Osteoclasts, macrophages, and the molecular mechanisms of bone resorption

Osteoclasts, macrophages, and the molecular mechanisms

Journal of Leukocyte Biology Volume 61, April 1997 381

of bone resorptionSteven L. Teitelbaum, M. Mehrdad Tondravi, and F. Patrick Ross

Department of Pathology, Washington University School of Medicine, St. Louis, Missouri

Abstract: The osteoclast is a physiological polykaryon

and the major if not exclusive resorptive cell of bone.

It participates in bone remodeling, repair, and growth

and mobilization of mineral to meet homeostatic de-

mands. Most importantly, osteoporosis, a disease en-

demic in Western society and Asia, is always a reflec-

tion of enhanced osteoclastic activity relative to bone

formation by osteoblasts. In fact, all forms of anti-

osteoporosis therapy proven successful involve inhi-

bition of osteoclastic bone resorption. Bone resorp-

tion is regulated either by altering recruitment of

osteoclast precursors into fully differentiated resorp-

tive polykaryons or modulating the rate at which ma-

hire osteoclasts degrade bone. With this in mind, our

laboratory has focused on the molecular mechanisms

of osteoclast differentiation and the means by which

the cell degrades bone matrix. J. Leukoc. Biol. 61:

381-388; 1997.

Key Words: integrins . H�-ATPase . cytokines . steroid hormones

OSTEOCLAST ONTOGENY

Before the last decade, little was known regarding osteo-

clast ontogeny or how the cell resorbs bone. For example,

as fundamental an issue as whether the osteoclast degrades

both the organic and inorganic phases of bone or mobilizes

only the mineral compartment was unresolved. We pro-

pose that this paucity of information reflected the lack of

meaningful in vitro models useful for evaluating osteoclast

differentiation and function. In contrast, recently devel-

oped systems permit addressing issues fundamental to os-

teoclast biology [1].

It is now known that the osteoclast is a member of the

monocyte/macrophage family. We believe initial confirma-

tion that the osteoclast is of hematopoietic origin comes

from observations, made in conjunction with our colleagues

at the University of Minnesota, involving a patient with os-

teopetrosis [21. This family of sclerotic bone diseases, dis-

cussed in this review, reflects failed osteoclastogenesis or

the inability of mature osteoclasts to resorb bone. Based

on seminal animal studies suggesting that osteoclast precur-

sors are hematopoietic [3, �1, we reasoned that bone mar-

row transplantation would be curative in circumstances of

defective osteoclast function. With this in mind we trans-

planted an osteopetrotic female infant with marrow of her

HLA/MLC-identical brother. Not only was the transplant

curative but, by following the Y chromosome, we estab-

lished that osteoclasts, but not osteoblasts, are donor (i.e.

marrow) derived. Ultimate proof that the osteoclast is of

myeloid ontogeny came with the capacity to generate these

resorptive cells in culture from pure populations of mono-

nuclear phagocytes 151.The osteoclast shares many features with other macro-

phage polykaryons but is a unique cell. Those characteris-

tics distinguishing the osteoclast are expression of calcito-

nm receptors, the capacity to degrade bone and in so doing

produce resorption lacunae, synthesis of abundant tartrate-

resistant acid phosphatase and distinctive polarization, the

latter eventuating in formation of a unique ruffled mem-

brane at the osteoclast-bone interface.

Osteoclast ontogeny predicts that absence of transcrip-

tion factors governing myeloid differentiation will prompt

osteopetrosis. In support of this hypothesis we find that

transgenic mice in which the myeloid and B lymphoid tran-

scription factor PU.1 (also called Spi-i or Sfpi-1) is deleted

fail to generate macrophages or osteoclasts and develop

this sclerotic bone disease 161. We successfully rescue the

mutant mice by marrow transplantation, with complete res-

toration of osteoclast and macrophage differentiation and

function. Our observations genetically support the com-

mon lineage of osteoclasts and macrophages and demon-

strate the PU.i mutation is intrinsic to hematopoietic cells.

To date only one other transcription factor, c-fos, has

been implicated in osteoclastogenesis. Whereas deletion of

c-fos also leads to osteopetrosis, in contrast to the PU.i ‘�

mouse, the c-fos knockout contains excess marrow macro-

phages [7] . The abundance of macrophages in the face

of absent osteoclasts suggests that c-fos promotes differ-

entiation of a bipotential macrophage/osteoclast precursor

toward the osteoclast pathway. Because both osteoclasts

and macrophages are absent in mice lacking the PU.1

gene product, PU.1 exerts its effect in osteoclastogenesis

Abbreviations: GM-CSF, granulocyte-macrophage colony-stimulating

factor; TNF-a, tumor necrosis factor a; LPS, lipopolysaccharide; IL-4,

interleukin-4.

Correspondence: Steven L. Teitelbaum, M.D., Department of Pathol-

ogy, Washington University School of Medicine, Barnes-Jewish Hospital,

North Campus, 216 South Kingshighway, St. Louis, MO 63110.

Received November 1, 1996; revised November 27, 1996; accepted

December 4, 1996.

L!�JL�JL�Jcarbonic

Anhydrase IIc-Src H�-ATPase

Determination Proliferation, Differentiation PolarizationSurvival

Resorption

382 Journal of Leukocyte Biology Volume 61, April 1997

Fig. 1. PU.! exerts its effect on osteoclast development earlier than other known osteopetrotic mutations. While PU.! and c-fos both affect os-

teoclastogenesis, PU.1 impacts the developmental pathway earlier. The mutations of c-src, carbonic anhydrase II, and H�-ATPase affect function

of the mature osteoclast. Absence of M-CSF in the op/op mouse is a mutation targeting stromal cells and osteoblasts that normally express the cytokine

and thus indirectly affects osteoclast progenitors.

earlier than c-fos. Other examples of the osteopetrotic phe-

notype include mice bearing the naturally occurring op/op

mutations 181, or those in which c-src has been deleted �

or humans lacking functional H�-ATPase [iOl or carbonic

anhydrase II genes [111. The op/op mutation leads to se-

cretion, by marrow stromal cells, of prematurely termi-

nated, nonfunctional CSF-i 1i2, i31. Although this muta-

tion is not intrinsic to hematopoietic cells, the cytokine

appears to be important in macrophage and osteoclast de-

velopment during early life. Given that PU.1 regulates ex-

pression of the CSF-i receptor, c-fms, in myeloid cells 1141,

we hypothesize that PU.i acts earlier than CSF-i in the cas-

cade leading to osteoclast formation. In contrast to osteo-

petrosis arising from a lack of osteoclasts, c-src mutants

have abundant multinucleated cells with the osteoclast

phenotype 1151. However, c-src� osteoclasts are incapa-

ble of normal bone resorption. A model consistent with the

findings summarized above suggests that PU.i represents

the earliest mutation in the pathway of osteoclast genera-

tion and function (Fig. 1).

ION TRANSPORT AND OSTEOCLASTIC BONERESORPTION

Although genetic approaches provide powerful tools to ana-

lyze ontogeny, exploration of the biochemical events of

bone resorption requires large numbers of functional osteo-

clasts. With this in mind we developed or adapted systems

for the isolation and/or generation, in vitro, of avian or mu-

rime osteoclasts 15’ i6]. Having techniques in hand for gen-

erating osteoclasts or isolating these cells from a variety of

animals and maintaining them in culture provided us with

the opportunity to explore the molecular mechanisms of

bone resorption. Our initial efforts established that osteo-

clasts degrade both bone mineral and collagen and do so

with a temporal asynchrony indicating that the inorganic

phase must be removed from collagen bundles prior to col-

lagenolysis [i7J. Bone mineral is removed by acidffication

of an isolated compartment, the osteoclast-matrix interface.

Following demineralization, the organic phase of bone is

degraded by collagenolytic enzymes with a pH optimum

approximating 4.5, reflecting that present in the resorptive

microenvironment [i7J. The fact that weak base arrests os-

teoclast activity indicates that acidification of the extracel-

lular microenvironment, at the osteoclast-bone interface, is

essential to the resorptive process. In fact, the magnitude

of acidification necessary to mobilize bone mineral estab-

lishes the osteoclast as the major proton-secreting cell.

Thus, we turned to the means by which osteoclasts secrete

protons into the resorptive microenvironment.

Using avian osteoclasts as our model, we established

that these cells contain an abundant vacuolar H�-ATPase

similar, if not identical, to the proton pump expressed by

intercalated cells of the renal tubule [181. Importantly,

when in contact with bone, this osteoclast proton pump po-

larizes to the osteoclast-bone interface where it is needed

to acidify the resorptive microenvironment. While there is

agreement that the resorptive proton pump of the osteoclast

is an electrogenic, vacuolar type H�-ATPase, controversy

has surrounded the subunit composition of this pump 1191.

To address this issue, we isolated the functional osteoclast

proton pump subunits and reconstituted their proton-

transporting activity in lipid bilayers. Those subunits essen-

tial to osteoclast proton transport are immunologically sim-

ilar to those within the renal H�-ATPase [201.

The massive proton transport essential to bone degrada-

tion raises the issue as to how the osteoclast maintains in-

tracellular pH in the face of potential accumulation of base

equivalents. To address this question we again adapted the

paradigm of the renal intercalated cell. We find that osteo-

clasts express, on their anti-resorptive surface, an anion ex-

changer similar to band 3 of the erythrocyte and renal tu-

bular cell. This transporter exchanges intracellular HCO3

for extracellular C1 in an energy-independent fashion

[21J. Thus, the osteoclast is polarized to acidify the resorp-

tive microenvironment and secrete base equivalents via the

anti-resorptive plasma membrane.

The model described thus far addresses massive proton

transport by osteoclasts and maintenance of intracellular

pH. This compendium of events does not account for dis-

sipation of charge due to anion accumulation. Because in-

cr

H2co3 H� cr. :#{149}#{149}#{149}:#{149}: #{149}. #{149} . #{149}#{149}� #{149}: #{149}: #{149}#{149}:� :#{149} #{149}:#{149}: #{149}#{149}#{149}:#{149}: #{149}� #{149}.� #{149}#{149}#{149}. Cathepsins

C02-4- .co2���H2O Pump \ADP + P1 ATP � Chloride

#{149}#{149} #{149} #{149}:#{149}: #{149}#{149} #{149}:#{149}#{149}#{149}#{149}#{149} #{149}#{149} #{149}:. #{149}.t Channel

#{149} #{149} #{149} #{149}: #{149} #{149}

Ruffled . #{149} #{149}: #{149} . #{149} #{149}

Membrane . #{149} #{149} #{149} . .

H� C1

BONE

ci-

Teitelbaum et al. Molecular mechanisms of bone resorption 383

Fig. 2. Model for the major steps in

osteoclastic bone resorption. The osteoclast

attaches to bone, which prompts formation

of a convoluted ruffled membrane and a

resorptive microenvironment beneath the

cell. Hydrochloric acid, the product of a

vacuolar-type H + -ATPase and charge-

coupled Cl- channel concentrated in the

ruffled membrane, is secreted, resulting

in mineral dissolution. Vesicles containing

acidic collagenolytic enzymes in the form

of cathepsins, fuse with the bone-apposed

membrane, leading to enzyme release and

consequent organic matrix degradation.

Intracellular pH balance is maintained by

a passive C1/HCO3 exchanger on the

contraresorptive surface of the cell.

tracellular anion excess in this circumstance would reflect

abundance of Cl, the anion exchanged for HC03, we

postulated that a mechanism exists in the membrane of the

osteoclast juxtaposed to bone, by which C1 passes into

the resorptive microenvironment. We find that the osteo-

clast contains a passive C1 permeability in its resorptive

membrane, which is charge coupled to its H�-ATPase

[221. Thus, the means by which osteoclasts acidify the re-

sorptive microenvironment involves secretion of HC1. We

have recently established that this C1 channel is outward-

ly rectifying and related to the renal microsomal chloride

channel p64. Interestingly, expression of the resorptive C1

channel is induced upon contact of osteoclast precursors

with bone, a step essential to development of the resorptive

phenotype [Schlesinger, P. H., Blair, H. C., Teitelbaum,

S. L., Edwards, J. C., unpublished resultsl. In summary,

acidification of the osteoclast resorptive microenvironment

consists of series of well-defined ion-transport events (Fig.

2). The process begins when, under the influence of car-

bonic anhydrase II, CO2 is hydrated to H2CO3, which dis-

sociates into protons and bicarbonate ions. Protons are Se-

creted in an energy-dependent fashion into an isolated

microenvironment located at the cell-bone interface and

HC03 exchanged for C1 at the cell’s anti-resorptive sur-

face. C1 entering the osteoclast passes through a resorp-

tive plasma membrane anion channel charge coupled to

the H�-ATPase.

Bone consists of mineral and an organic phase, 90% of

which is type I collagen [23j. Although acidification is

sufficient to mobilize bone mineral, organic matrix degra-

dation, which we established is also under the aegis of

the osteoclast [i7J, requires proteolytic activity. Given the

highly acidic resorptive milieu, we suspected the osteoclast

collagenolytic enzyme(s) must enjoy a low pH optimum.

We verified that such is the case by demonstrating that os-

teoclast lysates degrade authentic bone collagen most effec-

tively at pH 4.5 [i7, 241, that extant in the resorptive mi-

croenvironment. We established that the avian osteoclast

contains a cathepsin B-like acidic protease capable of de-

grading authentic fibrillar bone collagen [241. Recent

studies have identified the presence, in mammalian osteo-

clasts, of a uniquely expressed cathepsin homolog, desig-

nated cathepsin K [25, 261. Absence of this protein leads

to failure to resorb bone, attesting to the importance of this

family of proteases in osteoclast function [27J. While the

detailed pathway for delivery of cathepsins to the osteoclast

surface is incompletely understood, transport from the golgi

to the resorptive microenvironment involves the mannose-

6-phosphate receptor [28J. This observation indicates yes-

ide movement in the osteoclast contrasts with that of most

other cells. Whereas the majority of golgi-derived vesicles

are targeted in other cells to lysosomes, in the osteoclast

the default pathway of lysosomal enzyme transport, namely

targeting to plasma membrane, dominates.

OSTEOCLAST INTEGRINS

The requirement for an isolated extracellular resorptive

microenvironment with a pH distinctly different from the

general extracellular space indicates that physical intimacy

between the osteoclast and bone matrix is essential to the

resorptive process. With this in mind we explored the means

by which osteoclasts recognize and attach to matrix. Be-

cause of the pivotal role integrins play in cell-matrix attach-

ment we asked if members of this family of heterodimers

mediate osteoclast-bone recognition. To identify osteoclast

integrins participating in bone binding we coated wells

with isolated bone matrix proteins and asked which sup-

port attachment of avian osteoclasts. We find that only pro-

teins with the Arg-Gly-Asp (RGD) amino acid motif are

bound by osteoclasts, suggesting that the event is mediated

384 Journal of Leukocyte Biology Volume 61, April 1997

through the RGD-recognizing subfamily of a�-containing

integrins, particularly av�33. In fact, a blocking antibody

recognizing the external domain of the intact Uv�3 hetero-

dimer inhibits, in a dose-dependent fashion, the ability of

osteoclasts to attach to and degrade bone [291.Osteoporosis always reflects accelerated osteoclast-mediated

bone resorption relative to formation. Thus all successful

strategies to prevent or arrest this disease to date, have

involved osteoclast inhibition. Our finding that antibody

blockade of a433 arrests bone resorption, in vitro, encour-

aged us to search for an RGD peptide mimetic that recog-

nizes av�33 with high affinity. We reasoned that such a

molecule would blunt bone resorption in vivo and thus pre-

vent osteoporosis. We have identified a small organic mole-

cule that recognizes isolated av�33 in solid-phase assays

and prevents osteoclasts from attaching to and resorbing

bone in vitro. Most importantly, when administered to rats,

this av133 antagonist completely prevents the massive bone

loss occurring within 6 weeks of oophorectomy. Thus, av133

inhibition presents itself as a potential form of osteoporosis

prophylaxis [30].

Having established that Uv�33 plays a central role in os-

teoclastic bone resorption we turned to regulation of the in-

tegrin. We first examined the impact of the osteoclastogenic

steroids, vitamin D3 and retinoic acid. We had shown pre-

viously that the active metabolite of vitamin D3, namely

1,25 dihydroxyvitamin D3 [1,25(OH)2D3J, is a potent in-

ducer of differentiation of osteoclast precursors and other

macrophages [31-33]. We find the steroid, as a compo-

nent of osteoclast differentiation, enhances av�3 expres-

sion by marrow macrophages by transcriptional activation

of both av and 133 integrin gene [34, 35]. We extended

these studies to another osteoclastogenic steroid, retinoic

acid, and discovered that it too induces av�3 expression.

In this circumstance, however, appearance of the hetero-

dimer is regulated by enhanced transcription of the �33

subunit [361.The cascade of events inducing post-menopausal osteo-

porosis begins with a decline in physiological estrogen, ac-

celerating, in turn, osteoclastic bone resorption. We find

that while estrogen alone fails to impact Uv(33 expression by

osteoclast precursors, picomolar concentrations of the ste-

roid, namely those circulating in post-menopausal women,

enhance the integrin-inductive capacity of 1,25(OH)2D3

[371. In contrast, and in keeping with the anti-resorptiveeffects of estrogen, the sex steroid in nanomolar amounts,

which are present prior to menopause, fail to impact ct433.Similar to retinoic acid induction of av�3, the heterodimer

is regulated via the n-subunit.

We next addressed the mechanisms by which steroid hor-

mones transactivate the avian �33 gene. To this end we

cloned the �33 promoter and identffied a classical vitamin

D response element [38]. Perhaps of greater interest, we

characterized a novel steroid response element consisting

of three direct hexameric nucleotide repeats 1391. This mo-

tif is recognized by both the vitamin D and retinoic acid

receptors, each in complex with the RXR receptor. Inter-

estingly, each receptor heterodimer competes for the mid-

die half site, thereby modulating the other’s transactivating

capacity. We believe this represents the first example of ste-

roid hormone receptors modulating each other’s transcrip-

tional activity by competing for the same response element.

Having established regulation of av�3 expression by os-

teoclastogenic steroids, we asked if hematopoietic cytokines

also alter (i� integrin appearance on osteoclast precursors.

These experiments required a murine model of osteoclast

precursor differentiation. To this end we used pure popu-

lations of macrophage colony-stimulating factor-dependent

murine marrow macrophages which, when placed in ap-

propriate culture conditions, differentiate into bona fide

osteoclasts [5].

Our colleague Roberto Pacifici has demonstrated that

human CD34� cells, when cultured in vitro with the

proper combination of cytokines, including granulocyte-

macrophage colony-stimulating factor (GM-CSF), differen-

tiate into osteoclasts [40]. Given the central role of GM-CSF

in osteoclast formation, our first efforts were directed at de-

termining if the cytokine impacts a�[33. By a combination

of Northern analysis and immunoprecipitation studies on

murine osteoclast precursors, we demonstrated that GM-

CSF induces � mRNA and surface-expressed Uv�33 in a

time- and dose-dependent manner. Moreover, � transcrip-

tion is unaltered by GM-CSF, but stability of f33 mRNA is

substantially enhanced [Inoue, M., Teitelbaum, S. L., Ross,

F. P. , unpublished results]. Importantly, freshly isolated os-

teoclast precursors, while failing to express (1433, are still

capable of spreading on matrix, an event inhibited by our

RGD peptidomimetic. This finding led to us to examine

these precursors for the presence of other integrins capable

of ligating RGD. Our studies culminated in identification of

av�s as the integrin mediating matrix attachment of early

osteoclast precursors. Further experiments revealed that

GM-CSF decreases transcription of the �3s gene, diminish-

ing surface expression of av�s. The significance of our

findings on the ability of GM-CSF to regulate av integrin

expression is underscored by the fact that levels of av�3

and av1�5 change reciprocally during osteoclastogenesis in

vitro. Whereas a435 is present before multinucleation and

disappears with time, av[�3 initially absent, increases dur-

ing osteoclast formation [Inoue, M., Teitelbaum, S. L., Ross,

F. P., unpublished results]. These findings suggest a model

for the role of integrins in osteoclast formation in which the

osteoclast precursor expresses OtvI-35 and no av�3, with the

situation reversed in the mature cell (Fig. 3). Thus, av�35may be responsible for attachment of precursors, a pre-

requisite for their proliferation and differentiation. As av�5

disappears av�s, the functional integrin of the mature os-

teoclast, is expressed.

TUMOR NECROSIS FACTOR a (TNF-a)

Using murine osteoclastogenic cultures, we confirmed ear-

lier reports 141, 42] suggesting that TNF-a, a major secre-

tory product of activated macrophages, is among the most

potent of osteoclastogenic cytokines lunpublished data].

Differentiation

A� l�4�l�

Mature7steoclast

Teitelbaum et al. Molecular mechanisms of bone resorption 385

Fig. 3. Proposed scheme for the role of integ-

rins in osteoclast formation and function. The im-

mature osteoclast precursor, while arising in mar-

row, circulates in the blood. Attachment to

RGD-containing proteins in bone is mediated by

the integrin � Once adherent, the precursor

undergoes differentiation and fusion under the

influence of a range of hormones and cytokines

whose activities include decreasing expression of

� while enhancing that of a433, the functional

integrin of the mature osteoclast.

Bone

Multinucleationav)

BoneA = RGD-containing protein

This finding led us to hypothesize a possible mechanism

of implant osteolysis, the most frequent disabling compli-

cation following prosthetic replacement of diseased joints.

Thus, we suggested that TNF-a secretion, by macrophages

that have phagocytosed implant-derived particles, repre-

sents a critical first step in the accelerated bone resorption

characterizing this important clinical condition. Our initial

studies confirmed enhanced transcription of the TNF-a

gene by macrophages exposed to implant-derived particles

lunpublished data]. We then developed an in vivo modelin which either polymethylmethacrylate or polyethylene

particles (both found in tissues surrounding failed im-

plants) were placed under the external calvarial periosteum

of mice. Within 1 -2 weeks an osteolytic, osteoclast-rich le-

sion develops that is functionally and morphologically in-

distinguishable from that seen in humans. Resident macro-

phages contain high levels of TNF-a. Most importantly,

mice in which both TNF-a receptors have been deleted are

protected from implant particle osteolysis. This finding es-

tablishes the central role of TNF-a as an etiological agent

in post-implant osteolysis [unpublished data].

Periodontal disease, which is accompanied by the pres-

ence of bacterial lipopolysaccharide (LPS)-secreting bac-

teria, represents a second important clinical situation as-

sociated with accelerated bone loss. Cultures containing

osteoclast precursors derived from marrow of mice treated

in vivo with LPS, yield increased numbers of osteoclasts,

an event blocked once again by inhibition of TNF-a func-

tion [unpublished data]. In summary, TNF-a-stimulated

osteoclast formation represents the mechanism of two sep-

arate, clinically important conditions. Because TNF-a reg-

ulates osteoclast differentiation we wondered if the cytokine

also modulates av integrin expression, a process that paral-

lels generation of bone-resorbing cells. We find that treat-

ment of osteoclast precursors with TNF-a leads to a decline

in steady state �35 mRNA levels as a result of decreased

mRNA stability, with the overall result being diminished

surface expression of a435. In contrast to GM-CSF, which

induces av�33, TNFa does not impact this integrin recep-

tor [43].

INTERLEUKIN-4 (lL-4)

Regulation of osteoclast differentiation and integrin expres-

sion is not limited to hematopoietic cytokines. In this re-

gard, we reported several years ago that a transgenic mouse

overexpressing IL-4 develops a form of osteoporosis with

decreased osteoblast and osteoclast function [44] . We de-

termined that addition of IL-4 to our in vitro murine osteo-

clastogenic coculture results in dose-dependent decreased

multinucleation [45], a finding correlating with the in vivo

result. Furthermore, we find the target cell for IL-4 action

is the osteoclast precursor and not the osteoblast/stromal

component of the coculture [46] and that IL-4, in addition

to decreasing osteoclast formation, blunts the bone-resorbing

activity ofthe mature cell [47J. Turning to the action of IL-4

on integrins, we determined that the cytokine increases ex-

pression of a433 by stimulating transcription of the �33 sub-

unit, whereas steady state levels of a,, mRNA are unal-

tered [48]. In a finding reminiscent of that for GM-CSF,

IL-4 also accelerates disappearance of a�f35 from the cell

386 Journal of Leukocyte Biology Volume 61, April 1997

surface lunpublished data]. Although the role of IL-4 in

regulating integrin expression is of interest, a finding of

greater potential significance is that IL-4, by decreasing

transcription of the TNF-a gene, blocks secretion of the os-

teoclastogenic cytokine by activated macrophages [unpub-

lished data]. This observation may explain our earlier re-

port that IL-4 inhibits osteoclast formation [49].

Our studies on the molecular mechanisms whereby IL-

4, GM-CSF, and TNF-a regulate expression of the integrins

av�33 and av�3s reveal a variety of pathways are involved,

including both increases and decreases in the rate of tran-

scription and mRNA stability. Generally, it is the relevant

f3 and not av subunit that mediates heterodimer expres-

sion. To understand the molecular basis of these events we

have cloned both the murine � and �35 promoters [50,

51]. Initial examination reveals the presence of consensus

sequences for a number of basal and tissue-specific Iran-

scription factors. There are also consensus sequences for

STAT proteins, cytosol-residing latent transcription factors

known to mediate activation of genes following cytokine

treatment of cells [52, 53]. Direct proof that specffic se-

quences in the promoter regions of the � and �3s genes

are involved in cytokine regulation of a433 and a435 in os-

teoclast precursors will require experiments in which dele-

tion and/or mutation of the putative active sites is followed

by functional analysis.

OSTEOCLAST POLARIZATION

One of the major remaining unsolved issues in osteoclast

biology is the mechanism by which the cell polarizes. Fol-

lowing attachment to bone matrix a characteristic ruffled

membrane, containing a number ofcritical proteins, includ-

ing the vacuolar-type proton pump, is generated. An addi-

tional critical event is secretion of one or more cathepsins,

whose function is to degrade organic matrix in the acidic

bone-adjacent microenvironment. Although the detailed

events underlying ruffled membrane formation and regu-

lated exocytosis are unclear, the fact that acidifying vesicles

are randomly distributed in the cytoplasm of osteoclasts

not in contact with bone, but polarize to the bone-apposed

plasma membrane in the substrate-adherent cell, indicates

that in the resorptive polykaryon, matrix-derived signals

prompt directed vesicular movement. Based on informa-

tion derived from cells such as neurons, pancreatic beta,

mast, and pituitary cells [54-57], a reasonable model sug-

gests that a cell-specific signal (e.g., neuronal membrane

depolarization, recognition of glucose by its surface recep-

tor on the islet cell, activation of mast cells) triggers move-

ment of vesicles toward the cell surface, where fusion occurs

with the existing membrane, thereby leading to its expan-

sion. The overall process, as revealed by analysis of other

cell types, primarily neurons, involves a complex set of

events mediated by many proteins, including coatamer I

and II complexes, a series of docking-related proteins

called NSFs, VAMPS, SNAPs, SNAREs, and multiple mem-

bers of the small GTPase family (both ARFs and rabs).

Although outside the scope of this review, the detailed

interactions of the many molecules involved in membrane

targeting and fusion of specific vesicles are a matter of

intense research, summarized in a number of recent arti-

des [58-60].

There is no documentation, in osteoclasts or their pre-

cursors, of any proteins known to regulate exocytosis in

other systems. Thus, our finding that murine osteoclast pre-

cursors express two isoforms of the rab3 subfamily [61J,

proteins that mediate regulated exocytosis in a number of

other cell types, is potentially important. In the osteoclast,

regulated exocytosis (an event we predict is initiated by rec-

ognition of bone matrix by the osteoclast), would result in

ruffled membrane generation and secretion of collagenolytic

enzymes, critical events in bone resorption. The possible

significance of the presence of rab3 proteins in osteoclast

precursors is underscored by two additional observations.

First, the levels of the same two rab3 family members are

increased during osteoclastogenesis in our in vitro murine

system. Second, treatment of osteoclast precursors with a

range of hematopoietic cytokines, and most notably the po-

tent osteoclastogenic molecule TNF-a, results in enhanced

expression of both rab3 isoforms, raising the possibility

that GTPases participate in ruffled membrane formation.

The above events, although representing a reasonable

model for expansion of the ruffled membrane, fail to ex-

plain how vesicles are transported to the cell surface where

they fuse with the plasmalemma to form the ruffled mem-

brane. The first clue as to how vesicles move in osteoclasts

came from studies in which the protooncogene c-src was

deleted in mice by targeted recombination [91. The result-

ing animals exhibit osteopetrosis, whose cellular basis is

the inability of the differentiated osteoclasts to resorb bone

due to failed formation of a ruffled membrane. Based on

the reported localization of c-src in osteoclasts at both the

ruffled membrane [62] and within the intravesicular com-

partment [63J, the protooncogene may play a role in vesic-

ular transport. Because microtubules represent an impor-

tant pathway by which various proteins and vesicles migrate

through cells [64, 65] we hypothesized that proteins des-

tined for the ruffled membrane of the osteoclast associate

with intermediate filaments. A series of experiments utiliz-

ing a combination of confocal microscopy and co-immuno-

precipitation/immunoblot analysis demonstrate that adher-

ence of osteoclast precursors to matrix is followed by

co-association of c-src with microtubules and not mono-

meric tubulin. We extended the study to demonstrate that

the H�-ATPase, the hallmark and major functional protein

complex of the ruffled membrane, also decorates microtu-

bules. Furthermore, the osteoclast proton pump, c-src as

well as a rab3 isoform known to mediate cytoplasmic

vesicle-plasma membrane fusion, localize to the light golgi

fraction of osteoclast precursors [661.

Because of the central role played by c-src in osteoclast

polarization we asked if osteoclastogenic cytokines regulate

the protooncogene. We find that TNF-a is unique in that,

via accelerated transcription, it alone stimulates expression

of c-src by osteoclast precursors [67]. To our knowledge,

ACKNOWLEDGMENTS

Teitelbaum et a!. Molecular mechanisms of hone resorption 387

Fig. 4. Model ofosteoclast polarization. The osteoclast attaches to bone

via the integrin a433, resulting in tubulin polymerization. Unidentified

signals stimulate movement along microtubules of intracellular vesicles,

which bear functional proteins targeted to the bone-apposed plasma mem-

brane. Vesicle generation and targeting is a complex set of events involv-

ing a large number of facilitatory proteins. Fusion of vesicles results in

production of the characteristic ruffled membrane.

this represents the first report of transcriptional regulation

of c-src. Taken together, our findings suggest a model in

which movement of vesicles containing functionally impor-

tant proteins destined for the osteoclast ruffled membrane,

move along a track comprised of microtubules (Fig. 4).

SUMMARY

Recognition that the osteoclast is a member ofthe monocyte/

macrophage family has prompted development of meaning-

ful experimental models to study the cellular mechanisms

of bone resorption. These efforts have delineated molecu-

lar targets to inhibit the resorptive process and thus prevent

diseases such as osteoporosis. Many concerns remain to be

addressed. Among the most provocative is the means by

which the osteoclast polarizes and the impact of matrix rec-

ognition and cytokines on this event. The role of tissue-

specific transcription factors in osteoclast commitment is

also fertile area for investigation. The tools are at hand to

address these issues and one may expect progress in under-

standing osteoclast biology to continue to impact patient

care.

This study was supported in part by National Institutes of

Health Grants DE05413, AR32788 (S. L. T.), AR42404,

AR42378 (F. P. R.), AR44089 (M. M. T.) and a grant from

the Shriners Hospital for Crippled Children, St. Louis Unit,

St. Louis, MO (S. L. T.).

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