Expression And Locolization Of Activin Receptor-interacting Protein 1, 2 In Mouse Brain

Activin A, a member of the transforming growth factor-beta (TGF-β) superfamily, was named because it promote the secretion of pituitary follicle stimulating hormone (FSH). It can be secreted by Th2 cells, macrophages / microglial cells, also known as nerve cell survival molecule.In this study, the gene expression vector of activin receptor-interacting protein 1, 2 were constructed and anti-ARIP1, 2 antibodies were prepared. The distribution of ARIP1, 2 mRNA were analyzed by Northern hybridization in mouse tissuses, the results showed that expression of ARIP2 is widely, but the expression of ARIP1 was only in brain. The results of RT-PCR further showed that ARIP1 mRNA expressed in the brain stem, brain, cerebellum, pituitary, hypothalamus, testis, adrenal gland, and ARIP2 expressed in various tissues. The results of immunohistochemical staining showed that the ARIP1, 2 immunoreactivities were detected in the cerebral cortex, hippocampus, hypothalamus, choroid, pituitary and Purkinje cells. The localizations of ARIP1, 2 are the same with activin type IIA receptor (ActRIIA). In order to investigate bioactivities of ARIP1, 2, Neuro-2a cells were used to test the effects of ARIP1 on activin signaling. Then Neuro-2a cells were transfected with pcDNA3-ARIP1 or pcDNA3-ARIP2 together with a reporter plasmid CAGA-lux, then luciferase activity was measured. Both ARIP1 and ARIP2 can inhibit activin-induced transcriptional activities. ARIP1 overexpression in Neuro-2a cells also decreased the Smad3-mediated transcriptional activities, but ARIP2 overexpression could not induce. Further the electrophysiological method was used to observe the change of the whole-cell sodium current affected by the stable transformation for ARIP1, 2 in the Neuro-2a stimulated by activin A. Overexpression of ARIP1 decreased Activin A-induced INa and overexpression of ARIP2 did not influence INa. Through acute brain injury mouse models, ARIP1, 2 mRNA and protein were detected, and we found that ActivinA mRNA and protein expression was significantly increased in brain lesion tissues, while expression of ARIP1 decreased. The results suggest that the decrease of ARIP1 facilitate increased activin A.to play the neuroprotective effects in nerve cells.Part one: ARIP1, 2 expression plasmid constructionpGEX-4T-ARIP1C/2C, pcDNA3-ARIP1, pcDNA3-ARIP2 were constructed. All of the plasmids were automatically sequenced by DNA sequencing instrument.Part two: Expression of GST-ARIP1C/2C fusion protein and antibody preparation of anti-ARIP1 / 2 antibodiesIn order to detect the expression and locolization of ARIP1, 2, GST fusion proteins of ARIP1C and ARIP2C were prepared and purified, then used to immune rabbit. The rabbit anti-ARIP1 COOH-terminal (anti-ARIP1) and anti-ARIP2 COOH-terminal (anti-ARIP2) IgG polyclonal antibodies were purified by Protein A and anti-GST antibody was removed by GST-Sepharose 4B affinity chromatography. ELISA assay was used to detecte the cross-reaction of ARIP1, 2. The results showed that antibodies of ARIP1, 2 binded with not only the GST-ARIP1C/2C, but also recombinant ARIP1, 2, and anti-ARIP1, 2 antibodies had no cross-reaction with ARIP2, 1, respectively.Part three: Comparision of the expression of immunolocalization of ARIP1, 2 in mouse tissusesIn this study, RT-PCR and immunohistochemistry methods were used to study the expression and localization of ARIP1, 2 mRNA and protein in mouse tissues. The results showed ARIP1 mainly expressed in brain tissues. The expression of ARIP2 was detected in a variety of mouse tissues, included brain tissue, but highest expression were detected in striated muscle, kidney, testis. In the cerebral cortex, ARIP1 expressed mainly in parvicellular neurons, but ARIP2 expressed mainly in megacell neurons.In the cerebellar cortex, ARIP1 expressed mainly in parvicellular neurons, ARIP2 expressed mainly in megacell neurons. In the neurohypophysis and adenohypophysis, ARIP1 is highly expressed, ARIP2 is low expressed. However, ARIP1and ARIP2 both expressed in hippocampus, hypothalamus, choroid and Purkinje cells. These results showed that the localizations of ARIP1, 2 have histological difference, these may determine the biological effect of activin. The studies showed that ARIP1, 2 are different in the molecular structure and mode of action, but they both are negative regulatory factors in intracellular signal transduction.Part four: The role of ARIP1, 2 in the activin signal transductionThe mouse neuroblastoma cell line Neuro-2a cells were used to study the expression and bioactivities of ARIP1, 2. The immunocytochemical staining results showed that ARIP1, 2 were expressed in the Neuro-2a cells. After stimulated by ActivinA, the expression of ARIP1 reduced, the expression of ARIP2 has no changes. Stimulated by LPS, the expression of ARIP1, 2 were significantly enhanced. Tested with activin-specific signal transduction systems, the results showed that ARIP1, 2 can inhibit activin-induced transcription in Neuro-2a cells, further revealed that ARIP1 can inhibit Smad3-induced gene transcription, but ARIP2 could not affect the Smad3-induced gene transcription. The results showed that overexpression of ARIP1 inhibited Neuro-2a cell proliferation, but overexpression of ARIP2 could not affect Neuro-2a cell proliferation. The results suggest that both ARIP1 and 2 can inhibit activin signal transduction, which belong to the negative regulatory protein, but its signal transduction pathway may be different.Part five: Expressions of ARIP1, 2 mRNA and Protein in acute brain injury tissuesActivin A has a neuroprotective effect to injuried neurons. As the negative regulatory protein of activin A signal transduction, whether ARIP1, 2 can play a biological role in the nerve injury was unclear. So we set up the acute brain injury models of mouse, and observed the changes of ARIP1, 2 expression 24h after injury. The results showed that ARIP1 expression was significantly reduced at 24h after injury, ActivinA expression was significantly increased. These findings suggest that ARIP1 might be the key signal transduction molecules at early stage of acute brain injury, may be advantaged to play the role of Activin A.In summary, our data suggest that as negative regulators of activin signal transduction, the expressions and bioactivities of ARIP1, 2 are difference in brain tissues. Thus, ARIP1, 2 may be the key signal transduction molecules of activin A action in the nerve cells.