College of Podiatric Medicine

Approximately 30% of patients with renal cell carcinoma (RCC) develop bone metastasis, which is characterized by extensive osteolysis leading to severe bone pain and pathologic fracture. Although the mechanism of RCC-induced osteolysis is unknown, studies of bone metastasis have shown that tumor-induced changes in bone remodeling are likely mediated by alterations in the bone microenvironment. Here, we report the discovery of a novel osteoclast stimulatory factor secreted by RCC bone metastasis (RBM). Through microarray analysis, we found expression of the chemokine, macrophage inflammatory protein-1D (MIP-1D), to be increased in RBM versus patient-matched primary RCC tissues and confirmed this finding by quantitative reverse transcription-PCR (qRT-PCR) and ELISA (P < 0.05). Furthermore, MIP-1D expression in RBM tissues was significantly (P < 0.001) higher than in human bone marrow, suggesting a potential alteration of the bone microenvironment. The receptors for MIP-1D, CCR1 and CCR3, were expressed in both osteoclast precursors and mature, bone-resorbing osteoclasts as shown by qRT-PCR and Western analysis. In functional studies, MIP-1D stimulated chemotaxis of two osteoclast precursor cell types: murine bone marrow mononuclear cells (BM-MNC) and RAW 264.7 cells. Furthermore, MIP-1D treatment of murine calvaria caused increased bone resorption as determined by measurement of released calcium. Correspondingly, MIP-1D significantly enhanced osteoclast formation and activity in response to RANKL in both BM-MNC and RAW 264.7 cells. Taken together, these data suggest that MIP-1D expression is increased in RBM relative to RCC and bone marrow, and may promote RBM-induced osteolysis by stimulating the recruitment and differentiation of osteoclast precursors into mature osteoclasts.

Osteoactivin (OA) is a novel protein identified by mRl'lA differential display using bone from osteopetrotic \'ersus normal rats, Bioinformatic analysis showed that OA eDNA has an open reading frame of 1716 bp encoding a protein of 572 aa, the first 21 aa constitute a signal peptide. OA sequence analysis also demonstrated 13 putative N-glycosylation sites suggestive of a heavily glycosylated protein, In this study, we localized 0,-\ protein in primary osteoblast culture by immunofluorescent staining and \"/estern blot analysis, Primary osteoblast cultures pass through three stages: proliferation from day 1 to 7, matrL"X formation from day 7 to 1-1, and matrix mineralization from day 14 to 21. OA protein was detected at all stages examined, with maximal expression at .3 "'eeks "'hen osteoblasts are terminally differentiated. Using the Chariot transfection reagent as a vehicle to deli\'er anti-OA antibody into the cells, we demonstrated that anti-OA antibodv significantly inhibited osteoblast differentiation markers, including alkaline phosphatase activity, nodule formation, osteocalcin production, and calcium deposition, without affecting cell proliferation or viability. These data suggest that OA is an osteoblast-related protein that plays an important role in the regulation of osteoblast differentiation and function.

We previously identified osteoactivin (OA) as a novel secreted osteogenic factor with high expression in developing long bones and calvaria, and that stimulates osteoblast differentiation and matrix mineralization in vitro. In this study, we report on OA mRNA and protein expression in intact long bone and growth plate, and in fracture calluses collected at several time points up to 21 days post-fracture (PF). OA mRNA and protein were highly expressed in osteoblasts localized in the metaphysis of intact tibia, and in hypertrophic chondrocytes localized in growth plate, findings assessed by in situ hybridization and immunohistochemistry, respectively. Using a rat fracture model, Northern blot analysis showed that expression of OA mRNA was significantly higher in day-3 and day-10 PF calluses than in intact rat femurs. Using in situ hybridization, we examined OA mRNA expression during fracture healing and found that OA was temporally regulated, with positive signals seen as early as day-3 PF, reaching a maximal intensity at day-10 PF, and finally declining at day-21 PF. At day-5 PF, which correlates with chondrogenesis, OA mRNA levels were significantly higher in the soft callus than in intact femurs. Similarly, we detected high OA protein immunoexpression throughout the reparative phase of the hard callus compared to intact femurs. Interestingly, the secreted OA protein was also detected within the newly made cartilage matrix and osteoid tissue. Taken together, these results suggest the possibility that OA plays an important role in bone formation and serves as a positive regulator of fracture healing.

Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. We next examined whether the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a significant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Our findings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function.

A rare disease afflicts less than 200,000 individuals, according to the National Organization for Rare Diseases (NORD) of the United States. Over 6,000 rare disorders affect approximately 1 in 10 Americans. Rare genetic bone disorders remain the major causes of disability in US patients. These rare bone disorders also represent a therapeutic challenge for clinicians, due to lack of understanding of underlying mechanisms. This systematic review explored current literature on therapeutic directions for the following rare genetic bone disorders: fibrous dysplasia, Gorham-Stout syndrome, fibrodysplasia ossificans progressiva, melorheostosis, multiple hereditary exostosis, osteogenesis imperfecta, craniometaphyseal dysplasia, achondroplasia, and hypophosphatasia. The disease mechanisms of Gorham-Stout disease, melorheostosis, and multiple hereditary exostosis are not fully elucidated. Inhibitors of the ACVR1/ALK2 pathway may serve as possible therapeutic intervention for FOP. The use of ...

Osteoactivin (OA), a transmembrane protein, has recently emerged as a vital glycoprotein for the differentiation and function of bone forming osteoblasts. OA expression has been shown to increase during osteoblast development with maximal expression during the final stages of differentiation. Recent studies showed that OA is also expressed by osteoclasts and plays a role in their differentiation and function. In this study, we used a transgenic mouse model over-expressing OA under the control of CMV promoter (OATg) to determine the mechanisms by which OA contributes to bone formation and remodeling in vivo. Western blot analysis showed a three-fold increase in OA expression in OATg compared to wild-type (WT) osteoblasts. Micro-CT analysis of femurs from 12 week-old OATg showed increased trabecular bone volume (BV/TV) in OATg mice compared to WT mice. Given that OA is also over-expressed in osteoclasts in OATg mice, we evaluated bone resorption by histomophometry and ELISA. Serum RAN...

Introduction: Our laboratory previously showed that osteoactivin (OA) is a novel, osteoblast-related glycoprotein that plays a role in osteoblast differentiation and function. The purpose of this study was to examine the regulation of OA expression by BMP-2 and the role OA plays as a downstream mediator of BMP-2 effects in osteoblast function. Materials and Methods: Using primary osteoblast cultures, we tested different doses of BMP-2 on the regulation of OA expression during osteoblast development. Regulation of OA expression by BMP-2 was examined using real time-PCR and Western blot analysis, in cultures treated with 50 ng/ml of BMP-2 and terminated after 7, 14 and 21 days. To test whether Smad-1 signaling is responsible for BMP-2 regulation of OA expression, osteoblast cultures were transfected with Smad1 siRNA, treated with 50 ng/ml of BMP-2 and analyzed by Western blot. To examine the role of OA as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization, osteoblast cultures were transfected with OA antisense oligonucleotides and treated with 50 ng/ml of BMP-2 and analyzed by Western blot. Alkaline phosphatase activity, module formation and matrix mineralization were also measured. Results: BMP-2 treatment increased OA mRNA and protein expression in a dose-dependent manner and this upregulation was blocked in Smad1 siRNA transfected cultures. Cultures transfected with OA antisense oligonucleotides and treated with BMP-2 showed a reduction of OA expression associated with a signifi cant reduction in early and late differentiation markers induced by BMP-2. Therefore, OA acts, at least in part, as a downstream mediator of BMP-2 effects on osteoblast differentiation and matrix mineralization. Conclusions: Our fi ndings suggest that BMP-2 regulates OA expression through the Smad1 signaling pathway. Our data also emphasize that OA protein acts as a downstream mediator of BMP-2 effects on osteoblast differentiation and function.

Presentation Number: SA078 Osteoactivin (OA) is a novel osteoblast-related glycoprotein that plays an important role in osteoblast differentiation and function. TGF-β1, BMP-2 and calcitriol are important osteotrophic factors that regulate osteoblast proliferation and differentiation. In this study we first examined the effects TGF-β1, BMP-2 and calcitriol on OA expression. Primary osteoblast cultures treated with TGF-β1 (5 ng/ml), BMP-2 (50 ng/ml) and calcitriol (10-8 M) were terminated at different time points and examined for OA expression using Western blot analysis. TGF-β1 treatment showed minimal effect on OA expression, while BMP-2 and calcitriol showed an up-regulate of OA expression especially during the matrix maturation and mineralization stages in culture. BMP-2 maximally induced OA expression (5-fold) during the matrix mineralization stage in culture. Next, we treated primary osteoblast cultures with different doses of BMP-2 (10-200 ng/ml) for 24 hours under serum free conditions. OA protein expression levels reached a maximum at 50 ng/ml. In order to examine whether OA is a downstream mediator of BMP-2 effect on osteoblast differentiation, OA anti-sense oligonucleotide was used to inhibit OA expression in cultures treated with BMP-2. Primary osteoblast cultures treated with BMP-2 (50 ng/ml) stimulated nodule formation and matrix mineralization when compared to non-treated cultures. Cultures treated with OA anti-sense oligonucleotide (0.5 μM) showed a reduction of OA expression associated with decreased nodule formation and calcium deposition. BMP-2 (50 ng/ml) treatment rescued OA expression associated with an increase in nodule formation and matrix mineralization, when compared to OA anti-sense treated cultures. BMP-2 is known to stimulate osteoblast differentiation mainly through the phospho-Smad 1, 5 and 8 pathway. Cultures treated with BMP-2 showed an up-regulation of phospho-Smad 1 when compared to non-treated cultures. In OA anti-sense treated cultures, phospho-Smad 1 was down-regulated when compared to non-treated cultures, while BMP-2 rescued phospho-Smad 1 expression. These data suggest that OA might be a downstream mediator of BMP-2 in osteoblasts. Future studies are designed to use phospho-Smad 1 inhibitors, to examine whether OA expression is altered. We conclude that OA is regulated by different osteotrophic factors and may act as downstream mediator of BMP-2 effects on osteoblast differentiation.

Osteoactivin (OA)/gpnmb is a type I transmembrane protein (576 amino acids) containing an extracellular domain with an RGD motif, and intracellular domain with an immunotyrosine inhibitory motif (ITIM) and has been linked to osteoblast and osteoclast differentiation and function. Our laboratory previously showed that OA induces osteoblast differentiation and function in vitro and bone formation in vivo. In this study, we investigated the effects of recombinant OA (rOA) on osteoblast differentiation and signaling. First, we examined the effects of rOA treatment on osteoblast proliferation and survival. MC3T3-E1 osteoblast-like cells or primary osteoblast cultures were treated with different doses of rOA and proliferation and survival were evaluated at different time points. rOA treatment had no significant effects on proliferation and survival. Next, we examined the effects of rOA on markers of osteoblast differentiation. rOA treated cells showed a significant increase in Runx2, Oste...

Farnesoid X receptor (FXR), also known as NR1H4 (nuclear receptor subfamily 1, group H, member 4) is a bile acid receptor key in maintaining bile acids and cholesterol homeostasis. FXR is highly expressed in liver and small intestine, where bile acids are synthesized and secreted, respectively. As such, FXR plays an important role in fat metabolism. It has also been found that FXR is an upstream regulator of Fibroblast Growth Factor 21 (FGF-21), which has recently been shown to contribute to bone loss through the inhibition of osteoblastogenesis and stimulation of adipogenesis. While it has long been known that FXR is expressed in liver, intestine, kidney, and adipose tissue, it’s expression and function in bone are not well understood. In this study, we examined the expression of FXR in bone and demonstrate its key regulatory role in osteoblast and osteoclast differentiation and function. We performed qPCR on mRNA collected from long bone and calvaria of WT mice, demonstrating the ...

Background: This study elucidates exposure-response relationships between performance of repetitive tasks, grip strength declines, and fibrogenic-related protein changes in muscles, and their link to inflammation. Specifically, we examined forearm flexor digitorum muscles for changes in connective tissue growth factor (CTGF; a matrix protein associated with fibrosis), collagen type I (Col1; a matrix component), and transforming growth factor beta 1 (TGFB1; an upstream modulator of CTGF and collagen), in rats performing one of two repetitive tasks, with or without anti-inflammatory drugs. Methodology/Results: To examine the roles of force versus repetition, rats performed either a high repetition negligible force food retrieval task (HRNF), or a high repetition high force handle-pulling task (HRHF), for up to 9 weeks, with results compared to trained only (TR-NF or TR-HF) and normal control rats. Grip strength declined with both tasks, with the greatest declines in 9-week HRHF rats. Quantitative PCR (qPCR) analyses of HRNF muscles showed increased expression of Col1 in weeks 3-9, and CTGF in weeks 6 and 9. Immunohistochemistry confirmed PCR results, and also showed greater increases of CTGF and collagen matrix in 9-week HRHF rats than 9-week HRNF rats. ELISA, and immunohistochemistry revealed greater increases of TGFB1 in TR-HF and 6-week HRHF, compared to 6-week HRNF rats. To examine the role of inflammation, results from 6-week HRHF rats were compared to rats receiving ibuprofen or anti-TNF-a treatment in HRHF weeks 4-6. Both treatments attenuated HRHF-induced increases in CTGF and fibrosis by 6 weeks of task performance. Ibuprofen attenuated TGFB1 increases and grip strength declines, matching our prior results with anti-TNFa. Conclusions/Significance: Performance of highly repetitive tasks was associated with force-dependent declines in grip strength and increased fibrogenic-related proteins in flexor digitorum muscles. These changes were attenuated, at least short-term, by anti-inflammatory treatments.

Connective tissue growth factor (CTGF) is a secreted, extracellular matrix-associated protein that regulates diverse cellular functions in different cell types. CTGF gene belongs to a larger CCN gene family that also includes Cyr61 and NOV. It modulates many cellular functions, including proliferation, migration, adhesion, and extracellular matrix production, and it is involved in many biological and pathological processes. CTGF has special importance in skeletal development. During Meckel&#39;s cartilage development, CTGF acts as a down-stream molecule of TGFbeta to stimulate cell-cell interactions and the expression of condensation-associated genes. CTGF promotes endochondral ossification and articular cartilage regeneration. During the healing of experimental bone fracture, CTGF was expressed in periosteal cells and hypertrophic chondrocytes. It promotes the proliferation of chondrocytes and osteoblasts. CTGF is a down-stream mediator for prostaglandin E2 (PGE2) in osteoblast-ind...

We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb+). In these D2J mice, micro-CT and histomorphometric analyses revealed increased cortical thickness, while total porosity and eroded surface were significantly reduced in D2J mice compared to wild-type controls, and these results were corroborated by lower serum levels of CTX-1. Contrary to these observations, and counterintuitively, temporal gene expression analyses supported up-regulated osteoclastogenesis in D2J mice, and increased osteoclast differentiation rates ex vivo, marked by increased number and size. The finding that MAPK was activated in early differentiating and mature D2J osteoclasts, and survival of D2J osteoclasts was enhanced and mediated by activation of the AKT-GSK3β pathway supports this observation. Furthermore, this was abro...

We have previously identified osteoactivin (OA), encoded by Gpnmb, as an osteogenic factor that stimulates osteoblast differentiation in vitro. To elucidate the importance of OA in osteogenesis, we characterized the skeletal phenotype of a mouse model, DBA/2J (D2J) with a loss-of-function mutation in Gpnmb. Microtomography of D2J mice showed decreased trabecular mass, compared with wild-type mice [DBA/2J-Gpnmb+/SjJ (D2J/Gpnmb+)]. Serum analysis showed decreases in OA and in the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblast numbers were increased, compared with D2J/Gpnmb+ mice. We then examined the ability of osteoblasts in D2J mice to differentiate in culture; decreased differentiation and function in D2J mice were marked by decreased alkaline phosphatase activity and matrix mineralization. Gene expression analyses showed decreased differentiation marker expression in ...

Our lab was the first to report a key role of osteoactivin (OA/GPNMB) in osteoblast differentiation and bone formation. We reported that OA/GPNMB is expressed during osteoblast differentiation in vitro. We also showed that injection of recombinant (r) OA into the femur medullary space stimulated osteogenesis in vivo. Recent report showed that OA/GPNMB is expressed in osteoclasts (OC). In this study, we were interested to examine the functional role of OA/GPNMB during osteoclastogenesis. Immunofluorescent localization of OA/GPNMB in multinucleated osteoclasts (mOC) (≥ 3 nuclei) showed co-localization of OA/GPNMB protein with actin ring and β3 integrin. In addition, we tested the effects of rOA on osteoclastogenesis using bone marrow-derived hematopoietic cells. rOA protein enhanced RANKL-mediated osteoclastogenesis in a dose dependent manner measured by TRAP activity, total number and size of OC. Our laboratory obtained a mouse model with a natural mutation in the OA/GPNMB gene encod...

Osteoactivin/Glycoprotein nmb (OA/gpnmb) is a transmembrane glycoprotein. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. We have previously shown that OA/gpnmb expression in osteoblasts is regulated by BMP-2 through the Smad-1 signaling pathway. In this study, we used a mouse model with a naturally occurring mutation in the OA/gpnmb gene resulting from a premature stop codon that leads to the production of a truncated OA/gpnmb protein with no biological functions. OA/gpnmb mutant mice develop osteoporosis with age when compared to normal, wild type (WT) littermates. Histological and micro-CT measurements of femurs in mutant mice revealed a decrease in bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness in OA/gpnmb mutants compared to WT controls. Primary osteoblasts were generated from newborn OA/gpnmb and WT mice and examined for thei...

Intellectual disability (ID) is a serious disorder of the central nervous system with a prevalence of 1-3% in the general population. Recent studies identified different nonsense mutations in the trafficking protein particle complex (TRAPPC9) gene in consanguineous and nonconsanguineous families. These patients exhibit severe bone phenotypes such as short stature, polydactily, craniofacial deformity and microcephaly. TRAPPC9 is known to bind NFkB inducing kinase (NIK) and IkB kinasbeta (IKKbeta) and enhances cytokine-induced NFkB activation by increasing the kinase activities of IKK2 (IKKbeta) and NIK. It thus plays a role in the canonical and non-canonical NFkB signaling. Recent studies have identified NF-kB as a major negative regulator of osteoblast proliferation and differentiation that binds and suppresses Runx2 activation. Bone morphogenetic proteins (BMPs) are a group of growth factors and cytokines capable of inducing bone and cartilage formation. Two biochemical pathways ar...

Introduction: Osteoactivin (OA/Gpnmb) was first identified by differential gene display in mutant osteopetrotic rats (1). The novelty of OA/Gpnmb in bone regeneration has been characterized and reported by our group (2,3). Recent study demonstrated that OA/Gpnmb is highly expressed by osteoclasts in vitro (4). The same group showed that targeted overexpression of OA/Gpnmb in cells of the osteoclastic lineage demonstrated the importance of OA/Gpnmb in osteoclast activity and bone resorption (5). To specify the critical role and mechanism of action of OA/Gpnmb in osteoclastogenesis, we characterized the osteoclastic phenotype in a mouse model with natural mutation in the OA/Gpnmb gene. We hypothesized that mutation of OA/Gpnmb increases osteoclast formation and decreases its activity. Methods: For the purpose of the study, we used a genetically modified D2J mouse model at age of 8-week. D2J mice are mutant for OA/Gpnmb, characterized with nonsense mutation with early stop codon, gener...

Osteoactivin (OA) is a type I transmembrane protein (576 amino acids) that stimulates osteoblast differentiation and function. Our lab has previously shown that there are two isoforms of Osteoactivin, transmembrane and secreted forms. In addition, we also showed that overexpression of Osteoactivin in MC3T3-E1 increases osteoblast differentiation and matrix mineralization. In this study, we tested the effects of the recombinant Osteoactivin (rOA) on osteoblast function and determined the signaling pathways responsible for OA function in osteoblasts . MC3T3-E1 osteoblast-like cells and primary mouse osteoblasts were used in this study. Cells were treated with different concentrations of rOA and cell proliferation and survival were evaluated using CyQuant and MTT assays, respectively. rOA had no significant effects on cell proliferation or survival. Next, we examined the effects of OA treatment on osteoblast differentiation and function. Osteoblast treated with rOA showed significant i...