Vitamin D and Dexamethasone Inversely Regulate Parathyroid Hormone-Induced Regulator of G Protein Signaling-2 Expression in Osteoblast-Like Cells

Abstract

The PTH/PTHrP receptor stimulates both adenylate cyclase- and phospholipase C-dependent signaling pathways via different G proteins. The biological actions of PTH on bone are modified by steroid hormones. PTH induces expression of regulator of G protein signaling (RGS)-2, a putative preferential inhibitor of G(q)-mediated phospholipase C activation. We investigated whether steroid hormones interfere with PTH signaling by modulating PTH-induced RGS-2 expression in osteoblast-like UMR 106-01 cells. PTH (1-34) rapidly and transiently induced expression of RGS-2 mRNA and protein via the cAMP/protein kinase A pathway within 30 min, with maximal protein abundance after 2 h. PTH-induced RGS-2 preferentially bound to Galpha(q), compared with Galpha(s) protein. 1,25-(OH)(2)D(3) pretreatment enhanced PTH-induced RGS-2 mRNA and protein accumulation, whereas dexamethasone preincubation had an attenuating effect. These effects were due to modulation of the RGS-2 gene transcription rate, which increased by 35% with 1,25-(OH)(2)D(3) and decreased by 63% with dexamethasone pretreatment. RGS-2 mRNA half-life was not affected by either steroid. The transcriptional effects of dexamethasone and 1,25-(OH)(2)D(3) were independent of PTH/PTHrP receptor activation and were not explained by effects on cAMP accumulation, cAMP response element-binding protein expression or phosphorylation, or the abundance of the osteoblast-specific transcription factor core-binding factor alpha (CBFa1/Runx2), a known activator of RGS-2 expression. In conclusion, glucocorticoids and 1,25-(OH)(2)D(3) inversely modulate PTH-induced RGS-2 gene transcription. Regulation of RGS-2 may constitute a novel mechanism by which steroids modulate signaling via the PTH/PTHrP receptor and other G protein-coupled receptors in bone.

Differential expression of IGF system components in proliferating vs. differentiating growth plate chondrocytes: the functional role of IGFBP-5

“…The software provided by the company allowed the quantitative detection of fluorescence by the incorporation of the substance SYBR green into the amplification products. Amplification was performed in the presence of Universal Mastermix (PE Applied Biosystems, Darmstadt, Germany) with SYBR green to detect PCR products at the end of each amplification step, and results were analyzed as previously described (12,18).…”

Mechanisms of Mitogen-Activated Protein Kinase Inhibition by Parathyroid Hormone in Osteoblast-Like Cells

“…After incubation with the substances indicated, cells were washed once with cold PBS and lysed with ice-cold lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100) containing a cocktail of proteinase and phosphatase inhibitors (20 mM NaF, 2 mM EDTA, 1 mM EGTA, 1 mM Na 3 VO 4 , 1 mM PMSF, 10 g/ml leupeptin, 10 g/ml aprotinine, 3 mM benzamidine), vortexed, and centrifuged for 15 min at 15,000 ϫ g. For separation of nuclear and cytoplasmic proteins, we used the commercially available NE-Per Kit (Perbio Sciences, Bonn) and followed the manufacturer's instructions. The protein content of the supernatants was measured by the Bradford method (Protein Assay Kit; BioRad, Munich, Germany), and Western blot test was performed as described previously (16).…”

Signaling mechanisms leading to regulation of proliferation and differentiation of the mesenchymal chondrogenic cell line RCJ3.1C5.18 in response to IGF-I

“…For total cell homogenate cells were incubated with the indicated substances, scraped in 50 ml ice-cold lysis buffer containing a mixture of proteinase and phosphatase inhibitors, and cell extracts were treated as reported previously (Hömme et al 2003, Kiepe et al 2006. Cytosol membrane and nuclear factions of cells were prepared by previously described procedures (Hoeflich et al 2004).…”