RESUMO
The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the glucagon/vasoactive intestinal peptide (VIP) superfamily, stimulates cyclic AMP accumulation initiating a variety of biological processes such as: neurotropic actions, immune and pituitary function, learning and memory, catecholamine biosynthesis and regulation of cardiopulmonary function. Both osteoclasts and osteoblasts have been shown to express receptors for PACAP/VIP implicated in their role in bone metabolism. To further understand the role of PACAP/VIP family in controlling bone metabolism, we investigated differentiation model of MC3T3-E1 cells, an osteoblastic cell line derived from mouse calvaria. Quantitative RT-PCR analysis demonstrated that MC3T3-E1 cells expressed only VPAC2 receptor and its expression was upregulated during osteoblastic differentiation, whereas VPAC1 and PAC1 receptors were not expressed. Consistent with expression of receptor subtype, both PACAP and VIP stimulate cAMP accumulation in a time- and dose-dependent manner with the similar potency in undifferentiated and differentiated cells, while Maxadilan, a specific agonist for PAC1-R, did not. Furthermore, downregulation of VPAC2-R by siRNA completely blocked cAMP response mediated by PACAP and VIP. Importantly, PACAP/VIP as well as forskolin markedly suppressed the induction of alkaline phosphatase mRNA upon differentiation and the pretreatment with 2',5'-dideoxyadenosine, a cAMP inhibitor, restored its inhibitory effect of PACAP. We also found that PACAP and VIP stimulated IL-6 release, a stimulator of bone resorption, and VPAC2-R silencing inhibited IL-6 production. Thus, PACAP/VIP can activate adenylate cyclase response and regulate IL-6 release through VPAC2 receptor with profound functional consequences for the inhibition of osteoblastic differentiation in MC3T3-E1 cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , AMP Cíclico/farmacologia , Interleucina-6/metabolismo , Osteoblastos/citologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Peptídeo Intestinal Vasoativo/farmacologia , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genéticaRESUMO
Although the neuropeptide Y (NPY) family has been demonstrated to control bone metabolism, the role of pancreatic polypeptide (PP), which has structural homology with NPY and peptide YY (PYY) to share the NPY family receptors, in peripheral bone tissues has remained unknown. In the present study, we studied the regulatory roles of PP and its Y receptors using MC3T3-E1 cells, a murine transformed osteoblastic cell line, as a model for osteoblastic differentiation. We found that (1) PP mRNA was detected and increased during cell-contact-induced differentiation in MC3T3-E1 cells; (2) the immunoreactivity of PP was detected by radioimmunoassay and increased in culture medium during differentiation; (3) all the types of NPY family receptor mRNAs (Y1, Y2, Y4, Y5, and y6) were found to increase during differentiation; (4) PP stimulated differentiation in MC3T3-E1 cells in terms of ALP mRNA and BMP-2 mRNA. These findings suggested that MC3T3-E1 cells produce and secrete PP, which may in turn stimulate the differentiation of MC3T3-E1 through its specific receptors in an autocrine manner.
