Faculty Bibliography

Previously, we determined that the activator protein-1 (AP-1)-binding site and the runt domain (RD)-binding site and their binding proteins, c-Fos.c-Jun and Cbfa, regulate the collagenase-3 promoter in parathyroid hormone-treated and differentiating osteoblasts. Here we show that Cbfa1 and c-Fos.c-Jun appear to cooperatively bind the RD- and AP-1-binding sites and form ternary structures in vitro. Both in vitro and in vivo co-immunoprecipitation and yeast two-hybrid studies further demonstrate interaction between Cbfa1 with c-Fos and c-Jun in the absence of phosphorylation and without binding to DNA. Additionally, only the runt domain of Cbfa1 was required for interaction with c-Jun and c-Fos. In mammalian cells, overexpression of Cbfa1 enhanced c-Jun activation of AP-1-binding site promoter activity, demonstrating functional interaction. Finally, insertion of base pairs that disrupted the helical phasing between the AP-1- and RD-binding sites also inhibited collagenase-3 promoter activation. Thus, we provide direct evidence that Cbfa1 and c-Fos.c-Jun physically interact and cooperatively bind the AP-1- and RD-binding sites in the collagenase-3 promoter. Moreover, the AP-1- and RD-binding sites appear to be organized in a specific required helical arrangement that facilitates transcription factor interaction and enables promoter activation

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

Treatment of osteoblastic cells with PTH initiates dual signaling cascades resulting in activation of both PKA and PKC. It has been shown that PTH either inhibits or stimulates ERKs depending on dose of the hormone; nevertheless, the ability of PTH to regulate other members of the MAPK family is unknown. Another member of this family, c-Jun-NH(2)-terminal kinase (JNK), is preferentially activated by cytokines and cellular stresses and plays a key role in regulating the activity of various transcription factors. We demonstrate that treatment of UMR 106-01 cells and rat calvarial osteoblasts with PTH (10(-8) M), N-terminal peptides of PTH that selectively activate PKA, or 8-bromo-cAMP (activates PKA) results in the inhibition of JNK activity from high basal levels. Examination of the upstream members of the JNK cascade revealed that both stress-activated protein kinase/extracellular signal-related kinase kinase 1/MAPK kinase 4 and MAPK/extracellular signal-related kinase kinase kinase 1 activities were also inhibited after treatment with PTH (10(-8) M). We conclude that treatment of osteoblastic cells with PTH is sufficient to inhibit high basal JNK activity by activation of the PKA signaling cascade

The benefit of HMG-CoA reductase inhibitors (statins) to the cardiovascular system is now well established and these drugs are being used extensively to treat hypercholesterolaemia clinically. However, as clinical outcomes become available it appears that statins are proving more beneficial than expected and thus it is being proposed that the actions of statins go beyond their ability to lower serum cholesterol levels. The report that statins can interact directly with lymphocyte function-associated antigen (LFA)-1 and prevent it engaging with the intracellular adhesion molecule (ICAM)-1 receptor on T cells is a novel mechanism of statin action and provides convincing evidence that these compounds can regulate biological systems other than by the cholesterol synthesis pathway. Immunosuppression to prevent organ transplant rejection is one application for which statins are currently being assessed. The clinical evidence is conflicting and does not convincingly reflect whether statins are beneficial as immunomodulators. However, in vivo studies investigating the cellular actions of statins have identified two mechanisms by which statins can potentially modulate an in vivo immune response. Firstly, statins regulate inducible class II major histocompatibility complex (MHC) expression on macrophages and endothelial cells. Secondly, statins can inhibit LFA-1 adhesion to ICAM-1 and thus regulate T cell activation. These findings suggest that statins have the potential to regulate an immune response in vivo and that more investigation is essential in order to explain the opposing clinical data

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation

Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3beta (GSK-3beta) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal-activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects

Parathyroid hormone (PTH) is known to have both catabolic and anabolic effects on bone. The dual functionality of PTH may stem from its ability to activate two signal transduction mechanisms: adenylate cyclase and phospholipase C. Here, we demonstrate that continuous treatment of UMR 106-01 and primary osteoblasts with PTH peptides, which selectively activate protein kinase C, results in significant increases in DNA synthesis. Given that ERKs are involved in cellular proliferation, we examined the regulation of ERKs in UMR 106-01 and primary rat osteoblasts following PTH treatment. We demonstrate that treatment of osteoblastic cells with very low concentrations of PTH (10(-12) to 10(-11) m) is sufficient for substantial increases in ERK activity. Treatment with PTH-(1-34) (10(-8) m), PTH-(1-31), or 8-bromo-cAMP failed to stimulate ERKs, whereas treatment with phorbol 12-myristate 13-acetate, serum, or PTH peptides lacking the N-terminal amino acids stimulated activity. Furthermore, the activation of ERKs was prevented by pretreatment of osteoblastic cells with inhibitors of protein kinase C (GF 109203X) and MEK (PD 98059). Treatment of UMR cells with epidermal growth factor (EGF), but not PTH, promoted tyrosine phosphorylation of the EGF receptor. Transient transfection of UMR cells with p21(N17Ras) did not block activation of ERKs following treatment with low concentrations of PTH. Thus, activation of ERKs and proliferation by PTH is protein kinase C-dependent, but stimulation occurs independently of the EGF receptor and Ras activation

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts

Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane proteins that have been implicated in multiple physiological and pathological processes related to extracellular matrix turnover. Recent evidence strongly suggests a role for collagenase-3 (MMP-13) in tumor metastasis and invasion. We report here that collagenase-3 is constitutively expressed in the breast cancer cell line MDA-MB231 (MDA) and outline the molecular mechanism regulating its expression. Functional analysis of the collagenase-3 promoter showed that both the activator protein-1 (AP-1) site and the runt domain (RD) binding site were required for maximal constitutive expression of collagenase-3 in MDA cells. Determination of factors binding to those sites by Northern analysis and transient transfections identified the requirement of Fra-1, c-Jun, and Cbfa1 for basal collagenase-3 promoter activity in MDA cells

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo