Faculty Bibliography

BACKGROUND CONTEXT: The beneficial therapeutic effects of selected low energy, time varying pulsed electromagnetic fields (PEMFs) have been documented to treat therapeutically resistant problems of the musculoskeletal system. PEMFs promote fracture healing in non-unions. We have found that both bone morphogenetic protein-2 (BMP-2) and PEMF (Spinal-Stim by Orthofix, Inc.) separately stimulated proliferation of rat osteoblastic cells (Selvamurugan et al, 2007) in culture. This effect was additive when both agents were combined. Early osteoblastic differentiation genes were stimulated by PEMF treatment; whereas both the early and late differentiation genes were stimulated by BMP-2 treatment. When both treatments were combined, additive effects were found on both alkaline phosphatase and osteocalcin gene expression and this was more pronounced with daily PEMF treatment. Thus, PEMF has effects on both the proliferating and differentiating osteoblast. Our present work investigated the effects of PEMF (Cervical-Stim by Orthofix, Inc.) on human bone marrow mesenchymal stem cells (hBMMSCs) differentiated to osteoblastic cells and human bone marrow monocytic cells (hBMMs) differentiated to osteoclasts as well as some of the mechanisms of action of PEMF. METHODS: Fresh human bone marrow samples were obtained from the iliac crest of female subjects of different ages. Mononuclear cells were purified by running through Ficoll-Hypaque, and the cells then plated to allow the mesenchymal cells to adhere and expand in proliferation medium, aMEM with 15% fetal bovine serum. Colonies formed, and were expanded for experiments. Human BMMSCs from the second to sixth passages were used for experiments. To obtain human osteoclasts, the fresh mononuclear cells were incubated overnight and the next day non-adherent cells were plated into multiwell plates. All osteoclastic cultures were supplemented with 25 ng/ml M-CSF. Osteoclasts were induced by addition of 35 ng/ ml RANKL. After 9 days incubation multinuclear cells wer!

Parathyroid hormone (PTH) has a significant role as an anabolic hormone in bone when administered by intermittent injection. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when hPTH(1-34) is injected intermittently in rats. Through further in vivo studies, we found that rats treated with hPTH(1-34) showed a significant increase in serum MCP-1 levels 2 hours after PTH injection compared with basal levels. Using immunohistochemistry, increased MCP-1 expression in osteoblasts and osteocytes is evident after PTH treatment. PTH also increased the number of marrow macrophages. MCP-1 knockout mice injected daily with hPTH(1-34) showed less trabecular bone mineral density and bone volume compared with wild-type mice as measured by peripheral quantitative computed tomography (pQCT) and micro-computed tomography (microCT). Histomorphometric analysis revealed that the increase in osteoclast surface and osteoclast number observed with intermittent PTH treatment in the wild-type mice was completely eliminated in the MCP-1 null mice, as well as much lower numbers of macrophages. Consequently, the lack of osteoclast and macrophage activity in the MCP-1 null mice was paralleled by a reduction in bone formation. We conclude that osteoblast and osteocyte MCP-1 expression is an important mediator for the anabolic effects of PTH on bone.

Osteoblast differentiation is tightly regulated by post transcriptional regulators such as microRNAs (miRNAs). Several bioactive materials including nano-bioglass ceramic particles (nBGC) influence differentiation of the osteoblasts, but the molecular mechanisms of nBGC-stimulation of osteoblast differentiation via miRNAs are not yet determined. In this study, we identified that nBGC-treatment stimulated miR-30c expression in human osteoblastic cells (MG63). The bioinformatics tools identified its regulatory network, molecular function, biological processes and its target genes involved in negative regulation of osteoblast differentiation. TGIF2 and HDAC4 were found to be its putative target genes and their expression was down regulated by nBGC-treatment in MG63 cells. Thus, this study advances our understanding of nBGC action on bone cells and supports utilization of nBGC in bone tissue engineering.

Background: Matrix metalloproteinase-13 (Mmp-13) is an important enzyme for the modulation of bone turnover and gingival recession. Elevated levels of Mmp-13 are associated with alveolar bone resorption, periodontal ligament destruction, and gingival attachment loss, which are the clinical symptoms of periodontal disease. Continued evidence suggests periodontal disease contributes to oral tissue destruction and is linked to numerous systemic conditions. Triclosan is a long standing, proven antibacterial and anti-inflammatory agent found in the only FDA-approved dentifrice for the treatment of plaque and gingivitis. Methods: This study examined the inhibitory effects of triclosan on lipopolysaccharide (LPS), parathyroid hormone (PTH) and prostaglandin E(2) (PGE(2)) induced expression of Mmp-13 in UMR 106-01 cells, an osteoblastic osteosarcoma cell line. The cells were stimulated with PTH or PGE(2) to induce Mmp-13 mRNA expression and Real Time RT-PCR was performed to determine gene expression levels. Western blot analysis assessed the presence or absence of protein degradation or inhibition of protein synthesis. Mmp-13 Promoter Reporter Assay was utilized to explore possible direct effects of triclosan on the Mmp-13 promoter. Results: Triclosan significantly reduced PTH or PGE(2) elevated expression of Mmp-13 in osteoblastic cells without affecting basal levels of the mRNA. Surprisingly, triclosan enhanced the expression of c-fos and amphiregulin mRNA. A promoter assay indicated triclosan directly inhibits the activation of the PTH-responsive minimal promoter of Mmp-13. Conclusion: Our data appear to have identified a nuclear mechanism of action of triclosan which accounts for triclosan's ability to inhibit PTH or PGE(2) induced Mmp-13 expression in osteoblastic cells.

Parathyroid hormone (PTH) regulates the transcription in the osteoblast of many genes. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. Previously we reported that PTH induces MMP-13 transcription by regulating histone deacetylase 4 (HDAC4). HDAC4 interacts with Runx2, bound to the MMP-13 promoter, and suppresses basal MMP-13 gene transcription. In the rat osteoblastic cell line, UMR 106-01, after PTH treatment, HDAC4 dissociates from Runx2, which is then free to recruit histone acetyl transferases (HATs), especially p300, to the MMP-13 promoter. It is known that as well as Runx2, HDAC4 associates with the transcription factor, Mef2c, and represses its activity. It has been reported that zinc finger protein 521 (Zfp521) associates with Runx2 in chondrocytes, leading to a repression of Runx2 activity via an HDAC4- dependent mechanism. To determine whether Mef2c or Zfp521 are involved in the transcriptional regulation of MMP-13, their expression was transiently knocked down in UMR 106-01 cells by siRNA transfection. Knockdown of Mef2c repressed MMP-13 mRNA expression and promoter activity with or without PTH treatment. To determine whether Mef2c associates with the MMP-13 gene, we performed ChIP assays after 4 h of PTH treatment. The binding of Mef2c to the MMP-13 promoter was increased after PTH treatment. In addition, PTH treatment caused Mef2c protein to migrate more slowly at 2-4 h (indicating phosphorylation) and later degradation in the nucleus. On the other hand, knockdown of Zfp521 enhanced MMP-13 mRNA expression and promoter activity. To examine if Zfp521 is necessary for binding of HDAC4 on the MMP-13 promoter, we performed ChIP assays and found that depleting Zfp521 prevented HDAC4 binding. Immunoprecipitation showed that Mef2c interacts with Zfp521 and after PTH treatment the binding was decreased. Mef2c also binds to Runx2 and this binding was increased by PTH treatment. In conclusion, Me!

Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through causing its dissociation from Runx2. The aim of the present study was to determine the mechanism and signal transduction pathway by which this occurred. We found that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of cells of the rat osteoblastic cell line, UMR 106-01. In contrast, PKA is necessary for the translocation of HDAC4 into the nucleus. We demonstrated that PKA-dependent phosphorylated HDAC4 is released from Runx2 on the runt domain of the MMP-13 promoter in these cells. Point mutations of serines at 355, 576, 740 or threonine at 815 in rat HDAC4 showed that only mutation at serine 740 prevented the release of HDAC4 from Runx2 on the MMP-13 promoter and also the PTH stimulation of MMP-13 transcription. Using antibodies to the equivalent phosphorylation site in human HDAC4 (serine 632), we found that PTH transiently stimulated phosphorylation of this residue and this was abolished when PKA was inhibited. Thus, PTH-induced PKA phosphorylation of serine 740 in rat HDAC4 is crucial for regulating MMP-13 transcription in osteoblasts. The protein 14-3-3 beta is associated with the shuttling of HDAC4 between nucleus and cytoplasm. Colocalization of 14-3-3 beta and HDAC4 is increased after PTH stimulation and both accumulate in the cytoplasm. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA and trafficking and the control of MMP-13 transcription through association with Runx2