ILK-PI3K/AKT Pathway Participates In Cutaneous Wound Contraction By Regulating Fibroblast Migration And Differentiation To Myofibroblast

Wound contraction plays an important role in full thickness wound healing.Proper wound contraction can effectively reduce the wound area and shorten healing time.However,excessive wound contraction contributes to hypertrophic scar formation.Two main concepts explain wound contraction‘s mechanisms.One considers that the force of wound contraction is generated from the crawling of fibroblasts,which migrate from the periphery to the center.The other notion considers that wound contraction is related to myofibroblasts‘ contractile forces.It is generally accepted that these two notions are equally important.Fibroblast migration is required for the early phase of wound healing.At later stages,wound contraction is mainly attributed to myofibroblast contractile forces.Undoubtedly,the interactions between fibroblasts(or myofibroblasts)and the extracellular matrix(ECM)are necessary for wound contraction.Fibroblast-populated collagen lattice(FPCL)is a model for wound contraction study as well as studying cell-ECM interaction in vitro.In FPCL,fibroblasts are suspended in a collagen lattice to form a derma similar three-dimensional structure,which has visible contraction ability in vitro.The integrin signaling pathway plays a crucial role in mediating the interaction between cells and the ECM.The ECM‘s biological signals are transmitted in cells by binding to the transmembrane integrin receptors and activating the signaling pathway.Integrin-linked kinase(ILK)was first identified and cloned by Hannigan et al.in 1996 based on its interaction with the β1 integrin cytoplasmic domain.ILK is a serine/threonine kinase enzyme and has three highly conserved different domains.The ILK N-terminal ankyrin domain contains 4 ankyrin repeats,which mediate the crosstalk between ILK and other signaling pathways,such as the receptor tyrosine kinase(RTK)signaling pathway.ILK‘s central structure is a pleckstrin-homology(PH)-like domain that can combine phosphatidylinositol-3-phosphate(PIP3)and regulate ILK‘s catalytic activity.The ILK C-terminal is a catalytic domain that interacts with the cytoplasmic domains of the integrin‘s beta subunits.The ILK C-terminal can phosphorylate AKT on Ser473 in a phosphatidylinositol-3 kinase(PI3K)-dependent manner to regulate proliferation,migration,and other cell functions.Transforming growth factor-β1(TGF-β1)is one of the most important cytokines related to wound healing and scar formation.It is involved in multiple aspects of wound healing,including ECM synthesis promotion,fibroblast migration and proliferation regulation,and alpha smooth-muscle actin(α-SMA)expression induction.α-SMA is characteristically expressed by myofibroblasts,which are differentiated from fibroblasts,and it contributes to wound contraction.Recent studies have revealed that ILK is a key intracellular mediator for the ―cross talk‖ between an integrin and the TGF-β1 pathway.ILK over-expression can promote α-SMA expression as well as epithelial-mesenchymal transition(EMT)in renal tubular epithelial cells.In renal tubular epithelial cells,TGF-β1 can markedly up-regulate ILK expression in a time-and dose-dependent manner,and TGF-β1 effects promoting Fibronectin,snail 1,and MMP2 expressions can be blocked by ILK inhibitor QLT0267.Previous studies have found that,ILK may be involved in the regeneration of various tissues after injury.Overexpression of ILK in isolated stellate cells led to enhanced motility and adhesion as well as increases in α-SMA and type I collagen expression.ILK involves in stellate cell activation and fibrogenesis during liver injury.Besides,ILK–PI3K/AKT pathway also plays an important role in nerve growth factor(NGF)mediated neurite outgrowth and thymosin β4 mediated cardiomyocyte migration,survival and repair after damage,Thus,we hypothesis that ILK–PI3K/AKT pathway plays a critical role in the course of cutaneous wound healing as well.In the current study,we observed the effects of ILK inhibitor QLT0267 on rat wound contraction.Furthermore,we demonstrated the influences of ILK inhibitor QLT0267 and PI3K/AKT inhibitors LY294002 on FPCL contractile ability,cell proliferation,and migration,as well as TGF-β1 induced α-SMA expression.Meanwhile,we directly targeted ILK gene by ILK SiRNA transfection,then,the aforementioned effects were detected by us.Based on our results,we concluded that the ILK-PI3K/AKT pathway is involved in wound contraction by regulating fibroblast proliferation and migration as well as myofibroblast differentiation.