Comparison Of ARIP1 And ARIP2 Expression And Role In Neuro-2a Cells

Activin, a member of transforming growth factor-β superfamily, has various functions in the different cells. There is a new group of activin signaling proteins in cells, which are named as activin receptor-interacting proteins(ARIPs). ARIP1 and ARIP2 belong to the negative regulators of activin-induced signaling transduction. Previous studies have demonstrated that ARIP1 m RNA and protein are expressed in brain and ARIP2 m RNA and protein exist widely in various tissues. Our previous studies found that ARIP1 and ARIP2 are co-expressed in some nerve cells. However, the diversity of ARIP1 and ARIP 2 expression and function in the nerve cells are not well understood. On the other hand, our data have revealed that activin A can promote the release of 5-HT. However, the role of activin A in releasing other neurotransimitter is unclear. Therefore, this study was to investigate the expression and role of ARIP1 and ARIP2 in the primary cultured hippocampal neurons and Neuro-2a cells.1. Expression of ARIP1 and ARIP2 in the cultured hippocampal neurons ofmouse.The hippocampal neurons were cultured with neuron conditional medium, and incubated with actvin A for 12 h and 24 h. The results showed that activin A could promote expression of ARIP1 and ARIP2 m RNA in hippocampal neurons at 12 h. ARIP1 m RNA expression level was decreased obviously at 24 h, while ARIP2 m RNA expression was still higher than that of the control group.2. Comparison of ARIP1 and ARIP2 expression and role in Neuro-2a cells2.1 Expression of ARIP1 and ARIP2 in Neuro-2a cells.Firstly Neuro-2a cells were stimulated with activin A for 12 h and 24 h, respectively. It was found that activin A could increase the ARIP1 and ARIP2 m RNA expression at 12 h, but ARIP1 m RNA expression was decreased obviously at 24 h. Further studies showed that LPS could increase the expression of ARIP1 and ARIP2 m RNA, PMA could increase the expression of ARIP2 m RNA, but have no effect on ARIP1 m RNA; NGF could decrease the expression of ARIP1 m RNA, but did not alter the expression of ARIP2 m RNA.2.2 Expressions of Act RII and Smads m RNA in ARIP1, 2 overexpressing Neuro-2a cells.Neuro-2a cells were transfected with ARIP1 and ARIP2 overexpressing plasmids respectively. The results indicated that ARIP1 overexpressing could decrease Smad3,4 and, Act RIIA m RNA expression on cultured Neuro-2a cells, There was no effect on ARIP2 m RNA. ARIP2 overexpressing could significantly reduce the expression of ARIP1, At the same time it could inhibit the expression of smad3,4 and Act IIRA m RNA. Western blotting result showed that both ARIP1 and ARIP2 overexpressing could decrease the levels of both Smad3 and p-Smad3. Flow cytometry results showed that both ARIP1 and ARIP2 overexpressing could decrease the expression of Act RIIA and Act RIIB on neuro-2a surface.2.3 Effects of ARIP1 and ARIP2 overexpression on morphology and viability of Neuro-2a cells.Neuro-2a cells have the ability of differentiation and proliferation. Firstly, Neuro-2a cells were transfected with ARIP1 and ARIP2 overexpressing plasmids respectively, The results showed that ARIP1 overexpressing could decrease the cell viability, while ARIP2 overexpressing could increase the cell viability, both have no statistical difference. Overexpression of ARIP1 has no significant effect on the morphology of Neuro-2a cells, however irregular protruding cells increased significantly in the overexpressing of ARIP2.3. Effects of ARIP1, 2 overexpression on neurotransmitters release of Neuro-2a cells.In this research, we first detected the neurotransmitters. ELISA results showed that activin A could enhance significantly the levels of 5-HT, GABA, Glu in the supernatant of cultured Neuro-2a cells, while the level of Ach decreased. Either ARIP1 or ARIP2 overexpression could inhibit the levels of 5-HT, GABA, while there was no effect on the secretion of Ach.In conclusion, ARIP1 and ARIP2 are co-expressed in neurons cells, but expression dynamics and roles are different. Activin A can not only promote the release of 5-HT, but also can promote the secretion of GABA and Glu. Therefore, this study is contributed to further explaining the activin A in neurotrophic and neuroprotective effects and signal transduction mechanisms.