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CD44, MT1-MMP, and MMP9 are implicated in the migration of osteoclast and bone resorption. This study was designed to determine the functional relationship between CD44 and MT1-MMP in the activation of pro-MMP9. We used osteoclasts isolated from wild-type and CD44-null mice. Results showed that MT1-MMP is present in multiple forms with a molecular mass ~63, 55, and 45 kDa in the membrane of wild-type osteoclasts. CD44-null osteoclasts demonstrated a 55 kDa active MT1-MMP form in the membrane and conditioned medium. It failed to activate pro-MMP9 because TIMP2 binds and inhibits this MT1-MMP (~55 kDa) in CD44-null osteoclasts. The role of MT1-MMP in the activation of pro-MMP9, CD44 expression, and migration was confirmed by knockdown of MT1-MMP in wild-type osteoclasts. Although knockdown of MMP9 suppressed osteoclast migration, it had no effects on MT1-MMP activity or CD44 expression. These results suggest that CD44 and MT1-MMP are directly or indirectly involved in the regulation of pro-MMP9 activation. Surface expression of CD44, membrane localization of MT1-MMP, and activation of pro-MMP9 are the necessary sequence of events in osteoclast migration.

Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics.

CD44 is a glycosylated adhesion molecule and osteopontin is one of its ligand. CD44 undergoes alternative splicing to produce variant isoforms. Our recent studies have shown an increase in the surface expression of CD44 isoforms (sCD44 and v4-v10 variant CD44) in prostate cancer cells over-expressing osteopontin (PC3/OPN). Formation of CD44/MMP9 complex on the cell surface is indispensable for MMP9 activity. In this study, we have characterized the expression of variant CD44 using RT-PCR, surface labeling with NHS-biotin, and immunoblotting. Expression of variant CD44 encompassing v4-v10 and sCD44 at mRNA and protein levels are of the same levels in PC3 and PC3/OPN cells. However, an increase in the surface expression of v6, v10, and sCD44 in PC3/OPN cells suggest that OPN may be a ligand for these isoforms. We then proceeded to determine the role of sCD44 in MMP9 activation. Based on our previous studies in osteoclasts, we hypothesized that phosphorylation of CD44 has a role on its surface expression and subsequent activation of MMP9. We have prepared TAT-fused CD44 peptides comprising unphosphorylated and constitutively phosphorylated serine residues at positions Ser323 and Ser325. Transduction of phosphopeptides at Ser323 and Ser323/325 into PC3 cells reduced the surface levels of CD44, MMP9 activity, and cell migration; but had no effect on the membrane localization of MMP9. However, MMP9 knock-down PC3 cells showed reduced CD44 at cellular and surface levels. Thus we conclude that surface expression of CD44 and activation of MMP9 on the cell surface are interdependent., ((c) 2009 Wiley-Liss, Inc.)

CD44 and MMP-9 are implicated in cell migration. In the current study, we tested the hypothesis that actin polymerization is critical for CD44 surface expression and MMP-9 activity on the cell surface. To understand the underlying molecular mechanisms involved in CD44 surface expression and MMP-9 activity on the cell surface, osteoclasts were treated with bisphosphonate (BP) alendronate, cytochalasin D (Cyt D), and a broad-spectrum MMP inhibitor (GM6001). BP has been reported to block the mevalonate pathway, thereby preventing prenylation of small GTPase signaling required for actin cytoskeleton modulation. We show in this study that osteoclasts secrete CD44 and MMP-9 into the resorption bay during migration and bone resorption. Results indicate that actin polymerization is critical for CD44 surface expression and osteoclast function. In particular, the surface expression of CD44 and the membrane activity of MMP-9 are reduced in osteoclasts treated with alendronate and Cyt D despite the membrane levels of MMP-9 being unaffected. Although GM6001 blocked MMP-9 activity, osteoclast migration, and bone resorption, the surface levels of CD44 were unaffected. We suggest that the surface expression of CD44 requires actin polymerization. Disruption of podosome and actin ring structures by Cyt D and alendronate not only resulted in reduced localization of MMP-9 in these structures but also in osteoclast migration and bone resorption. These results suggest that inhibition of actin polymerization by alendronate and Cyt D is effective in blocking CD44/MMP-9 complex formation on the cell surface, secretion of active form of MMP-9, and osteoclast migration. CD44/MMP-9 complex formation may signify a unique motility-enhancing signal in osteoclast function., (2007 Wiley-Liss, Inc.)

Background: The expression level of osteopontin correlates with the metastatic potential of several tumors. Osteopontin is a well-characterized ligand for the alphavbeta3 integrin. The present study was undertaken to elucidate the possible role of osteopontin/alphavbeta3 signaling in prostate cancer cell migration., Results: We generated stable prostate cancer cell (PC3) lines that over-express osteopontin (PC3/OPN), mutant OPN in the integrin binding-site (PC3/RGDDeltaRGA), and null for OPN (PC3/SiRNA). The following observations were made in PC3/OPN cells as compared with PC3 cells: 1) an increase in multinucleated giant cells and RANKL expression; 2) an increase in CD44 surface expression, interaction of CD44/MMP-9 on the cell surface, MMP-9 activity in the conditioned medium, and cell migration; 3) western blot analysis of concentrated conditioned medium exhibited equal levels of MMP-9 protein in all PC3 cells. However, zymography analysis demonstrated that the levels of MMP-9 activity in the conditioned media reflect the CD44 surface expression pattern of the PC3 cell lines; 4) although MMP-9 and MMP-2 are secreted by PC3 cells, only the secretion of MMP-9 is regulated by OPN expression. A strong down regulation of the above-mentioned processes was observed in PC3/OPN (RGA) and PC3/SiRNA cells. PC3/OPN cells treated with bisphosphonate (BP) reproduce the down-regulation observed in PC3/OPN (RGA) and PC3/SiRNA cells., Conclusion: Rho signaling plays a crucial role in CD44 surface expression. BPs inhibits the mevalonate pathway, which in turn, prevents the prenylation of a number of small GTPases. Attenuation of Rho GTPase activation by BPs may have contributed to the down regulation of cell surface CD44/MMP-9 interaction, MMP-9 activation/secretion, and cell migration. Taken together, these observations suggest that CD44 surface expression is an important event in the activation of MMP-9 and migration of prostate cancer cells. The various steps involved in the above mentioned signaling pathway and/or the molecules regulating the activation of MMP-9 are potential therapeutic target.