Serum Response Factor Expedites Epithelial-mesenchymal Transition In Human Peritoneal Mesothelial Cells

Peritoneal fibrosis is a main complication in peritoneal dialysis, whichgreatly limits the effect of peritoneal dialysis as a long-term renal replacedtherapy. Epithelial-mesenchymal transition (EMT) of peritoneal mesothelialcells is considered as the beginning process of peritoneal fibrosis which isrelated to peritoneal early damage. Peritoneal fibrosis is characterized byaccumulation of myofibroblasts and extracellular matrix (ECM) undermesothelial cells. The process of EMT and fibrosis of peritoneum are similar tothat of other organs and tissues such as lung, heart, kidney and pleura. In theprocess of EMT, epithelial markers are downregulated along with upregulationof mesenchymal markers. Snail, Zeb and HLH family are importanttranscriptional repressors of E-cadherin and other genes about EMT. However,these factors can not fully explain the molecular mechanism of peritoneal EMT.So, new transcriptional factors about EMT will need to be found. Increasingevidences suggest that SRF plays an important role and activity in the process oftumor progression, especially in EMT of epithelial tumor cells. But how can SRF regulate EMT of HPMCs have not been known. Our study will investigatethis issue.【Objectives】1. To investigate SRF expression and function in EMT of primary andimmortalized HPMCs.2. To study SRF expression and its role in animal model of peritoneal fibrosis.【Methods】1. We collected and cultured HPMCs from peritoneal dialysis fluid (in PDpatients) by centrifugation and observed morphological changes of cells indifferent duration of dialysis. Then, we detected the expression andsubcellular location of E-cadherin, α-SMA, Snail and SRF byimmunofluorescence.2. We observed morphological change of HPMCs induced by HG. To verifycell model of EMT, we detected the expression of epithelial cell markerE-cadherin, mesenchymal markers α-SMA and Snail by Western-blot andimmunofluorescence.3. We examined the expression of SRF during HG-induced EMT of HPMCs byWestern blot and immunofluorescence.4. We up-regulated the expression of SRF by transfecting SRF plasmid intoHPMCs and investigated the expression of epithelial cell marker E-cadherin,mesenchymal markers α-SMA and Snail by Western-blot andimmunofluorescence.5. We down-regulated the expression of SRF by transfecting lentivirus intoHG-induced HPMCs. Then, we detected the expression of E-cadherin,α-SMAand Snail by Western-blot and immunofluorescence. 6. Sprague-Dawley rats were infused with4.25%glucose dialysis solution for6weeks to establish animal model of peritoneal fibrosis.We detectedperitoneal thickness by HE staining.Then, we detected the expression ofE-cadherin，α-SMA, Snail and SRF by immunohistochemistry.7. Infusing SRF inhibitor(CCG-1423) and4.25%glucose dialysis solution intorat peritoneal cavity, we detected the expression of E-cadherin, α-SMA andSnail.【Results】1. The expression of SRF in HPMCs from peritoneal dialysis fluid and inHG-stimulated immoral HPMCs.We have observed significant change of HPMCs from cobblestone-liketo fibroblast-like morphology with long time peritoneal dialysis (PD). Therewas also similar change in cell shape after stimulation with HG (60mmol/L)from0h to96h. Immunofluorescence and western-blot demonstratedgradual decrease of E-cadherin expression and increase of SRF, α-SMA andSnail expression. Nuclear localization of SRF was also observed in primarylong PD and HG-induced HPMCs.2. SRF expedites EMT of HPMCs in vitro.After transfecting SRF plasmid into HPMCs which could up-regulatedSRF expression, it resulted in decrease of epithelial cell marker E-cadherinexpression, associated with an increase in the expression of mesenchymalmarkers, α-SMA and Snail by immunofluorescence and Western-blot.Down-regulation of SRF expression via transfecting lentivirus intoHG-induced HPMCs showed decrease of α-SMA and Snail expression andincrease of epithelial cell marker E-cadherin expression. These resultsimplicated that SRF could promote EMT of HPMCs. 3. SRF enhanced peritoneal fibrosis in vivo.In animal model of peritoneal fibrosis, SRF was significantly expressedin the peritoneal tissue. However, the expression of E-cadherin wasdown-regulated and expression of α-SMA and Snail were up-regulated. AfterSRF inhibitor (CCG-1423) treatment, peritoneal thickness was significantlydecreased. Meanwhile, epithelial cell marker E-cadherin was up-regulated.【Conclusion】In this study, we found that transcription factor SRF were activated andtranslocated into nuclear during EMT of HPMCs. Overexpression of SRF inHPMCs could increase α-SMA expression and decrease E-cadherin expression,whereas down-regulation of SRF expression reversed EMT of HPMCs.Treatment of SRF inhibitor CCG-1423could protect EMT and peritonealfibrosis.