Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3',5'-monophosphate-mediated pathway in primary neonatal murine osteoblasts.
Parathyroid hormone (PTH) is a promising anabolic agent for the treatment of osteoporosis. However, PTH is also potently catabolic. To help delineate the molecular mediators of PTH's opposing effects on skeletal metabolism, we have examined PTH-induced regulator of G-protein signaling-2 (RGS-2) expression and function in murine osteoblasts. RGS proteins are GTPase-activating proteins (GAPs) that regulate GTP-binding protein-coupled receptor (GPCR) signaling by enhancing the intrinsic GTPase activity of Galpha subunits. We found that 10 nmol/L PTH maximally induced RGS-2 mRNA in murine MC3T3-E1 cells, rat Py1a and ROS-17/2.8 cells, primary mouse osteoblasts (MOB cells), and mouse calvariae organ culture at 1-2 h posttreatment. PTH signaling through its receptor, PTHR1, is coupled to cAMP-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling pathways. We examined the effect of selective signaling agonists and antagonists on RGS-2 expression in MOB cells to determine which pathway(s) mediates PTH-induced RGS-2 expression. Although selective activation of all three pathways led to RGS-2 expression, cAMP-PKA activation with 10 nmol/L PTH and 10 micromol/L forskolin elicited the strongest induction. Similarly, RGS-2 mRNA expression was most strongly inhibited by the PKA inhibitor, H89 (10-30 micromol/L). The phorbol ester, PMA (1 micromol/L), which activates the PKC pathway, and ionomycin (1 micromol/L), which activates the calcium pathway, produced small but detectable elevations in RGS-2 mRNA levels. Overnight treatment with 1 micromol/L PMA to deplete PKC did not affect subsequent RGS-2 induction by PTH, but significantly inhibited PMA-induced RGS-2 expression. Treatment with 1-100 nmol/L PTH(3-34), which does not activate cAMP-PKA signaling, did not induce RGS-2 expression. MOB cells pretreated with 3 microg/mL cycloheximide produced sustained RGS-2 mRNA levels 2 h after 10 nmol/L PTH treatment. Actinomycin D (5 microg/mL) completely blocked 10 nmol/L PTH-induced RGS-2 expression. Finally, we tested the effect of RGS-2 overexpression on PTH- and fluprostenol-induced interleukin (IL)-6 promoter activity in MOB cells. PTH induces IL-6 through PKA activation, whereas fluprostenol induces IL-6 through PKC activation. We found that RGS-2 overexpression significantly inhibited IL-6 promoter activity following fluprostenol treatment, but not following PTH treatment. We conclude that RGS-2 is a PTH-induced primary response gene in murine osteoblasts that is induced mainly through the cAMP-PKA pathway and specifically inhibits Galphaq-coupled receptors.
Tsingotjidou
A
A
Division of Diagnostic and Surgical Sciences, UCLA School of Dentistry, Los Angeles, CA 90095-1668, USA.
Nervina
J M
JM
Pham
L
L
Bezouglaia
O
O
Tetradis
S
S
D000818:Animals; D000831:Animals, Newborn; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D015850:Interleukin-6 / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D051379:Mice; D009924:Organ Culture Techniques; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000502:physiology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020710:RGS Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D018016:Receptors, Parathyroid Hormone / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D012886:Skull / Q000166:cytology / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D015850:Interleukin-6; D010281:Parathyroid Hormone; D011500:Protein Synthesis Inhibitors; D020710:RGS Proteins; D012333:RNA, Messenger; D018016:Receptors, Parathyroid Hormone; D011982:Receptors, Prostaglandin; C105345:Rgs2 protein, mouse; D003513:Cycloheximide; D000242:Cyclic AMP; D008156:Luciferases