Study Of Activin Signal Transduction On Macrophages Mediated By Activin Receptor Interacting Protein 2

Activin is a pluripotent growth and differentiation factor which belongs to the transforming growth factor-( (TGF-() superfamily. Two types of activin receptor have been identified, typeⅠandⅡ, both of which belong to the family of serine/threonine kinase receptors. Activin binds specifically to the typeⅡreceptor, resulting in the activation of the typeⅠreceptor. Then, the typeⅠreceptor interacts with intracellular signaling molecules, Smads, which regulate transcription of selected genes in a cell-specific manner. Recent investigations have suggested that activin-signal transduction by serine/ threonine kinase receptors is controlled by typeⅡreceptors by interacting with activin receptor interacting proteins(ActRIPs). Although different function to ActRIPs, all of ActRIPs can interact with the COOH-terminus of ActRⅡA. ARIP1 is a scaffolding protein that interacted with Smad3 as well as with ActRⅡA, and suppresses intercellular signal transduction mediated by Smad pathway. After interacting with ActRⅡA, ARIP2 also mediates endocytosis of ActRⅡA through the COOH-terminal region, and inhibits activin receptor signaling. To contrast, ARIP3 can increase activin-mediated signal transduction. By the observation of ActRIPs, the regulatory mechanism of signal transduction mediated by Ser/THr kinase receptor will be understood more clearly.On the basis of discovering and cloning the new gene of ActRIP2, the study clarified that the character of the interaction between ActRIP2 and activin receptor, the role of cellular signal transduction of activin receptor mediated by ActRIP2 in the macrophages, and confirmed that ActRIP2 is a important protein of signal transduction, which mediates activin response. It was further revealed that activin as a anti-inflammtory fator may have pro-or anti- function of regulation in macrophage. It will be estibalished the basis of theory and experiment that activin is applied the treatment of inflammatory diseases related with macrophages.Part one: Cloning, expression and characterization of a novel activin receptor-interacting protein2 geneTo study the interactions between the activin receptors and cytoplasmic proteins, the complete cDNA clone of a novel mouse activin receptor-interacting protein 2(ARIP2) that interacts with the activin type IIA receptor (ActRIIA) was isolated from a mouse brain cDNA library. ARIP2 had one activin receptor-interacting domain (ARID) in the NH2-terminal region and interacted with ActRIIA through the ARID by using Pull-down assay. ARIP2 can recognize the consensus sequence of the COOH-terminal residues of ActRIIA(SSL) by using immunoprecipitation analysis. Interestingly, the mRNA of ARIP2 was widely expressed in tissues by using Northern blot and RT-PCR analysis, for example that it was more abundant in the heart, liver, kidney, testis and pituitary tissues. However ARIP1 was more abundant only in the brain. The molecular weight of mature ARIP2 identified by western blot is about 26 000 in brain and liver. These findings suggest that ARIPs have a significant role in regulating signal transduction in various tissue cells. Part two: Effect of activin on secretion of IL-Iβ and NO in macrophages I．Activin enhances secretion of IL-Iβ and NO in non-activated macrophages.Masahiro Ohguchi revealed that activin inhibits sectetion of IL-Iβ in human monocytes activated with LPS, however, the effect of activin on sectetion of IL-Iβ in non-activated macrophages remains to be clarified. To investigate the effect of activin on secretion of IL-Iβ in mouse peritoneal macrophages, we examined the secretion of IL-Iβ in the culture supernatant of mouse peritoneal macrophages stimulated with activin. The results showed that a physiologic concentration of activin inhibited sectetion of IL-Iβ in mouse peritoneal macrophages activated with LPS; more importantly, activin in the same concentration stimulated sectetion of IL-Iβ in non-activated macrophages in the dose-dependent manner. RT-PCR analysis also revealed that non-activated macrop...