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In osteoblasts parathyroid hormone (PTH) stimulates the PTH/PTH-related peptide (PTHrP) receptor (PTH1R) that couples via Gs to adenylyl cyclase stimulation and via G11 to phospholipase C (PLC) stimulation. We have investigated the effect of increasing G11a levels in UMR 106-01 osteoblastic cells by transient transfection with cDNA encoding G11a on PTH stimulation of PLC and protein kinase C (PKC) as well as PTH regulation of mRNA encoding matrix metalloproteinase-13 (MMP-13). Transfection with G11a cDNA resulted in a 5-fold increase in PTH-stimulated PLC activity with no change in PTH-stimulated adenylyl cyclase. PTH-induced translocation of PKC-I, -d, and -? to the cell membrane and PKC-? to the nucleus was also increased. Increased G11a protein resulted in increased stimulation of MMP-13 mRNA levels at all doses of PTH. There was a 2.5??0.35 fold increase in maximal PTH-stimulation of c-jun mRNA and smaller but significant increases in c-fos accompanied by increased basal and PTH-stimulated AP-1 binding in cells expressing increased G11a. Runx-2 mRNA and protein levels were not significantly increased by increased G11a expression. The increase in PTH stimulation of c-jun, c-fos, and MMP-13 in G11a-transfected cells were all blocked by bisindolylmaleimide I, a selective inhibitor of PKC. These results demonstrate that regulation of the PLC pathway through the PTH1R is significantly increased by elevating expression of G11a in osteoblastic cells. This leads to increased PTH stimulation of MMP-13 expression by increased stimulation of AP-1 factors c-jun and c-fos. 2005 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Indian hedgehog (Ihh) is produced by growth plate pre-hypertrophic chondrocytes, and is an important regulator of endochondral ossification. However, little is known about the regulation of Ihh in chondrocytes. We have examined the role of integrins and mitogen-activated protein (MAP) kinases in Ihh mRNA regulation in CFK-2 chondrocytic cells. Cells incubated with the 1-integrin blocking antibody had decreased Ihh mRNA levels, which was accompanied by decreases of activated extracellular signal-regulated kinases (ERK1/2) and activated p38 MAPK. Ihh mRNA levels were also inhibited by U0126, a specific MEK1/2 inhibitor, or SB203580, a specific p38 MAPK inhibitor. Cells transfected with constitutively active MEK1 or MKK3 had increased Ihh mRNA levels, which were diminished by dominant-negative MEK1, p38a or p38. Stimulation of the PTH1R with 10-8 M rPTH (134) resulted in dephosphorylation of ERK1/2 that was evident within 15 min and sustained for 1 h, as well as transient dephosphorylation of p38 MAPK that was maximal after 25 min. PTH stimulation decreased Ihh mRNA levels, and this effect was blocked by transfecting the cells with constitutively active MEK1 but not by MKK3. These studies demonstrated that activation of ERK1/2 or p38 MAPK increased Ihh mRNA levels. Stimulation of the PTH1R or blocking of 1-integrin resulted in inhibition of ERK1/2 and p38 MAPK and decreased levels of Ihh mRNA. Our data demonstrate the central role of MAPK in the regulation of Ihh in CFK-2 cells. 2004 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]