Mechanism By Which Protein Kinase D2 Contributes To The Colitis Induced By Dextran Sulfate Sodium In Mice

BackgroundUlcerative colitis(UC),a major form of inflammatory bowel disease(IBD),is characterized by chronic inflammation of the gastrointestinal tract with a relapsing-remitting disease course requiring lifelong treatment.The disease affects mainly young people and the peak incidence is between the ages of 20 and 40.The constant alternation of relapses and remissions of UC can lead to perianal lesion,abdominal abscess,intestinal perforation and colectomy,which seriously impair the quality of life in UC patients.More importantly,it has been long established that patients with UC are at an increased risk of developing colorectal and extraintestinal cancers.UC has been regarded as an important problem threatening public health all over the world with a rising incidence and prevalence reported in recent years.So far,the aetiology of UC is still unclear.Genetic factors,immune factors,bacteria and diet are all involved in the initiation and progression of ulcerative colitis.Indeed,an altered epithelial barrier function and aberrant innate immune responses contribute to UC pathogenesis.The epithelial barrier consists of mucus layer covering the epithelium,intestinal epithelial cells(IECs),goblet cells and Paneth cells.Tight junctions(TJs)are the main components of the epithelial barrier and an altered expression and structural changes of TJs are closely associated with UC.MUC2,the main mucus protein secreted by goblet cells,plays a crucial role in intestinal barrier.It is reported that patients with UC have reduced goblet cell numbers and less mucus.And mice with MUC2 deficiency developed spontaneous colitis.Thus,the role of MUC2 has been implicated in the pathogenesis and development of UC.Available evidence suggests that an abnormal immune response has been considered to play a major role in the pathogenesis of UC.Harmful intestinal microbacteria break into the intestinal mucosa lamina propria to activate the mucosal effector T cells to produce large amounts of inflammatory cytokines and chemokines to trigger the onset of UC when epithelium barrier is impaired.During active inflammation,CD4+ ThO cells differentiate into T helper 1 cell type(Thl),Th2,Th17 and Treg cells.Thl cells,which are induced by 1L-12,release high amounts of interferon(IFN)-y and tumor necrosis factor(TNF)-a,whereas Th2 cells produce IL-4,IL-5 and IL-13.Th17 cells produce IL-17a,IL-17f,IL-21,IL-22 and IL-23 while IL-10 and TGF-β are secreted by Treg cells.A Th1 immune response is thought to cause intestinal inflammation in CD,while UC has been considered as a Th2-mediated disease with excessive production of IL-5 and IL-13.In addition,higher transcript and protein level of IL-17a,released by Th17 cells,have been detected in the lamina propria of patients with UC.PKD(protein kinase D)is serine/threonine protein kinase.There are currently three known PKD isoforms,PKD1/PKCμ,PKD2 and PKD3/PKCv.PKD is activated by various stimulis,including G protein coupled receptors,DAG,polypeptide growth factors and phorbol esters.PKD is involved in the signal transduction pathways induced by PKC or DAG.Recent research has implicated that PKD plays an important role in the regulation of diverse fundamental biological processes including cell survival,apoptosis,proliferation,differentiation,Golgi function,inflammation,cardiac diseases and cancers.Recently,PKD was found to participate in the regulation of epithelial barrier function and inflammatory response.It was reported that among three PKD isoforms,PKD3 knockdown was the most efficient one to disrupt the formation of apical intercellular junctions and their reassembly,impair the development of TEER and increase paracellular permeability to sodium fluorescein in airway epithelial monolayers.Overexpression of PKD3 markedly suppressed the mRNA and protein levels of claudin-1 but had only minor effects on the expression of other tight junctional proteins.Gan H et al found that PKD family kinases(PKD1,PKD2 and PKD3)were increased and activated in the hyperplastic and regenerative alveolar epithelial cells and macrophages in idiopathic pulmonary fibrosis lungs compared with normal controls.PKD was predominantly activated by poly-L-arginine,lysophosphatidic acid and thrombin in human lung epithelial cells and PKD promoted epithelial barrier dysfunction.Another study found that cytokines-induced epithelial barrier disruption in pancreatic epithelium was dependent on JAK and PKD signaling.PKD inhibitor,Go6976,had a protective effect on LPS/D:-GaIN-induced acute liverinjury in mice.It has been reported that PKD I is the major expression subtypes of pancreatic acinar cell.CID755673,a kind of specific PKD inhibitors,alleviated acute pancreatitis in mice effectively.Currently,the research on PKD2 and ulcerative colitis has not been reported.Therefore,it is unclear whether PKD2 is involved in the development of ulcerative colitis.It has been identified that PKD2 plays an important role in regulating the function of mature peripheral lymphocytes during adaptive immune responses.According to PKD2 knockin mice,Matthew et al found that PKD2 was the major PKD isoform expressed in lymphoid tissues of mice.Moreover,PKD2 catalytic activity was essential for efficient antigen receptor induced cytokine production in T-lymphocytes and for optimal T-cell-dependent antibody responses in vivo.Based on the above research,we hypothesize that PKD2 regulate the expression of tight junction proteins and inflammatory cytokines to affect the initiation and progression of ulcerative colitis.To test this hypothesis,we induced DSS-colitis in PKD2 knockin mice,transfected intestinal epithelial cells with siRNA and analyzed the expresson of PKD2 in clinical intestinal mucosal samples.This study focused on the combination of vitro and vivo from the following three aspects,assess the effect of PKD2 knockin on the DSS-induced colitis,the expression of TJs and inflammatory cytokines of NCM460 and Caco-2 cells treated with PKD inhibitors or siRNA,the association of PKD2 expression and clinical ulcerative colits.The prime limitations of anti-inflammatory medications and biological agents are significant side effects and at high cost.It is clear that there is a pressing need for new agents for the control of the clinical manifestations of UC.Better understanding of PKD2 in the pathogenesis of UC can lead to the development of novel therapeutic agents that target specific mediators of the inflammatory cascade.ObjectivesThis study is to explore the role and mechanism of protein kinase D2 in a transgenic mice model of DSS-induced colitis.The present study might provide a new therapeutic target for ulcerative colitis.Methods1.We successfully generated homozygous PKD2 knockin mice(PKD2SSAA/SSAA)by inter cross.The effect of knockin can be identified by PCR and western blotting.2.Colitis in mice was induced by DSS.The distal colon was fixed in 4%formaldehyde,embedded in paraffin,sectioned at 5μm and stained with haematoxylin and eosin(HE).3.The expression of multiple cytokines were analyzed by real time quantitative RT-PCR in vivo and vitro.4.The number of B cells in spleens and MLNs of mice were analyzed by flow cytometry.5.Serum IgA and IgG of mice were determined by ELISA.6.Biopsy specimens of intestinal mucosa from patients with UC and normal controls were analyzed by immunohistochemistry.7.The technology of siRNA was used to study the effect of gene knockout of PKD2 in intestinal cells on downstream target molecules.Data analysesAll the data were analyzed by SPSS 21.0 software.Data were expressed as mean±SD if they obeyed normality distribution or expressed as median if they did not obey normality distribution.Statistical significance was tested using One-Sample T Test,Independent-Samples T Test,Mann-Whitney U Test and ANOVA for Repeated Measures.Statistical significance was set at P values less than 0.05.Results1.PKD2 transgenic mice were generated successfully.We generated homozygous PKD2 knockin mice(PKD2SSAA/SSAA)by inter cross heterozygous PKD2 knockin mice(PKD2WT/SSAA)with heterozygote which was imported from Jackson Laboratory.Genotyping of mice was confirmed by PCR of genomic DNA.And western blotting showed that the expression of phosphor-PKD(Ser744/748)was decreased in all kinds of organs of homozygous PKD2 knockin mice.2.Dextran sulfate sodium(DSS)-colitis was successfully induced and PKD2 knockin mice experienced an enhanced inflammatory response.Chemical model of ulcerative colitis(UC)in mice was successfully induced by 5%DSS in our study.Then we preliminarily investigated the role of PKD2 in UC.And we found that after DSS-induction,both PKD2WT/WT mice and PKD2SSAA/SSAA mice had decreased body weitght and increased disease activity index.Apparently,PKD2 knockin mice experienced increased disease severity compared to wild-type controls as measured by weight loss(P<0.001),disease activity index(P<0.001)and histologic damage(P<0.05).In PKD2 knockin mice,there was a significant increase in lymphocytic infiltration,loss of goblet cells and thickening of vascular wall.In summary,phosphor-PKD deficiency exacerbates disease progression in mice with DSS-induced colitis.3.Multiple T cell-related inflammatory cytokines were up-regulated in PKD2 knockin mice with DSS induced colitis.Research has shown that T cell immunity response plays a crucial role in the occurrence and development of ulcerative colitis.To investigate whether PKD2 was involved in the immune regulation of colitis,we examined multiple cytokines of colon by real time quantitative RT-PCR test.The result showed that some important inflammatory cytokines,such as TNF-α,IL-13,IL-17f and IL-21(P<0.05),which were known to be involved in the pathogenesis of ulcerative colitis,were all significantly increased in PKD2 knockin mice.In summary,PKD2 might have an effect on mucosal immunity by regulating the production of inflammatory cytokines.4.PKD2 had no effect on the B-cell subtypes or the production of serum immunoglobulins of mice.There was no significant difference between the number of CD19+ B220+ B cells in the spleens and mesenteric lymph nodes of PKD2 knockin mice and wild-type mice(P>0.05).And further analysis of production of IgA and IgG in the serum of mice showed that PKD2 did not affect the secretion of immunoglobulins(P>0.05).5.PKD inhibitors increased the permeability of Caco-2 intestinal epithelial cell model.To assess the effect of PKD inhibitor on the permeability of intestinal epithelial cell,we incubated Caco-2 cells in Transwell chambers for 21 days.Then the cells were treated with different concentrations of specific PKD inhibitor,kb-NB 142-70 or CRT0066101.After that,the permeability of FITC-Dextran(4kDa)was examined.It was showed that PKD inhibitors increased the permeability of FITC-Dextran in Caco-2 intestinal epithelial cell(P<0.05).6.Inhibition of PKD activity or silencing of PKD2 in vivo or vitro both down-regulated TJs and mucins of intestinal epithelial cell monolayers.It has been reported that intestinal barrier,consisting of tight junction proteins,has been disrupted in ulcerative colitis.Western blotting of colonic lysates from PKD2 knockin mice showed significant down-regulation of ZO-1,claudin-1 and MUC2 compared with wild-type controls.Similarly,inhibition of PKD2 activity or silencing of PKD2 in intestinal epithelial cell monolayers also decreased the expression of TJs and mucins.These results suggest that PKD2 may regulate intestinal permeability by TJs and mucins.7.PKD2 negatively regulated the expression of inflammatory cytokines in NCM460 cells and Caco-2 cells.NCM460 cells or Caco-2 cells were transiently transfected with PKD2 siRNA(si-PKD2)or treated with specific PKD inhibitor,kb-NB 142-70 or CRT0066101.Real time quantitative RT-PCR analysis of cDNA samples from these cells showed that some important inflammatory cytokines,such as TNF-a,IL-1β,IFN-y,IL-8,IL-17a and CCL20,were remarkably increased(P<0.05).In summary,PKD2 negatively regulate the expression of inflammatory cytokines in vitro.8.Expression of PKD2 was down-regulated in patients with UC.We performed immunochemistry of PKD2 in colonic samples of patients with UC.The result showed that PKD2 immunostaining in epithelial cells of tissues with active disease was significantly down-regulated compared with those cells of normal tissues.In summary,our data show that PKD2 might be involved in the pathogenesis of ulcerative colitis.Conclusion1.PKD2 knockin mice are successfully generated and verified by PCR test and western blotting.2.In mice with DSS-induced colitis,PKD2 affects the occurrence and progression of colitis by regulating the expression of inflammatory cytokines,MUC2 and TJs.Conversely,PKD2 do not influence the number or function of B cells.3.In vitro,inhibition of PKD2 activity by specific inhibitors or silencing of PKD2 in intestinal epithelial cells results that the cell membrane permeability of Caco-2 is increased,the expression of inflammatory cytokines are up-regulated and the expression of TJs and MUC2 are decreased.4.PKD2 is down-regulated during intestinal inflammation.This study will provide some potential molecular targets for diagnosis and treatment of ulcerative colitis.