Faculty Bibliography

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr gene deletion in mice (DMP-GHRKO) to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate (Pi) and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.-Liu, Z., Kennedy, O. D., Cardoso, L., Basta-Pljakic, J., Partridge, N. C., Schaffler, M. B., Rosen, C. J., Yakar, S. DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH.

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

Activating transcription factor (ATF-3) is a stress response gene and is induced by transforming growth factor beta 1 (TGF-beta1) in breast cancer cells. In this study, we dissected the functional role of ATF-3 gene in vitro by knocking down its expression stably in human bone metastatic breast cancer cells (MDA-MB231). Knockdown of ATF-3 expression in these cells decreased cell number, altered cell cycle phase transition, and decreased mRNA expression of cell cycle genes. Knockdown of ATF-3 expression in MDA-MB231 cells also decreased cell migration, and the expression levels of invasive and metastatic genes such as MMP-13 and Runx2 were found to be decreased in these cells. Most importantly, ATF-3 was associated with Runx2 promoter in MDA-MB231 cells and knockdown of ATF-3 expression decreased its association with Runx2 promoter. Hence, our results suggested that ATF-3 plays a role in proliferation and invasion of bone metastatic breast cancer cells in vitro and we identified for the first time that Runx2 is a target gene of ATF-3 in MDA-MB231 cell line.

PTH is the only current anabolic treatment for osteoporosis in the USA. PTH stimulates expression of matrix metalloproteinase 13 (MMP13) in bone. Sirtuin 1 (SIRT1), an NAD-dependent deacetylase, participates in a variety of human diseases. Here we identify a role for SIRT1 in the action of PTH in osteoblasts. We observed increased Mmp13 mRNA expression and protein levels in bone from Sirt1 knockout mice compared with wild type mice. PTH-induced Mmp13 expression was significantly blocked by the SIRT1 activator, resveratrol, in osteoblastic UMR 106-01 cells. In contrast, the SIRT1 inhibitor, EX527, significantly enhanced PTH induced-Mmp13 expression. Two hours of PTH treatment augmented SIRT1 association with c-Jun, a component of the transcription factor complex, activator protein 1 (AP-1), and promoted SIRT1 association with the AP-1 site of the Mmp13 promoter. This binding was further increased by resveratrol, implicating SIRT1 as a feedback inhibitor regulating Mmp13 transcription. The AP-1 site of the Mmp13 promoter is required for PTH stimulation of Mmp13 transcriptional activity. When the AP-1 site was mutated, EX527 was unable to increase PTH stimulated Mmp13 promoter activity, indicating a role for the AP-1 site in SIRT1 inhibition. We further showed that SIRT1 deacetylates c-Jun and the cAMP pathway participates in this deacetylation process. These data indicate that SIRT1 is a negative regulator of MMP13 expression, SIRT1 activation inhibits PTH stimulation of Mmp13 expression and this regulation is mediated by SIRT1 association with c-Jun at the AP-1 site of the Mmp13 promoter.

Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper, we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3'UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation. J. Cell. Physiol. 229: 1236-1244, 2014. (c) 2014 Wiley Periodicals, Inc.

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 dissociation from Runx2. Here, we show that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of the rat osteoblastic cell line, UMR 106-01. We demonstrate that PKA-dependent phosphorylated HDAC4 is released from Runx2 bound to the MMP-13 promoter in these cells. Point mutation of Ser-740 in rHDAC4 prevents the release of HDAC4 from Runx2 on the MMP-13 promoter and also prevents the PTH stimulation of MMP-13 transcription. Thus, PTH-induced phosphorylation of rHDAC4 at Ser-740 is crucial for regulating MMP-13 transcription in osteoblasts. PTH causes degradation of HDAC4 and this product appears in the cytoplasm. The cytoplasmic degradation of HDAC4 is blocked by PKA and lysosomal inhibitors, but is not affected by proteasome, caspase-3, serine and aspartic protease inhibitors. In addition, the phosphatase inhibitor, okadaic acid, prevents degradation indicating that dephosphorylation is associated with degradation. 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, trafficking, partial degradation and the control of MMP-13 transcription through association with Runx2.