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Methylsulfonylmethane (MSM) is a naturally occurring anti-inflammatory compound that effectively treats multiple degenerative diseases such as osteoarthritis and acute pancreatitis. Our previous studies have demonstrated the ability of MSM to differentiate stem cells from human exfoliated deciduous (SHED) teeth into osteoblast-like cells. This study examined the systemic effect of MSM in 36-week-old aging C57BL/6 female mice in vivo by injecting MSM for 13 weeks. Serum analyses showed an increase in expression levels of bone formation markers [osteocalcin (OCN) and procollagen type 1 intact N-terminal propeptide (P1NP)] and a reduction in bone resorption markers [tartrate-resistant acid phosphatase (TRAP) and C-terminal telopeptide of type I collag (CTX-I)] in MSM-injected animals. Micro-computed tomographic images demonstrated an increase in trabecular bone density in mandibles. The trabecular bone density tended to be higher in the femur, although the increase was not significantly different between the MSM- and phosphate-buffered saline (PBS)-injected mice. In mandibles, an increase in bone density with a corresponding decrease in the marrow cavity was observed in the MSM-injected mice. Furthermore, immunohistochemical analyses of the mandibles for the osteoblast-specific marker - OCN, and the mesenchymal stem cell-specific marker - CD105 showed a significant increase and decrease in OCN and CD105 positive cells, respectively. Areas of bone loss were observed in the inter-radicular region of mandibles in control mice. However, this loss was considerably decreased due to stimulation of bone formation in response to MSM injection. In conclusion, our study has demonstrated the ability of MSM to induce osteoblast formation and function in vivo , resulting in increased bone formation in the mandible. Hence, the application of MSM and stem cells of interest may be the right combination in alveolar bone regeneration under periodontal or other related diseases that demonstrate bone loss., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aljohani, Senbanjo, Al Qranei, Stains and Chellaiah.)

Tumor necrosis factor-alpha (TNF-α) signaling regulates phosphorylation of L-plastin, which is involved in forming the nascent sealing zone, a precursor zone for the matured sealing ring. This study aimed to illustrate the molecular mechanisms of L-plastin phosphorylation and the subsequent formation of the nascent sealing zone in osteoclasts treated with TNF-α. Here, we report that anti-TNF-receptor 1, inhibitors of signaling proteins (Src, PI3-K, Rho, and Rho-kinase), and siRNA of TRAF-6 attenuated the phosphorylation of LPL and filamentous actin content significantly in the presence of TNF-α. An inhibitor of integrin αvβ3, PKC, or PKA did not inhibit TNF-α-induced L-plastin phosphorylation. Inhibitors of Src and PI3-K and not Rho or Rho-kinase reduced tyrosine phosphorylation of TRAF-6, suggesting that Src and PI3-K regulate TRAF-6 phosphorylation, and Rho and Rho-kinase are downstream of TRAF-6 regulation. Osteoclasts expressing constitutively active or kinase-defective Src proteins were used to determine the role of Src on L-plastin phosphorylation; similarly, the effect of Rho was confirmed by transducing TAT-fused constitutively active (V14) or dominant-negative (N19) Rho proteins into osteoclasts. Pull-down analysis with glutathione S-transferase-fused SH2 and SH3 domains of Src and PI3-K demonstrated coprecipitation of L-plastin and TRAF-6 with the SH3 and SH2 domains of the PI3-K and Src proteins. However, the actual order of the interaction of proteins requires further elucidation; a comprehensive screening should corroborate the initial findings of protein interactions via the SH2/SH3 domains. Ultimately, inhibition of the interaction of proteins with SH2/SH3 could reduce L-plastin phosphorylation and affect NSZ formation and bone resorption in conditions that display osteoclast activation and bone loss.

L-plastin (LPL) was identified as a potential regulator of the actin-bundling process involved in forming nascent sealing zones (NSZs), which are precursor zones for mature sealing zones. TAT-fused cell-penetrating small molecular weight LPL peptide (TAT- MARGSVSDEE, denoted as an inhibitory LPL peptide) attenuated the formation of NSZs and impaired bone resorption in vitro in osteoclasts. Also, the genetic deletion of LPL in mice demonstrated decreased eroded perimeters and increased trabecular bone density. In the present study, we hypothesized that targeting LPL with the inhibitory LPL peptide in vivo could reduce osteoclast function and increase bone density in a mice model of low bone mass. We injected aging C57BL/6 female mice (36 weeks old) subcutaneously with the inhibitory and scrambled peptides of LPL for 14 weeks. Micro-CT and histomorphometry analyses demonstrated an increase in trabecular bone density of femoral and tibial bones with no change in cortical thickness in mice injected with the inhibitory LPL peptide. A reduction in the serum levels of CTX-1 peptide suggests that the increase in bone density is associated with a decrease in osteoclast function. No changes in bone formation rate and mineral apposition rate, and the serum levels of P1NP indicate that the inhibitory LPL peptide does not affect osteoblast function. Our study shows that the inhibitory LPL peptide can block osteoclast function without impairing the function of osteoblasts. LPL peptide could be developed as a prospective therapeutic agent to treat osteoporosis.

Background: Lipopolysaccharide (LPS) is an endotoxin and a vital component of gram-negative bacteria's outer membrane. During gram-negative bacterial sepsis, LPS regulates osteoclast differentiation and activity, in addition to increasing inflammation. This study aimed to investigate how LPS regulates osteoclast differentiation of RAW 264.7 cells in vitro., Results: Herein, we revealed that RAW cells failed to differentiate into mature osteoclasts in vitro in the presence of LPS. However, differentiation occurred in cells primed with receptor activator of nuclear factor-kappa-Β ligand (RANKL) for 24 h and then treated with LPS for 48 h (henceforth, denoted as LPS-treated cells). In cells treated with either RANKL or LPS, an increase in membrane levels of toll-like receptor 4 (TLR4) receptor was observed. Mechanistically, an inhibitor of TLR4 (TAK-242) reduced the number of osteoclasts as well as the secretion of tumor necrosis factor (TNF)-α in LPS-treated cells. RANKL-induced RAW cells secreted a very basal level TNF-α. TAK-242 did not affect RANKL-induced osteoclastogenesis. Increased osteoclast differentiation in LPS-treated osteoclasts was not associated with the RANKL/RANK/OPG axis but connected with the LPS/TLR4/TNF-α tumor necrosis factor receptor (TNFR)-2 axis. We postulate that this is because TAK-242 and a TNF-α antibody suppress osteoclast differentiation. Furthermore, an antibody against TNF-α reduced membrane levels of TNFR-2. Secreted TNF-α appears to function as an autocrine/ paracrine factor in the induction of osteoclastogenesis independent of RANKL., Conclusion: TNF-α secreted via LPS/TLR4 signaling regulates osteoclastogenesis in macrophages primed with RANKL and then treated with LPS. Our findings suggest that TLR4/TNF-α might be a potential target to suppress bone loss associated with inflammatory bone diseases, including periodontitis, rheumatoid arthritis, and osteoporosis.

Background: Periodontitis is the inflammation of the tooth-supporting structures and is one of the most common diseases of the oral cavity. The outcome of periodontal infections is tooth loss due to a lack of alveolar bone support. Osteoclasts are giant, multi-nucleated, and bone-resorbing cells that are central for many osteolytic diseases, including periodontitis. Receptor activator of nuclear factor-kB ligand (RANKL) is the principal factor involved in osteoclast differentiation, activation, and survival. However, under pathological conditions, a variety of pro-inflammatory cytokines secreted by activated immune cells also contribute to osteoclast differentiation and activity. Lipopolysaccharide (LPS) is a vital component of the outer membrane of the Gram-negative bacteria. It binds to the Toll-like receptors (TLRs) expressed in many cells and elicits an immune response., Highlights: The presence of bacterial LPS in the periodontal area stimulates the secretion of RANKL as well as other inflammatory mediators, activating the process of osteoclastogenesis. RANKL, either independently or synergistically with LPS, can regulate osteoclastogenesis, while LPS alone cannot. MicroRNA, IL-22, M1/M2 macrophages, and memory B cells have recently been shown to modulate osteoclastogenesis in periodontal diseases., Conclusion: In this review, we summarize the mechanism of osteoclastogenesis accompanying periodontal diseases at the cellular level. We discuss a) the effects of LPS/TLR signaling and other cytokines on RANKL-dependent and -independent mechanisms involved in osteoclastogenesis; b) the recently identified role of several endogenous factors such as miRNA, IL-22, M1/M2 macrophages, and memory B cells in regulating osteoclastogenesis during periodontal pathogenesis., Competing Interests: Conflicts of interest The authors have no potential conflict of interest relevant to this article., (Copyright © 2020 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.)

Excessive bone loss occurs in inflammatory disorders such as periodontitis and osteoporosis. The underlying mechanism is related to the differentiation of macrophages into multinucleated giant osteoclasts and their bone resorptive activity. C-Phycocyanin (C-PC) is a phycobiliprotein extracted from the blue-green algae, which has been shown to have various pharmacological effects. The role of C-PC on bone metabolism needs revelation. In this study, we determined the effectiveness of C-PC as an inhibitor of osteoclast differentiation, activity, and survival in vitro. We found that C-PC strongly inhibited the differentiation of macrophages to TRAP-positive osteoclasts, distinctive osteoclast specific podosomal organization, and dentine matrix resorption without any cytotoxicity. Also, it suppressed the expression of osteoclast specific markers, such as cathepsin K and integrin β3 at mRNA and protein levels. RANKL mediated signaling utilizes reactive oxygen species (ROS) for the differentiation of osteoclasts. C-PC attenuated RANKL stimulated ROS. Mechanistic studies indicate that C-PC has the potential to reduce osteoclast formation via blocking the degradation of cytosolic IκB-α and hence, the activation of downstream markers such as c-Fos and NFATc1. However, it does not have any effect on osteoblast-mediated bone formation in vitro. Collectively, our data suggest that C-PC may be utilized as a therapeutic agent that can target bone loss mediated by excessive osteoclastic bone resorption without affecting osteoblastic activity in bone.

Bone resorption requires the formation of complex, actin-rich cytoskeletal structures. During the early phase of sealing ring formation by osteoclasts, L-plastin regulates actin-bundling to form the nascent sealing zones (NSZ). Here, we show that L-plastin knockout mice produce osteoclasts that are deficient in the formation of NSZs, are hyporesorptive, and make superficial resorption pits in vitro. Transduction of TAT-fused full-length L-plastin peptide into osteoclasts from L-plastin knockout mice rescued the formation of nascent sealing zones and sealing rings in a time-dependent manner. This response was not observed with mutated full-length L-plastin (Ser-5 and -7 to Ala-5 and -7) peptide. In contrast to the observed defect in the NSZ, L-plastin deficiency did not affect podosome formation or adhesion of osteoclasts in vitro or in vivo. Histomorphometry analyses in 8- and 12-week-old female L-plastin knockout mice demonstrated a decrease in eroded perimeters and an increase in trabecular bone density, without a change in bone formation by osteoblasts. This decrease in eroded perimeters supports that osteoclast function is attenuated in L-plastin knockouts. Micro-CT analyses confirmed a marked increase in trabecular bone mass. In conclusion, female L-plastin knockout mice had increased trabecular bone density due to impaired bone resorption by osteoclasts. L-plastin could be a potential target for therapeutic interventions to treat trabecular bone loss., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Aim: The Cluster of differentiation 44 (CD44) transmembrane protein is cleaved by γ-secretase, the inhibition of which blocks CD44 cleavage. This study aimed to determine the biological consequence of CD44 cleavage and its potential interaction with Runt-related transcription factor (RUNX2) in a sequence-specific manner in PC3 prostate cancer cells., Methods: Using full-length and C-terminal deletion constructs of CD44-ICD (D1-D5) expressed as stable green fluorescent protein-fusion proteins in PC3 cells, we located possible RUNX2-binding sequences., Results: Chromatin immunoprecipitation assays demonstrated that the C-terminal amino acid residues between amino acids 671 and 706 in D1 to D3 constructs were indispensable for sequence-specific binding of RUNX2. This binding was minimal for sequences in the D4 and D5 constructs. Correspondingly, an increase in matrix metalloprotease-9 (MMP-9) expression was observed at the mRNA and protein levels in PC3 cells stably expressing D1-D3 constructs., Conclusion: These results provide biochemical evidence for the possible sequence-specific CD44-ICD/RUNX2 interaction and its functional relationship to MMP-9 transcription in the promoter region., Competing Interests: Conflicts of interest All authors declared that there are no conflicts of interest.

Methylsulfonylmethane (MSM) is a naturally occurring, sulfate-containing, organic compound. It has been shown to stimulate the differentiation of mesenchymal stem cells into osteoblast-like cells and bone formation. In this study, we investigated whether MSM influences the differentiation of stem cells from human exfoliated deciduous teeth (SHED) into osteoblast-like cells and their osteogenic potential. Here, we report that MSM induced osteogenic differentiation through the expression of osteogenic markers such as osterix, osteopontin, and RUNX2, at both mRNA and protein levels in SHED cells. An increase in the activity of alkaline phosphatase and mineralization confirmed the osteogenic potential of MSM. These MSM-induced effects were observed in cells grown in basal medium but not osteogenic medium. MSM induced transglutaminase-2 (TG2), which may be responsible for the cross-linking of extracellular matrix proteins (collagen or osteopontin), and the mineralization process. Inhibition of TG2 ensued a significant decrease in the differentiation of SHED cells and cross-linking of matrix proteins. A comparison of mineralization with the use of mineralized and demineralized bone particles in the presence of MSM revealed that mineralization is higher with mineralized bone particles than with demineralized bone particles. In conclusion, these results indicated that MSM could promote differentiation and osteogenic potential of SHED cells. This osteogenic property is more in the presence of mineralized bone particles. TG2 is a likely cue in the regulation of differentiation and mineral deposition of SHED cells in response to MSM., Competing Interests: The authors have declared that no competing interests exist.

Background: Expression of CD44 receptor is associated with the onset of several tumors. The intracellular domain of CD44 (CD44-ICD) has been implicated as a co-transcription factor for RUNX2 in the regulation of expression of MMP-9 in breast carcinoma cells. Previous studies from our laboratory demonstrated the role of CD44 in migration and invasion of PC3 prostate cells through activation of MMP-9. CD44 signaling regulates the phosphorylation and hence the localization of RUNX2 in the nucleus. The role of CD44-ICD has not been studied in prostate cancer cells. This study aimed to explore the role of CD44-ICD and RUNX2 in the regulation of expression of metastasis-related genes., Methods: PC3 and PC3 cells overexpressing RUNX2 protein were analyzed for RUNX2/CD44-ICD interaction by immunoprecipitation, immunoblotting, and Immunofluorescence analyses. Wound healing and tumorsphere formation analyses were also done in these cells. The real-time PCR analysis was used to detect the expression levels of different genes., Results: Expression of CD44 and RUNX2 was observed only in PC3 cells (androgen receptor positive) and not in LNCaP or PCa2b cells (androgen receptor negative). Therefore, CD44-ICD fragment (~ 15-16 kDa) was observed in PC3 cells. Moreover, localization of CD44-ICD was more in the nucleus than in the cytoplasm of PC3 cells. Inhibition of cleavage of CD44 with a γ-secretase inhibitor, DAPT reduced the formation of CD44-ICD; however, accumulation of CD44-external truncation fragments (~ 20 and ~ 25 kDa) was detected. RUNX2 and CD44-ICD interact in the nucleus of PC3 cells, and this interaction was more in PC3 cells transfected with RUNX2 cDNA. Overexpression of RUNX2 augments the expression of metastasis-related genes (e.g., MMP-9 and osteopontin) which resulted in increased migration and tumorsphere formation., Conclusions: We have shown here a strong functional relationship between CD44-ICD and RUNX2 in PC3 cells. RUNX2 forms a complex with CD44-ICD as a co-transcriptional factor, and this complex formation not only activates the expression of metastasis-related genes but also contributes to migration and tumorsphere formation. Therefore, RUNX2 and CD44-ICD are potential targets for anti-cancer therapy, and attenuation of their interaction may validate the regulatory effects of these proteins on cancer migration and progression.

Studies have shown that a subgroup of tumor cells possess stemness characteristics having self-renewal capacity and the ability to form new tumors. We sought to identify the plausible stemness factor that determines the "molecular signature" of prostate cancer (PCa) cells derived from different metastases (PC3, PCa2b, LNCaP, and DU145) and whether androgen receptor (AR) influences the maintenance of stemness features. Here we show sex-determining region Y (SRY)-box 2 (SOX2) as a putative stem cell marker in PC3 PCa cells and not in DU145, PCa2b, or LNCaP cells. PCa2b and PC3 cells were derived from bone metastases. PCa2b cells which are positive for the AR failed to demonstrate the expression of either cluster of differentiation 44 (CD44) or SOX2. Knockdown (KD) of AR in these cells did not affect the expression of either CD44 or SOX2. Conversely, PC3 cells, which are negative for AR, expressed both CD44 and SOX2. However, the expression of AR downregulated the expression of both CD44 and SOX2 in PC3 cells. CD44 regulates SOX2 expression as KD of CD44 and reduces SOX2 levels considerably. SOX2 KD attenuated not only the expression of SNAIL and SLUG but also the migration and tumorsphere formation in PC3 cells. Collectively, our findings underscore a novel role of CD44 signaling in the maintenance of stemness and progression of cancer through SOX2 in AR-independent PC3 cells. SOX2 has a role in the regulation of expression of SNAIL and SLUG. SOX2 could be a potential therapeutic target to thwart the progression of SOX2-positive cancer cells or recurrence of androgen-independent PCa., (© 2018 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.)

The process of sealing ring formation requires major actin filament reorganization. We previously demonstrated that an actin-bundling protein L-plastin has a role in the cross-linking of actin filaments into tight bundles and forms actin aggregates (denoted as nascent sealing zones). These nascent sealing zones mature into fully functional sealing rings. We have shown here that TNF-alpha signaling regulates the phosphorylation of serine-5 and -7 in L-plastin which increases the actin bundling capacity of L-plastin and hence the formation of nascent sealing zones in mouse osteoclasts. Using the TAT-mediated transduction method, we confirmed the role of L-plastin in nascent sealing zones formation at the early phase of the sealing ring assembly. Transduction of TAT-fused full-length L-plastin peptide significantly increases the number of nascent sealing zones and therefore sealing rings. But, transduction of amino-terminal L-plastin peptides consisting of the serine-5 and -7 reduces the formation of both nascent sealing zones and sealing rings. Therefore, bone resorption in vitro was reduced considerably. The decrease was associated with the selective inhibition of cellular L-plastin phosphorylation by the transduced peptides. Neither the formation of podosomes nor the migration was affected in these osteoclasts. Phosphorylation of L- plastin on serine 5 and -7 residues increases the F-actin bundling capacity. The significance of our studies stands on laying the groundwork for a better understanding of L-plastin as a potential regulator at the early phase of sealing ring formation and could be a new therapeutic target to treat bone loss., (Published by Elsevier Inc.)

Sealing ring formation is a requirement for osteoclast function. We have recently identified the role of an actin-bundling protein L-plastin in the assembly of nascent sealing zones (NSZs) at the early phase of sealing ring formation in osteoclasts. TNF-α signaling regulates this actin assembly by the phosphorylation of L-plastin on serine -5 and -7 residues at the amino-terminal end. These NSZs function as a core for integrin localization and coordinating integrin signaling required for maturation into fully functional sealing rings. Our goal is to elucidate the essential function of L-plastin phosphorylation in actin bundling, a process required for NSZs formation. The present study was undertaken to determine whether targeting serine phosphorylation of cellular L-plastin would be the appropriate approach to attenuate the formation of NSZs. Our approach is to use TAT-fused small molecular weight amino-terminal L-plastin peptides (10 amino acids) containing phospho- Ser-5 and Ser-7. We used peptides unsubstituted (P1) and substituted (P2- P4) at serine-to-alanine residues. Immunoblotting, actin staining, and dentine resorption analyses were done to determine cellular L-plastin phosphorylation, NSZ or sealing ring formation, and osteoclast function, respectively. Immunoblotting for bone formation markers, Alizarin red staining and alkaline phosphatase activity assay have been done to determine the effect of peptides on the mineralization process mediated by osteoblasts. Transduction of unsubstituted (P1) and substituted peptides at either Serine 5 or Serine 7 with Alanine (P3 and P4) demonstrated variable inhibitory effects on the phosphorylation of cellular L-plastin protein. Peptide P1 reduces the following processes substantially: 1) cellular L-plastin phosphorylation; 2) formation of nascent sealing zones and sealing rings; 3) bone resorption. Substitution of both Serine-5 and -7 with Alanine (P2) had no effects on the inhibitory activities described above. Furthermore, either the L-plastin (P1-P5) or (P6) control peptides had a little or no impact on the a) assembly/disassembly of podosomes and migration of osteoclasts; b) mineralization process mediated by osteoblasts in vitro. Small molecular weight peptidomimetics of L-plastin inhibits bone resorption by osteoclasts via attenuation of NSZ and sealing ring formation but not bone formation by osteoblasts in vitro. The L-plastin may be a valuable therapeutic target to treat and prevent diseases associated with bone loss without affecting bone formation., Competing Interests: All authors declare that they have no conflicts of interest.

CD44 is a cell surface adhesion receptor that is highly expressed in many cancers and regulates metastasis via recruitment of CD44 to the cell surface. Its interaction with appropriate extracellular matrix ligands promotes the migration and invasion processes involved in metastases. It was originally identified as a receptor for hyaluronan or hyaluronic acid and later to several other ligands including, osteopontin (OPN), collagens, and matrix metalloproteinases. CD44 has also been identified as a marker for stem cells of several types. Beside standard CD44 (sCD44), variant (vCD44) isoforms of CD44 have been shown to be created by alternate splicing of the mRNA in several cancer. Addition of new exons into the extracellular domain near the transmembrane of sCD44 increases the tendency for expressing larger size vCD44 isoforms. Expression of certain vCD44 isoforms was linked with progression and metastasis of cancer cells as well as patient prognosis. The expression of CD44 isoforms can be correlated with tumor subtypes and be a marker of cancer stem cells. CD44 cleavage, shedding, and elevated levels of soluble CD44 in the serum of patients is a marker of tumor burden and metastasis in several cancers including colon and gastric cancer. Recent observations have shown that CD44 intracellular domain (CD44-ICD) is related to the metastatic potential of breast cancer cells. However, the underlying mechanisms need further elucidation.

Background: That citrate is a major indispensible component of bone in humans and in all osteovertebrates has been known for about seventy-five years. Yet, its role and importance in the structure and function of bone and bone formation have remained unknown. However, recent studies have identified that citrate is a major and essential component of the apatite/collagen structure of bone; and that the biomechanical properties of bone (e.g., stability, strength, resistance to fracture) depend on the appropriate incorporation of citrate in the structure of bone. The osteoblasts have recently been identified as citrate-producing cells that provide the citrate that is incorporated in the apatite/collagen structure during osteogenesis. Little is known regarding the factors and mechanisms involved in the regulation of citrate that is incorporated along with mineralization during the process of bone formation. Because of the importance of BMP2 in the initiation of osteogenesis and the development of the osteoblasts, it is essential to determine its possible implication in the development of the citrate-producing capability of the osteoblasts (i.e., "citration") during the formation of mineralized bone nodules., Methods: The goal of this study was to determine if BMP2 promotes the development of citrate-producing osteoblasts for increased citrate incorporation in the formation of mineralized bone nodules. The study employed MC3T3 mesenchyme stem cell osteogenic differentiation in the presence and absence of BMP2., Results: The results showed that BMP2 treatment increased the osteogenic development of mineralized bone nodules. In addition, BMP2 increased osteoblast citrate production and incorporation in the mineralized bone nodule. This was accompanied by increased ZIP1 transporter, which is an essential genetic/metabolic event for citrate-producing cells., Conclusions: The results demonstrate, for the first time, that BMP2 facilitates the osteoblast "citration" process in concert with mineralization during bone formation; and provide confirmation of the important role of osteoblasts as specialized citrate-producing cells in the process of bone formation. However, it is essential to determine if these in vitro effects will occur in vivo in BMP2-implant induction of bone formation. "Citration" is essential for osteoinductive bone to represent the chemical, structural, and biomechanical properties of "normal" bone.

Osteopontin and MMP9 are implicated in angiogenesis and cancer progression. The objective of this study is to gain insight into the molecular mechanisms underlying angiogenesis, and to elucidate the role of osteopontin in this process. We report here that osteopontin/αvβ3 signaling pathway which involves ERK1/2 phosphorylation regulates the expression of VEGF. An inhibitor to MEK or curcumin significantly suppressed the phosphorylation of ERK1/2 and expression of VEGF. MMP9 knockdown reduces the secretion but not the expression of VEGF. Moreover, MMP9 knockdown increases the release of angiostatin, a key protein that suppresses angiogenesis. Conditioned media from PC3 cells treated with curcumin or MEK inhibitor inhibited tube formation in vitro in human microvascular endothelial cells. Similar inhibitory effect on tube formation was found with conditioned media collected from PC3 cells expressing mutant-osteopontin at integrin-binding site and knockdown of osteopontin or MMP9. We conclude that MMP9 activation is associated with angiogenesis via regulation of secretion of VEGF and angiostatin in PC3 cells. Curcumin is thus a potential drug for cancer treatment because it demonstrated anti-angiogenic and anti-invasive properties.

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.

Background: Bone loss and pathological fractures are common skeletal complications associated with androgen deprivation therapy and bone metastases in prostate cancer patients. We have previously demonstrated that prostate cancer cells secrete receptor activator of NF-kB ligand (RANKL), a protein essential for osteoclast differentiation and activation. However, the mechanism(s) by which RANKL is produced remains to be determined. The objective of this study is to gain insight into the molecular mechanisms controlling RANKL expression in metastatic prostate cancer cells., Results: We show here that phosphorylation of Smad 5 by integrin αvβ3 and RUNX2 by CD44 signaling, respectively, regulates RANKL expression in human-derived PC3 prostate cancer cells isolated from bone metastasis. We found that RUNX2 intranuclear targeting is mediated by phosphorylation of Smad 5. Indeed, Smad5 knock-down via RNA interference and inhibition of Smad 5 phosphorylation by an αv inhibitor reduced RUNX2 nuclear localization and RANKL expression. Similarly, knockdown of CD44 or RUNX2 attenuated the expression of RANKL. As a result, conditioned media from these cells failed to support osteoclast differentiation in vitro. Immunohistochemistry analysis of tissue microarray sections containing primary prostatic tumor (grade2-4) detected predominant localization of RUNX2 and phosphorylated Smad 5 in the nuclei. Immunoblotting analyses of nuclear lysates from prostate tumor tissue corroborate these observations., Conclusions: Collectively, we show that CD44 signaling regulates phosphorylation of RUNX2. Localization of RUNX2 in the nucleus requires phosphorylation of Smad-5 by integrin αvβ3 signaling. Our results suggest possible integration of two different pathways in the expression of RANKL. These observations imply a novel mechanistic insight into the role of these proteins in bone loss associated with bone metastases in patients with prostate cancer.

Background: Osteopontin (OPN) has been shown to play many roles in the progression of cancer. We have recently demonstrated the activation of Akt by OPN. Integrin-linked kinase and PI3-kinase are integral proteins in OPN/AKT pathway in PC3 cells. To investigate the role of the extracellular receptors in OPN signaling, we have examined the spatio-temporal regulation of CD44 and integrin αvβ3 receptor in OPN-induced Akt activation in PC3 cells., Results: Here, our studies demonstrate that OPN can activate Akt either through the αVβ3 integrin or the CD44 cell surface receptor. Members of the Mitogen Activated Protein Kinase (MAPK) family have been shown to be up-regulated in a variety of human cancers and have been implicated in the metastatic behavior. Our studies have demonstrated an increase in the phosphorylation of c-Raf at Ser259 and Ser338 in PC3 cells over-expressing OPN. This increase matches up with the Erk1/2 phosphorylation at Thr202/204 and activation. However, the inhibition of Akt activity augments the phosphorylation state of ERK1/2 to two to three fold with a concomitant reduction in the phosphorylation state of c-Raf at Ser259., Conclusions: Regulation c-Raf phosphorylation at Ser259 has a role in the anti-apoptotic pathways mediated by Akt or Raf/MEK/ERK proteins. OPN may have dual effects in the activation of Erk1/2. We propose this based on the observations that while OPN activates c-Raf and Erk1/2; it also acts to inhibit c-Raf and Erk1/2 activation through Akt pathway. Our observations suggest that the activation of c-Raf-ERK cascade may promote cell cycle arrest in prostate cancer cells and OPN signaling has a role in the anti-apoptotic mechanism.

The aim of this study is to identify the exact mechanism(s) by which cytoskeletal structures are modulated during bone resorption. In this study, we have shown the possible role of different actin-binding and signaling proteins in the regulation of sealing ring formation. Our analyses have demonstrated a significant increase in cortactin and a corresponding decrease in L-plastin protein levels in osteoclasts subjected to bone resorption for 18 h in the presence of RANKL, M-CSF, and native bone particles. Time-dependent changes in the localization of L-plastin (in actin aggregates) and cortactin (in the sealing ring) suggest that these proteins may be involved in the initial and maturation phases of sealing ring formation, respectively. siRNA to cortactin inhibits this maturation process but not the formation of actin aggregates. Osteoclasts treated as above but with TNF-α demonstrated very similar effects as observed with RANKL. Osteoclasts treated with a neutralizing antibody to TNF-α displayed podosome-like structures in the entire subsurface and at the periphery of osteoclast. It is possible that TNF-α and RANKL-mediated signaling may play a role in the early phase of sealing ring configuration (i.e. either in the disassembly of podosomes or formation of actin aggregates). Furthermore, osteoclasts treated with alendronate or αv reduced the formation of the sealing ring but not actin aggregates. The present study demonstrates a novel mechanistic link between L-plastin and cortactin in sealing ring formation. These results suggest that actin aggregates formed by L-plastin independent of integrin signaling function as a core in assembling signaling molecules (integrin αvβ3, Src, cortactin, etc.) involved in the maturation process.

Secretion of osteopontin (OPN) by cancer cells is a known mediator of tumorigenesis and cancer progression in both experimental and clinical studies. Our work demonstrates that OPN can activate Akt, an important step in cancer progression. Both ILK and PI3K are integral proteins in the OPN/Akt pathway, as inhibition of either kinase leads to a loss of OPN-mediated Akt activation. Subsequent to OPN-induced Akt activation, we observe inactivation of GSK-3beta, a regulator of beta-catenin. Osteopontin stimulation leads to an overall increase in beta-catenin protein levels with a resultant transfer of beta-catenin to the nucleus. Through the nuclear import of beta-catenin, OPN increases both the transcription and protein levels of MMP-7 and CD44, which are known TCF/LEF transcription targets. This work describes an important aspect of cancer progression induced by OPN.

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.)

PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

The present study demonstrated that invadopodia are associated with invasion by degradation of matrix in prostate cancer cells PC3. To find out the presence of invadopodia in PC3 cells, we performed a few comparative analyses with osteoclasts, which utilize podosomes for migration. Our investigations indeed demonstrated that invadopodia are comparable to podosomes in the localization of Wiskott-Aldrich syndrome protein (WASP)/matrix metalloproteinase-9 and the degradation of matrix. Invadopodia are different from podosomes in the localization of actin/vinculin, distribution during migration, and the mode of degradation of extracellular matrix. Invadopodia enable polarized invasion of PC3 cells into the gelatin matrix in a time-dependent manner. Gelatin degradation was confined within the periphery of the cell. Osteoclasts demonstrated directional migration with extensive degradation of matrix underneath and around the osteoclasts. A pathway of degradation of matrix representing a migratory track was observed due to the rearrangement of podosomes as rosettes or clusters at the leading edge. Reducing the matrix metalloproteinase-9 levels by RNA interference inhibited the degradation of matrix but not the formation of podosomes or invadopodia. Competition experiments with TAT-fused WASP peptides suggest that actin polymerization and formation of invadopodia involve the WASP-Arp2/3 complex pathway. Moreover, PC3 cells overexpressing osteopontin (OPN) displayed an increase in the number of invadopodia and gelatinolytic activity as compared with PC3 cells and PC3 cells expressing mutant OPN in integrin-binding domain and null for OPN. Thus, we conclude that OPN/integrin alphavbeta3 signaling participates in the process of migration and invasion of PC3 cells through regulating processes essential for the formation and function of invadopodia.

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, verpolin-central, 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.

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.)

The activities of different kinases have been correlated to the phosphorylation of Wiscott-Aldrich syndrome protein (WASP) by studies in multiple cell systems. The purpose of this study was to elucidate the regulatory mechanisms involved in WASP phosphorylation and the resulting sealing ring formation in osteoclasts. The phosphorylation state of WASP and WASP-interacting proteins was determined in osteoclasts treated with osteopontin or expressing either constitutively active or kinase-defective Src by adenovirus-mediated delivery. In vitro kinase analysis of WASP immunoprecipitates exhibited phosphorylation of c-Src, PYK2, WASP, protein-tyrosine phosphatase (PTP)-PEST, and Pro-Ser-Thr phosphatase-interacting protein (PSTPIP). Phosphorylation of these proteins was increased in osteopontin-treated and constitutively active Src-expressing osteoclasts. Pulldown analysis with glutathione S-transferase-fused proline-rich regions of PTP-PEST revealed coprecipitation of WASP, PYK2, c-Src, and PSTPIP proteins with the N-terminal region (amino acids 294-497) of PTP-PEST. Similarly, interaction of the same signaling proteins, as well as PTP-PEST, was observed with glutathione S-transferase-fused proline-rich regions of WASP. Furthermore, osteopontin stimulation or constitutively active Src expression resulted in serine phosphorylation and inhibition of WASP-associated PTP-PEST. The inhibition of PTP-PEST was accompanied by an increase in tyrosine phosphorylation of WASP and other associated signaling proteins. Experiments with an inhibitor to phosphatase and small interference RNA to PTP-PEST confirmed the involvement of PTP-PEST in sealing ring formation and bone resorption. WASP, which is identified in the sealing ring of resorbing osteoclasts, also demonstrates colocalization with c-Src, PYK2, PSTPIP, and PTP-PEST in immunostaining analyses. Our findings suggest that both tyrosine kinase(s) and the tyrosine phosphatase PTP-PEST coordinate the formation of the sealing ring and thus the bone-resorbing function of osteoclasts.

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.

The level of integrin alpha(v)beta3 and its ligand osteopontin (OPN) has been directly correlated to tumorigenicity of melanoma and other cancer cells. We have previously shown an increase in pp(60c-Src) kinase activity associated with integrin alpha(v)beta3 in melanoma cells (M21) treated with soluble OPN. pp(60c-Src) kinase activity was not observed in melanoma cells expressing alpha(v) that lacks the cytoplasmic domain (alpha(v)995). Results of the current study demonstrate that the amino acid sequence '995RPPQEEQERE1004' in the beta-turn of alpha(v) chain is required for the interaction of pp(60c-Src). Our results suggest that the beta-turn of alpha(v) chain may be indispensable for alpha(v)-associated signaling complex formation and outside-in signaling. To further analyze the alpha(v)beta3 signaling in melanoma cells, we over expressed OPN in M21 cells (M21/OPN). CD44 surface expression and MMP-2 activity in the conditioned medium were increased to a greater extent in M21/OPN cells as compared with M21 or alpha(v)995 cells. Also, M21/OPN cells exhibit increased motility, which is markedly reduced upon treatment with inhibitors to alpha(v) and MMP-2. Our findings suggest that the increase in MMP-2 activity is integrin-dependent as MMP-2 activity is reduced in cells treated with an inhibitor to alpha(v) or in alpha(v)995 cells expressing mutant alpha(v).

In osteoclasts, polyphosphoinositides such as phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5 trisphosphate (PI(3,4,5)P3) are produced in response to integrin alphavbeta3 signaling and they have a critical role in actin cytoskeleton remodeling. The levels of PI(4,5)P2 and PI(3,4,5)P3 are regulated by Rho GTPase through the activation of phosphatidylinositol 4-phosphate 5-kinase (PI4P-5 kinase) and phospatidylinositol 3-kinase (PI3 kinase), respectively. Interaction of PI(4,5)P2 with gelsolin and Wiscott-Aldrich syndrome protein (WASP) is critical for podosome assembly/disassembly and actin ring formation in osteoclasts. Interaction of PI(3,4,5)P3 with gelsolin functions in orchestrating the podosome signaling complex consisting of several key signaling molecules. Gelsolin deficiency has been shown to block podosome assembly and motility in mouse osteoclasts. However, these osteoclasts are able to form a WASP-containing actin ring and retain their resorptive function. The TAT-mediated delivery of gelsolin phosphoinositide-binding domains into osteoclasts resulted in production of podosome clusters and disruption of actin ring formation. Hence, these osteoclasts were hypomotile and less resorptive. Our observations suggest that both PI(4,5)P2 and PI(3,4,5)P3 are involved in regulating osteoclast functions through modulation of severing, capping, and nucleating functions of actin-binding proteins.

Actin ring formation is a prerequisite for osteoclast bone resorption. Although gelsolin null osteoclasts failed to exhibit podosomes, actin ring was observed in these osteoclasts. Wiscott-Aldrich syndrome protein (WASP) was observed in the actin ring of gelsolin null osteoclast. Osteoclasts stimulated with osteopontin simulated the effects of Rho and Cdc42 in phosphatidylinositol 4,5-bisphosphate (PIP2) association with WASP as well as formation of podosomes, peripheral microfilopodia-like structures, and actin ring. To explore the potential functions of Rho and Cdc42, TAT-mediated delivery of Rho proteins into osteoclasts was performed. Although Rho and Cdc42 are required for actin ring formation, transduction of either one of the proteins alone is insufficient for this process. Addition of osteopontin to osteoclasts transduced with Cdc42Val12 or transduction of osteoclasts with both RhoVal14 and Cdc42Val12 augments the formation of WASP-Arp2/3 complex and actin ring. Neomycin, an antibiotic, blocked the effects of osteopontin or TAT-RhoVal14 on PIP2 interaction with WASP. WASP distribution was found to be cytosolic in these osteoclasts. Depletion of WASP by short interfering RNA-mediated gene silencing blocked actin polymerization as well as actin ring formation in osteoclasts. These results suggest that Rho-mediated PIP2 interaction with WASP may contribute to the activation and membrane targeting of WASP. Subsequent interaction of Cdc42 and Arp2/3 with WASP may enhance cortical actin polymerization in the process of actin ring formation in osteoclasts.

Background: Gelsolin, an actin capping protein of osteoclast podosomes, has a unique function in regulating assembly and disassembly of the podosome actin filament. Previously, we have reported that osteopontin (OPN) binding to integrin alphavbeta3 increased the levels of gelsolin-associated polyphosphoinositides, podosome assembly/disassembly, and actin filament formation. The present study was undertaken to identify the possible role of polyphosphoinositides and phosphoinositides binding domains (PBDs) of gelsolin in the osteoclast cytoskeletal structural organization and osteoclast function., Results: Transduction of TAT/full-length gelsolin and PBDs containing gelsolin peptides into osteoclasts demonstrated: 1) F-actin enriched patches; 2) disruption of actin ring; 3) an increase in the association polyphosphoinositides (PPIs) with the transduced peptides containing PBDs. The above-mentioned effects were more pronounced with gelsolin peptide containing 2 tandem repeats of PBDs (PBD (2)). Binding of PPIs to the transduced peptides has resulted in reduced levels of PPIs association with the endogenous gelsolin, and thereby disrupted the actin remodeling processes in terms of podosome organization in the clear zone area and actin ring formation. These peptides also exhibited a dominant negative effect in the formation of WASP-Arp2/3 complex indicating the role of phosphoinositides in WASP activation. The TAT-PBD gelsolin peptides transduced osteoclasts are functionally defective in terms of motility and bone resorption., Conclusions: Taken together, these data demonstrate that transduction of PBD gelsolin peptides into osteoclasts produced a dominant negative effect on actin assembly, motility, and bone resorption. These findings indicate that phosphoinositide-mediated signaling mechanisms regulate osteoclast cytoskeleton, podosome assembly/disassembly, actin ring formation and bone resorption activity of osteoclasts.

Osteoclasts from osteopontin-deficient mice exhibit decreased CD44 surface expression [corrected]. Osteopontin (OPN)/alphavbeta3 generated Rho signaling pathway is required for the surface expression of CD44. In this work we show the Rho effector, Rho kinase (ROK-alpha), to be a potent activator of CD44 surface expression. ROK-alpha activation was associated with autophosphorylation, leading to its translocation to the plasma membrane, as well as its association with CD44. ROK-alpha promoted CD44 surface expression through phosphorylation of CD44 and ezrin-radixin-moesin (ERM) proteins and CD44.ERM.actin complex formation. Osteoclasts from OPN-/- mice exhibited an approximately 55-60% decrease in basal level ROK-alpha phosphorylation as compared with wild type osteoclasts. Furthermore, RhoVal-14 transduction was only partially effective in stimulating ROK-alpha/CD44 phosphorylation, as well as CD44 surface expression, in these osteoclasts. Studies on the inhibition of Rho by C3 transferase or ROK-alpha by the specific inhibitor, Y-27632, showed a decrease in the phosphorylation mediated by ROK-alpha and CD44 surface expression. Neutralizing antibodies to alphav, beta3, or CD44 inhibited the migration and bone resorption of wild type osteoclasts. However, only anti-alphav or -beta3 antibodies blocked OPN-induced phosphorylation of ROK-alpha, CD44, and the ERM proteins. Our results strongly suggest a role for ROK-alpha in alphavbeta3-mediated Rho signaling, which is required for the phosphorylation events and CD44 surface expression. The functional deficiencies in the Rho effector(s) because of the lack of OPN were associated with decreased CD44 surface expression and hypomotility in the OPN-/- osteoclasts. Finally, we find that cooperativity exists between alphavbeta3 and CD44 for osteoclast motility and bone resorption.

We previously demonstrated that inhibition of Na-dependent phosphate (P(i)) transport in osteoclasts led to reduced ATP levels and diminished bone resorption. These findings suggested that Na/P(i) cotransporters in the osteoclast plasma membrane provide P(i) for ATP synthesis and that the osteoclast may utilize part of the P(i) released from bone resorption for this purpose. The present study was undertaken to define the cellular localization of Na/P(i) cotransporters in the mouse osteoclast and to identify the proteins with which they interact. Using glutathione S-transferase (GST) fusion constructs, we demonstrate that the type IIa Na/P(i) cotransporter (Npt2a) in osteoclast lysates interacts with the Na/H exchanger regulatory factor, NHERF-1, a PDZ protein that is essential for the regulation of various membrane transporters. In addition, NHERF-1 in osteoclast lysates interacts with Npt2a in spite of deletion of a putative PDZ-binding domain within the carboxy terminus of Npt2a. In contrast, deletion of the carboxy-terminal TRL amino acid motif of Npt2a significantly reduced its interaction with NHERF-1 in kidney lysates. Studies in osteoclasts transfected with green fluorescent protein-Npt2a constructs indicated that Npt2a colocalizes with NHERF-1 and actin at or near the plasma membrane of the osteoclast and associates with ezrin, a linker protein associated with the actin cytoskeleton, likely via NHERF-1. Furthermore, we demonstrate by RT/PCR of osteoclast RNA and in situ hybridization that the type III Na/P(i) cotransporter, PiT-1, is also expressed in mouse osteoclasts. To examine the cellular distribution of PiT-1, we infected mouse osteoclasts with a retroviral vector encoding PiT-1 fused to an epitope tag. PiT-1 colocalizes with actin and is present on the basolateral membrane of the polarized osteoclast, similar to that previously reported for Npt2a. Taken together, our data suggest that association of Npt2a with NHERF-1, ezrin, and actin, and of PiT-1 with actin, may be responsible for membrane sorting and regulation of these Na/P(i) cotransporters in the osteoclast.

In the studies reported here we demonstrate that osteopontin is secreted from the basolateral surfaces of osteoclasts where it binds to the avb3-integrin, suggesting that it may be an autocrine factor. Osteopontin stimulation of osteoclasts produced changes in cell shape by causing disruption of peripheral podosome structures and formation of actin filaments at the leading edge of the migrating osteoclasts. The latter was part of the assumption of a motile phenotype prior to cells reforming peripheral ring type podosome containing clear zones. It is well established in our laboratory as well as in others that osteopontin stimulated osteoclast motility and bone resorption. The effect of osteopontin was mimicked by RGD containing peptides and blocked by a avb3 antibody, demonstrating that signals generated by integrin ligation contributed to the actions of osteopontin. In addition, the migratory effects of osteopontin on osteoclasts were also mediated through CD44 receptors since blocking antibodies to CD44 blocked stimulation of motility. Our data strongly suggest that osteopontin is an osteoclast autocrine motility factor binding to avb3 and CD44 during stimulation of osteoclast migration.

Osteopontin (OPN) was expressed in murine wild-type osteoclasts, localized to the basolateral, clear zone, and ruffled border membranes, and deposited in the resorption pits during bone resorption. The lack of OPN secretion into the resorption bay of avian osteoclasts may be a component of their functional resorption deficiency in vitro. Osteoclasts deficient in OPN were hypomotile and exhibited decreased capacity for bone resorption in vitro. OPN stimulated CD44 expression on the osteoclast surface, and CD44 was shown to be required for osteoclast motility and bone resorption. Exogenous addition of OPN to OPN-/- osteoclasts increased the surface expression of CD44, and it rescued osteoclast motility due to activation of the alpha(v)beta(3) integrin. Exogenous OPN only partially restored bone resorption because addition of OPN failed to produce OPN secretion into resorption bays as seen in wild-type osteoclasts. As expected with these in vitro findings of osteoclast dysfunction, a bone phenotype, heretofore unappreciated, was characterized in OPN-deficient mice. Delayed bone resorption in metaphyseal trabeculae and diminished eroded perimeters despite an increase in osteoclast number were observed in histomorphometric measurements of tibiae isolated from OPN-deficient mice. The histomorphometric findings correlated with an increase in bone rigidity and moment of inertia revealed by load-to-failure testing of femurs. These findings demonstrate the role of OPN in osteoclast function and the requirement for OPN as an osteoclast autocrine factor during bone remodeling.

Podosomes are adhesion structures in osteoclasts and are structurally related to focal adhesions mediating cell motility during bone resorption. Here we show that gelsolin coprecipitates some of the focal adhesion-associated proteins such as c-Src, phosphoinositide 3-kinase (PI3K), p130(Cas), focal adhesion kinase, integrin alpha(v)beta(3), vinculin, talin, and paxillin. These proteins were inducibly tyrosine-phosphorylated in response to integrin activation by osteopontin. Previous studies have defined unique biochemical properties of gelsolin related to phosphatidylinositol 3,4,5-trisphosphate in osteoclast podosomes, and here we demonstrate phosphatidylinositol 3,4,5-trisphosphate/gelsolin function in mediating organization of the podosome signaling complex. Overlay and GST pull-down assays demonstrated strong phosphatidylinositol 3,4,5-trisphosphate-PI3K interactions based on the Src homology 2 domains of PI3K. Furthermore, lipid extraction of lysates from activated osteoclasts eliminated interaction between gelsolin, c-Src, PI3K, and focal adhesion kinase despite equal amounts of gelsolin in both the lipid-extracted and unextracted experiment. The cytoplasmic protein tyrosine phosphatase (PTP)-proline-glutamic acid-serine-threonine amino acid sequences (PEST) was also found to be associated with gelsolin in osteoclast podosomes and with stimulation of alpha(v)beta(3)-regulated phosphorylation of PTP-PEST. We conclude that gelsolin plays a key role in recruitment of signaling proteins to the plasma membrane through phospholipid-protein interactions and by regulation of their phosphorylation status through its association with PTP-PEST. Because both gelsolin deficiency and PI3K inhibition impair bone resorption, we conclude that phosphatidylinositol 3,4,5-trisphosphate-based protein interactions are critical for osteoclast function.

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.

Although osteopontin (OP) has been shown to play a role in bone mineralization and to mediate bone cell adhesion, its function in other tissues is not yet known. The sequences of OP from seven species have been reported; some of the sequences that are conserved in all seven species and their functions are mentioned. The biochemical structure of OP and the functional properties of its motifs make OP a strong candidate for regulating mineralization and/or mediating local cell dynamics. In addition to its role in mineralization, OP has also been shown to promote migration of smooth muscle cells and macrophages. OP expression is high in many tumors, and it correlates with the metastatic potential in some instances. Abundant OP has also been found in human tissue specimens from patients with clinical tuberculosis and in other granulomatous diseases. Experimental approaches in the authors' laboratory have focused on the role of OP as an autocrine motility factor in osteoclasts and human melanoma cell lines; their results suggest that posttranslational modification (phosphorylation) of OP is important in its biological functions.