Activated c-Fms recruits Vav and Rac during CSF-1-induced cytoskeletal remodeling and spreading in osteoclasts
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.
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These two individuals contributed equally to this manuscript.