Faculty Bibliography

The epidermal growth factor receptor (EGFR) has vital roles in developmental and pathological processes. However, its function in the skeletal system has not been well studied. Our previous research demonstrated that its ligands are potent mitogens for osteoprogenitors but strongly suppress osteoblast differentiation in vitro. To address the in vivo role of EGFR in bone, we constructed transgenic and pharmacological mouse models and used microCT and histomorphometry to analyze their bone phenotype. To eliminate the pre/osteoblastic EGFR activity, we generated 3.6 kb collagen1a1-Cre Egfr^Wa5/flox (Col-Cre Egfr^Wa5/f) mice harboring an EGFR dominant negative allele, Wa5. At 3-, 5- and 7-months old, these mice exhibited a remarkable 20-40% decrease in tibial trabecular bone volume (BV/TV) with a reduction in trabecular number (17-32%) and trabecular thickness (10%) in both genders. Histological analyses of 7-month old female mice revealed that bone formation and mineralization activities were reduced and bone resorption was elevated. These mice exhibited decreases in osteoblast surface (35%), osteoid surface (34%), mineral apposition rate (MAR, 15%), bone formation rate (BFR, 24%), and an increase in osteoclast surface (78%). Similarly, administration of an EGFR inhibitor into wild type mice caused a 25% reduction in trabecular BV/TV. Histomorphometric analyses suggested similar changes in bone formation and resorption parameters as Col-Cre Egfr^Wa5/fmice. In contrast, 3-month old Egfr^Dsk5/+ mice with an EGFR constitutively active allele displayed increases in trabecular BV/TV (36-43%) accompanied by an increase in trabecular number (28-35%) and a decrease in trabecular separation (9-17%) in both genders. These structural changes were due to increases in osteoblast surface (37%), osteoid surface (35%), MAR (18%) and BFR (34%) and a decrease in osteoclast surface (27%). Interestingly, we found that EGFR inhibitor dramatically reduced the number of CFU-F (52%!

Transforming growth factor-beta1 (TGF-beta1) is a crucial molecule for stimulation of breast cancer invasion and formation of bone metastases. The molecular mechanisms of how TGF-beta1 mediates these effects have yet to be completely determined. We have found that activating transcription factor-3 (ATF-3) is strongly stimulated and its level is sustained by TGF-beta1 in highly invasive and metastatic human breast cancer (MDA-MB231) and in mouse mammary pad tumor cells (r3T). ATF-3 is also overexpressed in human primary breast cancer tissue. Overexpression of ATF-3 increased normal human mammary epithelial cell number and DNA synthesis suggesting a role for ATF-3 in cell proliferation. The functional role of ATF-3 in breast cancer progression was determined by the RNA interference technique. Knockdown of ATF-3 by ATF-3 shRNA in MDA-MB231 cells decreased expression of cell cycle gene, cyclin A1 in MDA-MB231 cells. ATF-3 shRNA also decreased expression of an invasive and metastatic gene, matrix metalloproteinase-13 (MMP-13; collagenase-3) in these cells. Chromatin immunoprecipitation experiments identified the direct physical interaction of ATF-3 protein on the human MMP-13 promoter. Thus, the dysregulation of ATF-3 by TGF-beta1 is likely to activate cyclin A1 and MMP-13 genes in breast cancer cells and that would be key to the subsequent cancer cell invasion and metastasis

PTH regulates transcription of a number of genes involved in bone remodeling and calcium homeostasis. We have previously shown that the matrix metalloproteinase-13 (MMP-13) gene is induced by PTH in osteoblastic cells as a secondary response through the protein kinase A pathway requiring the runt domain and activator protein 1 binding sites of the proximal promoter. Here, we investigated the changes PTH causes in histone acetylation in this region (which contains the only deoxyribonuclease-hypersensitive sites in the promoter) leading to MMP-13 gene activation in these cells. Chromatin immunoprecipitation experiments revealed that PTH rapidly increased histone H4 acetylation followed by histone H3 acetylation associated with the different regions of the MMP-13 proximal promoter. The hormone also stimulated p300 histone acetyl transferase activity and increased p300 bound to the MMP-13 proximal promoter, and this required protein synthesis. Upon PTH treatment, Runx2, already bound to the runt domain site of the MMP-13 promoter, interacted with p300, which then acetylated histones H4 and H3. The knockdown of either Runx2 or p300 by RNA interference reduced PTH-induced acetylation of histones H3 and H4, association of p300 with the MMP-13 promoter, and resultant MMP-13 gene transcription. Overall, our studies suggest that without altering the gross chromatin structure, PTH stimulates acetylation of histones H3 and H4 via recruitment of p300 to Runx2 bound to the MMP-13 promoter, resulting in gene activation. This work establishes the molecular basis of transcriptional regulation in osteoblasts by PTH, a hormone acting through a G-protein coupled receptor

PURPOSE OF REVIEW: Parathyroid hormone (PTH) maintains a physiological balance of calcium and phosphate concentrations by binding to its receptor on the plasma membrane of cells in bone and kidney. It signals through multiple pathways, including protein kinase A and protein kinase C, although a preference for certain pathways is apparent in each organ and function. Here, we will review the recent advancements regarding PTH signaling in bone and kidney. RECENT FINDINGS: Wnt proteins have been reported as important regulators of bone metabolism in both PTH-dependent and independent pathways. Recent studies emphasize its role as a mediator of PTH signaling, as PTH treatment increased the expression of wnt4 and sfrp4 and decreased the expression of Wnt inhibitors such as Sost and sclerostin, leading to an increase in Wnt signaling. In kidney, sodium-hydrogen exchanger regulatory factor 1, originally known for its role in the retention of NaPi-IIa at the apical membrane, was shown to have multiple roles in PTH signaling, both as a mediator and regulator. SUMMARY: PTH activates a number of different signaling pathways by binding to a single receptor in bone and kidney. Recent studies demonstrate the involvement of novel factors as well as additional roles for previously identified downstream factors of PTH

A central effort in biomedical research concerns the development of materials for sustaining and controlling cell growth. Carbon nanotube based substrates have been shown to support the growth of different kinds of cells (Hu et al 2004 Nano Lett. 4 507-11; Kalbacova et al 2006 Phys. Status Solidi b 13 243; Zanello et al 2006 Nano Lett. 6 562-7); however the underlying molecular mechanisms remain poorly defined. To address the fundamental question of mechanisms by which nanotubes promote bone mitosis and histogenesis, primary calvariae osteoblastic cells were grown on single-walled carbon nanotube thin film (SWNT) substrates. Using a combination of biochemical and optical techniques we demonstrate here that SWNT networks promote cell development through two distinct steps. Initially, SWNTs are absorbed in a process that resembles endocytosis, inducing acute toxicity. Nanotube-mediated cell destruction, however, induces a release of endogenous factors that act to boost the activity of the surviving cells by stimulating the synthesis of extracellular matrix

Matrix metalloproteinase-13 (MMP-13) plays a critical role in parathyroid hormone (PTH)-induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP-13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8-bromo cyclic adenosine mono phosphate (8-Br-cAMP) treatment. We further demonstrate that in the presence of 8-Br-cAMP, the wild-type Runx2 construct stimulated MMP-13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP-13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling

TGF-beta (transforming growth factor-beta) plays a key role in osteoblast differentiation and bone development. While the ability of TGF-beta to inhibit the expression of osteoblast differentiation genes has been well documented, the mechanism of this inhibition is not yet completely characterized. Runx2, a transcription factor necessary for expression of osteoblast differentiation genes is a central target of inhibition by TGF-beta. In this study, we found that TGF-beta1 inhibits expression of osteoblast differentiation genes without altering expression of Runx2. Transient transfection experiments determined that TGF-beta1 inhibited osteocalcin promoter activity and this effect is mediated through Runx2. We further identified that there was no change in protein expression, cellular localization, or DNA binding affinity of Runx2 after TGF-beta1-treatment of osteoblasts, suggesting that Runx2 undergoes post-translational modifications following TGF-beta1 treatment. Co-immunoprecipitation experiments identified increased phosphorylation of Runx2 when differentiating osteoblasts were treated with TGF-beta1. Mitogen activated protein kinase (MAPK) inhibitors relieved the TGF-beta1-inhibitory effect of Runx2-mediated osteocalcin expression. Thus, our results suggest that TGF-beta1-inhibition of osteoblast differentiation is dependent on the MAPK pathway and this effect is most likely mediated by post-translational modification of Runx2 such as phosphorylation rather than other regulatory mechanisms

Interleukin-18 (IL-18) can regulate osteoblast and osteoclast function. We have identified, using cDNA microarray technology, that IL-18 expression is increased in UMR 106-01 rat osteoblastic cells in response to parathyroid hormone (PTH) treatment. Confirmation of these data using real-time reverse transcription-PCR showed that steady-state levels of IL-18 mRNA increased by 2 h (3-fold), peaked by 4 h (10-fold), and had diminished after 12 h (4.4-fold) and that this regulation was via the protein kinase A signaling pathway and did not involve activation of the PKC signal cascade. PTH regulation of IL-18 was confirmed at the protein level, and analysis of differentiating primary rat calvarial osteoblasts verified that both IL-18 mRNA and protein are regulated by PTH in primary rat osteoblasts. Promoter reporter assays revealed that PTH regulated the upstream IL-18 promoter and induced the exon 1 containing 1.1-kb IL-18 mRNA transcript in primary osteoblast cells. The in vivo physiological role of IL-18 in the anabolic actions of PTH on bone was then assessed using IL-18 knock-out mice. Female IL-18 null mice and wild-type littermate controls were injected with vehicle or 8 microg/100 g of human 1-38 PTH for 4 weeks. In IL-18 knock-out animals the anabolic effect of PTH (determined by bone mineral density changes in the proximal tibia) was abolished in trabecular bone but not in the cortical component. These data characterize the PTH regulation of IL-18 expression in osteoblastic cells and suggest that this cytokine is involved in the anabolic actions of PTH