Faculty Bibliography

Histone deacetylase 4 (Hdac4) is known to control chondrocyte hypertrophy and bone formation. We have previously shown that parathyroid hormone (PTH) regulates many aspects of Hdac4 function in osteoblastic cells in vitro; however, in vivo confirmation was previously precluded by pre-weaning lethality of the Hdac4 deficient mice. To analyze the function of Hdac4 in bone in mature animals, we generated mice with osteoblast lineage-specific knockout of Hdac4 (Hdac4^ob-/-) by crossing transgenic mice expressing Cre recombinase under the control of a 2.3kb fragment of the Col1a1 promoter with mice bearing loxP-Hdac4. The Hdac4^ob-/-mice survive to adulthood and developed a mild skeletal phenotype. At 12 weeks of age, they had short, irregularly-shaped and stiff tails due to smaller tail vertebrae, with almost no growth plates. The tibial growth plate zone was also thinned and Mmp13 and Sost mRNAs were increased in the distal femurs of Hdac4^ob-/-mice. Immunohistochemistry showed that sclerostin was elevated in Hdac4^ob-/-mice, suggesting that Hdac4 inhibits its gene and protein expression. To determine the effect of PTH in these mice, hPTH (1-34) or saline were delivered for 14 days with subcutaneously implanted devices in 8-week-old female Hdac4^ob-/-and wild type (Hdac4^fl/fl) mice. Serum CTX, a marker of bone resorption, was increased in Hdac4^ob-/-mice with or without PTH treatment. Tibial cortical BV/TV, Ct.Th, and relative cortical area (RCA) were decreased in Hdac4^ob-/-mice but PTH caused no further decrease in Hdac4^ob-/-mice. Tibial trabecular BV/TV and thickness were not changed significantly in Hdac4^ob-/-mice but decreased with PTH treatment. These results indicate that Hdac4 inhibits bone resorption and has anabolic effects via inhibiting Mmp13 and Sost/sclerostin expression. Hdac4 influences cortical bone mass and thickness and knockout of Hdac4 prevents the catabolic effect of PTH in cortical bone.

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.

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.