Faculty Bibliography

Parathyroid hormone (PTH) is the major hormone regulating calcium metabolism. It is also the only FDA-approved drug for osteoporosis treatment that stimulates bone formation when injected daily. However, continuous infusion of PTH causes severe bone loss in line with its known catabolic effects. Many studies to understand the dual effects of PTH have been carried out, and in recent years a growing number of molecular and cellular mechanisms underlying these effects have emerged. Here, we outline the present knowledge and conclude that the kinetics of administration and subsequent signaling probably account for the divergent actions of the hormone

Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 (MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) -148 to -38 and supports binding of numerous transcription factors, including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric(dA:dT) element (-119/-110 nt) that conforms to the consensus site for the novel transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ). This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5'-regulatory region, and overexpression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric(dA:dT) element enhances the MMP-13 response to PTH and PGE(2). Overexpression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric(dA:dT) element and the adjacent Runx2 site further augments the PTH response. On the basis of these data and previous studies, we propose that Nmp4/CIZ is a component of a multiprotein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy

Transforming growth factor-beta (TGF-beta) plays a key role in osteoblast differentiation and bone development and remodeling. Collagenase-3 (matrix metalloproteinase-13) is expressed by osteoblasts and seems to be involved in osteoclastic bone resorption. Here, we show that TGF-beta 1 stimulates collagenase-3 expression in the rat osteoblastic cell line UMR 106-01 and requires de novo protein synthesis. Dominant-negative Smad2/3 constructs indicated that Smad signaling is essential for TGF-beta 1-stimulated collagenase-3 promoter activity. Inhibitors of the ERK1/2 and p38 MAPK pathways, but not the JNK pathway, reduced TGF-beta 1-stimulated collagenase-3 expression, indicating that the p38 MAPK and ERK1/2 pathways are also required for TGF-beta 1-stimulated collagenase-3 expression in UMR 106-01 cells. These inhibitors did not prevent nuclear localization of Smad proteins, but they inhibited Smad-mediated transcriptional activation. We have shown for the first time that Runx2 (a bone transcription factor and a potential substrate for the MAPK pathway) is phosphorylated in response to TGF-beta 1 treatment in osteoblastic cells. Cotransfection of Smad2 and Runx2 constructs had a cooperative effect on TGF-beta 1-stimulated collagenase-3 promoter activity in these cells. We further identified ligand-independent physical interaction between Smad2 and Runx2. Taken together, our results provide an important role for cross-talk between the Smad and MAPK pathways and their components in expression of collagenase-3 following TGF-beta 1 treatment in UMR 106-01 cells

Taking advantage of the 'working draft' of the human genome and the MIT shotgun assembly of the mouse genome, we performed a comparative promoter analysis of human RefSeq mRNA (sequences from GenBank's RefSeq database). By combining this analysis with a transcription factor (TF) binding site analysis using a TRANSFAC position weight matrix (PWM) search, 86% of non-specific TF sites were removed. Using a set of genes that are regulated by parathyroid hormone (PTH), a statistical analysis was performed on the conserved TF binding sites among a set of eight human and mouse genes. From among the eight genes tested, we obtained a set of 31 TFs, suggesting possible roles for associated genes in PTH-mediated pathways. All three known PTH-responsive TFs (AP1, RUNX2, CREB) were correctly predicted by this analysis as well as two other potential TFs (VDR and CEBP Delta). Additionally, a model was made to describe the TF site characteristic module of PTH-regulated genes. This model was then used to search all human RefSeq gene promoters with established human-mouse ortholog relationships to identify other PTH-regulated genes. This comparative approach combined with statistical analysis proved to be sufficiently specific to decipher critical TFs involved in PTH-regulated pathways

OBJECTIVE: Collagenase-3, a matrix metalloproteinase (MMP-13) that can degrade collagen II and aggrecan, is produced by osteoarthritic (OA) chondrocytes and may contribute to matrix destruction in this disease. Our laboratory has previously identified a specific endocytotic receptor for collagenase-3 on osteoblastic and fibroblastic cells, which couples with the low-density lipoprotein receptor-related protein (LRP1) to mediate the internalization and degradation of this enzyme. We hypothesized that the activity of this receptor system is reduced in OA chondrocytes which may lead to increased local extracellular levels of collagenase-3 and increased destruction of the cartilage matrix at pericellular sites. METHODS: Human chondrocytes and synoviocytes were obtained from OA knees at the time of joint replacement surgery and from non-arthritic control specimens following autopsy or surgery. Enzyme-linked immunosorbant assay (ELISA) was used to measure collagenase-3 secreted from primary cultures. Iodinated collagenase-3 was used to analyze the cell-surface binding, internalization and intracellular degradation of collagenase-3. Reverse-transcriptase polymerase chain reaction was used to confirm chondrocyte phenotype and the expression of collagenase-3 and LRP1 mRNAs. RESULTS: OA chondrocytes and synoviocytes demonstrated significantly reduced (75-77%) binding of recombinant 125I collagenase-3. Internalization and degradation of the ligand was also significantly reduced (64-72%) in OA cells. Collagenase-3 removal was inhibited by the LRP1 receptor-associated protein (RAP). CONCLUSION: These results suggest a mechanism whereby impaired receptor-mediated removal of collagenase-3 in OA chondrocytes may lead to enhanced local degradation of the cartilage matrix. This work also implicates an LRP family member in endocytotic receptor-mediated collagenase-3 processing and suggests a novel therapeutic target for OA

Parathyroid hormone (PTH) binds to its receptor PTH1R (parathyroid hormone 1 receptor) in osteoblastic cells to regulate bone remodeling and calcium homeostasis. While prolonged exposure to PTH causes increased bone resorption, intermittent injections of PTH have an anabolic effect on bone. The molecular mechanisms regulating these processes are still largely unknown. Here, we present our results on gene expression profile changes in the PTH-treated osteoblastic cell line, UMR 106-01, using DNA microarray analysis. A total of 125 known genes and 30 unknown expressed sequence tags (ESTs) were found to have at least 2-fold expression changes after PTH treatment at 4, 12, and 24 h. 14 genes were previously known to be PTH-regulated but many were unknown to be regulated by PTH prior to our experiments. Real-time reverse transcriptase-PCR confirmed that 90 and 50% of the genes are regulated more than 2-fold by PTH in UMR 106-01 and rat primary osteoblastic cells, respectively. Most genes belong to the following protein families: hormones, growth factors, and receptors; signal transduction pathway proteins; transcription factors; proteases; metabolic enzymes; structural and matrix proteins; transporters; etc. These results provide a comprehensive and deeper knowledge about PTH regulation of osteoblastic gene expression. Next, we designed a computational method to extract information about transcription factors likely involved in regulating these genes. These factors include those previously known to be involved in PTH signaling (AP-1 and the cAMP response element-binding protein), those that were identified by microarray data (C/EBP), and some novel transcription factors (AP-2, AP-4, SP1, FoxD3, etc.). Our results suggest that a reliable bioinformatics approach can be easily applied for other systems

Transforming growth factor (TGF)-beta1, a crucial molecule in metastatic bone cancer, stimulates collagenase-3 expression in the human breast cancer cell line, MDA-MB231. Cycloheximide inhibited this stimulation, indicating that de novo protein synthesis was essential for this response. We examined whether mitogen-activated protein kinase (MAPK) and/or Smad pathways are involved in TGF-beta1-stimulated collagenase-3 expression in MDA-MB231 cells. Biochemical blockade of extracellular regulated kinase-1/2 and p38 MAPK pathways partially abolished TGF-beta1-stimulated collagenase-3 mRNA expression; whereas overexpression of a dominant negative form of Smad3 completely blocked the TGF-beta1-response. These data indicate that TGF-beta1-induced MAPK and Smad pathways are involved in TGF-beta1-stimulated collagenase-3 expression in MDA-MB231 cells

We have previously shown that PTH induction of c-fos expression in the rat osteoblastic cell line UMR 106-01 requires the phosphorylation of cAMP response element-binding protein (CREB) at serine 133. Here we show that this event is not sufficient for induced transcriptional activity in UMR cells. Serine 129, but not the casein kinase II sites (serines 108, 111, 114, 117, and 121), also plays a role in the activation of CREB. First, by metabolically labeling an epitope-tagged CREB, we determined that, in addition to serine 133, other residues are phosphorylated in vivo. Using mutational analysis of a GAL4-CREB reporter system we demonstrate that serines 129 and 133 are both required for PTH-induced transcriptional activity, whereas the casein kinase II sites are not. Furthermore, PTH failed to induce transcriptional activity of GAL4-CREB in cells treated with genistein, a general tyrosine kinase inhibitor known to inhibit glycogen synthase kinase-3 (GSK-3) activity, or LiCl, the most specific GSK-3-inhibiting agent known, strongly implicating GSK-3beta in this process. Importantly, although genistein and LiCl each inhibit GSK-3beta activity, neither prevented the phosphorylation of serine 133 induced by PTH. Lastly, when serine 129 is replaced with a negatively charged aspartic acid, LiCl has no effect on the PTH-induced trans-activation of CREB. We propose that GSK-3beta phosphorylates CREB at serine 129 and thus is required for the increased transcriptional activity of CREB in response to PTH