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 motilityenhancing signal in osteoclast function.

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In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac ␣-and ␤-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 3 Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.Although many important alterations have been described in the properties of hypertrophied myocardium, the mechanisms responsible for contractile dysfunction and many other maladaptive changes of cardiac muscle cells, or cardiocytes, have yet to be fully defined. Although most research in this area has focused on structural and regulatory changes within the myofilament, it has also been found that changes in the microtubule component of the extra-myofilament cytoskeleton may lead both to contractile dysfunction by increasing the internal resistance to sarcomere motion (1, 2) and to disordered cellular homeostasis by impeding cytoskeleton-based intracellular transport (3-6).Our major original finding was that, in severe pressure overload cardiac hypertrophy with increased ventricular wall stress, there is the early appearance and then the persistence of a dense microtubule network and associated contractile dysfunction (7,8). We have now found several synergistic bases for this dense microtubule network. First, during hypertrophy there is persistent tran...

Wiskott Aldrich Syndrome protein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. Here, using the TAT-transduction method, we show the possible role of WASP domain(s) in sealing ring formation and bone resorption. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption. Transduction of WASP peptides containing basic, verpolincentral, pTyr294, and proline-rich regions inhibited the processes listed above at various levels. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated the following: a) a considerable decrease in the interaction and phosphorylation of c-Src with endogenous WASP; b) total loss of sealing ring-like structures; c) formation of actin-rich patches at the peripheral edge that contains filopodia-like projections; d) reduced capacity for bone resorption in vitro. These findings suggest that modulation of phosphorylation state of pTyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring.

Prostate Cancer (PCa) is one of the most lethal diseases afflicting men. Although early cancer detection and treatment is often curative, subsequent metastatic spread of tumor cells renders the disease untreatable. Treatment failure is also due to a poor understanding of the contribution of the tumor microenvironment to disease progression. We find that a number of PCa cells secrete heat shock protein 90 (Hsp90). Extracellular Hsp90 (eHsp90) acts in a manner distinct from the intracellular chaperone and possesses ‘chaperokine’ properties. The ability of chaperones to buffer against cellular stress and promote survival of malignant cells led us investigate the potential role of eHsp90 in PCa progression. Interestingly, we find that eHsp90 expression correlates with PCa aggressiveness. Consistently, the more aggressive and metastatic PCa cells exhibit several fold higher eHsp90 secretion relative to their weakly tumorigenic matched counterparts. Interference with this pathway by antibody or drug-mediated neutralization dramatically impaired tumor cell migration. Concomitant with inhibition of eHsp90, the activation of several critical downstream signaling mediators of cell motility were attenuated. The multifunctional receptor LRP-1 (LDL-receptor Related protein-1) has been proposed as the receptor for eHsp90. The silencing of LRP1 dramatically suppressed PCa signaling and cell migration. The ability of LRP1 knockdown to recapitulate the drug dependent inhibition of eHsp90 strongly supports the requirement for an Hsp90-LRP1 signaling axis in PCa progression. A major component of the solid tumor microenvironment is the stromal fibroblast, which acts to potentiate both tumor growth and metastatic spread. Addition of Hsp90 to prostate stromal fibroblasts, which do not secrete Hsp90 protein, potently stimulates ERK activation and cell motility, demonstrating that the eHsp90 chaperokine possess paracrine effects upon the tumor stroma and induces properties associated with a reactive phenotype. The ability of eHsp90 to induce both ERK activation and cell motility was inhibited by an MMP inhibitor, suggesting that eHsp90 dependent modulation of MMP activity is a requirement for its downstream effects on stromal signaling and cell motility. Our data implicate that eHsp90 secretion may represent a critical component of PCa aggressiveness via its ability to initiate multiple signaling events in stromal fibroblasts that enable these cell to further drive tumor growth. Our studies raise the possibility that approaches designed to curtail eHsp90 activity may be a useful strategy to intercept tumor-stromal dynamic signaling, which is predicted to have a favorable impact upon the progression of prostate cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 545.