Tissue-specific Expression Of 2810430M08Rik And Its Function In T-cell Immune Responses

Tissue-specific expression of 2810430M08Rik and its function in T-cell immune responses2810430M08Rik is a novel gene with unknown functions, which was discovered in the mouse genome project in recent years. 2810430M08Rik encodes a protein of 281 amino acids. Its cDNA sequence has no significant homology with other known genes, and its peptide sequence carries no conserved domains with proven function. By far, there's no report concerning the expression pattern or the possible function of this new gene. Based on our previous work, this gene was noted to be changed during T-cell activation, suggesting a possible role in T-cell related immune functions. This study was conducted to investigate the tissue-specific distribution and ontogenetic expression pattern of 2810430M08Rik transcripts, and its function in T-cell development, activation and related T-cell immune responses.Objectives(1) To confirm the change of gene expression of 2810430M08Rik during T-cell activation in detail.(2) To observe the ontogenetic expression pattern and its tissue-specific distribution of gene transcripts in different stages of C57BL/6J mouse development.(3) To evaluate the effect of increased expression of 2810430M08Rik on general function of major systems.(4) To investigate the influence of increased expression of 2810430M08Rik on T-celldevelopment, activation and related T-cell immune responses, and to explore its possible mechanisms if necessary.Methods(1) Northern Blot was conducted to determine the change of gene expression.(2) In situ hybridization was applied to explore its mRNA expression pattern.(3) Transgenic mice over-expressing 2810430M08Rik were created by micro-injection of plasmid containing target cDNA sequence which was driven by β-actin promoter. Phenotype of adult mice and expression level of the trans-gene was verified by Southern blot, regular PCR and Real-time RT-PCR respectively.(4) In vivo and in vitro T-cell development and function was assayed by means of proliferation test, flow cytometry, ELISA, trans-well assay and DTH test.Results(1) Expression of 2810430M08Rik was up-regulated after T-cell activation, which peaked at 5h and declined at 24h.(2) 2810430M08Rik showed a temporal expression during embryonic development, and in adulthood it selectively distributed in the center nervous system and some other tissues with a robust proliferation programme, e.g. thymus, etc.(3) Two lines of transgenic mice were successfully created, with 4～6-fold increase of 2810430M08Rik expression in lymphoid organs compared to littermate controls.(4) No significant phenotype or abnormality of T-cell functions was detected in all the in vivo or in vitro tests performed in the transgenic model.Conclusions(1) The tissue-specific expression pattern of 2810430M08Rik indicates a possible role in embryonic development and related tissue functions, such as proliferation. It is a necessary and useful tool for further investigation of this gene.(2) Expression of 2810430M08Rik was up-regulated during T-cell activation. However, experiments based on transgenic mice showed normal T-cell development, activation and related T-cell immune responses.(3) Further work is required to draw a more complete picture of this new gene.Proteasome inhibitor PS-341 can effectively inhibit proliferation and trigger apoptosis in vascular smooth muscle cellProteasome, the core component of mammalian protein degradation system, is responsible for about 80～90% of total protein hydrolysis within the cell. Its function is vital for maintaining environmental balance and the regulation of cell growth and metabolism. Proteasome inhibitors are small molecular chemicals, which can selectively inhibit proteolysis activity of proteasome. Proteasome inhibitor is one of the most promising categories of immunosuppressants due to its significant anti-tumor and anti-inflammation effect. Increasing evidence indicates that, proteasome inhibitors impact on various strands of malignant cells, as well as normal cells like smooth muscle cell and cardiomyocyte. PS-341 is the first proteasome inhibitor applied in clinical treatment on multiple myeloma patients. In this study, PS-341's role in proliferation and apoptosis of normal vascular smooth muscle cell was evaluated, and possible mechanism was explored and compared to other known proteasome inhibitors in literature.Objectives(1) To evaluate inhibitory effect of PS-341 on tissue specific proteasome in normal mouse vascular smooth muscle and heart tissue.(2) To study effect of PS-341 on aortic smooth muscle cell proliferation in vitro.(3) To study effect of PS-341 on aortic smooth muscle cell apoptosis in vitro and explore the possible mechanisms.(4) To study effect of PS-341 on TNF-α stimulated NF-kB translation in aortic smooth mscle cell.Methods(1) Fresh heart tissue and aorta smooth muscle layer was isolated and chymotrysin-like proteolytic activity was measured by site-specific cleavage of Suc- Leu- Leu-Val-Tyr-AMC.(2) Aortic smooth muscle cells were harvest and cultured and effect of PS-341 on proliferation was assayed by [³H]-thymidine uptake test.(3) Smooth muscle apoptosis was assayed by flow cytometric Annexin-V staining. Protein and mRNA level of some anti-/pro-apoptotic regulators were determined by western blotting and real-time RT-PCR.(4) Nuclear NF-kB level and cytoplasmic IκB-α level in smooth muscle cell was measured by western blotting after TNF-a stimulation.Results(1) PS-341 inhibited chymotrypsin-like activity of proteasome in vascular smooth muscle and heart tissue in a dose-dependent manner with similar potency (P>0.05).(2) PS-341 effectively inhibited smooth muscle cell proliferation in a rather low dosage (IC₅₀<10nM).(3) PS-341 significantly enhanced cell apoptosis at higher concentrations (>100nM at 24h, P<0.01). PS-341 down-regulated mRNA expression of protective factor Bcl-2, Bcl-xL and Flip (P<0.01), but not the protein level.(4) Not like other inhibitors, PS-341 had no significant effect on inhibiting NF-kB translocation in normal smooth muscle cells when stimulated with TNF-α.ConclusionOther than in tumor tissues, PS-341 can effectively inhibit proteasome activity inmajor cardiovascular tissues, including vascular smooth muscle and heart. It can inhibit smooth muscle proliferation and trigger cell apoptosis in a dose-dependent manner. Although there's a transcriptional down-regulation of anti-apoptotic regulators Bcl-2, Bcl-xL and Flip, the protein concentration of these factors is not changed during PS-341 triggered apoptosis. PS-341 has minimum inhibiton on IkB-α degradation or NF-kB translocation upon TNF-α stimulation, which indicates a lack of ability as an good anti-inflammation reagent in smooth muscle compared to other proteasome inhibitors. Further study will be required to explore the possibility of applying proteasome inhibitor in cardiovascular diseases.