α 1-Adrenergic receptor stimulation induces the expression of receptor activator of nuclear factor κB ligand gene via protein kinase C and extracellular signal-regulated kinase pathways in MC3T3-E1 osteoblast-like cells

Abstract

The receptor activator of nuclear factor κB ligand (RANKL) produced by bone marrow stromal/osteoblast cells is a crucial regulator of osteoclastgenesis and bone resorption. Osteoblastic cells have been demonstrated to express α 1-adrenergic receptors. Objective The purpose of this study was to test the hypothesis that α 1-adrenergic receptor stimulation induces the expression of RANKL gene via protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways in osteoblastic cells. Design The steady-state mRNA levels of RANKL and activation of ERK in mouse MC3T3-E1 osteoblast-like cells were analyzed by semi-quantitative RT-PCR and Western blotting, respectively. Results In three α 1-adrenergic receptor subtype mRNAs, α 1b- and α 1d-subtypes were expressed in MC3T3-E1 cells. The mRNA levels of RANKL were increased by phenylephrine (α 1-agonist) in time- and dose-dependent manners. Prazosin (α 1-antagonist) suppressed the phenylephrine-induced RANKL mRNA expression, but yohimbine (α 2-antagonist) and propranolol (β-antagonist) did not. Phorbol 12-myristate 13-acetate (PMA, PKC activator) increased RANKL mRNA expression and GF109203X (PKC inhibitor) suppressed the phenylephrine-induced RANKL mRNA expression. Both phenylephrine and PMA stimulated the phosphorylation of ERK, while both prazosin and GF109203X inhibited phenylephrine-induced ERK activation. Pretreatment with PD98059 (ERK kinase inhibitor) inhibited both the phosphorylation of ERK and the expression of RANKL gene induced by phenylephrine in MC3T3-E1 cells. Conclusion These results show that α 1b- and α 1d-adrenergic receptor subtype genes are expressed and the expression of RANKL mRNA may be regulated by α 1-adrenergic receptor stimulation in osteoblastic cells. The induction of RANKL mRNA by activating the α 1-adrenergic receptor is probably mediated via PKC and ERK signalling pathways in osteoblastic cells.