Parathyroid Hormone Induces Receptor Activity Modifying Protein-3 (RAMP3) Expression Primarily Via 3′,5′-Cyclic Adenosine Monophosphate Signaling in Osteoblasts

Abstract

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.

The Many Faces of H89: A Review

“…It is wellestablished that the PKA signalling cascade is of major importance for cellular function and accounts for the intense interest in a specific, high affinity inhibitor. A recent survey of the literature showed that H89 has been used extensively for evaluation of the role of PKA in heart muscle (63,80), osteoblasts (41,61), hepatocytes (23,82), smooth muscle cells (24,70), neuronal tissue (42,44), epithelial cells (11,69), to name but a few. No fewer than 1500 articles have appeared on this subject.…”

CBP/p300-Interacting Protein CITED1 Modulates Parathyroid Hormone Regulation of Osteoblastic Differentiation

“…As shown in Fig. 2, CHX pretreatment did not inhibit the increase in CITED1 mRNA induced by 4 h of PTH treatment, whereas CHX enhanced PTH induction of RAMP3 mRNA, as expected (18). Actinomycin D (5 g/ml), an inhibitor of RNA synthesis, completely blocked PTHinduced expression of CITED1 (and RAMP3), indicating that PTH increases CITED1 mRNA expression via an effect on transcription (Fig.…”

“…To determine whether CITED1 might be a PTHinduced primary response gene, Wt9 cells were treated with cycloheximide (CHX; 5 g/ml) for 1 h to inhibit protein synthesis before addition of hPTH(1-34) at 30 nm. The effect on CITED1 mRNA induction was compared with that on receptor activity modifying protein-3 (RAMP3), which we and others have found to be strongly induced in osteoblasts by PTH (13,18). As shown in Fig.…”

Post-transcriptional regulation of the Type IIA sodium-phosphate cotransporter by parathyroid hormone.

“…Activation of both the PKA and PKC pathways has been shown to downregulate expression of Npt2a at the apical membrane in proximal tubule cells 77,143 . Several studies using cell-based and animal-based models have demonstrated that the two signaling pathways both contribute to PTH- 123 used PTH analogues to demonstrate a dominant role for PKA signaling, Carpenter et al 149 showed preservation of the phosphaturic 153 . Given the evidence for downregulation of Npt2a protein expression by PTH, and the sustained cAMP signaling generated by prolonged PTHR1 stimulation, we hypothesize that PKA is the primary mediator of PTH-induced Npt2a mRNA destabilization.…”