Expression and Regulation of RAB3 Proteins in Osteoclasts and Their Precursors

Authors

  • Yousef Abu-Amer,

    1. Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, U.S.A.
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  • Steven L. Teitelbaum,

    1. Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, U.S.A.
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  • Jean C. Chappel,

    1. Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, U.S.A.
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  • Paul Schlesinger,

    1. Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri, U.S.A.
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  • F. Patrick Ross

    Corresponding author
    1. Department of Pathology, Washington University School of Medicine, St. Louis, Missouri, U.S.A.
    • Address reprint requests to: F. Patrick Ross, Ph.D. Department of Pathology Washington University School of Medicine Barnes-Jewish Hospital North 216 South Kingshighway St. Louis, MO 63110 U.S.A.
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Abstract

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c–src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

INTRODUCTION

Rabs, a group of low molecular weight GTPases constituting part of the ras superfamily, regulate intracellular and plasma membrane fusion and expansion.(1–3) More than 30 Rabs have been identified, with each executing a unique role in membrane generation.(4,5) The Rab3 subfamily, consisting of four isoforms,(4–8) controls regulated exocytosis in neurons,(9–11) neuroendocrine,(12–14) epithelial,(15) pancreatic,(16,17) and mast cells.(18) Thus, Rab3A selectively localizes to secretory granules in adrenal chromaffin cells(19) and the surface of synaptic vesicles.(20) The same molecular switch participates in recruitment of exocytic synaptic vesicles.(20–23) Rab3B and Rab3C are expressed by anterior pituitary,(24) insulin-secreting,(25,26) and epithelial cells.(13,15) The final member of the family Rab3D is important for regulated secretion by the exocrine pancreas(8,14,16,17) and gastric chief cells.(27)

Nonadherent bone marrow macrophages (BMMs) are early precursors of monocytes and tissue macrophages. The same immature myeloid cells can differentiate into osteoclasts, in vitro(28–30) and in vivo.(31) Given the likely importance of Rab3 proteins in secretion by macrophages at all stages of maturation, we examined expression and distribution of Rab3 isoforms in BMMs differentiating along the osteoclastogenic pathway. We find BMMs and mature osteoclasts contain several Rab3 isoforms, whose expression is enhanced by a range of osteoclastogenic cytokines. We also document differential localization of individual rab3 isoforms with pp60c–src (c-src) and the vacuolar H+ATPase, two markers of the ruffled membrane, the resorptive organelle of the osteoclast.(32–34) These experiments document, for the first time, regulation of Rab proteins and their expression by marrow-residing macrophages and osteoclasts.

MATERIALS AND METHODS

Materials

Monoclonal antibodies that recognize Rab3A (42.1) or Rab3A/B/C (42.2) were kindly provided by Dr. Reinhard Jahn (New Haven, CT, U.S.A.). The antibodies against Rab3A, Rab3B, and Rab4 was purchased from Santa Cruz Biologicals (Santa Cruz, CA, U.S.A.), while the monoclonal antibody 327,(35) directed against the c-src protein, was a gift of Dr. A. Shaw (Department of Pathology, Washington University of St. Louis, MO, U.S.A.). Enhanced chemiluminescence kits were obtained from Amersham Co. (Arlington Heights, IL, U.S.A.). All other chemicals were from Sigma Chemicals (St. Louis, MO, U.S.A.).

Cell culture

Mouse osteoclast precursors, in the form of BMMs, were isolated from C3H mice, as described previously.(36) Briefly, BMMs were isolated from whole bone marrow of 4- to 6-week-old mice and incubated in tissue culture plates, at 37°C in 5% CO2, in the presence of CSF-1 (1000 μ/ml).(36) After 24 h in culture, the nonadherent cells were collected and layered on a Ficoll-Hypaque gradient and the cells at the gradient interface were collected and plated in alpha modified essential medium, supplemented with 10% heat-inactivated fetal bovine serum, at 37°C in 5% CO2 in the presence of CSF-1 (1000 μ/ml), and plated according to each experimental conditions. Coculture of BMMs and ST2 cells, which results in formation of osteoclasts in vitro, was performed as described.(37) Pure BMMs were cultured with ST2 cells (ratio 10:1) at a final density of 105 cells/cm2, in the presence of 10 nM 1,25(OH)2D3 and 100 nM dexamethasone.(37) Cocultured cells were supplemented with fresh media and steroids on the fourth day. A typical monolayer of multinucleated osteoclasts was obtained between days 7–9 of culture. ST2 cells were collagenase-removed,(29) and remaining osteoclasts and their precursors were used according to the experimental conditions. Avian BMM precursors which differentiate into osteoclasts were isolated from calcium-deprived laying hens.(28)

Immunoblotting

Whole cell lysates were boiled in the presence of SDS-sample buffer (0.5 M Tris-HCl, pH 6.8, 10% [w/v] SDS, 10% glycerol, 0.05% [w/v] bromophenol blue, distilled water) for 5 minutes and subjected to electrophoresis on 12% SDS-PAGE.(38) Proteins were transferred to nitrocellulose membranes using a semidry blotter (Bio-Rad, Richmond, CA, U.S.A.) and incubated in blocking solution (10% skim milk prepared in phosphate-buffered saline (PBS) containing 0.05% Tween-20), to reduce nonspecific binding. Membranes were washed with PBS/Tween buffer and exposed to primary antibodies, washed again four times and incubated with secondary goat anti-mouse horse radish peroxidase–conjugated antibody. Membranes were washed extensively, and an enhanced chemiluminescence detection assay was performed following manufacturer's directions.

Sucrose density gradient fractionation

Avian osteoclasts were homogenized in buffer A (150 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 10 mM HEPES pH 7.4, supplemented with a cocktail of protease inhibitors (Leupeptin, Pepstatin, Aprotinin, and Chymostatin {all 10 ng/ml}, AEBSF {1 mM}, O-phenanthroline {1 μg/ml}, and Benzamidine {1 μM}) and spun at 1000g for 5 minutes. Nuclei and large cell debris (P1) were discarded and supernatant (S0) was collected and spun at 27,000g for 35 minutes. Supernatant from the last step was spun at 127,000g for 1.5 h. Pellets (P3) enriched with Golgi and small intracellular membranes were collected, adjusted to 1.22 M sucrose, and loaded at the bottom of a discontinuous sucrose gradient.(39,40) The gradient was spun for 3 h at 100,000g and interface bands collected and pelleted. Pellets were lysed in sample buffer and analyzed by immunoblot.

Preparation of osteoclast membrane vesicles

Osteoclasts were isolated from the long bones of calcium-deprived laying hens.(41) Membrane vesicles from freshly isolated cells were prepared using the method of Gluck and Al-Awqati(42) with slight modifications.(43) In brief, cell fragmentation was achieved via nitrogen cavitation in 250 mM sucrose, 1 mM EGTA, 1 mM dithiothreitol, and 10 mM Tris pH 7.0 (lysis buffer). Disruption of 20 × 106 cells in 20 ml of lysis buffer, contained in a 50-ml centrifuge tube placed within the cavitation bomb chamber, was initiated by pressurization to 40 atm N2 for 30 minutes at 4°C. Decompression occurred over 2 s through a 5-mm-diameter orifice. For some experiments, sucrose was iso-osmotically replaced with 140 mM KCl. Nuclei and large cell fragments were removed by centrifugation at 1000g for 5 minutes, a mitochondrial fraction by centrifugation at 4700g for 10 minutes, and the vesicular fraction by centrifugation at 49,000g for 40 minutes. The last pellet was frozen at –80°C at ∼900 μg total protein per aliquot in lysis buffer.

Fluorescence microscopy

Murine osteoclasts were generated by treatment of pure populations of BMMs with recombinant macrophage colony-stimulating factor (M-CSF) (10 ng/ml; R&D, Minneapolis, MN, U.S.A.) and hOPGL (20 ng/ml; Amgen, Thousand Oaks, CA, U.S.A.) for 5 days. Cells were fixed with 0.25% glutaraldehyde in PBS, and fluorescence microscopy was performed as described in Abu-Amer et al.(44) using rabbit polyclonal Rab3A and Rab3B antibodies, visualized with Texas red-labeled goat anti-rabbit immunoglobulin G.

RESULTS

Murine BMM precursors express several Rab3 isoforms

Rab3 expression by BMMs was assessed by immunoblot. Using monoclonal antibody 42.1, which recognizes Rab3A, Rab3B, and Rab3C, major and minor bands appear at 27 kDa and 25 kDa, respectively (Fig. 1). The identity of the faster migrating band as Rab3A is established with monoclonal antibody 42.2 specific for this isoform. Based on the intensity of the common epitope present in all three isomers, BMMs express a preponderance of Rab3B/C, with less Rab3A.

Figure FIG. 1.

Expression of Rab3 proteins by BMMs. BMMs were maintained in culture alone for 3 days and then lysed in sample buffer. Equal amounts of whole cell lysates were analyzed by Western immunoblot for the presence of Rab3 isoforms using anti-Rab3 antibodies.

Hematopoietic cytokines stimulate expression of several Rab3 isoforms by BMMs

BMMs, cultured for 3 days, were untreated for an additional 18 h or exposed to individual hematopoietic cytokines, followed by immunoblot analysis of equal amounts of total protein lysates. As can be seen in Fig. 2, the quantities of Rab3A and Rab3B/C isoforms are substantially and individually increased by a number of cytokines. In contrast, the antiosteoclastogenic cytokine interleukin-13 (IL-13) does not affect expression of Rab3 proteins.

Figure FIG. 2.

Cytokine-regulation of Rab3 protein expression by BMMs. Cells were maintained in culture for 3 days, then treated with the indicated cytokines for 18 h. BMMs were then lysed and equal amounts of whole cell lysates (see actin blot) were analyzed by Western immunoblot using anti-Rab3 antibodies. All Interleukins (IL), tumor necrosis factor (TNF), and oncostatin M (OSM) were used at 10 ng/ml, with leukemia inhibitory factor (LIF) at 20 ng/ml and stem cell factor (SCF) at 50 ng/ml.

Rab3 expression is induced during murine osteoclastogenesis

A number of cytokines, such as tumor necrosis factor, IL-3, IL-1, and IL-6, which induce Rab3 expression, promote differentiation of BMMs into osteoclasts,(45–48) raising the possibility Rab3 may be a hallmark of the osteoclast phenotype. To determine if this is the case, we cultured BMMs with murine marrow stromal cells, a circumstance in which they differentiate into bona fide osteoclasts. BMMs maintained in similar circumstances, but in the absence of stromal cells, form adherent macrophages which do not express the osteoclast phenotype. Immunoblot analysis of equal amounts of cell lysate (notice equal amounts of actin as control) shows that purified murine osteoclasts contain considerably more Rab3 than do macrophages, with the major isoform being Rab3B/C (Fig. 3A). In contrast, BMMs cultured in the presence of stromal cells, under nonosteoclastic conditions (absence of vitamin D3 and dexamethasone), fail to express high levels of Rab3 proteins (Fig. 3B).

Figure FIG. 3.

Induction of Rab3 expression during murine osteoclastogenesis. BMMs were grown alone or cocultured with ST2 cells, in the presence (A) (to form osteoclasts) or absence (B) of steroids (see Materials and Methods). BMMs or in vitro generated multinucleated osteoclasts were lysed and equal amounts of whole cell lysates were subjected to Western immunoblot analysis using anti-Rab3 antibodies.

Rab3 isoforms are located in vesicular fractions in avian osteoclasts and their precursors

The process of bone resorption involves ruffled membrane-residing proteins including a vacuolar proton pump, required for matrix degradation and, c-src, central to ruffled membrane formation.(32,34) Because Rab3 participates in exocytosis, a process analogous to ruffled membrane development, and in light of the abundance of the GTPase in osteoclasts, we asked if Rab3 localizes with resorptive proteins. To this end, we turned to a model in which marrow macrophages derived from calcium-deprived laying hens differentiate into osteoclast-like polykaryons in vitro.(28) The abundance of osteoclast-like cells obtained by this approach lends itself to biochemical analyses.

Density gradient analysis demonstrates that Rab isoforms differentially localize within these cells (Fig. 4). Specifically, Rab3B/C and Rab3A are present in the Golgi light and heavy fractions, respectively. c-src and H+ATPase, two proteins destined for the ruffled membrane, also distribute selectively. While c-src is present almost exclusively in the same light Golgi fraction as Rab3B/C, the proton pump complex is equally distributed between light and heavy vesicles. The macrophage-specific plasma membrane protein, serving as a negative control F4/80,(49) is absent in intracellular fractions.

Figure FIG. 4.

Subcellular colocalization of Rab3A/B/C with vacuolar H+ATPase and c-src. Cells maintained on tissue culture plates for 3 days were lysed (S0) and the lysate centrifuged at 1000g to remove nuclei and large cell debris. Supernatant was centrifuged at 27,000g, and the membrane pellet (P3) was fractionated on a discontinuous sucrose density gradient. An equal amount of protein from each gradient faction was subjected to immunoblot using antibodies to Rab3A, Rab3B/C, c-src, H+ATPase, and F4/80. GLF, Golgi light; GHF, Golgi heavy; CVCF, crude vesicle fractions.

Having established that Rab3 localizes within osteoclast-like cells with resorptive proteins, we asked if the same is true in authentic avian osteoclasts. Because the ruffled membrane represents acidifying vesicle inserted into the bone-apposed plasmalemma, enriched populations of these vesicles, derived largely from ruffled membrane, were isolated from osteoclasts resident in medullary bone of calcium-deprived laying hens. Immunoblot analysis of this fraction reveals the presence of not only the expected c-src and H+ATPase, but also both the Rab3A and, especially, RabB/C isoforms (Fig. 5). In contrast, Rab4, a protein found in endocytic but not exocytic vesicles,(4) is absent from acidifying vesicles. More importantly, using fluorescence microscopy on authentic murine osteoclasts, we demonstrate the presence of abundant Rab3A and Rab3B, both at the cell periphery and in intracellular vesicles (Fig. 6).

Figure FIG. 5.

Rab3 localization with c-src and vacuolar H+ATPase in acidifying vesicle preparation from authentic osteoclasts. Acidifying membrane vesicles were isolated from authentic osteoclasts, lysed, and the lysate probed, by immunoblot, with anti-Rab3, Rab4, c-src, and H+ATPase antibodies. An equal amount of total cell lysate was used as control for Rab4 (last lane).

Figure FIG. 6.

Rab3 isoforms are present at the surface of authentic murine osteoclasts. Pure populations of BMMs were cultured for 5 days with 20 ng/ml OPGL and 10 ng/ml M-CSF. Following fixation, fluorescence microscopy was performed using standard techniques. Staining with Rab3B antibody (right panel) or nonimmune rabbit IgG as control (left panel). Similar results were obtained with an anti-Rab3A antibody (not shown).

DISCUSSION

Regulated exocytosis, a multicomponent, evolutionarily conserved event,(5,22) involves targeted vesicular movement and fusion. These processes involve a family of membrane-bound proteins collectively known as SNAREs (soluble N-ethylmaleimide-sensitive attachment protein receptors). During membrane fusion, vesicular SNAREs (v-SNAREs) interact with their counterparts on target membranes (t-SNAREs).(50) While association of these various SNAREs is necessary, it is not sufficient to initiate and control vesicle trafficking and/or fusion, events also requiring cytosolic proteins, including Rabs.(4–5,7)

Bone resorption by osteoclasts, which derive from macrophages, requires polarization of the polykaryon following matrix attachment. The most dramatic feature of the polarized osteoclast is its ruffled membrane. This complex infolding of the bone-apposed plasmalemma forms by its fusion with intracellular acidifying vesicles containing both membrane-bound and soluble proteins, many of which participate in the resorptive process. Thus, the membrane-residing protein, c-src, is required for osteoclast polarization,(34) while the vacuolar-type H+ATPase is critical for acidification of the bone-resorbing compartment leading to inorganic matrix mobilization.(32) Secreted cathepsin K degrades the organic phase of bone,(51,52) while osteopontin is a phosphorylated extracellular matrix protein capable of supporting bone–cell attachment.(53) Furthermore, in a process catalyzed by secreted tartrate-resistant acid phosphatase, dephosphorylation of osteopontin results in diminished cell binding capacity.(54) Finally, formation and targeting of vesicles involves the action one or more members of the phosphatidyl inositol-3 kinase family which, in their activated form, are associated with vesicular proteins.(55–57) A similar role for this family of enzymes is suggested from studies demonstrating their localization in the osteoclast ruffled membrane(58) and the fact that PI3 kinase-specific inhibitors block osteoclast function.(59)

The similarities between regulated exocytosis, as manifest by neuroneal and neuroendocrine cells, and ruffled membrane formation by the osteoclast, raises the possibility that Rab proteins, particularly those of the Rab3 family, participate in plasma membrane polarization in the resorptive polykaryon. If such was the case, one would expect these proteins to be expressed by osteoclasts and regulated, in precursor cells, by osteoclastogenic agents. In fact, we establish for the first time, the presence of at least two Rab3 proteins in generated murine osteoclasts and their precursors, avian osteoclast-like cells and the ruffled membrane of primary avian osteoclasts.

Consistent with our posture that these GTPases may be important to the resorptive process, Rab3 isoforms are regulated by osteoclastogenic cytokines. We point out that this observation represents the first documentation that Rab protein expression is regulated in any circumstance. Importantly, Rab3 subfamily members, in osteoclasts, are localized primarily to fractions obtained from the Golgi apparatus, where they localize with the vacuolar proton pump and c-src, ruffled membrane-residing proteins critical to osteoclast function. Absence of Rab4, an endocytic GTPase, in this fraction, supports our contention that vesicles containing Rab3, the H+ATPase and c-src are specifically destined for the ruffled membrane. A substantial role for Rab proteins in osteoclastogenesis is further supported by recent findings, demonstrating Rab7 localization to the ruffled border of osteoclasts.(60)

We have shown that both c-src and the vacuolar H+ATPase, probably in the form of packaged post-Golgi vesicles, associate with microtubules.(44) Given that exocytic Rab proteins interact with the cytoskeleton,(61) Rab3 family members may polarize microtubule-delivered exocytic vesicles to cytoskeletal components adjacent to the bone-apposed osteoclast surface, thereby facilitating vesicular fusion and plasmalemma expansion.

Acknowledgements

We would like to thank Drs. Reinhard Jahn and Andrey Shaw for providing antibodies.

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