Effects Of BNIP3 On Keratinocyte Migration Under Hypoxia And Its Possible Mechanisms

BackgroundWound healing is a well ordered process that requires the orchestration of a variety of tissues and cells.Re-epithelialization is a biological process that starts at the early stage of wound healing,and plays a very important role in the reconstitution of the intact epidermal mechanical barrier to prevent infection and excessive moisture loss,throughout which keratinocyte migration is an essential step,and keratinocyte migration has become the focus of wound healing study.Hypoxic microenvironment is an important factor in the regulation of keratinocyte migration,but the underlying molecular mechanism is not fully elucidated.As a member of Bcl-2 protein family that contains atypical BH3 domain only,BNIP3?Bcl-2 nineteen-kilodalton interacting protein 3?is a hypoxia-reactive protein.It is well documented that BNIP3 involves in the regulation of many biological behaviors such as cell proliferation,apoptosis,autophagy and differentiation,and plays an important role in tumor development,cardiac hypertrophy and atrophy,cerebral hypoxic/ischemic damage and many other processes of disease.BNIP3 is expressed in human keratinocytes and involved in regulating their differentiation and homeostasis.But no reports were found for BNIP3 in the regulation of keratinocyte migration.Upon the formation of wound,many signaling pathways are activated to mobilize wound repair cells and promote wound re-epithelialization.Focal adhesion kinase?FAK?,a cytoplasmic protein tyrosine kinase,is the core molecule of integrin associated signaling pathways that regulate cell motility.FAK is mainly activated through autophosphorylation of tyrosine at 397 site.It has been widely known that FAK participates in regulation of keratinocyte migration during wound healing.Inhibition of integrin activity by specific antibodies could effectively reduce the activation of FAK and inhibited keratinocyte migration;Wound healing significantly delayed in a FAK deletion mouse model,which was highly related with blocking of FAK activation.In the studies associated with tumors,FAK and its downstream signaling molecules have been found to involve in regulating the polymerization,depolymerization and reconstruction of cellular actin cytoskeleton,which are important parts of cell migration.In fact,BNIP3 can regulate the remodeling of cellular actin cytoskeleton,thus we speculate that FAK may play a role in BNIP3 directed keratinocyte migration.Among a variety of researches of mammalian cells,BNIP3 was found to rely on its BH3 domain to disturb the interaction of Bcl-2 and Beclin-1 so as to start autophagy,which is a highly conserved lysosomal degradation pathway and plays an important role in maintaining body homeostasis.Functionally,autophagy involved in the regulation of many physiological and pathological processes such as growth and development,inflammation,tumor invasion and metastasis.Recently,the biological significance of keratinocyte autophagy has also been widely concerned.It is suggested that certain physical and chemical factors or substances with biological activity can induce keratinocyte autophagy;and autophagy can regulated the differentiation of keratinocytes;moreover,autophagy reduces damage of keratinocytes caused by ultraviolet radiation and promote their survival.However,there still lack evidence of autophagy in keratinocyte migration.Therefore,we assumed that BNIP3 promoted keratinocyte migration through the activation of FAK activity or by modulating autophagy under hypoxia.Then studies were undertaken to detect the effects of BNIP3 on keratinocyte migration under hypoxic conditions,and to explore its regulation of FAK activity and autophagy.Methods1.To observe the changes of keratinocyte motility and migration and BNIP3 expression,we used primary mouse keratinocytes to establish an in vitro hypoxia model in which keratinocytes were subjected to hypoxia?2% O₂: 3,6,9h?to simulate the hypoxic wound microenvironment.2.To elucidate whether BNIP3 involves in the regulation of keratinocyte motility and migration under hypoxia,we performed siRNA transfection to reduced the expression of BNIP3 under hypoxia?2% O₂: 6h?.Firstly,we detected the validity of siRNA transfection through WB;then we observed the impacts of BNIP3-downregulation on keratinocyte motility under live cell imaging workstation;lastly,we observed the impacts of BNIP3-downregulation on keratinocyte migration by in vitro wound healing assay.3.To determine whether FAK and autophagy involve in the BNIP3-induced keratinocyte migration under hypoxia,we firstly detected changes of p-FAK activity and autophagy after keratinocytes were subjected to hypoxia?2% O₂: 3,6,9h?through western blot,then we tested the effects of BNIP3 down-regulation by si RNA transfection on FAK activity and autophagy under hypoxia.After that we evaluated the effects of selective FAK inhibition by TAE226 on keratinocyte motility and migration under hypoxia,and the effects of autophagy activation by rapamycin on keratinocyte motility and migration under normoxia,respectively.In this way,our findings provide new insights into the underlying mechanism surrounding the biological function of BNIP3 in keratinocyte migration during wound healing.Results1.It was found that hypoxia significantly promoted the motility and migration of primary mouse keratinocytes and remarkably elevated BNIP3 protein expression with the prolonged upward trend,which was basically fit with variation of keratinocyte motility under hypoxia.2.In our study,BNIP3 was successfully downregulated by si RNA transfection.And downregulation of BNIP3 effectively reversed keratinocyte motility and migration promoted by hypoxia through the single cell motility assay and in vitro wound healing assay,indicating BNIP3 involved in regulation of keratinocyte migration induced by hypoxia;3.Hypoxia activated FAK pathway and significantly increased the expression of the autophagy marker LC3-II;BNIP3 downregulation with siRNA transfection largely inhibited FAK signaling pathway under hypoxia,and inhibition of the activity of FAK reversed hypoxia induced keratinocyte migration,indicating that FAK signaling pathway was involved in the BNIP3 mediated keratinocyte migration under hypoxic conditions.It is suggested that BNIP3 regulated keratinocyte autophagy because BNIP3 downregulation apparently suppressed the elevation of LC3-II expression under hypoxia.However,despite rapamycin could inhibit keratinocyte migration under normoxia,the autophagy simulated by rapamycin is different from hypoxia-induced one,thus whether BNIP3 regulate keratinocyte migration through autophagy under hypoxia still remains to be elucidated in further study.ConclusionsWe revealed that hypoxia increased BNIP3 expression and activation of FAK pathway,thereby promoting keratinocyte migration;despite hypoxia can activate autophagy,but whether BNIP3 regulated keratinocyte migration through autophagy under hypoxia still remains to be elucidated in further study.This study identified the role of BNIP3 in wound epithelialization,preliminarily elucidated its mechanism,which may provide a potential new molecular target for the clinical treatment.