Signal transduction oflipopolysaccharide-inducedosteoclast differentiation

NO B U Y U K I UD A G A W A, NO B U A K I SA T O, SH U H U A YA N G, MI D O R I NA K A M U R A,TE R U H I T O YA M A S H I T A, HI R O S H I NA K A M U R A & TO S H I H I D E NO G U C H I

Osteoclasts, the multinucleated cells that resorb

bone, originate from monocyte/macrophage lineage

cells. Osteoblasts (or bone marrow stromal cells) are

involved in osteoclastogenesis (27, 30). Macrophage

colony-stimulating factor, produced by osteoblasts, is

an essential factor for osteoclast formation (39).

Receptor activator of nuclear factor-kappa B (NF-jB)

ligand (RANKL) is another cytokine essential for

osteoclastogenesis and expressed by osteoblasts as

a membrane-associated cytokine (3, 12). Osteoclast

precursors express RANK (a receptor of RANKL),

recognize RANKL expressed by osteoblasts through

cell–cell interaction and differentiate into osteoclasts

in the presence of macrophage colony-stimulating

factor. Osteoprotegerin, produced mainly by osteo-

blasts, is a soluble decoy receptor for RANKL (31).

Osteoprotegerin blocks osteoclastogenesis by inhib-

iting the RANKL–RANK interaction. Bone resorption-

stimulating hormones and cytokines enhance the

expression of RANKL in osteoblasts. Mature osteo-

clasts also express RANK, and RANKL supports the

survival and stimulates the bone-resorbing activity of

osteoclasts (3, 12).

Lipopolysaccharide, a major constituent of the cell

wall of gram-negative bacteria, is proposed to be a

potent stimulator of bone resorption in inflammatory

diseases (21). CD14 is a membrane-anchored glyco-

protein that functions as a member of the lipopoly-

saccharide receptor system. Toll-like receptor-4 is a

critical receptor and signal transducer for lipopoly-

saccharide (2, 38). Bacterial lipoprotein/lipopeptides

have pathogen-specific molecular patterns. The

complex of Toll-like receptor-6 and Toll-like recep-

tor-2 recognizes diacyl lipopeptide (2, 7). It has been

reported that lipoproteins derived from Mycoplasma

salivarium, a member of the human oral microbial

flora, and a synthetic diacyl lipopeptide (FSL-1)

activate human gingival fibroblasts to induce

inflammatory cytokine production via p38 mitogen-

activated protein kinase (MAPK)-mediated signals

(22).

Toll-like receptor family members have an intracy-

toplasmic region, called the Toll/interleukin-1 recep-

tor homology domain. Through the homophilic

interaction of Toll/interleukin-1 receptor domains,

myeloid differentiation factor 88 (MyD88) is asso-

ciated not only with cytokine receptors for inter-

leukin-1 and interleukin-18, but also with various

Toll-like receptors (1, 2). MyD88-deficient (MyD88)/))

mice showed resistance to lipopolysaccharide-in-

duced responses, including cytokine production by

macrophages, B-lymphocyte proliferation and endo-

toxin shock (1, 17). MyD88)/) mice did not respond to

interleukin-1, interleukin-18 or other microbial cell

wall components such as peptidoglycan and lipopep-

tides (29). However, MyD88-deficient macrophages

showed a delayed activation of NF-jB and MAPK

cascades in response to lipopolysaccharide (17). In

addition, lipopolysaccharide induced the functional

maturation of MyD88-deficient dendritic cells, inclu-

ding the up-regulation of co-stimulatory molecules

(16). These results indicate the existence of a MyD88-

independent pathway through Toll-like receptor-4.

Toll/interleukin-1 receptor domain-containing

adapter-inducing interferon-b (TRIF) was identified as

an adopter involved in MyD88-independent signaling

pathways (33). TRIF plays essential roles in Toll-like

receptor-4- and Toll-like receptor-3-mediated path-

ways (11, 34). TRIF-related adaptor molecule (TRAM)

was identified as an adopter specifically involved in the

Toll-like receptor-4-mediated MyD88-independent

signaling pathway (6, 35). Using TRIF-deficient

(TRIF)/)) mice and TRAM-deficient (TRAM)/)) mice,

it was shown that both MyD88-dependent and

56

Periodontology 2000, Vol. 43, 2007, 56–64

Printed in Singapore. All rights reserved

� 2007 The Authors.

Journal compilation � 2007 Blackwell Munksgaard

PERIODONTOLOGY 2000

TRAM–TRIF-dependent pathways were required for

lipopolysaccharide-induced pro-inflammatory cyto-

kine production in macrophages and for lipopolysac-

charide-induced activation of B lymphocytes (35). In

addition, p38 MAPK- and MAPK/ERK (extracellular

signal-regulated kinase) (MEK)/ERK-mediated signals

are shown to be involved in lipopolysaccharide-in-

duced pro-inflammatory cytokine production in

human osteoblastic cells (18). Using MyD88)/) mice

and TRIF)/) mice, we explored the roles of MyD88 and

TRIF in osteoclast differentiation and function

induced by lipopolysaccharide, interleukin-1a and

diacyl lipopeptide (24).

Muramyl dipeptide, the essential structure respon-

sible for the immunoadjuvant activity of peptidogly-

can, is present in gram-positive and gram-negative

bacterial cell walls. Muramyl dipeptide alone is unable

to induce osteoclast formation in the coculture, but

synergistically stimulated is induced by lipopolysac-

charide and interleukin-1a, but not by 1a,25-dihyd-

roxy vitamin D3 [1a,25(OH)2D3] or prostaglandin E2.

We have explored the roles of muramyl dipeptide in

osteoclastogenesis and the function of mature osteo-

clasts (37).

MyD88 is an essential signal forosteoclast differentiation inducedby lipopolysaccharide andinterleukin-1

Lipopolysaccharide, interleukin-1a and diacyl lipo-

peptide, as well as 1a,25(OH)2D3 plus prostaglandin

E2, stimulated the formation of tartrate resistant acid

phosphatase-positive osteoclasts in cocultures of pri-

mary osteoblasts and bone marrow-derived hemo-

poietic cells obtained from wild-type mice (Fig. 1A). In

contrast, lipopolysaccharide, interleukin-1a and dia-

cyl lipopeptide did not induce osteoclast formation in

the coculture of MyD88)/)-derived osteoblasts and

hemopoietic cells (Fig. 1A). The number of osteoclasts

that formed in response to 1a,25(OH)2D3plus prosta-

glandin E2 in cocultures prepared from MyD88)/)

mice was always significantly smaller than that from

wild-type mice (Fig. 1A). This suggests that MyD88 is

involved in osteoblast function, including the support

of osteoclasts, in response to 1a,25(OH)2D3 plus

prostaglandin E2. Osteoprotegerin was reported to

completely inhibit the osteoclast formation induced

by lipopolysaccharide, interleukin-1a, diacyl lipopep-

tide and 1a,25(OH)2D3 plus prostaglandin E2 in wild-

type cocultures. Treatment of wild-type osteoblasts

with lipopolysaccharide, interleukin-1a and diacyl

lipopeptide stimulated the expression of RANKL

messenger ribonucleic acid within 24 h. These results

suggest that MyD88-mediated signals are important

to osteoblasts, but not osteoclast precursors, in

osteoclast formation induced by lipopolysaccharide,

interleukin-1a and diacyl lipopeptide in the coculture

system (24) (Fig. 4B).

Both MyD88-dependent and TRIF-dependent

pathways are essential for pro-inflammatory cytokine

production induced by lipopolysaccharide in perito-

neal macrophages (11, 34). Using TRIF)/) mice, we

examined the importance of TRIF-mediated signals in

lipopolysaccharide-induced osteoclast formation.

Lipopolysaccharide stimulated osteoclast formation

in cocultures prepared from TRIF)/) mice as well as

wild-type mice. Similarly, interleukin-1a and diacyl

lipopeptide stimulated osteoclast formation in

cocultures prepared from TRIF)/) mice. These results

suggest that the TRIF-mediated pathway is not

involved in osteoclast formation induced by interleu-

kin-1 and Toll-like receptor ligands (24) (Fig. 4B).

Role of MyD88 inpro-inflammatory cytokineproduction induced bylipopolysaccharide

We next examined pro-inflammatory cytokine pro-

duction in osteoblasts and macrophages prepared

from TRIF)/) and MyD88)/) mice. Treatment with

lipopolysaccharide for 24 h stimulated interleukin-6

production in TRIF)/) and wild-type osteoblasts, but

not in MyD88)/) osteoblasts (Fig. 1B). Lipopolysac-

charide stimulated interleukin-6 production in wild-

type bone marrow macrophages, but not in TRIF)/) or

MyD88)/) bone marrow macrophages (Fig. 1C). These

results suggest that the TRIF-dependent pathway is

involved in lipopolysaccharide-induced interleukin-6

production in macrophages but not in osteoblasts (24)

(Fig. 4A). At present, it is not known why immune cells

such as macrophages and B-lymphocytes required

both MyD88 and TRIF signaling in response to

lipopolysaccharide. We previously reported that

lipopolysaccharide stimulated the production of

pro-inflammatory cytokines, such as interleukin-1b,

tumor necrosis factor-a and interleukin-6, in bone

marrow macrophages, but not in osteoclasts (14).

Thus, osteoclasts respond to lipopolysaccharide

through Toll-like receptor-4, but the characteristics of

osteoclasts are quite different from those of their

57

Signal transduction of lipopolysaccharide-induced osteoclast differentiation

precursors, bone marrow macrophages. These results

suggest that TRIF is important for the function of

immune cells but not that of nonimmune cells, such as

osteoblasts and osteoclasts. Loss of immune respon-

siveness to lipopolysaccharide in osteoclasts must be a

requirement for performing essential roles in physio-

logical bone turnover. Further studies will elucidate

the significance of the requirement of TRAM–TRIF

signals in immune cells.

MyD88 is involved in the survivalof osteoclasts supported bylipopolysaccharide andinterleukin-1

We previously reported that purified osteoclasts

spontaneously died as a result of apoptosis within

36 h, and lipopolysaccharide and interleukin-1apromoted the survival of osteoclasts (15). Purified

osteoclasts were prepared from cocultures of osteo-

blasts and bone marrow cells obtained from wild-type,

MyD88)/), Toll-like receptor-4)/) and TRIF)/) mice.

Most of the osteoclasts died spontaneously and dis-

appeared within 24 h. RANKL promoted the survival of

osteoclasts derived from MyD88)/), Toll-like receptor-

4)/) and TRIF)/) mice. Lipopolysaccharide and inter-

leukin-1a supported the survival of wild-type and

TRIF)/) osteoclasts, but not of MyD88)/) osteoclasts

(Fig. 2A). Interleukin-1a and RANKL, but not lipo-

polysaccharide, promoted the survival of osteoclasts

derived from Toll-like receptor-4)/) mice. Diacyl

lipopeptide (a ligand for the Toll-like receptor-2 plus

Toll-like receptor-6 complex) did not support the

survival of osteoclasts derived from any of the mice

(Fig. 2A). Takami et al. (28) reported that mature

A

Control 1,25D3+

PGE2

LPS IL-1α Diacyllipopeptide

Control 1,25D3+

PGE2

LPS IL-1α Diacyllipopeptide

WT MyD88-/-

0

200

400

600

800

1000

Ost

eocl

asts

/ wel

lOsteoblasts

IL-6

(ng

/ml)

Control LPS Control LPS Control LPSWT MyD88-/-TRIF-/-

B C Bone marrow macrophages

IL-6

(ng

/ml)

Control LPS Control LPS Control LPSWT MyD88-/-TRIF-/-

Fig. 1. Myeloid differentiation factor 88 (MyD88) is

essential for osteoclastogenesis induced by lipopolysac-

charide, interleukin-1a (IL-1a), and diacyl lipopeptide. (A)

Effects of 1a,25(OH)2D3 (1,25D3) plus prostaglandin E2,

lipopolysaccharide, IL-1a and diacyl lipopeptide on oste-

oclast formation in cocultures of osteoblasts and hemo-

poietic cells prepared from male wild-type (WT) and

MyD88)/) mice. Calvarial osteoblasts and bone-marrow-

derived hemopoietic cells prepared from wild-type and

MyD88)/) mice were cocultured for 7 days in a 48-well

plate in the presence or absence of lipopolysaccharide

(1 lg/ml), IL-1a (10 ng/ml), diacyl lipopeptide (10)8M)

and 1a,25(OH)2D3 (10)8M) plus prostaglandin E2 (PGE2)

(10)6M). Cells were then fixed and stained for TRAP.

TRAP-positive multinucleated cells containing three or

more nuclei were counted as osteoclasts. Values were

expressed as the mean ± standard deviation (SD) of three

cultures. (B) Effects of lipopolysaccharide on the produc-

tion of IL-6 in osteoblasts prepared from wild-type (WT),

Toll/IL-1 receptor domain-containing adapter-inducing

interferon-b)/) (TRIF)/)) and MyD88)/) mice. Osteoblasts

were incubated for 24 h in the presence or absence of

lipopolysaccharide (100 ng/ml). The conditioned medium

was collected, and the concentration of IL-6 in the med-

ium was measured using an enzyme-linked immuno-

sorbent assay (ELISA). Values are expressed as the

mean ± SD of quadruplicate cultures. (C) Effects of lipo-

polysaccharide on the production of IL-6 in macrophage

colony-stimulating factor-treated bone marrow macr-

ophages prepared from wild-type (WT), TRIF)/) and

MyD88)/) mice. Bone marrow-derived macrophages were

incubated for 24 h in the presence or absence of lipo-

polysaccharide (100 ng/ml). The conditioned medium was

collected, and the concentration of IL-6 in the medium

was measured using an ELISA. Values are expressed as the

mean ± SD of quadruplicate cultures.

58

Udagawa et al.

osteoclasts expressed the messenger ribonucleic acid

of Toll-like receptor-2 and Toll-like receptor-4, but not

Toll-like receptor-6. These results suggest that diacyl

lipopeptide did not support the survival of osteoclasts

because of the lack of Toll-like receptor-6 in osteo-

clasts. Thus, MyD88-mediated signals, but not TRIF-

mediated signals, were essential for the survival of

osteoclasts supported by lipopolysaccharide and

interleukin-1a (24) (Fig. 4C).

TRAM is not expressed inosteoblasts and osteoclasts

TRAM was shown to be involved in the lipopolysac-

charide-induced TRIF-mediated signaling pathway

(6, 35). We examined the expression of TRIF and

TRAM messenger ribonucleic acids in osteoblasts,

bone marrow macrophages and osteoclasts prepared

from MyD88)/), TRIF)/) and wild-type mice. TRIF

messenger ribonucleic acid was expressed in osteo-

blasts, macrophages and osteoclasts derived from

wild-type and MyD88)/) mice (Fig. 2B). Interestingly,

TRAM was expressed in macrophages but not in

osteoblasts or mature osteoclasts (Fig. 2B). Osteo-

blasts and osteoclasts expressed TRIF but not

TRAM, suggesting that TRAM expression is required

for TRIF-mediated action in osteoblasts and osteo-

clasts (Fig. 4). Our results also suggest that TRAM

may be an important key adaptor in the Toll-

like receptor-4-mediated pathway of cell-specific

functions.

OsteoblastsBone marrowmacrophages

WT

MyD

88-/-

TRIF-/-

WT

MyD

88-/-

TRIF-/-

WT

MyD

88-/-

TRIF-/-

Osteoclasts

GAPDH

TRIF

TRAM

MyD88

B

0

10

20

30

40

50

60

Contro

l

RANKL

Lipopo

lysac

chari

de

IL-1

αDiac

yl

lipop

eptid

e

Contro

l

RANKL

Lipopo

lysac

chari

de

Lipopo

lysac

chari

deIL

-1αDiac

yl

lipop

eptid

e

Contro

l

RANKL

IL-1

α

Sur

viva

l of

oste

ocla

sts

(%) 70

WT MyD88-/- TRIF-/-A

Diacyl

lipop

eptid

e

Fig. 2. Effects of receptor activator of nuclear factor-

kappa B (NF-jB) ligand (RANKL), lipopolysaccharide,

interleukin-1a (IL-1a) and diacyl lipopeptide on the sur-

vival of osteoclasts prepared from wild-type (WT), myeloid

differentiation factor 88)/) (MyD88)/)) and Toll/IL-1

receptor domain-containing adapter-inducing interferon-

b)/) (TRIF)/)) mice. (A) Purified osteoclasts were prepared

in cocultures of osteoblasts and bone marrow cells ob-

tained from WT, MyD88)/) and TRIF)/) mice. WT,

MyD88)/) and TRIF)/) osteoclasts were treated with or

without RANKL (100 ng/ml), lipopolysaccharide (1 lg/ml),

IL-1a (10 ng/ml) and diacyl lipopeptide (10)8M). After

culture for 24 h, cells were fixed and stained for TRAP.

TRAP-positive multinucleated cells containing three or

more nuclei were counted as viable osteoclasts. Values

were expressed as the mean ± standard deviation (SD) of

three cultures. (B) Expression of MyD88, TRIF and TRIF-

related adaptor molecule (TRAM) in osteoblasts, macr-

ophages and osteoclasts prepared from WT, TRIF)/) and

MyD88)/) mice. Total cellular RNA was extracted from

osteoblasts, macrophage colony-stimulating factor-in-

duced bone marrow macrophages and osteoclasts, reverse

transcribed and amplified by polymerase chain reaction

for mouse MyD88)/), TRIF, TRAM or glyceraldehyde-3-

phosphate dehydrogenase (GAPDH).

59

Signal transduction of lipopolysaccharide-induced osteoclast differentiation

MyD88)/) mice exhibited profoundosteopenia with reduced boneresorption and formation

Histomorphometric measurements of vertebrae

showed that MyD88)/) mice exhibited osteopenia

with reduced bone resorption and formation. Bone

resorption-related functional parameters, such as

osteoclast surface/bone surface and osteoclast

number/bone surface, were 37.4% and 46.8% lower

in MyD88)/) mice than in wild-type mice, respect-

ively (Fig. 3). Bone formation-related functional

parameters, such as osteoid volume/tissue volume

and osteoblast surface/bone surface, were also sig-

nificantly reduced in MyD88)/) mice (Fig. 3). Both

trabecular bone volume (bone volume per tissue

volume) and trabecular number were significantly

decreased in 14-week-old MyD88)/) mice in com-

parison with the wild-type mice (Fig. 3). No signifi-

cant differences in body size and shape were

observed between MyD88)/) and wild-type mice.

Histological analysis showed that a loss of trabecular

bone in the tibiae was evident in MyD88)/) mice.

These results suggest that MyD88 is involved in the

physiologic regulation of bone resorption and for-

mation (24) (Fig. 4).

Not only bone resorption-related parameters, but

also bone formation-related parameters, were signi-

ficantly decreased in MyD88)/) mice in comparison

with wild-type mice (Fig. 3). Mice deficient in bone

matrix proteins, such as osteonectin and biglycan,

similarly developed profound osteopenia with a

decrease of bone formation and resorption (5, 8, 32).

Deficiency of osteoprotegerin in mice induced severe

osteoporosis caused by enhanced bone resorption,

but accelerated bone formation was also observed in

these mice (4, 19). These findings suggest that bone

formation is tightly coupled with bone resorption.

Muramyl dipeptide enhancesosteoclast formation induced bylipopolysaccharide, interleukin-1aand tumor necrosis factor-athrough nucleotide-bindingoligomerization domain2-mediated signaling in osteoblasts

Muramyl dipeptide, the minimal essential structural

unit responsible for the immunoadjuvant activity of

peptidoglycans, is distributed ubiquitously in cell

walls of both gram-negative and gram-positive bac-

teria (23) (Fig. 5). Muramyl dipeptide has been shown

to exert diverse biological effects on immunocom-

petent cells (20). We have shown that injection of

muramyl dipeptide into mice resulted in endotoxin

hypersensitivity: enhanced production of tumor

necrosis factor-a (26) and lethal shock (25) upon

challenge injection of lipopolysaccharide. We also

showed that muramyl dipeptide synergistically

enhanced lipopolysaccharide-induced pro-inflam-

matory cytokine production in human monocytic

WT MyD88-/-0

10

20

30

(%)

* *

0

0.4

0.8

1.2

1.6

WT MyD88-/-

(%)

* *

WT MyD88-/-0

5

10

15

20

25(%

)* * *

WT MyD88-/-0

2

4

6

(num

ber/

mm

)

* * *

0

0.5

1

1.5

2

2.5

WT MyD88-/-

(num

ber/

mm

)

*

0

4

8

12

16

(%)

WT MyD88-/-

* *

Osteoblast surface/bone surface

Osteoid volume/tissue volume

Bone volume/tissue volume Trabecular number

Osteoclast number/bone surface

Osteoclast surface/bone surface

Fig. 3. Histomorphometric analysis of vertebrae in wild-

type (WT) and myeloid differentiation factor 88)/)

(MyD88)/)) mice. The seven male MyD88)/) and WT

(14 weeks old) mice were killed for bone histomorpho-

metric analysis. Vertebrae were removed from the mice,

fixed in 70% ethanol, and embedded in glycol/methacry-

late without decalcification. The sections were prepared

and stained with Villanueva Goldner to discriminate

between mineralized and unmineralized bone and to

identify cellular components. Quantitative histomorpho-

metric analysis was performed in a double-blind manner.

Values were expressed as the mean ± SD of seven mice.

Statistical analysis was performed using the Student’s

t-test. Significantly different between WT and MyD88)/)

mice (*P < 0.005, **P < 0.05, ***P < 0.01).

60

Udagawa et al.

cells (36). Recently, it was proposed that nucleotide-

binding oligomerization domain 2 (Nod2), a member

of the Apaf1/Nod protein family, is an intracellular

sensor of muramyl dipeptide (9, 10, 13). Nod2 con-

sists of an N-terminal caspase-recruitment domain, a

centrally located nucleotide-binding domain and

C-terminal leucine-rich repeats, and acts as a signal-

transducing adaptor. Nod2 expression is enhanced by

pro-inflammatory cytokines and lipopolysaccharide

(10). Recent studies have established that a frameshift

mutation of Nod2, which results in a deficiency in

muramyl dipeptide-mediated NF-jB activation, is

involved in susceptibility to Crohn’s disease, a chro-

nic inflammatory disorder of the intestinal tract (13).

Thus, muramyl dipeptide plays roles in many aspects

of inflammatory responses.

In mouse cocultures of primary osteoblasts and

hemopoietic cells 1a,25(OH)2D3, prostaglandin E2,

lipopolysaccharide and interleukin-1a stimulate

osteoclast formation. Muramyl dipeptide alone could

not induce osteoclast formation in the coculture,

but enhanced osteoclast formation induced by

lipopolysaccharide, interleukin-1a or tumor necrosis

factor-a, but not 1a,25(OH)2D3 or prostaglandin E2

(37). Muramyl dipeptide failed to enhance osteoclast

formation from osteoclast progenitors induced by

RANKL or tumor necrosis factor-a. Muramyl dipeptide

upregulated RANKL expression in osteoblasts treated

with lipopolysaccharide or tumor necrosis factor-a,

but not 1a,25(OH)2D3. Osteoblasts expressed mes-

senger ribonucleic acid of Nod2, an intracellular sen-

sor of muramyl dipeptide, in response to lipopolysac-

charide, interleukin-1a or tumor necrosis factor-a but

not 1a,25(OH)2D3. Induction of Nod2 messenger ri-

bonucleic acid expression by lipopolysaccharide, but

not by tumor necrosis factor-a, in osteoblasts was

dependent on Toll-like receptor-4 and MyD88. Mura-

myl dipeptide also enhanced tumor necrosis factor-

a-induced osteoclast formation in cocultures prepared

from Toll-interleukin-1 receptor domain-containing

adaptor protein-deficient mice through the upregula-

tion of RANKL messenger ribonucleic acid expression

in osteoblasts, suggesting that Toll-like receptor-2 is

not involved in the muramyl dipeptide-induced

osteoclast formation (37). The depletion of intra-

cellular Nod2 by small interfering RNA blocked

TLR4

LPS

MyD88TRAM

TRIF

IL-6production

MacrophagesA B

TLR4

LPS

IL-1R

IL-1

TLR2 TLR6

Diacyllipopeptide

PKC

MEK/ERK

RANKL

Osteoclastformation

MyD88

Osteoblasts

MyD88

IL-6production

TRAMTRIF

C

TLR4

LPS

IL-1R

IL-1

MyD88

Survival ofMature

osteoclasts

Osteoclasts

RANK

RANKL

TRAMTRIF

Fig. 4. Roles of myeloid differentiation factor 88)/)

(MyD88) and Toll/IL-1 receptor domain-containing

adapter-inducing interferon-b (TRIF)/TRIF-related adap-

tor molecule (TRAM) (i.e. TRAM–TRIF) signaling pathways

in macrophages, osteoblasts and osteoclasts exposed to

lipopolysaccharide (LPS), interleukin-1 (IL-1), diacyl

lipopeptide and receptor activator of nuclear factor-kappa

B (NF-jB) ligand (RANKL). (A) Role of MyD88- and TRAM-

TRIF-mediated signaling in IL-6 production in macroph-

ages. Macrophages express CD14 and Toll-like receptor

(TLR)-4. Both MyD88-dependent and TRAM–TRIF-

dependent pathways mediated by TLR-4 were essential

for IL-6 production in macrophages. (B) Roles of

MyD88-mediated signals in IL-6 production and osteo-

clast formation in osteoblasts. Osteoblasts express CD14,

TLR-2, TLR-4, TLR-6 and IL-1R. Lipopolysaccharide sti-

mulates IL-6 production through MyD88 signaling. Lipo-

polysaccharide, IL-1 and diacyl lipopeptide stimulate

RANKL expression in osteoblasts through the respective

receptor systems. TLR- and IL-1R-induced RANKL mRNA

expression in osteoblasts is mediated through MyD88

signaling followed by protein kinase C (PKC) and ME-

K/ERK signaling. (C) Role of MyD88-mediated signals in

osteoclast function. Mature osteoclasts express CD14,

TLR-4 and IL-1R as well as RANK. Lipopolysaccharide,

IL-1 and RANKL stimulate the survival of osteoclasts

through TLR-4, IL-1R and RANK, respectively. MyD88 is

involved in the survival of osteoclasts supported by lipo-

polysaccharide and IL-1, but not by RANKL.

61

Signal transduction of lipopolysaccharide-induced osteoclast differentiation

muramyl dipeptide-induced upregulation of RANKL

messenger ribonucleic acid in osteoblasts. Lipopoly-

saccharide and RANKL stimulated the survival of

osteoclasts, and this effect was not enhanced by

muramyl dipeptide. These results suggest that mura-

myl dipeptide synergistically enhances osteoclast for-

mation induced by lipopolysaccharide, interleukin-1aand tumor necrosis factor-a through RANKL expres-

sion in osteoblasts, and that Nod2-mediated signals

are involved in the muramyl dipeptide-induced

RANKL expression in osteoblasts (37) (Fig. 6).

In conclusion, muramyl dipeptide enhanced osteo-

clast formation induced by lipopolysaccharide, inter-

leukin-1a or tumor necrosis factor-a in cocultures via

stimulation of RANKL expression in osteoblasts.

Lipopolysaccharide and interleukin-1a stimulated

Nod2 messenger ribonucleic acid expression in

osteoblasts through via MyD88 signaling, whereas

tumor necrosis factor-a induced Nod2 messenger

ribonucleic acid expression in osteoblasts via MyD88-

independent signaling (Fig. 6). The reduction of Nod2

expression by small interference RNAs blocked mur-

amyl dipeptide-induced upregulation of RANKL mes-

senger ribonucleic acid in osteoblasts. These results

suggest that Nod2 acts as an intracellular receptor of

muramyl dipeptide to induce RANKL expression in

osteoblasts (Fig. 6). Muramyl dipeptide has been

shown to synergistically enhance the production of

pro-inflammatory cytokines by monocytic cells in the

presence of lipopolysaccharide. These results suggest

that muramyl dipeptide might play a key role in oste-

oclastic bone resorption in inflammatory diseases

such as periodontitis.

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