Effect Of BNIP3-mediated Autophagy On Keratinocyte Migration And Its Mechanism

BackgroundBNIP3(Bcl-2 nineteen-kilodalton interacting protein 3)is a single transmembrane Bcl-2 family protein containing only atypical BH3 domain.It is mainly distributed on the outer membrane of mitochondria and is a hypoxia-reactive protein.Recent studies have shown that BNIP3 is involved in the regulation of biological behaviors such as cell survival,apoptosis,and differentiation,and has been reported to be invovled in various disease models like tumor development,myocardial injury and repair,and cerebral ischemia and hypoxia damage.Our previous studies have shown that hypoxic microenvironment can induce high expression of BNIP3 in mouse skin keratinocytes and play a positive regulatory role in their migration.Emerging studies have shown that BNIP3 regulates autophagy,and the role of autophagy on cell migration is receiving increasing attention.Studies have shown that BNIP3 is distributed in human skin keratinocytes and has a regulatory effect on its homeostasis and differentiation.The distribution of BNIP3 is accompanied with a significant up-regulation of autophagy.Therefore,we hypothesized that BNIP3-mediated autophagy may regulate the migration of keratinocytes and play an important role in wound re-epithelialization.Autophagy was originally described basing on its ultrastructure of bilayer membranes that encapsulate cytoplasm and organelles,which are named autophagosomes.This process is triggered by the formation of two complexes,the ULK1 complex and the PIK3C3/VPS34 complex,which contains Atg14 L and Beclin-1.These complexes are located the upstream of two ubiquitin-like conjugation systems,namely Atg12-Atg5 and LC3-phosphatidylethanolamine,both of which extend and block the autophagosome membrane.During elongation of the phagophore membrane,Atg7 mediates the formation of the Atg12-Atg5 complex,which then binds to Atg16L1 and produces an Atg16L1 complex.The autophagosome then fuses with the endocytic compartment and acquires acidic and degradative properties prior to fusion with the lysosome.Eventually,the autophagy cargo is degraded in the lysosome.Autophagy is an evolutionarily highly conserved catabolic process that is activated in response to changes in hunger,hypoxia,or nutritional conditions.In recent years,more and more studies have found that autophagy is closely related to cell migration.However,whether autophagy leads to an increase or decrease in cell migration ability is controversial.Induction of autophagy under certain conditions has a pro-migration effect,whereas activation of autophagy is associated with inhibition of cell migration under other conditions.BNIP3 competes with Beclin-1 for the binding site of BH3 domain on Bcl-2,thereby disrupting the stable binding of Bcl-2 to Beclin-1,releasing Beclin-1 and initiating autophagy.Recent studies have shown that BNIP3 induces autophagy to differentiate keratinocytes and protect keratinocytes from UVB-induced apoptosis.However,the role of BNIP3 in the induction of autophagy in keratinocytes during wound healing and its role in keratinocyte migration remains unclear.Although BNIP3 has been widely reported to be upregulated by hypoxia-inducible factor(HIF-1)under hypoxic conditions,the HIF-1-independent mechanism of the overexpression of BNIP3 and its post-transcriptional regulation are also crucial for understanding the expressions of BNIP3 in different cells and tissues.It is well known that the hypoxic microenvironment activates a variety of signal transduction pathways,including AMP-activated protein kinase(AMPK),HIF-1,and mitogen-activated protein kinase(MAPK)signaling pathways.Among them,MAPK is an evolutionarily conserved serine/threonine protein kinase that plays an important role in the basic life behaviors of cells,such as proliferation,differentiation,apoptosis,survival and migration.The p38 kinase pathway,extracellular signal-regulated kinase(ERK),and c-Jun N-terminal kinase(JNK)are the three basic components of the mammalian MAPK pathway.The ERK pathway is primarily activated by growth factors(such as epidermal growth factor);while the JNK and p38 signaling pathways are activated by a variety of stress stimuli,such as hypoxia,UVB radiation,and inflammatory cytokines like tumor necrosis factor(TNF)-?.In addition,hypoxia has been shown to stimulate the accumulation of reactive oxygen species(ROS),which are also involved in the activation of the MAPK signaling pathway.Therefore,this study focused on the mechanism of action of BNIP3 as a pro-migratory molecule during wound healing.Our study found that hypoxia-mediated ROS accumulation promotes the activation of p38 MAPK and JNK MAPK in human immortalized keratinocytes HaCaT cells,and up-regulates BNIP3-induced autophagy.In addition,we have also found that inhibition of autophagy significantly impairs hypoxia-induced keratinocyte migration.These results reveal a previously unreported mechanism by which ROS activates p38 MAPK and JNK MAPK signaling pathways under hypoxic conditions,sequentially promotes up-regulation of BNIP3 and activation of autophagy,thereby promoting keratinocyte migration.Methods1.Using mouse full-thickness skin defect wound model,we collected wound specimens at different time points during wound healing,and detected the expressions of autophagy-related proteins such as BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62 in keratinocytes at wound by immunofluorescence and Western Blot.The interaction between LC3 and BNIP3 was detected by immunoprecipitation(CO-IP)in order to verify the possible regulation of BNIP3 on autophagy of keratinocytes during wound healing.2.Using the in vitro hypoxic culture model of human immortalized keratinocyte HaCaT cell line,we detected the expressions of autophagy-related proteins such as BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62 in keratinocytes at wound by immunofluorescence and Western Blot.The autophagosomes under hypoxic conditions were detected by transmission electron microscopy and immunofluorescence.We analyzed the autophagic flow in keratinocytes under hypoxic conditions by autophagy inhibitors,chloroquine(CQ),bafilomycin(BafA1)and autophagy-related protein Atg5-specific interference(siAtg5).Keratinocyte migration aupon autophagy intervention was detected by live cell workstation and in vitro wound-healing assays.3.The HaCaT cell line with stable and low expression of BNIP3 was established by recombinant adenovirus transfection.Changes in autophagy of HaCaT cells upon BNIP3 deletion were analyzed by Western Blot and immunofluorescence.Changes in keratinocyte migration after knockdown of BNIP3 were analyzed using live cell workstation and in vitro wound-healing assays.4.ROS levels in the epidermal layer at wound edge were analyzed by reactive oxygen species detection kit.We established a hypoxic culture model of newborn mouse in vitro,and detected ROS levels in epidermal layer after hypoxia treatment by active oxygen detection kit.Then the hypoxic culture model of newborn mouse in vitro was treated with the active oxygen scavenger N-acetylcysteine(NAC),and immunofluorescence and Western Blot were performed to analyze the expressions of autophagy-related proteins such as BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62.Using the in vitro hypoxic culture model of HaCaT cells,we analyzed the expressions of autophagy-related proteins such as BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62,and the alterations of the activity of p38 MAPK,JNK MAPK,HIF-1? and other signaling pathways,as well as the migratory capacities in cells after NAC treatment.We analyzed the expressions of autophagy-related proteins BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62,and the migratory capacities in cells after NAC treatment.Thus,we revealed a possible mechanism by which hypoxia promotes keratinocyte BNIP3,autophagy and migration both in vivo and ex vivo.Results1.Results of immunofluorescence showed that the expressions of BNIP3,LC3 and Atg5 were significantly increased in the keratinocytes at wound edge in mice.Results of Western Blot further confirmed that the expression of autophagy-related proteins such as BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1 and P62 were increased in keratinocytes at wound edge in mice.Results of CO-IP showed an interaction between LC3 and BNIP3 in mouse wound tissue.These results suggest that BNIP3 may have a regulatory effect on keratinocyte autophagy during wound healing;2.Western Blot assay showed significantly increases in the expressions of autophagy-related proteins BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1 and P62 in hypoxic HaCaT cells,which was in a time-dependent manner.The results of transmission electron microscopy(TEM)and immunofluorescence(IF)showed an increase in the formation of autophagosomes in hypoxic keratinocytes.Autophagic intervention was performed using autophagy inhibitors chloroquine(CQ),bafilomycin(BafA1)and Atg5-specific siRNA(siAtg5),and the autophagic influx of hypoxic keratinocytes was found unobstructed.Moreover,the migration of keratinocytes under hypoxic conditions upon autophagy inhibition was significantly inhibited.These results indicate that autophagy elevates in hypoxic keratinocytes and promotes their ability to move and migrate.3.The HaCaT cell line with stable low BNIP3 expression was successfully constructed.It was found that autophagy of hypoxic HaCaT cells decreased significantly upon BNIP3 knockdown,and the capacity of cell movement and migration was also significantly decreased.These results further indicated that BNIP3-mediated autophagy can be involved in the regulation of keratinocyte migration under hypoxic conditions.4.ROS in the epidermal layer at wound edge significantly increased during wound healing.Epidermal layer in the in vitro hypoxic culture model of newborn mouse showed a significant increase in ROS production,thus it can simulate the hypoxic wound edge in vivo.NAC can effectively eliminate ROS induced by hypoxia,and inhibited the expressions of autophagy-related proteins BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62.The expressions of autophagy-related proteins BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62 notably increased in hypoxic HaCaT cells,as well as the activities of p38 MAPK,JNK MAPK,HIF-1?,and the migratory capacities.While the addition of NAC remarkably reversed it.It was found that expressions of autophagy-related proteins BNIP3,LC3,Atg5,Atg7,Atg16L1,Beclin-1,and P62 as well as migratory capacities in HaCaT cells treated with p38 MAPK and JNK MAPK chemical inhibitors(SB203580,SP600125)was significantly inhibited.These data revealed that BNIP3-induced autophagy occurs through hypoxia-induced ROS-mediated p38,JNK MAPK and HIF-1? activation and supports the migration of epidermal keratinocytes during wound healing.ConclusionsThese data from in vivo and ex vivo clarified that wound-induced hypoxic microenvironment promotes ROS generation,activates p38 and JNK MAPK signaling pathway,which in turn elevates the BNIP3-induced autophagy,thereby promoting the migration of keratinocytes.This study is the first to explore the important role of BNIP3-mediated autophagy on wound re-epithelialization,and provides a new perspective on the function of BNIP3 in wound healing,which is expected to provide a new possible target for clinical wound treatment.