The Mechanism Of IL-6Expression And Regulation In Retinal Müller Cells

Purpose: IL-6plays an important role in various inflammatory ocular diseases，including diabetic retinopathy, uveitis etc.. Retinal Müller cells, spanning throughout thewhole retinal layers, are one of the main sources of inflammatory mediators, including IL-6,in the retina. However, the mechanism of regulating IL-6production in these cells remainsunclear. The purpose of this experiment is to employ human retinal Müller cell line, MIO-M1cells, to further study of the secretion of IL-6in retinal Müller cells and its regulationmechanism.Methods: To find out the potential cause of IL-6production in retinal Müller cells,MIO-M1cells were treated with different stimulus: IL-1β（10ng/mL）, TNF-α（10ng/mL）,IL-6（10ng/mL）, IL-8（10ng/mL）,VEGF（10ng/mL）, IFN-γ（10ng/mL）, Glucose（50mM）or Mannito（l50mM）for24hours（h）, Western Blot was employed to detect IL-6protein expression in MIO-M1cells.Aim to further clarify IL-6regulation induced by IL-1β in the mNRA and protein level inretinal Müller cells, MIO-M1cells were treated with different concentrations of IL-1β（0,0.1,1,10ng/mL）for24h, or IL-1β（10ng/mL）for different time（s0,2,6,12,24h）. Then WesternBlot and RT-PCR were employed to detect intracellular IL-6mRNA and protein expression,respectively. MIO-M1cells were also treated with different concentrations of IL-6（0,0.1,1,10ng/mL）for24h, or IL-6（10ng/mL）for different times（0,2,6,12,24h）. Then WesternBlot was employed to detect intracellular IL-1β precursor protein expression.To find out the relevant pathways those involved in the IL-1β-induced IL-6production inretinal Müller cells, Protein Pathway Array（PPA）was employed to widespreadly screen theglobal protein spectrum changes after IL-1β（10ng/mL） treatment in MIO-M1cells for24h.At the same time, MIO-M1cells were treated with IL-1β（10ng/mL）for different times（0,2,6,12,24h）, then Western Blot was employed to validate the PPA results.To further explore the specific pathways involved in IL-1β-induced IL-6production inretinal Müller cells, MIO-M1cells were pretreated with p38MAPK pathway inhibitorSB20358（05μM and10μM）, JNK pathway inhibitor SP600125（5μM and10μM）, ERK1/2 pathway inhibitors U0126（5μM and10μM）, JAK2/STAT3pathway inhibitor AG490（20μM and40μM）, NF-κB pathway inhibitor BAY11-7082（5μM and10μM）for1h, and thentreated with IL-1β（10ng/mL）for24h. Western Blot was employed to detect the IL-6proteinexpression. MIO-M1cells were pretreated with SB203580（10μM）or U0126（10μM）for1h, and then treated with IL-1β（10ng/mL）for6h, RT-PCR was employed to detect IL-6mRNA expression.To explore the specific elements in IL-6promoter region responsible for IL-1β-inductionof IL-6expression, a series of plasmids carrying various IL-6promoter mutations linked to aluciferase gene were employed: parantal （pIL-6-Luc651）, NF-κB mutation（pIL-6-Luc651△NF-κB）,AP-1mutation （pIL-6-Luc651△AP-1） and C/EBP mutations（pIL-6-Luc651△C/EBP）. Plasmids were transiently transfected into MIO-M1cells culturedin the presence or absence of IL-1β（10ng/mL）for24h, then the luciferase activity wasdetermined. To further explore the role of p38MAPK and ERK1/2pathway in regulating IL-6promoter activity of the retinal Müller cells, MIO-M1cells were transiently transfected withthe plasmids for24h, then treated with SB203580（10μM）or U0126（10μM）for1h, thenwith IL-1β（10ng/mL） for additional24h, then the luciferase activity was determined.Results: Compared with TNF-α（10ng/mL）, IL-6（10ng/mL）, IL-8（10ng/mL）, VEGF（10ng/mL）, IFN-γ（10ng/mL）, Glucose（50mM）, Mannito（l50mM）, IL-1β（10ng/mL）increased IL-6production most significantly in MIO-M1cells, indicating that IL-1β was themost potent stimulator of IL-6production in retinal Müller cells. IL-1β increased IL-6mNRAand protein significantly（P <0.001）in MIO-M1cells, both in a dose and time dependentmanner. In opposite, IL-1β is not induced by IL-6simulation in MIO-M1cells. PPA foundthat NF-κB p50, p-P38MAPK, p-c-Jun, p-ERK1/2, IL-1β precursor proteins were increased,while STAT3, JAK2proteins were decreased after IL-1β（10ng/mL）treatment in MIO-M1cells for24h.Western Blot showed that IL-1β increased p-P38MAPK, p-c-Jun, p-ERK1/2,NF-κB p50proteins, reduced JAK2, STAT3in a time dependent manner, which showed thesimilar tendency with PPA. It indicated that p38MAPK, JNK1/2, ERK1/2, NF-κB,JAK2/STAT3pathways may involve in IL-1β-induced IL-6production in MIO-M1cells.Western Blot showed that p38MAPK, ERK1/2and NF-κB pathway inhibitor significantlyreduced IL-1β induced IL-6production in MIO-M1cells, with the effect of p38MAPKinhibitor（SB203580）> ERK1/2inhibitor（U0126）> NF-κB inhibitor（BAY11-7082）. Real Time RT-PCR result showed that p38MAPK inhibitor（SB203580）and ERK1/2inhibitor（U0126）significantly reduced IL-1β induced IL-6mRNAproduction in MIO-M1cells, withthe effect of p38MAPK inhibito（rSB203580）> ERK1/2inhibito（rU0126）. Luciferase resultsshowed that IL-6promoter activity of the parent pIL-6-Luc651was significantly enhanced byIL-1β, suggesting that the binding sites regulating IL-1β-induced IL-6mRNA were locatedwithin-651bp fragment of the IL-6promoter. IL-6promoter activity was significantlyattenuated by SB203580（p<0.01）, but not by U0126（10μM）, suggesting that p38MAPKwas involved in IL-1β-induced IL-6mRNA, although other regulatory binding sites may alsoinvolve in the ERK1/2pathway-regulating IL-1β induction of IL-6mRNA expression.Furthermore, the IL-6promoter activity was also reduced upon deletion of NF-κB, AP-1orC/EBP binding sites, with NF-κB deletion being the greatest. Finally, the p38MAPK inhibitor（SB203580）exerted further inhibitory effect on pIL-6-Luc651NF-κB, which resulted inadditional reduction of luciferase activity（p<0.001）. This confirmed that IL-1β predominantlystimulated IL-6through activation of the p38MAPK/NF-κB pathways.Conclusion: These results are the first demonstration that IL-1β induces IL-6productionin Müller cells by activation of IL-6promoter activity predominantly through the p38MAPK/NF-κB pathway. These results laid a theoretical basis for the future medicationdevelopment targeted at IL-6related ocular dieases.