Journal of Jiangsu University Medicine Edition. sublytic C5b-9 glomerular mesangial cell GMC. sir- NA small hair RNA shrna. TSP-1 shrna shtsp-1 GMC

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1 Journal of Jiangsu University Medicine Edition Vol. 21 No. 5 Sept TSP-1 C5b thrombospondin-1 TSP-1 sublytic C5b-9 glomerular mesangial cell GMC TSP-1 sir- NA small hair RNA shrna TSP-1 shrna shtsp-1 GMC C5b-9 PCR GMC TSP-1 D2 cyclin D2 proliferative cell nuclear antigen PCNA mrna 3 H- 3 H-thymidine incorporation 3 H-TdR CCK-8 cell counting kit-8 GMC DNA GMC α- α-smooth muscle actin α-sma shtsp-1 TSP-1 C5b-9 GMC 18 h cyclin D2 PCNA DNA shtsp-1 C5b-9 GMC 54 h C5b-9 GMC α-sma shtsp-1 GMC TSP-1 C5b-9 GMC α-sma shtsp-1 GMC α-sma C5b-9-1 α- R A Roles of thrombospondin-1 gene in glomerular mesangial cell proliferation induced by sublytic C5b-9 WU Yi-qin CHE Nan FENG Xue-feng QIU Wen WANG Ying-wei Department of Microbiology and Immunology Nanjing Medical University Nanjing Jiangsu China Abstract Objective To explore the effects of silencing thrombospondin-1 TSP-1 gene on sublytic C5b-9 complex-induced glomerular mesangial cell GMC proliferation. Methods Small hair RNA shr- NA against TSP-1 gene was designed and the corresponding expression plasmids were constructed. Cultured rat GMC was transfected with TSP-1 shrna shtsp-1 and then stimulated with sublytic C5b-9 complexes. The expression of TSP-1 cyclin D2 and proliferative cell nuclear antigen PCNA at mrna and protein levels in different groups of GMC was assessed by real-time PCR and Western blot respectively. In addition DNA synthesis and GMC number were detected by using 3 H-thymidine incorporation 3 H-TdR and cell counting kit-8 CCK-8. Finally the expression of α-smooth muscle actin α-sma in GMC was examined in different groups. Results TSP-1 gene knockdown by using shtsp-1 in rat GMC had no significant effect on the expression of cyclin D2 and PCNA and the synthesis of DNA in the GMC induced by sublytic C5b-9 for 18 h. But shtsp-1 could suppress the increase of GMC number due to sublytic C5b-9 stimulation for 54 h and production of α-sma at 18 h after sublytic C5b-9 stimulation. Conclusion Silencing TSP-1 gene in GMC with shtsp-1 can inhibit sublytic C5b-9-induced GMC proliferation and α-sma production. The proliferation of GMC treated with sublytic C5b-9 was possibly mediated in part by α-sma KJB NJMUM003 09JC qiuwen@ njmu. edu. cn wangyw1508@ njmu. edu. cn

2 production induced by TSP-1 gene activation. Key words glomerular mesangial cell sublytic C5b-9 complex thrombospondin-1 proliferation α-smooth muscle actin mesangial proliferative glomerulonephritis MsPGN GMC 9 Congote 10 1 TSP-1 glomerular mesangial cell GMC extracellular matrix ECM C5b-9 GMC TSP-1 MsPGN GMC TSP-1 C5b-9 GMC MsPGN GMC MsPGN TSP-1 sirna small hair RNA shrna ECM GMC C5b-9 MsPGN Thy-1 Thy-1 nephritis Thy-1N anti-thymocyte serum nephritis ATSN MsPGN MsPGN 1 1 MPGN C5b-9 Thy-1 2 C5b GMC HBZY-1 C5b-9 3 C5b-9 ATS Thy-1 Ab 10 4 normal human serum NHS C8 5 ReverTra Ace-α- TM TOYOBA C9 C5b-9 TSP-1 D2 cyclin D2 Santa Cruz C5b-9 sub- proliferative cell nuclear antigen PCNA lytic C5b-9 6 C5b-9 α-smooth muscle actin α-sma CCK-8 cell counting kit-8 7 Thy-1 pgcsi. U6. neo. RFP GMC GMC C5b GMC 2 GMC GMC 50 ml C5b-9 3 ml MEM 37 5% CO 2 C5b-9 GMC GMC 80% ~ 90% -1 thrombospondin-1 TSP-1 GMC TSP-1 C5b-9 GMC TSP-1 GMC Thy-1 C5b-9 GMC Thermo α- 5 min r /min Hanks TSP-1 shrna shtsp-1 Tuschl TSP-1 shrna

3 5. TSP-1 C5b '-CTCAATGAACGAGACAACT-3' DNA 5'-CTTGCATTGCATCCT- CATT-3' shrna shctr pgcsi. U6. neo. RFP DNA Xho I EcoR I ± s SPSS13. 0 Bonfferoni P < T 4 DNA 2 DH5α LB TSP-1 shrna shtsp-1 GMC TSP shtsp-1 GMC 50% shtsp-1 shctr C5b-9 18 h TSP-1 GMC GenEscort TM III shtsp-1 48 h C5b-9 TSP-1 red fluorescence protein RFP shrna shctr TSP-1 GMC TSP-1 1 C5b-9 shtsp-1 + C5b-9 shctr + C5b-9 MEM shtsp-1 + C5b-9 shctr + C5b-9 shtsp-1 shctr GMC 48 h C5b-9 5% Thy-1 Ab 4% NHS C5b-9 18 h mrna PCR mrna PCR GMC cyclin D2 PCNA α-sma mrna Trizol RNA 1 μg cdna PCR mrna 50 2 min min s 60 1 min GMC TSP-1 Fig 1 Protein level of TSP-1 in GMC in various groups GMC TSP-1 cyclin D2 PCNA α-sma GMC TSP-1 GMC cyclin D2 PC- g 10 min SDS-PAGE NA PVDF 3% BSA Cyclin D2 PCNA mrna 4 TBST 3 shtsp-1 GMC C5b-9 HRP 1 h ECL GMC cyclin D2 PCNA GMC h C5b-9 18 h h GMC 450 nm D ± x TSP-1 shrna shtsp-1 GMC 48 h A 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 B * P < C5b-9 Δ P < shctr + C5b-9 PCR shtsp-1 C5b-9 cyclin D PCNA mrna 2 3 H-TdR 18 h Cyclin D2 PCNA count per minute cpm mrna h h shtsp-1 GMC C5b-9 cyclin D2 PCNA CCK-8 2 h

4 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < ** P < MEM 2 GMC D2 PCNA mrna Fig 2 The mrna level of cyclin D2 and PCNA in GMC in various groups A cyclin D2 a 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 b B PCNA ** P < MEM Fig 3 3 GMC D2 PCNA Levels of cyclin D2 and PCNA protein in GMC with different treatments 2. 3 TSP-1 C5b-9 GMC DNA 3 H-TdR GMC DNA cpm 18 h shtsp-1 + C5b-9 DNA shctr + C5b-9 C5b-9 cpm MEM shtsp-1 GMC C5b-9 DNA shtsp-1 GMC TSP-1 GMC CCK-8 GMC D 36 h 54 h 54 h 18 h 36 h shtsp C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < * * P < MEM 4 Fig 4 GMC DNA Detection of DNA synthesis in GMC in varying groups C5b-9 C5b-9 shtsp-1 + C5b-9 GMC shctr + C5b-9 C5b-9 C5b-9 GMC shctr + shtsp-1 GMC

5 5. TSP-1 C5b C5b-9 54 h GMC 5 sublytic 11 C5b-9 3 sublytic C5b-9 GMC TSP-1 12 * P < C5b-9 Δ P < shctr + C5b-9 P < MEM Fig 5 5 GMC The numbers of GMC in different groups 2. 5 TSP-1 α-sma α-sma mrna PCR GMC α-sma shtsp-1 C5b-9 α-sma 6 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 * P < C5b-9 Δ P < shctr + C5b-9 6 Fig 6 GMC α-sma mrna The mrna levels of α-sma in GMC with different treatments α-sma TSP-1 15 shtsp-1 GMC α-sma TSP-1 C5b-9 GMC mrna 7 18 h shtsp-1 + RNAi RNA dsrna C5b-9 α-sma shctr + mrna 16 RNAi C5b-9 C5b-9 RNAi 3 TSP-1 CDS sir- MsPGN GMC ECM A 1 C5b-9 2 shtsp-1 + C5b-9 3 shctr + C5b-9 4 MEM B * P < C5b-9 Δ P < shctr + C5b-9 pgcsi. U6. neo. RFP Thy-1 GMC TSP-1 TSP-1 MsPGN C5b-9 GMC 2 shtsp-1 GMC C5b-9 lytic TSP-1 C5b-9 18 h 7 Fig 7 GMC α-sma Levels of α-sma protein in GMC with different treatments TSP-1 TSPs TSP-1 13 TSP-1 14 NA TSP-1 shrna shtsp-1

6 TSP-1 C5b-9 cyclin D2 PCNA 3 H-TdR GMC DNA 7 Fosbrink M Niculescu F Rus V et al. C5b-9-induced TSP-1 GMC endothelial cell proliferation and migration are dependent CCK-8 GMC on Akt inactivation of forkhead transcription factor 18 h 36 h shtsp-1 + C5b-9 GMC shctr + C5b-9 C5b-9 54 h shtsp-1 + C5b-9 GMC Commun Signal shctr + C5b-9 C5b-9 9 Daniel C Schaub K Amann K et al. Thrombospondin- TSP-1 C5b-9 GMC 1 is an endogenous activator of TGF-beta in experimental diabetic nephropathy in vivo J. Diabetes α-sma 10 Congote LF DiFalco MR Gibbs BF. Thrombospondin α-sma GMC 1 produced by endothelial cells under the action of GMC α-sma α-sma GMC erythropoietin stimulates thymidine incorporation into erythroid cells and counteracts the inhibitory action of in- GMC sulin-like growth factor binding protein 3 J. Cytokine TSP GMC α-sma TSP-1 11 Kunchithapautham K Rohrer B. Sublytic membrane-at- GMC TSP-1 tack-complex MAC activation alters regulated rather α-sma GMC than constitutive VEGF secretion in retinal pigment epithelium monolayers J. J Biol Chem Gao L Qiu W Wang Y et al. Sublytic complement 1 Nazeer K Janech MG Lin JJ et al. Changes in protein profiles during course of experimental glomerulonephritis J. Am J Physiol Renal Physiol F Qiu W Che N Feng X et al. Apoptosis of glomerular mesangial cells induced by sublytic C5b-9 complexes in rats with Thy-1 nephritis is dependent on Gadd45gamma upregulation J. Eur J Immunol Fosbrink M Cudrici C Tegla CA et al. Response gene to complement 32 is required for C5b-9 induced cell cycle activation in endothelial cells J. Exp Mol Pathol Helske S Oksjoki R Lindstedt KA et al. Complement system is activated in stenotic aortic valves J. Atherosclerosis Lee HM Oh BC Lim DP et al. Role of complement regulatory proteins in the survival of murine allo-transplanted Sertoli cells J. J Korean Med Sci Liu H Tian N Arany I et al. Cytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy J. J Biol Chem FOXO1 J. J Biol Chem Di Guglielmo GM. TSP-1 in lung fibrosis J. J Cell C5b-9 complexes induce thrombospondin-1 production in rat glomerular mesangial cells via PI3-k /Akt association with activation of latent transforming growth factor-beta1 J. Clin Exp Immunol El Btaouri H Morjani H Greffe Y et al. Role of JNK / ATF-2 pathway in inhibition of thrombospondin-1 TSP- 1 expression and apoptosis mediated by doxorubicin and camptothecin in FTC-133 cells J. Biochim Biophys Acta Lu Z Kipnis J. Thrombospondin 1 a key astrocyte-derived neurogenic factor J. Faseb J Lu A Miao M Schoeb TR et al. Blockade of TSP1- dependent TGF-beta activity reduces renal injury and proteinuria in a murine model of diabetic nephropathy J. Am J Pathol Badiga AV Chetty C Kesanakurti D et al. MMP-2 sirna inhibits radiation-enhanced invasiveness in glioma cells J. PLoS One e