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 to that in wild-type mice [DBA/2J-Gpnmb(+)/SjJ (D2J/Gpnmb(+))]. Serum analysis showed decreases in OA and the bone-formation markers alkaline phosphatase and osteocalcin in D2J mice. Although D2J mice showed decreased osteoid and mineralization surfaces, their osteoblasts were increased in number, compared to D2J/Gpnmb(+) mice. We then examined the ability of D2J osteoblasts to differentiate in culture, where their differentiation and function were decreased, as evidenced by low alkaline phosphatase activity and matrix mineralization. Quantitative RT-PCR analyses confirmed the decreased expression of differentiation markers in D2J osteoblasts. In vitro, D2J osteoblasts proliferated and survived significantly less, compared to D2J/Gpnmb(+) osteoblasts. Next, we investigated whether mutant OA protein induces endoplasmic reticulum stress in D2J osteoblasts. Neither endoplasmic reticulum stress markers nor endoplasmic reticulum ultrastructure were altered in D2J osteoblasts. Finally, we assessed underlying mechanisms that might alter proliferation of D2J osteoblasts. Interestingly, TGF-β receptors and Smad-2/3 phosphorylation were up-regulated in D2J osteoblasts, suggesting that OA contributes to TGF-β signaling. These data confirm the anabolic role of OA in postnatal bone formation.

Current osteoinductive protein therapy utilizes bolus administration of large doses of bone morphogenetic proteins (BMPs), which is costly, and may not replicate normal bone healing. The limited in vivo biologic activity of BMPs requires the investigation of growth factors that may enhance this activity. In this study, we utilized the C3H10T1/2 murine mesenchymal stem cell line to test the hypotheses that osteoactivin (OA) has comparable osteoinductive effects to bone morphogenetic protein-2 (BMP-2), and that sustained administration of either growth factor would result in increased osteoblastic differentiation as compared to bolus administration. Sustained release biodegradable hydrogels were designed, and C3H10T1/2 cells were grown on hydrogels loaded with BMP-2 or OA. Controls were grown on unloaded hydrogels, and positive controls were exposed to bolus growth factor administration. Cells were harvested at several time points to assess osteoblastic differentiation. Alkaline phosphatase (ALP) staining and activity, and gene expression of ALP and osteocalcin were assessed. Treatment with OA or BMP-2 resulted in comparable effects on osteoblastic marker expression. However, cells grown on hydrogels demonstrated osteoblastic differentiation that was not as robust as cells treated with bolus administration. This study shows that OA has comparable effects to BMP-2 on osteoblastic differentiation using both bolus administration and continuous release, and that bolus administration of OA has a more profound effect than administration using hydrogels for sustained release. This study will lead to a better understanding of appropriate delivery methods of osteogenic growth factors like OA for repair of fractures and segmental bone defects.

Background Phosphatidylcholine Transfer Protein (PCTP) regulates the intermembrane transfer of phosphatidylcholine (PC). Higher platelet PCTP expression is associated with increased platelet responses upon activation of protease-activated receptor 4 (PAR4) thrombin receptors noted in black subjects as compared to white subjects. Little is known regarding regulation of platelet PCTP. Haplodeficiency of RUNX1, a major hematopoietic transcription factor, is associated with thrombocytopenia and impaired platelet responses upon activation. Platelet expression profiling of a patient described by us with a RUNX1 loss-of-function mutation revealed a 10-fold downregulation of PCTP gene compared with healthy controls. Methods We pursued the hypothesis that PCTP is regulated by RUNX1 and that PCTP expression is correlated with cardiovascular events. We studied RUNX1 binding to PCTP promoter using DNA-protein binding studies and human erythroleukemia (HEL) cells, and promoter activity using luciferase reporter studies. We assessed the relationship between RUNX1 and PCTP in peripheral blood RNA and PCTP and death or myocardial infarction (MI) in two separate patient cohorts (587 total patients) with cardiovascular disease. Results Platelet PCTP protein in the patient was reduced by ~50%. DNA-protein binding studies showed RUNX1 binding to consensus sites in ~1 kB of PCTP promoter. PCTP expression was increased with RUNX1 overexpression and reduced with RUNX1 knockdown in HEL cells, indicating that PCTP is regulated by RUNX1. Studies in two cohorts of patients showed that RUNX1 expression in blood correlated with PCTP gene expression; PCTP expression was higher in black compared to white subjects, and associated with future death/myocardial infarction after adjusting for age, sex, and race (odds ratio 2.05, 95% CI [1.6–2.7], P-value < 0.0001). RUNX1 expression is known to initiate at two alternate promoters, a distal P1 and a proximal P2 promoter. In patient cohorts there were differential effects of RUNX1 isoforms on PCTP expression with a negative correlation in blood between RUNX1 expressed from the P1 promoter and PCTP expression. Conclusions PCTP is a direct transcriptional target of RUNX1. PCTP expression is associated with death/myocardial infarction in patients with cardiovascular disease. RUNX1 regulation of PCTP may play a role in the pathogenesis of platelet-mediated cardiovascular events.

Osteoactivin (OA) is required for the differentiation of osteoblast cells. OA expression is stimulated by bone morphogenetic protein-2 (BMP-2). BMP-2 recruits homeodomain transcription factors Dlx3, Dlx5, and Msx2 to selectively activate or repress transcription of osteogenic genes and hence tightly regulate their transcription during osteoblast differentiation. Considering the key roles of Dlx3, Dlx5, and Msx2 in osteoblast differentiation, here we hypothesize that homeodomain proteins regulate BMP-2-induced OA transcription during osteoblast differentiation. Four classical homeodomain binding sites were identified in the proximal 0.96 kb region of rat OA promoter. Deletions and mutagenesis studies of the OA promoter region indicated that all four homeodomain binding sites are crucial for BMP-2-induced OA promoter activity. Simultaneous disruption of homeodomain binding sites at -852 and -843 of the transcription start site of OA gene significantly decreased the BMP-2-induced OA transcription and inhibited binding of Dlx3, Dlx5, and Msx2 proteins to the OA promoter. Dlx3 and Dlx5 proteins were found to activate the OA transcription, whereas, Msx2 suppressed BMP-2-induced OA transcription. Using chromatin immunoprecipitation assays, we demonstrated that the OA promoter is predominantly occupied by Dlx3 and Dlx5 during the proliferation and matrix maturation stages of osteoblast differentiation, respectively. During the matrix mineralization stage, BMP-2 robustly enhanced the recruitment of Dlx5 and to a lesser extent of Dlx3 and Msx2 to the OA promoter region. Collectively, our results show that the BMP-2-induced OA transcription is differentially regulated by Dlx3, Dlx5, and Msx2 during osteoblast differentiation.

Involvement of the human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) protein in neuronal deregulation and in the development of HIV-1 associated neurocognitive disorders (HAND) has been amply explored; however the mechanisms involved remain unclear. In search for the mechanisms, we demonstrated that Tat deregulates neuronal functions through a pathway that involved p73 and p53 pathway. We showed that Tat uses microRNA-196a (miR-196a) to deregulate the p73 pathway. Further, we found that the Abelson murine leukemia (c-Abl) phosphorylates p73 on tyrosine residue 99 (Tyr-99) in Tat-treated cells. Interestingly, Tat lost its ability to promote accumulation and phosphorylation of p73 in the presence of miR-196a mimic. Interestingly, accumulation of p73 did not lead to neuronal cell death by apoptosis as obtained by cell viability assay. Western blot analysis using antibodies directed against serine residues 807 and 811 of retinoblastoma (Rb) protein was also used to validate our data regarding lack of cell death. Hyperphosphorylation of RB (S807/811) is an indication of cell neuronal viability. These results highlight the key role played by p73 and microRNA in Tat-treated neurons leading to their deregulation and it deciphers mechanistically one of the pathways used by Tat to cause neuronal dysfunction that contributes to the development of HAND.