Identification and characterization of Runx2 phosphorylation sites involved in matrix metalloproteinase-13 promoter activation

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.     

Distinct roles for mitogen-activated protein kinase phosphatase-1 (MKP-1) and ERK-MAPK in PTH1R signaling during osteoblast proliferation and differentiation

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) activate one single receptor (PTH1R) which mediates catabolic and anabolic actions in the bone. Activation of PTH1R modulates multiple intracellular signaling responses. We previously reported that PTH and PTHrP down-regulate pERK1/2 and cyclin D1 in differentiated osteoblasts. In this study we investigate the role of MAPK phosphatase-1 (MKP-1) in PTHrP regulation of ERK1/2 activity in relation to osteoblast proliferation, differentiation and bone formation. Here we show that PTHrP increases MKP-1 expression in differentiated osteoblastic MC3T3-E1 cells, primary cultures of differentiated bone marrow stromal cells (BMSCs) and calvarial osteoblasts. PTHrP had no effect on MKP-1 expression in proliferating osteoblastic cells. Overexpression of MKP-1 in MC-4 cells inhibited osteoblastic cell proliferation. Cell extracts from differentiated MC-4 cells treated with PTHrP inactivate/dephosphorylate pERK1/2 in vitro; immunodepletion of MKP-1 blocked the ability of the extract to dephosphorylate pERK1/2; these data indicate that MKP-1 is involved in PTHrP-induced pERK1/2 dephosphorylation in the differentiated osteoblastic cells. PTHrP regulation of MKP-1 expression is partially dependent on PKA and PKC pathways. Treatment of nude mice, bearing ectopic ossicles, with intermittent PTH for 3 weeks, up-regulated MKP-1 and osteocalcin, a bone formation marker, with an increase in bone formation. These data indicate that PTH and PTHrP increase MKP-1 expression in differentiated osteoblasts; and that MKP-1 induces growth arrest of osteoblasts, via inactivating pERK1/2 and down-regulating cyclin D1; and identify MKP-1 as a possible mediator of the anabolic actions of PTH1R in mature osteoblasts.     

Regulation of matrix Gla protein by parathyroid hormone in MC3T3-E1 osteoblast-like cells involves protein kinase A and extracellular signal-regulated kinase pathways

Inhibition of osteoblast-mediated mineralization is one of the major catabolic effects of parathyroid hormone (PTH) on bone. Previously, we showed that PTH induces matrix gamma-carboxyglutamic acid (Gla) protein (MGP) expression and established that this induction is critical for PTH-mediated inhibition of osteoblast mineralization. In the present study, we focus on the mechanism through which PTH regulates MGP expression in osteoblastic MC3T3-E1 cells. Following transient transfection of these cells with a -748 bp murine MGP promoter-luciferase construct (pMGP-luc), PTH (10 (-7) M) induced promoter activity in a time-dependent manner with a maximal four- to six fold induction seen 6 h after PTH treatment. Both H-89 (PKA inhibitor) and U0126 (MEK inhibitor), suppressed PTH induction of MGP promoter activity as well as the MGP mRNA level. In addition, forskolin (PKA activator) stimulated MGP promoter activity and mRNA levels confirming that PKA is one of the signaling molecules required for regulation of MGP by PTH. Co-transfection of MC3T3-E1 cells with pMGP-luc and MEK(SP), a plasmid encoding the constitutively active form of MEK, led to a dose-dependent increase in MGP promoter activity. Both MGP promoter activity and MGP mRNA level were not affected by the protein kinase C (PKC) inhibitor, GF109203X. However, phorbol 12-myristate 13-acetate (PMA), a selective PKC activator induced MGP mRNA expression through activation of extracellular signal-regulated kinase (ERK). Taken together, these results indicate that PTH regulates MGP via both PKA- and ERK-dependent pathways.     

Repression of 92-kDa type IV collagenase expression by MTA1 is mediated through direct interactions with the promoter via a mechanism,which is both dependent on and independent of histone deacetylation

Although the expression of the metastases-associated gene MTA1 correlates with tumor metastases, its role in regulating type IV collagenase expression is unknown. Enforced MTA1 expression in HT1080 cells reduced basal and 12-myristate 13-acetate-induced 92-kDa type IV collagenase (MMP-9) protein/mRNA levels. DNase I hypersensitivity and PstI accessibility assays revealed multiple regions of the MMP-9 promoter (-650/-450 and -120/+1), showing reduced hypersensitivity in the MTA1-expressing cells. Chromatin immunoprecipitation assays demonstrated MTA1 binding to the distal region, which spans several regulatory cis elements. Co-immunoprecipitation and chromatin immunoprecipitation assay experiments revealed histone deacetylase 2 (HDAC2)-MTA1 protein-protein interactions and the MTA1-dependent recruitment of HDAC2 to the distal MMP-9 promoter region, yielding diminished histone H3/H4 acetylation. However, HDAC2 binding and H3/H4 acetylation at the proximal MMP-9 region were unaffected by MTA1 expression. Furthermore, trichostatin treatment only partially relieved MTA1-repressed MMP-9 expression, indicating a HDAC-insensitive component possibly involv ing the nucleosome-remodeling Mi2 activity, which was recruited to the promoter by MTA1. In summary, (a) MMP-9 adds to a short list of MTA1-regulated genes, which so far only includes c-myc and pS2, and (b) MTA1 binds to the MMP-9 promoter, thereby repressing expression of this type IV collagenase via histone-dependent and independent mechanisms.