Faculty Bibliography

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

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF-like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real-time RT-PCR and promoter reporter assays revealed that EGF-like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR-dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR-induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co-repressors HDAC4 and 6 and over-expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down-regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage.

CONTEXT: Chronic high levels of PTH may be associated with up-regulation of proteases and cytokines. Monocyte chemoattractant protein-1 (MCP-1) is an inflammatory cytokine, produced predominantly by macrophages and endothelial cells, and is expressed in adipose tissue. More recently it has been shown that PTH administration increases MCP-1 expression in osteoblasts. OBJECTIVES: Because both PTH and MCP-1 levels are higher in obesity, the goal was to determine whether the high MCP-1 occurs only in the presence of high serum PTH and is independent of adiposity and examine its relationship with bone mineral density (BMD) and turnover. DESIGN, SETTING, AND PARTICIPANTS: In this case-control clinical design, 111 eligible women were categorized into four groups: leaner women [body mass index (BMI) 23 +/- 2 kg/m(2)] with normal or higher PTH and obese (BMI 44 +/- 7 kg/m(2)) with normal or higher PTH. RESULTS: Serum MCP-1 levels were higher (P < 0.01) in the high (PTH = 74.9 +/- 27.0 pg/ml, MCP-1 = 421.5 +/- 157.0 pg/ml) compared with normal PTH (PTH = 32.5 +/- 10.4 pg/ml, MCP-1 = 322.5 +/- 97.8 pg/ml) group, independent of BMI. C-reactive protein and adiponectin were influenced only by BMI and not PTH. MCP-1 was positively associated with osteocalcin and propeptide of type 1 collagen in the leaner (r > 0.3, P < 0.05) but not the obese women and was not associated with BMD in either group. CONCLUSIONS: Together these data suggest that MCP-1 is higher only in the presence of increased PTH and that adiposity alone cannot explain the higher MCP-1 levels in obesity.

While the epidermal growth factor receptor (EGFR)-mediated signaling pathway has been shown to have vital roles in many developmental and pathologic processes, its functions in the development and homeostasis of the skeletal system has been poorly defined. To address its in vivo role, we constructed transgenic and pharmacologic mouse models and used peripheral quantitative computed tomography (pQCT), micro-computed tomography (microCT) and histomorphometry to analyze their trabecular and cortical bone phenotypes. We initially deleted the EGFR in preosteoblasts/osteoblasts using a Cre/loxP system (Col-Cre Egfr(f/f)), but no bone phenotype was observed because of incomplete deletion of the Egfr genomic locus. To further reduce the remaining osteoblastic EGFR activity, we introduced an EGFR dominant-negative allele, Wa5, and generated Col-Cre Egfr(Wa5/f) mice. At 3 and 7 months of age, both male and female mice exhibited a remarkable decrease in tibial trabecular bone mass with abnormalities in trabecular number and thickness. Histologic analyses revealed decreases in osteoblast number and mineralization activity and an increase in osteoclast number. Significant increases in trabecular pattern factor and structural model index indicate that trabecular microarchitecture was altered. The femurs of these mice were shorter and smaller with reduced cortical area and periosteal perimeter. Moreover, colony-forming unit-fibroblast (CFU-F) assay indicates that these mice had fewer bone marrow mesenchymal stem cells and committed progenitors. Similarly, administration of an EGFR inhibitor into wild-type mice caused a significant reduction in trabecular bone volume. In contrast, Egfr(Dsk5/+) mice with a constitutively active EGFR allele displayed increases in trabecular and cortical bone content. Taken together, these data demonstrate that the EGFR signaling pathway is an important bone regulator and that it primarily plays an anabolic role in bone metabolism.

Parathyroid hormone (PTH) regulates the transcription of many genes involved in bone remodeling in osteoblasts. One of these genes is matrix metalloproteinase-13 (MMP-13), which is involved in bone remodeling and early stages of endochondral bone formation. We have previously shown that Mmp-13 gene expression is highly induced by PTH treatment in osteoblastic UMR 106-01 cells, as well as primary osteoblasts. Here, we show that p300/CBP-associated factor (PCAF), in addition to p300 and Runx2, is required for PTH activation of Mmp-13 transcription. PCAF was increasingly recruited to the MMP-13 proximal promoter region after PTH treatment, and this was associated with an increase in RNA polymerase II recruitment and histone acetylation. In addition, PTH treatment increased the acetylation of PCAF, a process that required p300. Knockdown of PCAF, p300, or Runx2 by siRNA decreased Mmp-13 mRNA expression after PTH treatment in both UMR 106-01 cells and primary osteoblasts. We found that there is a mutual dependence between p300 and PCAF to be recruited to the Mmp-13 promoter after PTH treatment. In promoter-reporter assays, p300 and PCAF had an additive effect on PTH stimulation of MMP-13 promoter activity, and this required their histone acetyltransferase activity. Our findings demonstrate that PCAF acts downstream of PTH signaling as a transcriptional coactivator that is required for PTH stimulation of MMP-13 transcription. PCAF cooperates with p300 and Runx2 to mediate PTH activation of MMP-13 transcription.

Physiological bone remodeling is a highly coordinated process responsible for bone resorption and formation and is necessary to repair damaged bone and to maintain mineral homeostasis. In addition to the traditional bone cells (osteoclasts, osteoblasts, and osteocytes) that are necessary for bone remodeling, several immune cells have also been implicated in bone disease. This minireview discusses physiological bone remodeling, outlining the traditional bone biology dogma in light of emerging osteoimmunology data. Specifically discussed in detail are the cellular and molecular mechanisms of bone remodeling, including events that orchestrate the five sequential phases of bone remodeling: activation, resorption, reversal, formation, and termination

The bacterial infection is one of the major problems associated with implant and reconstructive surgery of bone. Hence, the aim of this study was to develop biomaterials having antibacterial activity for bone tissue engineering. The hydroxyapatite nanoparticles (nHAp) improve the mechanical properties and incorporate nanotopographic features that mimic the nanostructure of natural bone. We report here for the first time the synthesis and characterization of nHAp and nHAp soaked with copper (nHAp-Cu) using SEM, AFM, FTIR and XRD. The antibacterial activity of nHAp and nHAp-Cu was determined using Gram-positive and Gram-negative bacterial strains. To have accelerated antibacterial activity, polyethylene glycol 400 (PEG 400), a synthetic biodegradable polymer was also added along with nHAp-Cu. The nHAp-Cu/PEG 400 had increased antibacterial activity towards Gram-positive than Gram-negative bacterial strains. The cytotoxicity of nHAp-Cu/PEG 400 was determined using MTT assay with rat primary osteoprogenitor cells and these biomaterials were found to be non-toxic. Hence, based on these results we suggest that the biomaterials containing nHAp-Cu/PEG 400 can be used as antibacterial materials in bone implant and bone regenerative medicine.

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Parathyroid hormone (PTH) is the major hormone regulating calcium homeostasis, but it also has a significant role as an anabolic hormone in bone when administered by intermittent injection. The mechanisms for the bone anabolic effect of PTH are not understood. However, it has been established that the anabolic effects of PTH require osteoclast activity. PTH acts through a single receptor on the osteoblast to regulate many known genes. 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 PTH(1-34) is injected intermittently in rats for 14 days. Through further in vivo studies, we found that rats treated with PTH(1-34) for 14 days showed a significant increase in serum MCP-1 levels 2 h after PTH injection (159 pg/ml) compared to basal levels obtained prior to the last injection (65 pg/ml). Furthermore, serum MCP-1 levels increase with the duration of treatment. Compared to vehicle treatment, PTH(1-34) treatment in rats for 1, 7 and 14 days showed a 24%, 53% and 79% increase in serum MCP-1 levels 2 h after injection, respectively. Using immunohistochemistry, increased MCP-1 expression is evident in trabecular osteoblasts by the second PTH(1-34) injection with peak staining occurring between 3 and 7 days of treatment. Male and female MCP-1 knockout mice injected daily with 80 ug/kg PTH(1-34) for 6 weeks showed significantly reduced anabolic effects of PTH compared to wild type mice as measured by pQCT and micro-CT. PTH-treated male MCP-1 null mice have significantly less trabecular bone mineral density (means, mg/cm³, vehicle: 454; PTH: 486) than the corresponding PTH-treated wild type mice (means, mg/cm³, vehicle: 452; PTH: 521). Trabecular bone volume (BV/TV) increased 27% in PTH-treated wild type mice with a corresponding increase in trabecular thickness (Tb.Th), however no increase in BV/TV or Tb.Th was observed in the PTH-treated MCP-1 null mice. Hi!