Mitogen activated protein kinase-dependent inhibition of osteocalcin gene expression by transforming growth factor-beta1.

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.

Transforming growth factor-beta1 regulation of ATF-3 and identification of ATF-3 target genes in breast cancer cells.

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.

Effects of BMP-2 and pulsed electromagnetic field (PEMF) on rat primary osteoblastic cell proliferation and gene expression.

Bone morphogenetic proteins (BMPs) strongly promote osteoblast differentiation. Pulsed electromagnetic fields (PEMFs) promote fracture healing in non-union fractures. In this study, we hypothesized that a combined BMP-2 and PEMF stimulation would augment bone formation to a greater degree than treatment with either single stimulus. BMP-2 maximally increased the proliferative activity of rat primary osteoblastic cells at 25 ng/ml concentration. Real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that BMP-2 stimulated mRNA levels of alkaline phosphatase (ALP), alpha(1) (I) procollagen, and osteocalcin (OC) in the differentiation phase and only OC mRNA expression in the mineralization phase after 24-h treatment. Both BMP-2 and PEMF (Spinal-Stim) increased cell proliferation, which was additive when both agents were combined. PEMF alone or together with BMP-2 increased only ALP mRNA expression and only during the differentiation phase 24 h after one 4-h treatment. This effect was additive when both agents were combined. Continuous daily 4-h treatment with PEMF alone or together with BMP-2 increased expression of all three osteoblast marker genes during the differentiation phase and increased the mineralized matrix. This effect was additive when both agents were combined, suggesting that the two interventions may be working on different cellular pathways. Thus, a combined effect of BMP-2 and PEMF in vitro could be considered as groundwork for in vivo bone development that may support skeletal therapy.

Overexpression of Runx2 directed by the matrix metalloproteinase-13 promoter containing the AP-1 and Runx/RD/Cbfa sites alters bone remodeling in vivo.

The activator protein-1 (AP-1) and runt domain binding (Runx/RD/Cbfa) sites and their respective binding proteins, c-Fos/c-Jun and Runx2 (Cbfa1), regulate the rat matrix metalloproteinase-13 (MMP-13) promoter in both parathyroid hormone (PTH)-treated and differentiating osteoblastic cells in culture. To determine the importance of these regulatory sites in the expression of MMP-13 in vivo, transgenic mice containing either wild-type (-456 or -148) or AP-1 and Runx/RD/Cbfa sites mutated (-148A3R3) MMP-13 promoters fused with the E. coli lacZ reporter were generated. The wild-type transgenic lines expressed higher levels of bacterial beta-galactosidase in bone, teeth, and skin compared to the mutant and non-transgenic lines. Next, we investigated if overexpression of Runx2 directed by the MMP-13 promoter regulated expression of bone specific genes in vivo, and whether this causes morphological changes in these animals. Real time RT-PCR experiments identified increased mRNA expression of bone forming genes and decreased MMP-13 in the tibiae of transgenic mice (14 days and 6 weeks old). Histomorphometric analyses of the proximal tibiae showed increased bone mineralization surface, mineral apposition rate, and bone formation rate in the transgenic mice which appears to be due to decreased osteoclast number. Since MMP-13 is likely to play a role in recruiting osteoclasts to the bone surface, decreased expression of MMP-13 may cause reduced osteoclast-mediated bone resorption, resulting in greater bone formation in transgenic mice. In summary, we show here that the 148 bp upstream of the MMP-13 transcriptional start site is sufficient and necessary for gene expression in bone, teeth, and skin in vivo and the AP-1 and Runx/RD/Cbfa sites are likely to regulate this. Overexpression of Runx2 by these regulatory elements appears to alter the balance between the bone formation-bone resorption processes in vivo.

Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells.

Parathyroid hormone (PTH) regulates bone remodeling and calcium homeostasis by acting on osteoblasts. Recently, the gene expression profile changes in the rat PTH (1-34, 10(-8)M)-treated rat osteoblastic osteosarcoma cell line, UMR 106-01, using DNA microarray analysis showed that mRNA for LTBP-1, a latent transforming growth factor (TGF-beta)-binding protein is stimulated by PTH. Latent TGF-beta binding proteins (LTBPs) are required for the proper folding and secretion of TGF-beta, thus modifying the activity of TGF-beta, which is a local factor necessary for bone remodeling. We show here by real time RT-PCR that PTH-stimulated LTBP-1 mRNA expression in rat and mouse preosteoblastic cells. PTH also stimulated LTBP-1 mRNA expression in all stages of rat primary osteoblastic cells but extended expression was found in differentiating osteoblasts. PTH also stimulated TGF-beta1 mRNA expression in rat primary osteoblastic cells, indicating a link between systemic and local factors for intracellular signaling in osteoblasts. An additive effect on LTBP-1 mRNA expression was found when UMR 106-01 cells were treated with PTH and TGF-beta1 together. We further examined the signaling pathways responsible for PTH-stimulated LTBP-1 and TGF-beta1 mRNA expression in UMR 106-01 cells. The PTH stimulation of LTBP-1 and TGF-beta1 mRNA expression was dependent on the PKA and the MAPK (MEK and p38 MAPK) pathways, respectively in these cells, suggesting that PTH mediates its effects on osteoblasts by several intracellular signaling pathways. Overall, we demonstrate here that PTH stimulates LTBP-1 mRNA expression in osteoblastic cells and this is PKA-dependent. This event may be important for PTH action via TGF-beta in bone remodeling.

Smad3 interacts with JunB and Cbfa1/Runx2 for transforming growth factor-beta1-stimulated collagenase-3 expression in human breast cancer cells.

We have previously shown that 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. To understand the molecular mechanisms responsible for TGF-beta1 response on collagenase-3 promoter activity, a functional analysis of the promoter region of the collagenase-3 gene was carried out, and we identified the distal runt domain (RD) and proximal RD/activator protein-1 (AP-1) sites as necessary for full TGF-beta1-stimulated collagenase-3 promoter activity. Gel shift, real time reverse transcriptase-PCR, and Western blot analyses showed increased levels of c-Jun, JunB, and Cbfa1/Runx2 upon TGF-beta1 treatment in MDA-MB231 cells. Co-immunoprecipitation in vitro studies identified no physical interaction between JunB and Cbfa1/Runx2, whereas Smad3 interacted with both. Chromatin immunoprecipitation experiments confirmed interaction of Smad3 with JunB and Cbfa1/Runx2. Under basal conditions, Cbfa1/Runx2 bound to both the proximal RD/AP-1 and distal RD sites. In response to TGF-beta1, Cbfa1/Runx2 was seen only at the distal RD site, whereas JunB occupied the proximal RD/AP-1 site. An assemblage of Smad3, JunB, and Cbfa1/Runx2 at the distal RD site of the collagenase-3 promoter occurred in response to TGF-beta1 in MDA-MB231 cells. Co-transfection of Smad3, JunB, and Cbfa1/Runx2 constructs along with a constitutively active TGF-beta type I receptor construct identified functional interaction of these proteins and transcriptional activation of the collagenase-3 gene by TGF-beta1. Taken together, our results suggest that TGF-beta1 stimulated JunB and Cbfa1/Runx2 to bind to their respective DNA consensus sites and that Smad3 is likely to stabilize their interaction to confer functional TGF-beta1-stimulation of collagenase-3 expression in MDA-MB231 cells.

Transforming growth factor-beta 1 regulation of collagenase-3 expression in osteoblastic cells by cross-talk between the Smad and MAPK signaling pathways and their components, Smad2 and Runx2.

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.