Mechanism Study Of Retinoic Acid Facilitating The Tighnt Junction Of Caco-2Cells

Part1Retinoic acid improves the tight junction of Caco-2cellsObjective This study aimed to clarify the role of retinoic acid (RA) onimproving tight junction of Caco-2cells, and explore the RARs signalingpathway associated with TLRs and tight junction proteins throughscreening optimal concentration of RA.Methods The changes of transepithelial resistance (TER) were detectedby digital voltage-resistance meter after RA treated to the Caco-2cells.Caco-2cells were treated by the five different concentrations (0.5μmol/L,1μmol/L,5μmol/L,10μmol/L,20μmol/L) of RA. The expression levels ofRARs, TLRs and tight junction associated proteins in the Caco-2cells weretested with real-time PCR and western blot following RA treatment,respectively. The cellular localization of key receptor RAR in the Caco-2cells were observed through immunofluorescence staining.Results The RA treatment significantly increased TER of Caco-2cells compared to the untreatment group (P0.01). The levels of RAR, TLR4and ZO-2mRNA expression were significantly up-regulated following thefive different RA treatments, and the RA concentrations at1-10μmol/Lwere remarkable (P0.05， P0.001). The changes of these proteinsexpression were completely consistent with the levels of their mRNAexpression. However, the RA treatment couldn’t change expression levelsof RAR, RAR, TLR2, Occludin and ZO-1in the Caco-2cells.Immunofluorescence staining showed that the RAR expression waslocated near the nuclear membrane in the Caco-2cells without RAtreatment, and then it was higher expressed in the cytoplasm from thenuclear membrane with the treatment of RA concentration increase.Conclusions RA increases TER of Caco-2cells to enhance the tightjunction among cells, which mechanism maybe up-regulate the expressionlevels of TLR4and ZO-2through RAR signaling pathway to improve theintestinal epithelial barrier function. Part2RARβ facilitates the expression of ZO-2in the Caco-2cells through regulating TLR4Objective This study aimed to investigate RARβ regulating RA signal pathway to improve tight junction of Caco-2cells by Ad-RARP and siRARβ adenovirus, and to explore the interactions among the RARP, TLR4and ZO-2proteins by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), in order to find the specific regulatory mechanisms of RARβ improving ZO-2expression.Methods After infected by the Ad-RARP and siRARβ adenovirus, real-time PCR and western blot analyzed the changes of RARP, TLR4and ZO-2expression levels in the Caco-2cells. Using co-immunoprecipitation (Co-IP) technique, the RARβ and ZO-2antibodies were respectively used to immunoprecipitation the binding target proteins in the Caco-2cells. With chromatin immunoprecipitation (ChIP) technique, RARβ antibody precipitated DNA-protein complexes and the expression differences of DNA target sequences in the complexes of RARP antibody enriched were detected by real-time PCR.Results The recombinant Ad-RARβ adenovirus infection significantly increased the levels of RARP mRNA and protein expression in the Caco-2cells compared to the RFP control group (P<0.001), and the expression levels of TLR4and ZO-2of Caco-2cells were also obviously up-regulated. Meanwhile, the levels of TLR4and ZO-2expression were significantly decreased in the Caco-2cells compared to the RFP control group when the RARβ expression was blocked by the siRARβ adenovirus (P0.05，P0.01). The results of co-IP showed that RARβ interacted with TLR4, andTLR4combined with ZO-2in the Caco-2cells, but the RARβ did not bindto ZO-2. What’s more, the RAR bound on the promoter sequence ofTLR4in the Caco-2cells via ChIP, but no binding in the promoter of ZO-2sequence.Conclusions Using Ad-RARβ and siRARβ, we demonstrate that RAup-regulates the expressions of TLR4and ZO-2through activating RARβpathway to facilitate tight junction in the Caco-2cells. The mechanism isthat nuclear receptor RARβ can bind to the TLR4promoter region toregulate the expression level of TLR4, whereas TLR4and ZO-2may occurprotein-protein interactions. In other words, RARβ induces the expressionof ZO-2by regulating TLR4to improve intestinal epithelial barrierfunction.