Moreover, suppression of HIF-1 pathway by small-interfering RNAs significantly inhibited hypoxia-induced BNIP3 expression. and cell migration, suggesting a positive role of BNIP3-induced autophagy in keratinocyte migration. Furthermore, these results indicated that the accumulation of reactive oxygen species (ROS) by hypoxia triggered the activation of p38 and JNK mitogen-activated protein kinase (MAPK) in human immortalized keratinocyte HaCaT cells. In turn, activated p38 and JNK MAPK mediated the activation of BNIP3-induced autophagy and the enhancement of keratinocyte migration. These data revealed a previously unknown mechanism that BNIP3-induced autophagy occurs through hypoxia-induced Citicoline ROS-mediated p38 and JNK MAPK activation and supports the migration of epidermal keratinocytes during wound healing. Introduction Wound healing is a complex multistep process involving three partially overlapping phases as follows: inflammation, re-epithelialization, and tissue remodeling. During re-epithelialization, epidermal keratinocytes migrate into the wound site, proliferate, and differentiate to reconstruct the epidermal barrier1. Defects in keratinocyte migration usually result in unsatisfied wound repair2,3. It has been implicated that wound-induced hypoxia promotes keratinocyte motility and enhances keratinocyte migration4,5, while the underlying mechanisms remain largely unclear. BNIP3 (Bcl-2 and adenovirus E1B 19-kDa interacting protein 3) is a single transmembrane protein that is mainly located in the outer membrane of mitochondria. Previously, we determined that BNIP3 is upregulated by hypoxic exposure and plays a critical role in hypoxia-induced keratinocyte motility and migration6. Others have elucidated that BNIP3 significantly triggers cell Citicoline autophagy in hypoxic cardiomyocytes7 and that autophagy clearly promotes cell migration in some cases8. Thus, we speculated that autophagy induced by BNIP3 might serve a pro-migratory function. Macroautophagy (hereafter referred to as autophagy) was initially described based on its ultrastructural Rabbit Polyclonal to RPL26L features of double-membraned structures that surround the cytoplasm and organelles in cells, which are known as autophagosomes9. Many studies have reported that autophagy is involved in cell migration. However, whether autophagy leads to the enhancement or impairment of cell migration is controversial. The induction of autophagy has a pro-migratory effect in some conditions, whereas it is associated with the inhibition of cell migration in other conditions10,11. Recently, the induction of autophagy by BNIP3 has been demonstrated to be essential for the differentiation of keratinocytes and the protection of keratinocytes from UVB-induced apoptosis12,13, whereas the role of BNIP3-induced autophagy in keratinocytes during wound healing remains unclear. Although BNIP3 is known to be highly upregulated under hypoxia through the hypoxia-inducible factor (HIF-1), the HIF-1-independent mechanisms and posttranscriptional mechanisms are also crucial for BNIP3 expression in different cells and tissues14C17. Hypoxia is well established to stimulate several signaling pathways, Citicoline including the AMP-activated protein kinase (AMPK), HIF-1, and mitogen-activated protein kinase (MAPK) signaling pathways. MAPK is an evolutionary conserved serine/threonine protein kinase playing an important role in fundamental cellular processes, such as proliferation, differentiation, apoptosis, survival, and migration18. The extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 kinase pathways are the three components of the MAPK pathway in mammals. The ERK pathway is primarily activated by growth factors, such as epidermal growth factor, whereas the JNK and p38 signaling pathways are activated by various stress stimuli, including hypoxia, UVB radiation, and Citicoline inflammatory cytokines, such as tumor necrosis factor (TNF)-19,20. Additionally, hypoxia has been shown to cause the accumulation of reactive oxygen species (ROS), which are also involved in activating MAPK signaling pathways21. The present study investigated the molecular mechanisms by which BNIP3 acts as a pro-migratory factor in response to hypoxia during wound healing. The present data demonstrated that ROS accumulation mediated by hypoxia exposure triggered the activation of p38 and JNK MAPK in human immortalized keratinocyte HaCaT cells, in turn upregulating BNIP3-induced autophagy. Moreover, the present results also indicated that autophagy inhibition significantly impairs Citicoline hypoxia-induced keratinocyte migration. These data revealed a previously unknown mechanism that BNIP3-induced autophagy occurs through hypoxia-induced ROS-mediated p38 and JNK MAPK activation and supports the migration of epidermal keratinocytes during wound healing. Materials and Methods Ethics Statement All animal experiments were performed in accordance with the guidelines of the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Pub. No. 85C23, revised 1996) and approved by the Animal Experiment Ethics Committee of the Third Military Medical University in Chongqing, China. In vivo wound closure assay For wounding experiments, 8-.