Parathyroid Hormone-related Protein-induced Epithelial-to-mesenchymal Transition Of Intestinal Epithelial Cells

BackgroundCrohn's disease(CD)is a chronic progressive disease of the gastrointestinal tract characterized by transmural granulomatous inflammation,muscle layer overgrowth and collagenous fibrosis that cause the characteristic symptoms of abdominal pain,vomit,nausea and diarrhea.At the last stage of this disease,which can develop to intestinal obstruction.More than one-third of patients with CD require surgery to relieve obstructions related to fibrotic strictures,and many require repeat interventions because of recurrence of the disease.Diet?environment?immune?infection?genetic and other factors may contribute to CD,but the real pathogenesis in Crohn' s disease patients is babely understood.Intestinal fibrosis(IF)is a serious complication of inflammatory bowel diseases(IBD).Recent findings suggest that intestinal fibrosis follows a commonly used pathway of fibrosis,involving a progressive and chronic process characterized by an excessive deposition of extracellular matrix(ECM)components,such as a-smooth muscle actin(a-SMA),collagens and fibroblast-specific protein 1(FSP1).Intestinal epithelial cells,with fibroblasts are a major origin of extracellular matrix(ECM).Mesenchymal cells such as myofibroblasts,fibroblasts and smooth muscle cells are primarily responsible for intestinal fibrosis,but where are them from?Recent findings suggest that intestinal epithelial cells maybe through EMT leading to mesenchymal cells under the process of infection and injury etc.Epithelial-to-mesenchymal transition(EMT),a multistep process that requires the integration of multiple extrinsic and intrinsic pathways,can activate myofibroblasts in multiple organs including the heart,lung,liver,kidney and the intestines,eventually lead to fibrosis.In addition,a decrease in the expression of certain proteins—those that maintain basolateral polarity,namely cytokeratin,and intercellular junctions,including the adherent junction protein E-cadherin—takes place in rat intestine cells during EMT,and hence promote cell growth,migration and invasion.Parathyroid hormone-related protein(PTHrP also known as PTHLH),originally identified as a factor associated with humoral hypercalcemia of malignancy,was later found to be widely distributed in most fetal and adult tissues,and on account of the similar of function and gene structure with parathyroid hormone(PTH)and named PTHrP.Widespread expression of PTHrP and PTH/PTHrP receptor genes and proteins has been found in intestinal villus epithelium.It was recently found that PTHrP promote fibrosis in the obstructed mouse kidney,with the cooperation of TGF-?1,endothelial growth factor(EGF),and vascular endothelial growth factor(VEGF).Meanwhile,it was reported that PTHrP in tubulointerstitial apoptosis and fibrosis after folic acid-induced acute renal injury.In addition,PTHrP can promote in pancreas and liver.In view of PTHrP multiple organ fibrosis effect,and studies have demonstrated that PTHrP promotes death of cells of the rat small intestinal cell line IEC-6.We thus think that PTHrP may be a mediator for intestinal fibrosis.We have reasons to believe that PTHrP may be through the process of EMT to induce intestinal fibrosis.The role of EMT in intestinal fibrosis,however,has not to be investigated.It has previously been suggested that transforming growth factor-?1(TGF-?1),in its target tissues such as liver and kidney,act as a modulator of at least some PTHrP functions through the PTH receptor 1(PTHR1),which is common to PTH and PTHrP.In clinic PTH has been used as a treatment for osteoporosis,and have done a great job for prevention of osteoporosis,PTHrP is the next target for prevention of fracture in postmenopausal women and has entered the phase ?,and we need pay attention to the side effect of promote fibrosis during the therapy.The signaling mechanisms by which PTHrP induces cellular phenotypic effects have been widely investigated.PTHrP signals through a G-protein-coupled receptor that activates protein kinases A and C.In addition,activation of different signaling pathways results from EMT intrinsic regulators,which include mitogen-activated kinases(MAPKs),namely extracellular-signal-regulated kinases(ERKs)1/2,p38,and N-terminal c-Jun kinase(JNK),as well as Wnt and smad proteins.Multiple signaling pathways may contribute to the complex pathological of intestinal fibrosis,and lead to the deposition of ECM,at last result in intestinal fibrosis.ObjectivesDue to the severe complication and bad treatment effect of inflammatory bowel disease(IBD),coupled with the increase of incidence,intestinal fibrosis has become a hot spot.PTHrP impact on small intestinal epithelial cells may provide us a new starting point to the treatment of IBD.In this study,the interaction of PTHrP with TGF-?1,it can offer theoretical basis in treatment of IBD.Materials and methods1.The rat small intestinal cell line IEC-6 was obtained from our laboratory.To determine whether PTHrP induces EMT in IEC-6 cells,these cells were starved overnight and exposed to 10 nM of three bioactive subdomains of PTHrP in culture media containing 1%serum for 48 h,or treated with different concentration(0.1nM,1nM,10nM,100nM),different time period(6h,12h,24h,36h,48h,60h,72h)of PTHrP,pre-treated with different inhibitors for 2h.Then the total protein and total RNA were collected for further experiment;2.We used relative quantitative reverse transcriptase polymerase chain reaction(RT-qPCR)to detecte the following factor that related to the change of EMT:E-cadherin,collagen I,TGF-?1,FSP1,and Western-blot to detect the following factor:E-cadherin,collagen I,TGF-?1,FSPl,p-PKCa/?/PKC?/?,PKA C-a;3.Immunofluorescence was performed to identify the expression of E-cadherin,collagen I,TGF-?1,FSP1 after treated with only PTHrP(10 nM)for 48h,or pretreated with H-89(l0uM),Go6983(lOuM),PMA(1uM),SD208(10uM),LY2157299(10uM),PTHrP(7-34)(1?M)or an equivalent amount of DMSO for 2 h and then added in PTHrP;4.IEC-6 cells were treated with PTHrP(1-40)at various concentrations or for different durations,and cell viability was measured using CCK8.Or use the EdU incorporation assay to detect cell proliferation.5.We used the Tanswell assay to investigate whether PTHrP(1-40)is associated with migration and invasion.6.The data were shown as mean ± standard error of the mean(S.E.M.)based on experiments repeated in triplicate.Multiple comparisons were analyzed using one-way analysis of variance(ANOVA)with Statistical Package for the Social Sciences(SPSS)13.0 software(Chicago,IL).Probability(p)-value less than 0.05 were considered statistically significant.The results of Ctrl group were set to 1 in Western-blot and qPCR.Results1.To determine whether PTHrP induces EMT in IEC-6 cells,these cells were starved overnight and exposed to 10 nM of three bioactive subdomains of PTHrP in culture media containing 1%serum for 48 h.Only the portion of PTHrP(1-40)resulted in EMT in the IEC-6 cells and the relative are assessed by western-blot and comparative real time PCR,found that Collagen I was increased in the concentration of 1nM for 48h with 3.4-fold(P<0.01),the FSP1 protein was significant decreased to one third of non-treat control cells(P<0.01),in response to TGF-?1 at 10nM for 48h,FSP1 was signigicant increased,as assessed by protein levels with 2.4-fold campared with Ctrls(P<0.01).Pre-incubation with PTHrP(7-34),an inhibitor of a PTH 1R,PTHrP(1-40)could not induce EMT.TGF-?1 of 4 ng/ml as positive control.2.PTHrP(1-40)could induce an increase in levels of TGF-?1 mRNA and protein,pre-incubation with PTHrP(7-34)indeed abolished the increase of TGF-?1 levels.Pre-incubation with inhibitor of TGF-? receptors:SD 208(10?M)the inhibitor of T?R I,LY2157299(10?M)the inhibitor of T?R I and ?.They were shown no significant differences in LY2157299(10?M)+ PTHrP 1-40(10nM)or SD 208(10?M)+ PTHrP 1-40(10nM)group with control group.3.PTHrP(1-40)induce EMT in IEC-6 cells at a dose-response,in response to PTHrP(1-40)at 10-100Nm for 48 h,the protein and mRNA levels of E-cadherin,collagen I,TGF-?1and FSP1 were significant changed.Under condition of PTHrP(1-40)at lOnM for 0-72 h,the protein and gene expression of collagen I,TGF-?1and FSP1 first rises then falls,contrary the levels of E-cadherin first falls then rises.4.PKA and PKC signaling pathways were acivated after treated with PTHrP(1-40)at 10nM at same time.The following results were compared with non-treated Ctrl cells.PKA and PKC were stimulated at 7min.IEC-6 cells were pre-incubated with H-89(which is a PKA inhibitor)(10uM)or Go6983(which is a PKC inhibitor)(10uM)for 2 h and then treated with PTHrP(1-40)(10 nM)or phorbol-12-myristate-13-acetate(PMA)(which is a PKC activator)(1 uM)for 48 h.Western blot showed that pre-incubation of the IEC-6 cells with inhibitor significantly abolished these EMT-related changes.Pre-incubation with H-89 could block the PTHrP(1-40)induction of TGF-?1,but Go6983 could not inhibit the action of PTHrP(1-40),and PMA can not cause the rise of TGF-?1.5.Used cell immunofluorescence technique to detect the level of protein under different processing consistent with the results of western blot.6.PTHrP(1-40)can promote cell growth,migration and invasion.ConclusionPTHrP(1-40)induces EMT through TGF-?1 in rat intestine epithelial cells.PTHrP(1-40)promotes EMT depended on dose and time.PTHrP(1-40)influences TGF-?1 through the PKA(but not the PKC)signaling pathway.The PTHrP(1-40)-induced EMT maybe through the PKA and PKC pathways in IEC-6 cells.In summary,PTHrP(1-40)could induce EMT during intestinal fibrosis.