Alpha1-adrenergic receptor stimulation induces the expression of receptor activator of nuclear factor kappaB ligand gene via protein kinase C and extracellular signal-regulated kinase pathways in MC3T3-E1 osteoblast-like cells.
The receptor activator of nuclear factor kappaB 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 alpha(1)-adrenergic receptors.


OBJECTIVE
The purpose of this study was to test the hypothesis that alpha(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.


METHODS
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 alpha(1)-adrenergic receptor subtype mRNAs, alpha(1b)- and alpha(1d)-subtypes were expressed in MC3T3-E1 cells. The mRNA levels of RANKL were increased by phenylephrine (alpha(1)-agonist) in time- and dose-dependent manners. Prazosin (alpha(1)-antagonist) suppressed the phenylephrine-induced RANKL mRNA expression, but yohimbine (alpha(2)-antagonist) and propranolol (beta-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.


CONCLUSIONS
These results show that alpha(1b)- and alpha(1d)-adrenergic receptor subtype genes are expressed and the expression of RANKL mRNA may be regulated by alpha(1)-adrenergic receptor stimulation in osteoblastic cells. The induction of RANKL mRNA by activating the alpha(1)-adrenergic receptor is probably mediated via PKC and ERK signalling pathways in osteoblastic cells.
Nishiura
Toshihiro
T
Section of Biochemistry, Department of Functional Bioscience, Fukuoka Dental College, 2-15-1 Tamura, Sawara-ku, Fukuoka 814-0193, Japan. nishiura@college.fdcnet.ac.jp
Abe
Kimio
K
D016475:3T3 Cells; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*; D005419:Flavonoids / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology / Q000378:metabolism*; D010656:Phenylephrine / Q000494:pharmacology; D011224:Prazosin / Q000494:pharmacology; D011433:Propranolol / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000096:biosynthesis* / Q000187:drug effects; D018340:Receptors, Adrenergic, alpha-1 / Q000187:drug effects / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015016:Yohimbine
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D000319:Adrenergic beta-Antagonists; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007211:Indoles; D008301:Maleimides; D053246:Receptor Activator of Nuclear Factor-kappa B; D018340:Receptors, Adrenergic, alpha-1; D010656:Phenylephrine; D015016:Yohimbine; D011433:Propranolol; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D011224:Prazosin