|
Study of P2X4 receptor function during in vitro neuronal differentiation of P19 and PC12 cells through gene knockdown induced by RNAi
Lopes, C. G. ; Trujillo, C.A. ; Resende, R.R. ; Silva, R.L. ; Ulrich, H
Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil.
The development of the nervous system of vertebrates depends on the development and growth of neuroepithelial cells accordant to the temporal standard of gene expression. This process is heavily controlled and involves the action of differentiation factors as peptides, proteins, and receptors that induce morphological and molecular changes, originating a functional nervous system. Purinergic receptors, activated by ATP and other nucleotides, contain P2Y receptors, which transmit their cellular response by G-protein activation, and P2X receptors composed of three receptor subunits, which are ligand-gated ion channels. From the seven members of a structurally related protein family (P2X1-7), P2X2, P2X4, and P2X6 subunits predominately form functional receptors in the brain.
Using P19 embryonal carcinoma and PC12 pheochromocytoma cells as in vitro models for neuronal differentiation, we detected gene and protein expression at the onset of the differentiation process. Following induction of differentiation, P2X4 receptor expression decreases during neuronal differentiation of P19 and PC12 cells, as observed by RT-PCR experiments. RNAi (RNA interference) experiments were performed to generate a knockdown of gene expression of the P2X4 receptor in an attempt to deduce its function during neuronal differentiation. Interfering sequences siRNA 21 and siRNA 32 were chosen from the complete codifying sequence for the P2X4 receptor. Cells transiently transfected with these siRNA showed an evident reduction in mRNA and protein concentration of P2X4 receptors, compared to cells transfected with scrambled siRNA, in which no silencing of gene expression occurred. Patch-clamp whole-cell recording experiments performed in PC12 cells transfected with the interfering sequences revealed decreased whole-cell currents in the presence of ATP compared to untransfected control cells. The fact that the same percentage of reduction in whole-cell currents was obtained in the presence of alpha,beta-methylen-ATP which only inhibits P2X4 receptor function, indicates the specificity of P2X4 receptor mRNA down regulation in the presence of RNAi. Down regulation of P2X4 receptor gene expression in the onset of differentiation resulted in a reduction of the size of embryonic bodies which are prerequisites for further neuronal differentiation. During later differentiation neurite outgrowth was affected, indicating principle functions of P2X4 receptors during neuronal differentiation
Supported by FAPESP, CNPq and CAPES
|