α1-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
Introduction
Bone marrow stromal/osteoblast cells induce osteoclastic differentiation and resorptive activity through the expression of the receptor activator of nuclear factor κB ligand (RANKL, also known as ODF, TRANCE or OPGL). RANKL is a member of the tumour necrosis factor (TNF) family and binds to its receptor, RANK, on osteoclasts, thereby initiating a cascade of signalling events that leads to the differentiation and activation of osteoclasts.1, 2, 3, 4, 5, 6 Thus, osteoclast differentiation and activation are affected by RANKL produced by marrow stromal/osteoblast cells.
The adrenergic receptors are a family of G protein-coupled signalling proteins and can be functionally and structurally divided into three main classes, designated as α1-, α2- and β-adrenergic receptors. The α1-adrenergic receptors are generally coupled to stimulation of phospholipase C. The α2- and β-adrenergic receptors are coupled to inhibition of adenylyl cyclase and to activation of adenylyl cyclase, respectively. Furthermore, multiple subtypes have been identified through molecular cloning within each class: three α1-adrenergic receptors (α1a, α1b and α1d), three α2-adrenergic receptors (α2A, α2B and α2D) and three β-adrenergic receptors (β1, β2 and β3).7 It has been demonstrated that osteoblastic cells are equipped with adrenergic receptors, suggesting the regulation of bone metabolism by the adrenergic receptors.8, 9, 10, 11 Togari et al.9, 11 investigated the expression of adrenergic receptor subtype genes in human periosteum-derived (SaM-1) and osteosarcoma-derived osteoblastic cells (SaOS-2, HOS and MG-63) by RT-PCR, and showed that α1b-, α2B- and β2-adrenergic receptor subtype genes were expressed in SaM-1 and HOS cells, and that α1b-, α2A- and β2-subtype mRNAs were expressed in SaOS-2 and MG-63 cells. Kellenberger et al.10 examined the expression of β-adrenergic receptor subtype genes in four different human osteosarcoma cell lines, SaOS-2, TE-85, MG-63 and OHS-4 cells, and in primary human osteoblast cultures by Northern blot and RT-PCR analyses, and showed that β1-subtype expression was found in SaOS-2, TE-85 and OHS-4, but not in MG-63 cells and primary osteoblast cultures, and that β2-subtype gene was expressed in MG-63, SaOS-2, TE-85 cells and primary osteoblast cultures, but not in OHS-4 cells. Moore et al.8 reported that adrenergic receptor agonists stimulate bone resorption in neonatal mouse calvariae in organ culture. These findings led us to suppose that the stimulation of bone resorption by adrenergic receptor agonists may possibly be mediated through the expression of RANKL in the osteoblasts. However, little information is available on the expression of RANKL mRNAs by adrenergic receptor stimulation in the osteoblasts. In this study, we investigated the expression of α1-adrenergic receptor subtype genes and the effect of α1-adrenergic receptor agonist on the mRNA expression of RANKL in mouse calvariae-derived MC3T3-E1 osteoblast-like cells, using the RT-PCR method. We found that α1-adrenergic receptor stimulation induced RANKL gene expression in the cells. Furthermore, our results demonstrated that PKC and ERK signalling pathways were involved in the α1-adrenergic receptor agonist-induced RANKL mRNA expression in MC3T3-E1 cells.
Section snippets
Materials
Phenylephrine (α1-agonist), prazosin (α1-antagonist), yohimbine (α2-antagonist), propranolol (β-antagonist), bisindolylmaleimide I hydrochloride (GF109203X, PKC inhibitor), phorbol 12-myristate 13-acetate (PMA, PKC activator) and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059, ERK kinase inhibitor) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Anti-ERK and anti-phospho-ERK rabbit polyclonal antibodies were purchased from Cell Signalling Technology (Beverly, MA, USA).
Expression of α1-adrenergic receptor subtype genes
We examined the expression of α1-adrenergic receptor subtype genes in MC3T3-E1 cells by RT-PCR. In three α1-adrenergic receptor subtype mRNAs, α1b- and α1d-subtypes were expressed in MC3T3-E1 cells at 26 cycles, but α1a-subtype mRNA was not detected at 40 cycles (Fig. 1).
Induction of RANKL mRNA expression by α1-adrenergic receptor stimulation
We investigated the effect of phenylephrine (α1-agonist) on the expression of RANKL mRNA in MC3T3-E1 cells by semi-quantitative RT-PCR. As shown in Fig. 2A, phenylephrine rapidly increased RANKL mRNA expression, its expression
Discussion
Osteoblastic cells have been demonstrated to express the adrenergic receptor.8, 9, 10, 11 Togari 11 investigated the expression of adrenergic receptor subtype genes in human periosteum-derived (SaM-1) and osteosarcoma-derived osteoblastic cells (SaOS-2, HOS and MG-63) by RT-PCR. Their findings showed that the α1b-adrenergic receptor subtype gene was expressed in all these cells, but α1a- and α1d-subtype mRNAs were not detected. In the present study, we examined the expression of α1-adrenergic
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