Comparative Study Of Arip1 And ARIP2 Actions On Activities Of Neuro-2a Cells

Activin A as a multifunctional cytokine of the transforming growth factor-β(TGF-β)superfamily has neurotrophic and neuroprotective effects on neurons.Activin receptor-interacting proteins(ARIPs)are a group of regulatory proteins of activin signaling pathway.In previous study,we found that ARIP1 was mainly expressed in nerve cells,while ARIP2 was widely distributed in brain,pituitary,adrenal gland,pancreas and other tissues,and ARIP1 and ARIP2 could be co-expressed in nerve cells of mouse brain.Although ARIP1 and ARIP2 are both negative regulatory proteins of activin signaling,any possible biological differences in the same nerve cell remain to be identified.Therefore,mouse neuroblastoma Neuro-2a cells were used to compare the effects of ARIP1 and ARIP2 on Neuro-2a cells by fluorescence-labeled ARIP1 and ARIP2 expression vector in this study.1.Methods:Green fluorescence-labeled ARIP1 and red fluorescence-labeled ARIP2 expression plasmids were constructed.MTT assay was used to determine proliferation of Neuro-2a cells,hoechst staining and flow cytometry were used to examine apoptosis of ARIP1 and ARIP2 overexpressed Neuro-2a cells.Giemsa staining was used to observe the morphologiy of Neuro-2a cells,and transwell chamber and RTCA were performed to assay the migration and adhesion of Neuro-2a cells.Smads m RNA and protein expression levels in Neuro-2a cells were examined by RT-PCR and Western blotting.2.Results:(1)Activin A had no obvious effect on the secretion of nitric oxide from Neuro-2a cells and proliferation of Neuro-2a cells,but increased the migration of Neuro-2a cells.(2)RT-PCR and immunocytochemical staining showed that ARIP1 and ARIP2 were expressed in Neuro-2a cells.After treatment with activin A,the expression of ARIP1 was significantly increased,and ARIP2 was slightly increased in Neuro-2a cells.(3)ARIP1 overexpression inhibited proliferation of Neuro-2a cells and promoted apoptosis of Neuro-2a cells,while ARIP2 overexpression had no significant effect on proliferation and apoptosis of Neuro-2a cells.ARIP1-overexpressed Neuro-2a cells become morphologically rounded,while ARIP2-overexpressed Neuro-2a cells indicate more irregular in shape.(4)Both ARIP1 and ARIP2 overexpression promoted Neuro-2a cell adhesion,and downregulated the inhibitory effect of activin A on Neuro-2a cell adhesion.(5)Both ARIP1 and ARIP2 overexpression downregulated the expression level of Smad3 protein significantly.3.Conclusion:In summary,ARIP1 and ARIP2 were expressed in Neuro-2a cells,and overexpression of ARIP1 and ARIP2 inhibited cell migration,but promoted cell adhension of Neuro-2a cells.ARIP1 and ARIP2 may inhibit the classical activin/Smad signaling transduction pathway by downregulating Smad3 expression,but the detailed mechanism needs to be further explored.This study revealed the differences of the roles and mechanisms of ARIP1 and ARIP2 in regulation of neural cell activity,which laid a research foundation for further search for targets for regulation of neural cell activity.