Analysis of regulator of G-protein signaling-2 (RGS-2) expression and function in osteoblastic cells.
Regulator of G-protein signaling-2 (RGS-2) belongs to a novel family of GTPase-activating proteins that rapidly turn-off G-protein coupled receptor signaling. RGS proteins contain a characteristic RGS domain by which they interact with the alpha-subunit of G-proteins and drive them into their inactive GDP-bound forms. Previously, we have reported that RGS-2 mRNA is rapidly and transiently increased by PTH in rat bone and in osteoblast cultures in vitro. In this study, we further explored the molecular basis for the regulation of RGS-2 by cloning and functionally characterizing the RGS-2 gene promoter. We cloned 2.3- and 2.8-kb fragments of the 5'-flanking regions of the rat and mouse RGS-2 genes, respectively, and generated a stable clone of UMR106 osteoblastic cells containing the rat RGS-2 promoter driving the beta-gal reporter gene (p2.3RGS-2-beta-gal). Treatment of the stable clone with PTH resulted in a maximal 2.2- to 3.6-fold increase in promoter activity at 8 h, reminiscent of the early response observed with endogenous RGS-2 mRNA regulation. Further, PTH (1-38), (1-31), PTHrP (1-34), and forskolin, which elevate cAMP levels, stimulated the promoter, while PTH (3-34) and (7-34), which do not readily stimulate cAMP accumulation, and PMA that directly activates protein kinase C, had no effect on promoter activity. Taken together, these results implicate the involvement of the Galpha(s)-adenylate cyclase-protein kinase A pathway in stimulating RGS-2 expression. Maintenance of a hyperphosphorylated state via the inhibition of type 2A protein phosphatases by okadaic acid, resulted in a strong dose-dependent increase in transcriptional activity of the RGS-2 promoter as well as that of the endogenous RGS-2 gene. Furthermore, overexpression of the osteoblast-specific transcription factor Runx2 also led to a stimulation of RGS-2 promoter activity. Functional analysis using RGS-2 overexpression suggests the potential negative regulatory effects of RGS-2 on PTH- and forskolin-induced cAMP production in osteoblastic cells. In summary, our data suggest that PTH treatment results in a direct transcriptional stimulation of RGS-2 that in turn may play a role in modulating the duration/intensity of PTH receptor signaling.
Thirunavukkarasu
Kannan
K
Gene Regulation, Bone and Inflammation Research, Lilly Research Labs, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
Halladay
David L
DL
Miles
Rebecca R
RR
Geringer
Chad D
CD
Onyia
Jude E
JE
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D050659:Core Binding Factor Alpha 1 Subunit; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D019319:Okadaic Acid / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011401:Promoter Regions, Genetic / Q000187:drug effects; D020710:RGS Proteins / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D050659:Core Binding Factor Alpha 1 Subunit; D018076:DNA, Complementary; D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D010446:Peptide Fragments; D020710:RGS Proteins; C105345:Rgs2 protein, mouse; D014157:Transcription Factors; C035747:parathyroid hormone (1-38); D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D001616:beta-Galactosidase