). This result suggested that Akt is a possible site

of interaction/cross-talk between ecdysone and insulin in the regulation of growth.

The effects of insulin and ecdysone on pAkt are due at least in part to changes in

total Akt (Fig. 14). There was a significant increase in total Akt in response to ecdysone

treatment combined with insulin (Ec+Ins) (Dunnett Multiple Comparison test, p<0.05).

Though not statistically significant, both insulin (Ins) and ecdysone (Ec) showed a trend

in increasing total Akt. IRsiRNA significantly inhibited total Akt expression. As

expected, samples treated with control RNAi directed against GFP (COsiRNA, same as

GFPsiRNA) did not show a change in total Akt expression (Fig. 14).

Figure 14. Total Akt in wing discs after 48 hour incubation. Western blots of total Akt were quantified using ImageJ (Abramoff et al., 2004). Samples were treated with ecdysone (Ec), insulin (Ins), ecdysone and insulin (Ec+Ins), or ecdysone and insulin and siRNA against the insulin receptor (Ec+Ins+IRsiRNA) or LY294002 (LY). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).

b b

a a a a a

a a a

a

a a

63

Effects of PI3 kinase and TOR inhibitors on disc growth

Disc growth was successfully inhibited via knockdown of the insulin receptor

which also led to a decrease in activation of downstream signaling proteins. Given the

importance of PI3K in the insulin signaling pathway, we anticipated the PI3K inhibitor

LY294002 (LY) would have effects similar to siRNA. As expected, LY blocked growth

stimulated by ecdysone and insulin (Ec+Ins) (Fig. 11), as well as accompanying changes

in pAkt, total Akt, and p4EBP (Figs. 13 and 14).

We next examined effects of the TOR inhibitor, rapamycin, on disc growth.

Rapamycin significantly decreased growth in discs treated with ecdysone and insulin

(Ec+Ins+Rapamycin) (Fig. 11). Rapamycin also decreased the size of discs treated with

insulin alone (Ins) or ecdysone alone (Ec) (Fig. 11). As expected, rapamycin

significantly decreased the phosphorylation of 4EBP (Fig. 15A). Non-phosphorylated

4EBP serves as a potent inhibitor of translation (refer to Fig. 2, Introduction). Together,

results shown in Figs. 11 through 15 argue strongly that ecdysone and insulin enhance

growth through the activation of IR, PI3K, pAkt, TOR, and 4EBP.

64

Figure 15. Effects of rapamycin on p4EBP and EcR in wing discs after 48 hour incubation. Western blots of A. p4EBP and B. EcR were quantified using Image J (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05). Effects of insulin on ecdysone receptor protein content

We next examined whether insulin could in turn affect the ecdysone receptor.

Antibodies were available for the Manduca ecdysone receptor (isoform A, EcR-A) and

the transcription factor with which it heterodimerizes, ultraspiracle (USP). The same

Western blots that were previously used to detect insulin pathway proteins were stripped

and re-probed for EcR and USP. As shown in Fig. 16, combined treatment with

ecdysone and insulin (Ec+Ins) led to increased EcR and USP relative to controls.

Ecdysone (Ec) significantly stimulated EcR content on its own, but insulin (Ins) did not

(Fig. 16). USP was only slightly stimulated by ecdysone (Ec), and not at all by insulin

(Ins). All three inhibitors discussed previously, RNAi directed against the

bombyxin/insulin receptor (IRsiRNA), LY294002 (LY), and rapamycin, significantly

decreased disc content of EcR and USP (Fig. 15B, Fig. 16). Together, these results

65

suggest that the insulin receptor, PI3K, and TOR affect EcR, even though insulin does

not enhance cellular content of EcR directly.

Figure 16. Ecdysone receptor proteins in wing discs after 48 hour incubation. Western blots of ecdysone receptor (EcR) and untraspiracle (USP) were quantified using ImageJ (Abramoff et al., 2004). All data was normalized to actin and significant values are designated by “b” (Dunnett Multiple Comparison test, p<0.05).

b b

a a a a

a a a a a a a

a a a a a a a a a

b b

b

66

DISCUSSION

In Manduca wing discs, growth in culture occurs only in the presence of both

ecdysone and insulin. In our initial study of Manduca discs (Chapter 1), replicated in the

present chapter, we found that ecdysone stimulates elements of the insulin pathway,

specifically pAkt, but has the greatest effect when combined with insulin. We used

siRNA directed against the bombyxin/insulin receptor (IRsiRNA), to determine if the

synergistic effects of insulin and ecdysone on growth of the wing discs were in fact

occurring through the insulin receptor. The IRsiRNA successfully inhibited growth in

ecdysone- and insulin-treated discs and confirmed that the receptor was a requisite player

in the signaling pathway. A central focus of Akt was also suggested by the inhibitory

effects of upstream and downstream inhibitors (LY294002 and rapamycin, respectively).

Our findings with regard to the enhancement of Akt by members of the nuclear

receptor superfamily are in keeping with what others have found in several human

diseases including cancer. For example, in vertebrate thyroid cancer, the thyroid

hormone receptor interacts with PI3K and activates Akt to increase cell proliferation and

motility (Furuya et al., 2006; Furuya et al., 2007; Furuya et al., 2009). Cheng et al.

(1996) found that Akt was overexpressed estrogen-sensitive ovarian tumor cell lines and

subsequently showed that siRNA silencing of Akt blocked transformation of these cell

lines.

Furthermore, Akt has also been shown to activate steroid receptors. The androgen

receptor plays an important role in early prostate cancer. Studies have shown that

androgen receptor transcriptional activity and expression are regulated by Akt (Paliouras

and Diamandis, 2008). The mechanism by which Akt influences steroid receptor

67

activation is not entirely clear, but a recent study by Morelli et al. (2010) offers a

potential model. Morelli et al. (2010) showed that estrogen receptor and insulin-like

growth factor I receptor (IGF-IR) pathways engage in cross-talk in breast cancer.

FOXO3a in these tissues inhibits estrogen receptor expression. Activation of IGF-I/IGF-

IR and concomitant activation of Akt leads to phosphorylation of FOXO3a, reducing its

nuclear content and enhancing estrogen receptor expression. Morelli et al. (2010)

observed a strong decrease in estrogen receptor expression, at both RNA and protein

levels, and a decrease in growth, in FoxO3a-overexpressing cells (Morelli et al., 2010).

On the flip side, in FoxO3a-silenced cells, Morelli et al. (2010) noted an increase in cell

proliferation. The experiments were repeated in two additional estrogen-receptor-

positive cell lines. Our own results are in keeping with an inhibitory effect of FOXO on

EcR transcription. Insulin may remove this inhibition via its effects on Akt and FOXO,

although in wing discs, insulin does not, alone, increase EcR content.

We have generated a model for the stimulatory effects of insulin and ecdysone, in

keeping with our results (Fig. 17A). Ecdysone stimulates growth by increasing cellular

content of its own receptor, as well as the phosphorylation and activation of Akt. The

stimulatory effects of insulin are also mediated by activation of Akt. Akt phosphorylates

FOXO, preventing its entry into the nucleus. A reduction in nuclear FOXO augments

EcR expression. Akt also activates TOR, enhancing growth through the phosphorylation

and inhibition of 4EBP, and the phosphorylation and activation of 70S6K. However,

activation of Akt alone is insufficient to stimulate growth in the absence of ecdysone,

which enhances cellular content and activity of the activated ecdysone receptor. Thus,

growth is stimulated by the combined effects of both hormones (Fig. 17A).

68

This model also explains what we see in the presence of siRNA, and inhibitors of

the insulin signaling pathway (Fig. 17B). siRNA against the insulin receptor (IRsiRNA),

prevents increases in pAkt and total Akt. LY294002 (LY) and rapamycin also cause a

reduction in Akt phosphorylation and action. Reduced Akt activity allows FOXO into

the nucleus, causing a decrease in EcR transcription. The reduction in EcR transcription

blocks growth-promoting effects of ecdysone and further reduces Akt activity and

growth.

Figure 17. Suggested mechanism through which ecdysone and insulin synergistically interact to modulate EcR expression and activity. A. In the presence of ecdysone and insulin EcR is liganded (1) and enhances the activation of Akt (2), allowing Akt to effectively phosphorylate and thus inhibit FOXO transcription factor (3), and also activate TOR (4), promoting the nuclear exclusion of and retention of FOXO in the cytoplasm so FOXO can no longer inhibit EcR transcription and expression (5). B. Akt inhibition (1) is a result of siRNA directed against the bombyxin/insulin receptor or via LY294002 inhibiting PI3K, leading to the activation and subsequent nuclear recruitment of FOXO (2). Akt inhibition causes a reduction in TOR (or direct inhibition of TOR by rapamycin) allowing non-phosphorylated 4EBP to block protein synthesis (3) while FOXO causes EcR transcriptional repression, reduction of liganded-EcR retention in the nucleus and consequently, less EcR recruitment on the promoter leading to downregulation (4).

69

Our results focus new attention on changes in ecdysone receptor content,

particularly as regulated by Akt/FOXO, in steroid-dependent growth. FOXO proteins are

evolutionarily conserved across species and are involved in diverse cellular and

physiological processes including cell proliferation, cancer, and cellular response to

reactive oxygen species (ROS). Though insect growth has been studied extensively in

Drosophila, that animal model does not seem to share the antagonistic relationship

between insulin and nuclear FOXO seen in other animal models. As mentioned earlier,

reduced insulin signaling in Drosophila reduces body size. Recently, Slack et al. (2011)

showed that removal of FOXO failed to return normal function to flies with decreased

insulin signaling. By contrast, in C. elegans, removing DAF-16, the worm FOXO

transcription factor, suppressed all negative phenotypes in insulin-deficient worms (Slack

et al., 2011). This suggests that there is evolutionary divergence in the mechanisms that

control effects of insulin signaling in Drosophila relative to other organisms.

It may be of interest to see if growth of Manduca wing discs is sensitive to

reactive oxygen species (ROS). FOXO is important for defensive response to cellular

stress, including increased oxidative stress levels (Greer and Brunet, 2005). In mammals,

there are four FOXO family members: FOXO1, FOX3, FOXO4, and FOXO6.

Phosphorylation of FOXO1, FOXO3, and FOXO4 by Akt leads to their retention in the

cytoplasm, thereby inhibiting their transcriptional activities (Brunet et al., 1999; Rafalski

and Brunet, 2011). When FOXO6 is phosphorylated, subcellular localization is not

affected but transcription activity is still inhibited (van der Heide et al., 2005; Rafalski

and Brunet, 2011). In neural stem cells, FOXO3 is involved in the transcriptional

regulation of genes involved in cellular response to hypoxia (low oxygen) (Renault et al,

70

2009; Rafalski and Brunet, 2011). For example, genes known to be upregulated in

hypoxic brains are downregulated in FOXO3-null neural stem cells (Renault et al, 2009).

In the liver, accumulation of lipid metabolites leads to a reduction in Akt activity, and

enhances the entry of FOXO into the nucleus, which then activates transcription of redox

enzymes (Goldstein et al., 2005; Papconstantinou, 2009; Rafalski and Brunet, 2011).

Hypoxia may be a stronger regulator of growth than previously seen. Under hypoxic

conditions, FOXO would be highly active, and EcR transcription would be down-

regulated, leading to less active Akt and a decrease in growth.

Creating a FOXO siRNA would be an interesting future experiment to test our

model. A FOXO knockdown would reduce the amount of FOXO in the nucleus, leading

to an increase in EcR, and more active Akt to remove even more FOXO from the

nucleus, enhancing growth. FOXO knockdown might even promote/sustain growth in

the absence of insulin or ecdysone. FOXO knockdown may also reveal other players

involved in ecdysone-stimulated growth, or in the response of growing tissues to cellular

stress. Likewise, an Akt knockdown would provide useful information relative to our

model for disc growth. We would expect an elevation in nuclear FOXO following Akt

knockdown, regardless of the presence of ecdysone or insulin. A resulting decrease in

EcR transcription would lead to a drastic decline in growth.

To summarize, the results of the present study shed new light on the roles of

ecdysone and insulin in insect disc growth. Insulin appears to permit a requisite increase

in ecdysone receptor content. Only when discs are fully sensitive to ecdysone can

maximal growth occur, through insulin- and ecdysone-stimulated activation of Akt. The

same pathways are likely to regulate post-embryonic development in other organisms,

71

with our results placing a fresh focus on the importance of insulin in steroid-driven

growth.

72

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Positive cross-talk between ecdysone and insulin signaling ...597/fulltext.pdf · 3 POSITIVE CROSS-TALK BETWEEN ECDYSONE AND INSULIN SIGNALING IN INSECT GROWTH A dissertation presented