Elsevier

Bone

Volume 39, Issue 6, December 2006, Pages 1290-1301
Bone

Activated c-Fms recruits Vav and Rac during CSF-1-induced cytoskeletal remodeling and spreading in osteoclasts

https://doi.org/10.1016/j.bone.2006.06.012Get rights and content

Abstract

Colony-stimulating factor-1 (CSF-1) induces osteoclast spreading that requires activation of c-Src and phosphatidyl inositol 3-kinase (PI3-K), both of which are recruited to activated c-Fms, the CSF-1 receptor. The present report provides evidence that the hemopoietic guanine nucleotide exchange factor (GEF), Vav, and its target GTPase, Rac, lie downstream from this initial signaling complex. CSF-1 treatment of osteoclast-like cells induced translocation of Vav to the plasma membrane, an increase in its phosphotyrosine content, and a concomitant decline in the amount of phosphoinositol 4,5-bisphosphate bound to Vav, changes known to induce Vav's GEF activity. CSF-1 induced the association of Vav and Rac and increased Rac's GTPase activity. CSF-1 also induced rapid translocation of Rac to the periphery of spreading neonatal rat osteoclasts where it co-localized primarily with Vav3 and to a lesser extent with Vav1. Wortmannin, an inhibitor of PI3-K, blocked CSF-1-induced Rac translocation and prevented CSF-1-induced spreading and actin reorganization in osteoclasts. CSF-1-induced osteoclast spreading was not significantly reduced in osteoclasts isolated from Vav1 knock-out mice and Vav1 knock-out mice had normal bone density. Microinjection of constitutively active Rac, but not constitutively active Cdc42 or RhoA, induced lamellipodia formation and osteoclast spreading, mimicking the effects of CSF-1. Dominant-negative Rac blocked CSF-1-induced osteoclast spreading, whereas neither dominant-negative Cdc42 nor C3, an inhibitor of RhoA, affected the response to CSF-1. These data demonstrate that Vav and Rac lie downstream from activated PI3-K in CSF-1-treated osteoclasts and that Rac is required for CSF-1-induced cytoskeletal remodeling in these cells.

Introduction

Bone remodeling is a complex, highly orchestrated process that is initiated by a cycle of bone resorption, mediated, at the cellular level, by osteoclasts [1]. A distinguishing feature of the resorbing activity of osteoclasts is their ability to move along the bone surface. After excavating a resorbing pit, the sealing zone of an osteoclast is disassembled and the cells move to a new site of resorption. The signals that attract osteoclasts to new sites of resorption are unknown. One possible signal may be colony-stimulating factor-1 (CSF-1).

CSF-1 clearly affects osteoclastogenesis and must therefore have osteoclast progenitors as one of its targets in bone. However, the CSF-1 receptor, c-Fms, is most highly expressed on mature osteoclasts [2]. We and others have reported that CSF-1 induces cell spreading, motility, and actin reorganization in mature osteoclasts [3], [4], [5]. The signaling cascade that regulates these cytoskeletal events is not clear. C-src appears to be required for CSF-1's cytoskeletal effects because mature osteoclasts derived from src−/− mice do not spread in response to CSF-1 [6]. However, when plated on vitronectin, src−/− pre-osteoclasts can respond to CSF-1 [7]. Phosphatidylinositol 3-kinase (PI3-K) is also important for this process because inhibition of this enzyme prevents CSF-1-induced osteoclast spreading and motility [5].

Available evidence suggests that the substrate, phosphoinositol 4,5-bisphosphate (PIP2), and product, phosphoinositol 3,4,5-triphosphate (PIP3), of PI3-K play important signaling roles. For example, binding of these phospholipids to a conserved protein motif called the pleckstrin homology (PH) domain in guanine nucleotide exchange factors (GEFs) markedly influences the activity of these molecules [8]. Thus, the PI3-K substrate, PIP2, when bound to the PH domain of the GEF, Vav, inhibits its activity, whereas binding of the product, PIP3, activates Vav. In this way, activation of PI3-K results in activation of Vav in part by facilitating exchange of an inhibitory molecule PIP2, for a stimulatory molecule PIP3, on Vav's PH domain.

Vav and other GEFs lie upstream of the Rho GTPase family of proteins Rho, Rac, and Cdc42 [9], [10], [11], [12], [13]. The Rho GTPase family of proteins is known to be critically important in regulating the cytoskeleton [13]. In macrophages, activated Rac causes the development of lamellipodia, broad apron-like cytoplasmic extensions, whereas activated Cdc42 stimulates filopodia formation, small finger-like or spike-like cytoplasmic extensions [14]. Activation of Rho causes cytoplasmic retraction [14].

In the current study, we sought to determine whether the Rho GTPases participate in mediating CSF-1's cytoskeletal effects in osteoclasts. We report that the GTPase Rac is required for CSF-1-induced osteoclast spreading and that Vav and Rac appear to lie downstream from PI3-K in a signaling cascade that leads from activated c-Fms to the actin cytoskeleton in these cells.

Section snippets

Materials

Recombinant human colony-stimulating factor-1 (CSF-1) was a generous gift from Genetics Institute (Cambridge, MA). Alpha-MEM cell culture medium and fetal bovine serum were from Sigma (St. Louis, MO). Antisera to phosphoinositol 4,5-bisphosphate (PIP2) [15] was kindly provided by Kiyoko Fukami, PhD, Department of Molecular Oncology, University of Tokyo, Tokyo, Japan. Wortmannin and FluorSave were purchased from Calbiochem (La Jolla, CA). Antibodies to the p85 subunit of PI3-K, c-Fms, Rac,

CSF-1-induced actin cytoskeleton reorganization is blocked by wortmannin, a PI3-K inhibitor

We have previously shown that CSF-1 induces PI3-K activation and actin cytoskeletal reorganization [5], [6]. To determine if these two effects were causally linked, mature osteoclasts were pretreated with 50 nM wortmannin for 30 min and subsequently treated with or without CSF-1 for an additional 20 min. As shown in Fig. 1A and consistent with our previous observations [5], CSF-1-induced osteoclast spreading, lamellipodia formation, and actin remodeling. Pretreatment with wortmannin completely

Discussion

The two principal findings in this study are as follows: (1) Rac is required for CSF-1-induced cytoskeletal remodeling in osteoclasts. (2) Vav is activated by CSF-1 treatment in osteoclasts and serves as the GEF for Rac. Of the three Vav isoforms, Vav3 seems to act as the principal GEF although Vav1 may play a minor role.

The data supporting the first conclusion are as follows: CSF-1 stimulates rapid translocation of Rac to the plasma membrane and on-loading of GTP to Rac in osteoclasts.

Acknowledgments

This work was supported by a grant from NIDCR to KI (DE12459) and in part by a P30 Core Center Award from NIAMS to KI (AR46032). The authors wish to thank Dr. Marcjanna Bartkiewicz for her critical review of the manuscript and helpful editorial suggestions.

References (52)

  • B. Aghazadeh et al.

    Structural basis for relief of autoinhibition of the Dbl homology domain of proto-oncogene Vav by tyrosine phosphorylation

    Cell

    (2000)
  • Y.G. Yeung et al.

    Colony-stimulating factor-1 stimulates the formation of multimeric cytosolic complexes of signaling proteins and cytoskeletal components in macrophages

    J. Biol. Chem.

    (1998)
  • M. Deckert et al.

    Functional and physical interactions of Syk family kinases with the Vav proto-oncogene product

    Immunity

    (1996)
  • F. Michel et al.

    Fyn and ZAP-70 are required for Vav phosphorylation in T cells stimulated by antigen-presenting cells

    J. Biol. Chem.

    (1998)
  • I. Hornstein et al.

    Vav proteins, masters of the world of cytoskeleton organization

    Cell. Signal.

    (2004)
  • S. Razzouk et al.

    Rac-GTPase, osteoclast cytoskeleton and bone resorption

    Eur. J. Cell Biol.

    (1999)
  • M.A. Chellaiah et al.

    Rho-A is critical for osteoclast podosome organization, motility, and bone resorption

    J. Biol. Chem.

    (2000)
  • G.D. Roodman

    Advances in bone biology: the osteoclast

    Endocr. Rev.

    (1996)
  • E.C. Weir et al.

    Macrophage colony-stimulating factor release and receptor expression in bone cells

    J. Bone Miner. Res.

    (1993)
  • K. Fuller et al.

    Macrophage colony-stimulating factor stimulates survival and chemotactic behavior in isolated osteoclasts

    J. Exp. Med.

    (1993)
  • A. Grey et al.

    Evidence for a functional association between phosphatidylinositol 3-kinase and c-src in the spreading response of osteoclasts to colony-stimulating factor-1

    Endocrinology

    (2000)
  • K. Insogna et al.

    Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src- dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts

    J. Clin. Invest.

    (1997)
  • I. Nakamura et al.

    Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase C gamma in prefusion osteoclasts

    J. Cell Biol.

    (2001)
  • J. Han et al.

    Role of substrates and products of PI 3-kinase in regulating activation of Rac-related guanosine triphosphatases by Vav

    Science

    (1998)
  • M. Turner et al.

    VAV proteins as signal integrators for multi-subunit immune-recognition receptors

    Nat. Rev., Immunol.

    (2002)
  • X.R. Bustelo

    Regulatory and signaling properties of the Vav family

    Mol. Cell. Biol.

    (2000)
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