The colony stimulating factor 1 (CSF-1) regulates osteoclastogenesis and bone resorption. Mutations in the CSF-1 gene cause an osteopetrosis characterized by the absence of osteoclasts. Mature osteoclasts respond to CSF-1 with inhibition of bone resorption and an increment of cell spreading. Herein we demonstrate that CSF-1-induced osteoclast spreading depends on the substrate the osteoclast interacts with and requires integrity of the vitronectin receptor and of the c-src proto-oncogene. Rabbit osteoclasts were allowed to attach to glass, serum, osteopontin, and bone substrates, and were treated with 10 ng/ml human recombinant CSF-1 for 4 h. In osteoclasts plated on glass, the cytokine induced 70% inhibition of bone resorption and 1.8-fold stimulation of cell spreading, without changes in podosome expression and microfilament array. In contrast, CSF-1 induced a 2.5-fold increase of osteoclasts showing filopodia, and a 9.5-fold increase of osteoclasts presenting lamellipodia, indicating that membrane motility was required for cell spreading. Osteoclasts plated on serum substrates showed a 50% reduction of spontaneous spreading. However, in this circumstance, CSF-1 still stimulated an increase of osteoclast area. In osteoclasts cultured on osteopontin substrate or on bone slices, an inhibition of CSF-1-induced osteoclast spreading was observed. To establish involvement of the vitronectin receptor and c-src proto-oncogene, cells were treated with the ␣ v ␤ 3 integrin neutralizing antibody, LM609, or c-src antisense oligonucleotides, which reduced CSF-1-induced osteoclast spreading by 57% and 60%, respectively. The results demonstrate that CSF-1-induced osteoclast spreading requires both the vitronectin receptor and the c-src proto-oncogene and that this action is modulated by the adhesion substrata. (J Bone Miner Res 1998;13:50-58)

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Giant cell tumours (GCT) of bone are locally aggressive and highly bone lytic. Tumours are composed of multinucleated giant cells interspersed with mononuclear tumour and stromal cells, the former cell type exhibiting phenotypic similarity to authentic osteoclasts. Mineralised bone resorption by GCTs has been attributed to the multinucleated, osteoclast-like giant cell component, although the persistence of lytic bone lesions may also involve impaired mineralised bone deposition involving other cellular elements (Dahlin et al., 1970;Roodman, 1996).Bone is composed largely of type I collagen (Gehron Robey, 1989), and type I collagenolytic enzymes have, therefore, been implicated in its degradation. Physiological osteoclast-mediated bone resorption is currently considered to result from the combined action of acidic collagenolytic cystein proteases (cathepsins) and neutral metalloproteinases (MMP) (Baron, 1989;Delaisse and Vaes, 1993;Hill et al., 1995;Roodman, 1996). Within the neutral MMP family, classical type I collagenase, MMP-1, and gelatinases which include MMP-2 and MMP-9 have been implicated in osteoclast-dependent bone resorption (Hill et al., 1995). These enzymes are expressed by GCT cells in vitro and by intraosseous GC tumours in vivo (Wucherpfennig et al., 1994;Rao et al., 1995), suggesting potential roles in GCT pathology.MMPs are secreted in latent form and require N-terminal sequence removal for activity (Cottam and Rees., 1993). Therefore, the physiological mechanisms that control MMP activation are central to the subsequent involvement of MMPs in biological processes. Many MMPs, such as MMP-1 and MMP-9, are activated by proteases such as plasmin, cathepsin G and trypsin-like enzymes. MMP-2, however, is resistant to activation by many of these enzymes (Mackay et al., 1990;Okada et al., 1990;Cottam and Rees, 1993). Activation of MMP-2 has been attributed to autocatalysis or to the action of other MMPs including MMP-1, MMP-7 and MT-MMP or to the serine proteases urokinase and thrombin (Atkinson et al., 1995; Crabbe et al., 1994a,b;Reith and Rucklidge., 1992;Zucker et al., 1995). Recently, a membraneassociated, MMP-dependent, MMP-2 activation mechanism has been described (Brown et al., 1993;Strongin et al., 1995;Ward et al., 1994) that possibly involves members of a novel membrane type MMP (MT-MMP) family (Sato et al., 1994).Bone matrix influences physiological and pathological bone cell behaviour. GCT cells and osteoclasts exhibit ␣V␤3 integrin and RGD sequence-dependent adherence to bone matrix components such as osteopontin (OPN) and bone sialoprotein (BSP) (Butler, 1989;Gehron Robey, 1989;Denhardt and Xiaojia, 1993;Horton et al., 1985Horton et al., , 1991Horton et al., , 1993Helfrich et al., 1992; Grano et al., 1994a,b;Roodman, 1996), and a close relationship has been reported between bone adherence and mineralised bone resorption (Horton et al., 1993;Grano et al., 1994a). Soluble RGD peptides inhibit RGD-dependent adherence to and resorption of mineralised bone in vitro (Horton et al., 1991(Hort...

Prostate cancers (PRCAs) frequently metastasize to bone. We show here that this process is facilitated by osteoblastmediated tumor cell recruitment. Transforming growth factor-␤1 (TGF-␤1) is produced by osteoblasts in a latent form and is activated by proteases in a cell-dependent manner. This cytokine exhibits pleiotropic effects on cell-extracellular matrix (ECM) interactions and may influence tumor cell invasion and metastasis. Our purpose was to identify the potential molecular mechanisms involved in osteoblastmediated cell recruitment and to characterize the effect of TGF-␤1 on adhesion, motility and invasiveness of a human prostate cancer cell line with high bone metastatic potential (PC3 cell line) in vitro. Conditioned media from osteoblast cultures (OB CM) enhanced PC3 cell chemotaxis and invasion of reconstituted basement membrane. These effects were blocked by a neutralyzing TGF-␤1 polyclonal antibody but not by elution of the OB CM in agarose-heparin columns, suggesting that TGF-␤1, but not EGF-like proteins, contribute to PC3 cell recruitment. In addition, TGF-␤1 directly induced chemotaxis and invasion of PC3 cells in a dosedependent manner. The TGF-␤1-mediated invasion and motility were accompanied by increased PC3 cell adhesion, spreading and ␣2␤1 and ␣3␤1 integrin expression. These events are involved in the cell adhesion to several components of basement membrane and ECM and in the selective invasion of metastatic tumor cells. Our results suggest that TGF-␤1 can influence cellular recognition of ECM components by prostatic cancer cells and can modulate cell adhesion and invasion leading to increased invasive potential. Given the widespread tissue distribution of TGF-␤1, and the high levels present in the bone, this cytokine may be an important autocrine-paracrine modulator of the bone invasive phenotype in vivo. Int. J. Cancer 81:395-403, 1999. Wiley-Liss, Inc.Tumor progression to the stage of metastasis may result in part from the selection of primary tumor cell clones which are phenotypically competent for survival, invasion and growth at secondary sites (Rak and Kerbel, 1993). The ability of malignant tumor cells to invade host tissues contributes to their locally destructive nature and can play an important role in their metastatic spread. At a molecular level, a variety of processes contribute to the invasive and metastatic phenotypes. Tumor cell adhesion to extracellular matrix proteins, degradation of these proteins by tumor cell-produced proteolytic enzymes and tumor cell migration through proteolytically modified extracellular matrix represent major events in the invasive process (Liotta et al., 1991;Cottam and Rees, 1993). These events occur repeatedly during tumor invasion and show changes in the adhesive and/or migratory phenotype of tumor cells, with modifications in the proteolytic enzyme profile, which may be expected to modulate tumor cell invasion.Selection for traits such as loss of growth inhibitory responses, acquisition of increased adhesiveness, increased local immunosuppr...