Parathyroid hormone induces receptor activity modifying protein-3 (RAMP3) expression primarily via 3',5'-cyclic adenosine monophosphate signaling in osteoblasts.
Parathyroid hormone (PTH) has significant anabolic and catabolic effects on bone. We hypothesize that PTH-induced primary response genes are important determinants of osteoblast function. PTH induces osteoblastic gene expression through PTHR1, a heptahelical receptor that triggers cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA), protein kinase C (PKC), and calcium signaling. By using representational difference analysis we found that receptor activity modifying protein-3 (RAMP3) is a PTH-induced primary response gene in osteoblastic cells. RAMP3 is a coactivator that directs calcitonin receptor (CTR) and CTR-like receptor (CRLR) glycosylation, trafficking, and ligand-binding specificity. Our purpose was to characterize PTH-induced RAMP3 messenger ribonucleic acid (mRNA) levels in primary mouse osteoblasts (MOBs) and to determine which signaling pathway mediates this effect. 10 nM PTH maximally induced RAMP3 mRNA levels in MOBs at 4 hours. Protein synthesis inhibition with 3 microg/mL cycloheximide did not affect PTH-induced RAMP3 mRNA levels. Selective activation of cAMP-PKA signaling with, 10 microM forskolin (FSK) and PKC signaling with 1 microM phorbol 12-myristate 13-acetate (PMA) significantly increased RAMP3 mRNA levels, whereas 1 microM ionomycin (a calcium ionophore) had no effect. Pretreatment with 30 microM H89, a PKA inhibitor, significantly blocked PTH- and FSK-induced RAMP3 mRNA levels. Pretreatment with 1 microM PMA, which depletes PKC, had no effect on PTH- and FSK-induced RAMP3 mRNA levels but blocked PMA-induced RAMP3 mRNA levels. 100 nM PTH (3-34), which activates PKC and calcium but not PKA, had no effect on RAMP3 mRNA levels. These findings indicate that RAMP3 is a PTH-induced primary response gene in primary MOBs and that PTH regulates RAMP3 gene expression primarily through the cAMP-PKA pathway.
Phelps
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Section of Orthodontics, UCLA School of Dentistry, Los Angeles, CA 90095-1668, USA.
Bezouglaia
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O
Tetradis
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Nervina
J M
JM
D000818:Animals; D015152:Blotting, Northern; D002417:Cattle; D000242:Cyclic AMP / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects*; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D015398:Signal Transduction; D012886:Skull / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019379:Teriparatide / Q000494:pharmacology*; D013997:Time Factors
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C545507:Ramp3 protein, mouse; D058264:Receptor Activity-Modifying Protein 3; D058261:Receptor Activity-Modifying Proteins; D019379:Teriparatide; D000242:Cyclic AMP