The Relationship Between Syndecan-1Shedding And Inflammatory Regulation In Intestinal Epithelial Cells

Background&objectivesSyndecan-1(CD138)which mainly expressed on the surface of epithelial cells physiologically is a member of heparin sulfate proteoglycan family. It is composed of transmembrane core protein,heparan sulfate(HS) chains and chondroitin sulfatase (CS) chains which both bind with extracellular region of Syndecan-1core protein. It can be shed from the cell surface and become a soluble or insoluble component of the extracellular matrix. Syndecan-1with integral HS chains regulates complex cell behaviors such as adhesion, motility, invasion and intracellular signaling by acting as a co-receptor for a variety of cytokines、 growth factor、chemockine and extracellular matrix. It is known that shedding of Syndecan-1controls diverse pathophysiological responses in cancer, wound healing, inflammation and immunity.Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparin sulfate must be attached to the core protein to suppress shedding. The cleavage site of PMA-stimulated or constitutively shed Syndecan-1is localized to amino acids A243and S244. Change of the cleavage site can inhibit the shedding of HS chain. The etiology of inflammatory bowel diseases (IBDs) is unknown, but hereditary,environmental factors and a deregulated mucosal immune response are thought to be involved. The prevalence of IBD is increasing year by year in China.Recent studies were mainly focused on the etiology.And we have found a relationship between Syndecan-1shedding and IBD in our previous studies.The NF-κB family of transcription factors plays a central role in coordinating the expression of a wide variety of genes controlling immune responses. Activation of NF-κB leads to the gene expression of inflammatory cytokines and other mediators involved in many kinds of pathogenesis. In many cell types, the most abundant form of NF-κB is the P50/P65heterodimer, which remains in an inactive state in the cytoplasm, forming a ternary complex with the inhibitory protein IκB-α. Upon simulation of LPS,TNF-a and IL-1β, IκB-α is rapidly phosphorylated by IκB kinase (IKK), allowing translocation of P50/P65to the nucleus. P50is responsible for DNA binding, P65is responsible for transcriptional activation. NF-κB plays a crucial role in determine the outcome of inflammation in IBD. inhibitors of NF-κB signaling caused a mitigation in inflammation.Tumor Necrosis Factor alpha (TNF-a) is a cytokine that has a wide variety of functions including immune cell activation and proliferation. TNF-a is produced primarily by macrophages in response to bacterial byproducts such as lipopolysaccharide (LPS). The excessive production of TNF-a can lead to cytotoxicity and tissue damage associated with inflammation, we found increased levels of TNF-a in faeces from IBD patients and the levels of TNF-a associate with the severity of inflammation. TNF-a can induce the secretion of proinflammatory factors and activative NF-κB signaling pathway. Interleukin-lbeta (IL-1β),a proinflammatory factor, play a major role in several autoinflammatory and chronic immune diseases, including inflammatory bowel disease (IBD) it also has the ability to stimulate the production of other powerful cytokines like IL-6,suggest that IL-1β is a viable candidate mediator that accompanies colonic inflammation. IL-1β has received considerable attention as a potential mediator of inflammatory cell infiltration and mucosal barrier disruption that accompanies gut inflammation. IL-1β also appears to promote inflammation by stimulating the production of other cytokines (eg, IL-6) and chemokines. Neutrophils are proinflammatory cells with the capacity to ingest and kill invading pathogens, by generating reactive oxygen intermediates together with other cytokine released during the process of degranulation. Neutrophils migrate from the blood to the intestinal tissues is highly increased and show a high activities in patients with UC. Increased levels of neutrophils detected in intestinal lamina propria is consider as the maker of inflammation. KC, a CXC chemokine, is bound to shed syndecan-1, and the syndecan-1/KC complexes directs and confines neutrophil influx to sites of injury.We speculated that shedding of Syndecan-1probably participate in intestinal inflammation. But limited report about unshedding Syndecan-1is found. The inflammatory process may directly increase by activation of NF-κB pathway, or may by the release of inflammatory cytokines.However,the relationship among Syndecan-1、NF-κB pathway and inflammatory cytokines in the inflammatory process is still unkown. Therefore, we construct the expressing vector pcDNA3.0-wildtype-syndecan-1(WT-sdc-1) and pcDNA3.0-unshedding-syndecan-1(uS-sdc-1), and stimulate intestinal epithelial cells by LPS after transfection to explore the effect on the expression of NF-κB and inflammatory cytokines in vitro.Materials and Methods1.The mouse WT-sdc-1DNA was successfully amplified by PCR and then cloned into pcDNA3.0. uS-sdc-1was construct by Gene splicing by overlap extension-PCR(SOE-PCR) to induce the mutation for the243site(A)in Syndecan-1protien into (T), the244site(S)in into (W) on the basis of WT-sdc1. The two vectors confirmed by DNA sequencing.2. Control group、Vector transfected group、WT-sdc-1transfected group and uS-sdc-1transfected group were established. Each vecter was transfected into IEC-6and Caco-2cells by Lipofectamine2000. RT-PCR、Immunofluorescence and Dot-blot were performed to detect the expression of Syndecan-1before and after simulation of phorbol12-myristate13-acetate (PMA) for15min.3.There were5groups in our research:Control group,LPS group,Vector+LPS group,WT-sdc-1+LPS group and uS-sdc-1+LPS group. The transfected cells were then cultrured for24h. After stimulated with LPS(lug/ml) for24h, Nucleoprotein、 Plasma protein and total protein were isolated from cell lines respectively. P65in3kinds of protein and IκB in total protein were detected through Western blot.4.5groups of IEC-6and Caco-2cells was transfected and then cultrured for24h as above, then stimulated with LPS of lug/ml.The supernatant of different group was collected at the Oth、6th、12th、24th、48th hour, respectively. The levels of TNF-a accumulated in the conditioned media were assessed by enzyme-linked immunosorbent assay (ELISA) using an ELISAkit.5.Neutropils was isolated from peripheral blood of human and rat respectively. Neutropils transmigration stimulated with Supernatant of different LPS treated group model were established using transwells inserts.6. Data analyses:All the data were analyzed by SPSS13.0. Data are expressed as mean±SD. If samples obeyed normality distribution. Statistical significance was tested using One-way ANOVA, LSD, Dunnett’s T3, if samples did not obey normality distribution K Independent-Samples Nonparametric Test was used. Statistical significance was set at P values less than0.05.Results:l.The vector WT-sdc-1and uS-sdc-1were successfully constructed although an none-sense mutation was in uS-sdc-1.2. Compared to Control and Vector transfected groups,WT-sdc-1and uS-sdc-1groups showed a significant increase in the expression of Syndecan-1in both mRNA and protein levels. In response to the simulation of PMA, the expression of Syndecan-1was down-regulated at the protein levels but not mRNA levels.3. P65in total protein of2cell lines in LPS treated group were higher than that in Control group(the group did not add in LPS)(P＜0.05). Compared to LPS and Vector+LPS groups, WT-sdc-1+LPS and uS-sdc-1+LPS groups showed a significant increase in the expression of P65in protein levels. P65in uS-sdc-1+LPS groups was lower than that of WT-sdc-1+LPS groups(P<0.05). IκBα of the two cell lines in total protein had the same trend with P65.4. The differences of P65in plasma protein were statistically significance among the five groups of two cell lines (P<0.05). Only a weak stripe was found in Control group, The differences were not statistically significance between the LPS group and Vector+LPS group (P>0.05), P65in LPS group was higher than that in uS-sdc-1+LPS group but there was no difference compared with WT-sdc-1+LPS group. There was no difference between WT-sdc-1+LPS group and uS-sdc-1+LPS group.5.In these2cell lines,there were significant differences in P65of nucleoprotein among different treated group.(P<0.05). Only a weak stripe was found in Control group, The differences were not statistically significance between LPS and Vector+LPS groups.P65in LPS group was higher than that in WT-sdc-1+LPS group and uS-sdc-1+LPS group. There was no difference between WT-sdc-1+LPS group and uS-sdc-1+LPS group.6.TNF-a in LPS treated group were higher than that in Control group (P<0.001). TNF-a expression and Syndecan-1expression was negatively correlated; Expression of TNF-a showed a significant increase in LPS and Vector+LPS groups,while WT-sdc-1+LPS group and uS-sdc-1+LPS group do not. TNF-a in uS-sdc-1+LPS groups was lower than that of WT-sdc-1+LPS groups. But there were no significant difference of TNF-a among groups in IEC-6cell at Oth hour and Caco-2cell at Oth,48th hour.TNF-a in Supernatant obtained at0-6h was low, it at12h and24h was the highest, which gradually decreased with time.7.After simulation with LPS for12h and48h, IL-lβ in uS-sdc-1+LPS group of IEC-6cell was the highest (P＜0.001). WT-sdc-1+LPS group was the second, while IL-1β in Control group was the lowest. There was no difference between groups of IEC-6cell at0th、6thu、24th hour and groups of Caco-2cell of any time.(P>0.05)8.The expression of CINC-1among groups in IEC-6of any time (except0th hour) were significant difference (P<0.05),and so does Caco-2cell at24th and48th hour. Expression of CINC-1showed a significant increase in LPS and Vector+LPS groups comparing to WT-sdc-1+LPS group and uS-sdc-1+LPS group. CINC-1in uS-sdc-1+LPS groups was lower than that of WT-sdc-1+LPS groups (P＜0.05). There were no significant difference in expression of CINC-1among groups in Caco-2at0th、6th、12th hour. For IEC-6cells, up-regulation of CINC-1was time-dependent. For Caco-2cells, expression of CINC-1increased among0-24h,but among24-48h,expression of CINC-1decreased.Conclusion:1.The WT-sdc-1and uS-sdc-1are successfully constructed,which lays thefoundation for further studying of Syndecan-1in Gastrointestinal inflammation.2.High expression of Syndecan-1may reduce the expression of NF-κB especially the unshedding Syndecan-1, Syndecan-1may regulate the inflammatory processes by down-regulating the NF-κB signaling pathway.3.TNF-α CINC-1are decreased in cells with high expression of Syndecan-1especially high expression of unshedding Syndecan-1. Integral HS chains may play an important role in regulationg of inflammation by inhibiting these cytokine.4.There are no difference in IL-1β among groups of different expression levels of Syndecan-1.Complex regulatory mechanism may existence in IL-1β regulation when inflammation occur. Syndecan-1may not play a role in regulationg of IL-1β in inflammation.5.High expression and unshedding of Syndecan-1may regulate the inflammatory processes by reducing the inflammatory factors and down-regulate the activity of NF-κB...