Faculty Bibliography

Pulsed electromagnetic fields (PEMFs) can be effective in promoting the healing of delayed union or nonunion fractures. We previously reported that PEMF (Spinal-Stim(R) by Orthofix, Inc., Lewisville, TX) stimulated proliferation, differentiation and mineralization of rat calvarial osteoblastic cells in culture. In the present work we investigated the effects of PEMF (Physio-Stim(R) by Orthofix, Inc., Lewisville, TX) on human bone marrow macrophages (hBMMs) differentiated to osteoclasts. PEMF had striking inhibitory effects on formation of osteoclasts from hBMMs from both younger and older women. There were significantly greater changes in gene expression as ascertained by RNAseq from cells from older women. Interestingly, all of the genes identified by RNAseq were upregulated, and all were genes of mesenchymal or osteoblastic cells and included members of the TGF-beta signaling pathway and many extracellular matrix proteins, as well as RANKL and osteoprotegerin, indicating the mixed nature of these cultures. From these results, we suggest that PEMF can inhibit osteoclast formation via action on osteoblasts. Thus, PEMF may be very effective for bone mass maintenance in subjects with osteoporosis.

The bone catabolic actions of parathyroid hormone (PTH) are seen in patients with hyperparathyroidism, or with infusion of PTH in rodents. We have previously shown that the chemokine, monocyte chemoattractant protein-1 (MCP-1), is a mediator of PTH's anabolic effects on bone. To determine its role in PTH's catabolic effects, we continuously infused female wild-type (WT) and MCP-1-/- mice with hPTH or vehicle. Microcomputed tomography (microCT) analysis of cortical bone showed that hPTH-infusion induced significant bone loss in WT mice. Further, muCT analysis of trabecular bone revealed that, compared with the vehicle-treated group, the PTH-treated WT mice had reduced trabecular thickness and trabecular number. Notably, MCP-1-/- mice were protected against PTH-induced cortical and trabecular bone loss as well as from increases in serum CTX (C-terminal crosslinking telopeptide of type I collagen) and TRACP-5b (tartrate-resistant acid phosphatase 5b). In vitro, bone marrow macrophages (BMMs) from MCP-1-/- and WT mice were cultured with M-CSF, RANKL and/or MCP-1. BMMs from MCP-1-/- mice showed decreased multinucleated osteoclast formation compared with WT mice. Taken together, our work demonstrates that MCP-1 has a role in PTH's catabolic effects on bone including monocyte and macrophage recruitment, osteoclast formation, bone resorption, and cortical and trabecular bone loss.

Parathyroid hormone (PTH) regulates the transcription of many genes in the osteoblast. One of these genes is Mmp13, which is involved in bone remodeling and early stages of endochondral bone formation. Previously we reported that PTH induces Mmp13 transcription by regulating the dissociation of HDAC4 from Runx2, and the association of the HATs, p300 and P/CAF. It is known that as well as Runx2, HDAC4 binds to the transcription factor, MEF2C, and represses its activity. In this work, we investigated whether MEF2C participates in PTH-stimulated Mmp13 gene expression in osteoblastic cells and how it does so. Knockdown of Mef2c in UMR 106-01 cells repressed Mmp13 mRNA expression and promoter activity with or without PTH treatment. ChIP assays showed that MEF2C associated with the Mmp13 promoter, and this increased after 4 h of PTH treatment. ChIP-reChIP results indicate that endogenous MEF2C associates with HDAC4 on the Mmp13 promoter and after PTH treatment this association decreased. From gel shift, ChIP, and promoter-reporter assays, MEF2C was found to associate with the AP-1 site without directly binding to DNA and had its stimulatory effect through interaction with c-FOS. In conclusion, MEF2C is necessary for Mmp13 gene expression at the transcriptional level and participates in PTH stimulated Mmp13 gene expression by increased binding to c-FOS at the AP-1 site in the Mmp13 promoter. This is the first observation of MEF2C interacting with a member of the AP-1 transcription factor family and provides new knowledge of the functions of HDAC4, c-FOS and MEF2C.

The intermittent administration of recombinant parathyroid hormone (PTH 1-34), or teriparatide, remains the only FDA-approved osteoanabolic therapy for the treatment of osteoporosis. However, prolonged use of teriparatide causes levels of bone resorption markers to rise to that of formation, thus limiting its usefulness. This "anabolic window" justifies the search for therapies that maximize anabolism, without incurring the resorptive effects of teriparatide. Miller et al. (2016) describe the phase III trial results of abaloparatide (ABL), a novel analog of parathyroid hormone-related protein (PTHrP 1-36), where similar anabolic effects are observed, but a lesser stimulation of resorption is induced with ABL compared with teriparatide or placebo (1). This study aims to elucidate the mechanisms that underlie the actions of PTHrP (1-36) and ABL in the osteoblast. Here, we show that in primary murine calvarial osteoblasts, administration of PTHrP (1-36) or ABL results in an attenuated cyclic AMP (cAMP) response compared to PTH (1-34). Time course and dose response curves revealed a maximal difference at 30 minutes and 1/2 max values of 9.3x10^-9M and 2.52x10^-8M for PTH (1-34) and PTHrP (1-36), respectively, whereas cAMP stimulation was nearly undetectable upon treatment with ABL, even at 10^-6M. Consequently, the activation of protein kinase A (PKA) upon PTHrP (1-36) or ABL administration was also attenuated and this difference was maximally observed as soon as 60 sec. post-treatment. These events resulted in the differential subcellular localization of the transcriptional repressor, histone deacetylase 4 (HDAC4), where PTH (1-34) led to its nuclear export, while PTHrP (1-36) and ABL did not. Lastly, real-time quantitative PCR revealed differential expression of key osteoblastic genes, receptor activator of nuclear factor kappa-B ligand (RANKL) and transcription factor, c-fos, where PTH (1-34) treatment resulted in increased RANKL expression 3-fold greater and c-fos expression 2-fold greater compared with PTHrP (1-36) and ABL (n=3, p<0.05). These data suggest that PTH (1-34) utilizes the cAMP/PKA arm of this pathway at a much greater degree compared with PTHrP (1-36) and ABL and that the latter two peptides may employ alternative modes of signaling. Taken together, this study may provide a possible explanation for the effects of PTHrP (1-36) and ABL with respect to bone remodeling and illuminate a potentially novel therapy in the treatment of osteoporosis

Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co-factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR-590-5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR-590-5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR-590-5p and its mRNA and protein expression decreased after miR-590-5p mimic transfection in human osteoblast-like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR-590-5p. However, Runx2 protein, but not mRNA expression, increased after miR-590-5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3'-untranslated region of Smad7 was directly targeted by miR-590-5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2-mediated Runx2 degradation. Hence, based on our results, we suggest that miR-590-5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression

Chemokines are small molecules that play a crucial role as chemoattractants for several cell types, and their components are associated with host immune responses and repair mechanisms. Chemokines selectively recruit monocytes, neutrophils, and lymphocytes and induce chemotaxis through the activation of G protein-coupled receptors. Two well-described chemokine families (CXC and CC) are known to regulate the localization and trafficking of immune cells in cases of injury, infection, and tumors. Monocyte chemoattractant protein 1 (MCP-1/CCL2) is one of the important chemokines from the CC family that controls migration and infiltration of monocytes/macrophages during inflammation. CCL2 is profoundly expressed in osteoporotic bone and prostate cancer-induced bone resorption. CCL2 also regulates physiological bone remodeling in response to hormonal and mechanical stimuli. Parathyroid hormone (PTH) has multifaceted effects on bone, depending on the mode of administration. Intermittent PTH increases bone in vivo by increasing the number and activity of osteoblasts, whereas a continuous infusion of PTH decreases bone mass by stimulating a net increase in bone resorption. CCL2 is essential for both anabolic and catabolic effects of PTH. In this review, we will discuss the pharmacological role of PTH and involvement of CCL2 in the processes of PTH-mediated bone remodeling.

Pulsed electromagnetic fields (PEMFs) have been documented to promote bone fracture healing in nonunions and increase lumbar spinal fusion rates. However, the molecular mechanisms by which PEMF stimulates differentiation of human bone marrow stromal cells (hBMSCs) into osteoblasts are not well understood. In this study the PEMF effects on hBMSCs were studied by microarray analysis. PEMF stimulation of hBMSCs' cell numbers mainly affected genes of cell cycle regulation, cell structure, and growth receptors or kinase pathways. In the differentiation and mineralization stages, PEMF regulated preosteoblast gene expression and notably, the transforming growth factor-beta (TGF-beta) signaling pathway and microRNA 21 (miR21) were most highly regulated. PEMF stimulated activation of Smad2 and miR21-5p expression in differentiated osteoblasts, and TGF-beta signaling was essential for PEMF stimulation of alkaline phosphatase mRNA expression. Smad7, an antagonist of the TGF-beta signaling pathway, was found to be miR21-5p's putative target gene and PEMF caused a decrease in Smad7 expression. Expression of Runx2 was increased by PEMF treatment and the miR21-5p inhibitor prevented the PEMF stimulation of Runx2 expression in differentiating cells. Thus, PEMF could mediate its effects on bone metabolism by activation of the TGF-beta signaling pathway and stimulation of expression of miR21-5p in hBMSCs.

Histone deacetylase 4 (Hdac4) regulates chondrocyte hypertrophy.Hdac4-/- mice are runted in size and do not survive to weaning.This phenotype is primarily due to the acceleration of onset of chondrocyte hypertrophy and, as a consequence, inappropriate endochondral mineralization.Previously, we reported that Hdac4 is a repressor of matrix metalloproteinase-13 (Mmp13) transcription, and the absence of Hdac4 leads to increased expression of MMP-13 both in vitro (osteoblastic cells) and in vivo (hypertrophic chondrocytes and trabecular osteoblasts).MMP-13 is thought to be involved in endochondral ossification and bone remodeling.To identify whether the phenotype of Hdac4-/- mice is due to up-regulation of MMP-13, we generated Hdac4/Mmp13 double knockout mice and determined the ability of deletion of MMP-13 to rescue the Hdac4-/- mouse phenotype.Mmp13-/- mice have normal body size. Hdac4-/-/Mmp13-/- double knockout mice are significantly heavier and larger than Hdac4-/- mice, they survive longer, and they recover the thickness of their growth plate zones.In Hdac4-/-/Mmp13-/- double knockout mice, alkaline phosphatase (ALP) expression and TRAP-positive osteoclasts were restored (together with an increase in Mmp9 expression) but osteocalcin (OCN) was not.Micro-CT analysis of the tibiae revealed that Hdac4-/- mice have significantly decreased cortical bone area compared with the wild type mice.In addition, the bone architectural parameter, bone porosity, was significantly decreased in Hdac4-/- mice.Hdac4-/-/Mmp13-/- double knockout mice recover these cortical parameters.Likewise, Hdac4-/- mice exhibit significantly increased Tb.Th and bone mineral density (BMD) while the Hdac4-/-/Mmp13-/- mice significantly recovered these parameters toward normal for this age.Taken together, our findings indicate that the phenotype seen in the Hdac4-/- mice is partially derived from elevation in MMP-13 and may be due to a bone remodeling disorder caused by overexpression of this enzyme.

Bone remodeling is essential for adult bone homeostasis. It comprises two phases: bone formation and resorption. The balance between the two phases is crucial for sustaining bone mass and systemic mineral homeostasis. This review highlights recent work on physiological bone remodeling and discusses our knowledge of how systemic and growth factors regulate this process.

Direct application of histone-deacetylase-inhibitors (HDACis) to dental pulp cells (DPCs) induces chromatin changes, promoting gene expression and cellular-reparative events. We have previously demonstrated that HDACis (Valproic acid, Trichostatin A) increase mineralization in dental papillae-derived cell-lines and primary DPCs by stimulation of dentinogenic gene expression. Here, we investigated novel genes regulated by the HDACi, suberoylanilide hydroxamic acid (SAHA), to identify new pathways contributing to DPC differentiation. SAHA significantly compromised DPC viability only at relatively high concentrations (5 muM); while low concentrations (1 muM) SAHA did not increase apoptosis. HDACi-exposure for 24 h induced mineralization-per-cell dose-dependently after 2 weeks; however, constant 14d SAHA-exposure inhibited mineralization. Microarray analysis (24 h and 14d) of SAHA exposed cultures highlighted that 764 transcripts showed a significant >2.0-fold change at 24 h, which reduced to 36 genes at 14d. 59% of genes were down-regulated at 24 h and 36% at 14d, respectively. Pathway analysis indicated SAHA increased expression of members of the matrix metalloproteinase (MMP) family. Furthermore, SAHA-supplementation increased MMP-13 protein expression (7d, 14 d) and enzyme activity (48 h, 14d). Selective MMP-13-inhibition (MMP-13i) dose-dependently accelerated mineralization in both SAHA-treated and non-treated cultures. MMP-13i-supplementation promoted expression of several mineralization-associated markers, however, HDACi-induced cell migration and wound healing were impaired. Data demonstrate that short-term low-dose SAHA-exposure promotes mineralization in DPCs by modulating gene pathways and tissue proteases. MMP-13i further increased mineralization-associated events, but decreased HDACi cell migration indicating a specific role for MMP-13 in pulpal repair processes. Pharmacological inhibition of HDAC and MMP may provide novel insights into pulpal repair processes with significant translational benefit