The Effects Of Intergrin-linked Kinase Inhibition On Human Fibroblast In Migration And Differentiation

BackgroudWound healing is a pathophysiological process for reparing by migration,proliferation, remodeling in local tissue after rupture and defect of skin. This processneeds to be regulated through the interaction between various reparing cells and aseries of cytokines. Among them, the fibroblast is the main reactive cell forreconstructing dermal. Therefore, a large number of sophisticated biological reactionsoccurred between fibroblasts and extracellular matrix (extracellular matrix, ECM),which is an important part of exploring pathological process for wound healing.Integrin is a main receptor on the cell membrane for ECM. The studies showedhigh expression of multiple integrins during wound healing process. It was speculatedthat integrins may participate in the regulation of wound healing process.Integrin-linked kinase (ILK) links the cytoplasmic domain of integrin and promotesthe cascade reactions of cell signals. Experiments showed that reducing the expressionof ILK with small interfering RNA (siRNA) may inhibit the invasion and deterioratedof tumor cells. Studies also showed that ILK may play an important role in theprogress of the mice renal fibrosis induced by TGF-β1. Granulation tissue formationdue to ECM deposition and fibroblasts proliferation starts fibrosis process in woundhealing. Our previous studies found that ILK expression was closely related to woundhealing process, but it needs to further explore the mechanism of ILK in woundhealing.The synthetic small molecule QLT0267could specifically bind and inhibits ILKactivity in the cytoplasm by blocking the Ser473phosphorylation site, according tothe amino acids sequence of ILK. The ILK inhibitor can supress protein kinase B(PKB/AKT) phosphorylation and reduce the phosphorylation of AKT，by whichregulats biological changes of cells. Research shows that QLT0267can effectivelyinhibit the activity of ILK, and consequently influence the migration, differentiation,proliferation and apoptosis in tumor cells and renal tubular epithelial cells, whichsuggesting that ILK may be involved in the regulation of cellular physiological function in the ILK-PKB/AKT pathway. The application of ILK inhibitorQLT0267points out a new direction for study on the function of ILK in themechanism of wound healing.In this study, normal human skin fibroblasts as the research object were inhibitedwith the QLT0267to prevent ILK phosphorylation activity or transfected with smallmolecule interference RNA to silence the expression of ILK. The migration,differentiation and proliferation functions of the cells were observed for thepreliminary investigation on the role of ILK in wound healing processPART Ⅰ The affect of QLT0267on human skin fibroblast cellswith different concentrationsObjective To investigate the biological changes of fibroblasts by usingdifferent concentrations of ILK inhibitor QLT0267in different time point anddetermine the optimal concentration and observed time point for the subsequentexperiments.Methods Skin fibroblasts were isolated from redundant grafts of plasticsurgery patients by mechanical method combining with enzyme digestion. Accordingto different concentrations of ILK inhibitor QLT0267, the cells were divided into sixgroups: A. bank control group, B.5nM group, C.10nM group, D.20nM group, E.30nM group and F. DMSO group (QLT0267solution). Firstly we observed the cellmorphology under inverted phase contrast microscope at12,24,48,72h, then cellproliferation was detected at24,48,72h by CellTiter kit, at last flow cytometry andWestern Blot detection was used to detect the apoptosis of cells and pAKT, tAKTprotein expression of the cell at48h. Data were analyzed using LSD-t and repeatedmeasurement ANOVA to determine significance between samples.Result It was more than10nM inhibitors of ILK could make fibroblast swellingand expand roundly, nuclear pyknosis or disintegration, even the apoptotic bodiesappeared, with the longer time of drug stimulation and higher concentration of drugthe more obvious. The result of CellTiter kit showed that at least10nM the ILK specific inhibitor QLT0267could inhibit the cell proliferation (p <0.05), and as theconcentration increase and stimulation time extend the inhibition rate of proliferationwould ascensus (p <0.05). Flow cytometry test results showed that fibroblastapoptosis could be induced by ILK specific inhibitor with above10nM concentrationcompared with control group, ILK inhibitor could induce inhibitors (P <0.05). Fromhigh to low concentrations of the QLT0267, groups expressed less pAKT than thecontrol group(p <0.05), with no significant difference expression of tAKT in the test(p>0.05).Conclusion ILK specific inhibitor QLT0267can induce the biologicalcharacteristics changes of fibroblasts. It could inhibit the cell proliferation and startcell apoptosis by down-regulating the amount of pAKT. As higher the concentrationand longer stimulating time, its effect become more obvious. According to theresults of the experiments and requirements of subsequent experiments, theapplication of10nM QLT0267on fibroblasts for48h is most suitable for thefollowing experiments.PART Ⅱ Affection of ILK on migration of normal human skinfibroblast cellsObjective To explore the function and mechanism of ILK on the migration inhuman skin fibroblast.Methods Human dermal fibroblasts were cultured in vitro. The target siRNAsequence of ILK gene was designed and synthesized. ILK-siRNA was thentransfected into the fiberoblasts by using Lipofectamine2000. Fibroblasts have beentreated with10nM QLT0267for48h. There were six groups in the present study: A.bank control group, B. transfection vehicles group (Lipofectamine2000transfectionreagent), C. con-siRNA group, D. ILK-siRNA group, E.QLT0267group and F.DMSO group (QLT0267solution).The in vitro scratch adhesion test was conducted toobserve the healing rate of fibroblasts at12、24h by comparing the areas which thefibroblasts migrated to the scratch aere at0. Real-time quantitative PCR was used todetect the expression of ILKmRNA. and Western Blot was used to detect the expression of protein pAKT, tAKT, ILK. The data analysis was conducted by ANOVAand LSD-t test.Result The scratch adhesion test In vitro showed the cell healing rates of theILK-siRNA group and QLT0267group were significantly lower than that of thecontrol group at12h (p <0.01), but no significance difference was found betweenthem(p>0.05). The findings in RT-PCR showed the expression of ILK gene wassignificantly inhibited by siRNA by comparison of control group (p <0.01). Thefindings in Western Blot also showed that expression of ILK in the ILK-siRNA groupwas significantly lower than that the other five groups (p <0.01); the expression ofpAKT in ILK-siRNA group and QLT0267group decreased significantly than that ofbank control group(p<0.05). but no significance difference between them(p>0.05).Moreover, there were no significant differences of the tAKT expression in each group(p>0.05).Conclusion siRNA could inhibit the expression of ILK protein. The decreaseof ILK protein and the inhibition of phosphorylation activity could reduce theexpression of pAKT protein. All the finding in the current study suggested that ILKmay regulate the migration of fibroblasts and consequently influencing the woundhealing through the ILK-AKT pathway.PART Ⅲ Influence of ILK on differentiation of normal humanskin fibroblast cells with TGFβ1Objective To investigate the effect of ILK on expression of alpha-smooth muscleactin with TGFβ1, further study the role of ILK in the differentiation of normal humanskin fibroblast cells.Methods The cells were divied based on the rationale in part two. Then theexpression of α–SMA protein was detected by the Western Blot. Clutured cells weredivided into7groups:1. TGF-β1group,2. transfection vehicles+TGF-β1group,3.con-siRNA+TGF-β1group,4. ILK-siRNA+TGF-β1group,5. QLT0267+TGF-β1group,6. DMSO+TGF-β1group,7. blank control group. First,1~6group wereprocessed as the second part, after that stimulated by5ng/ml TGF-β1for24h. each group α–SMA and ILK protein was detected by the Western Blot. One-way ANOVAand Independent t test were applied in the data analysis.Result The expression of α-SMA protein in ILK-siRNA group and QLT0267group was significant lower than that in the control group respectively (p<0.05), butno significance difference between them(p>0.05). The synthesis of α-SMA protein inTGF-β1group is higer than the bank control group(p<0.05). α-SMA expression inboth of ILK-siRNA+TGF-β1group and QLT0267+TGF-β1group were lower thanthat in TGF-β1group (p<0.05),but no significance difference between them(p>0.05).Conclusion The down-regulated ILK protein expression or inhibition of itsphosphorylation activity decreased the synthesis of α-SMA protein in fibroblast.TGF-β1could speed up the differentiation of fibroblasts into myofibroblast, but thedecrease of ILK expression or inhibition of ILK activity limited the induction of thefibroblast differentiation with TGF-β1. The findings of the present study suggestedthat ILK could participate in fibroblast differentiation with TGF-β1by PKB/AKTpathway, and hence affecting the wound healing.