Intestinal epithelia are critical for maintaining gastrointestinal homeostasis. Epithelial barrier injury, causing inflammation and vascular damage, results in inflammatory hypoxia and thus healing occurs in an oxygen-restricted environment. The transcription factor hypoxia inducible factor (HIF)-1 regulates genes important for cell survival and repair, including the cell adhesion protein β₁-integrin. Integrins function as αβ-dimers and α-integrin-matrix binding is critical for cell migration. We hypothesized that HIF-1 stabilization accelerates epithelial migration through integrin-dependent pathways. We aimed to examine functional and post-translational activity of α-integrins during HIF-1-mediated intestinal epithelial healing. Wound healing was assessed in T84 monolayers over 24 hours with/without prolyl-hydroxylase inhibitor (PHDi) (GB-004), which stabilizes HIF-1. Gene and protein expression were measured by RT-PCR and immunoblot, α-integrin localization was assessed by immunofluorescence. α-integrin function was assessed by antibody-mediated blockade and integrin-α6 regulation was determined by HIF-1α chromatin immunoprecipitation. Models of mucosal wounding and TNBS-induced colitis were used to examine integrin expression and localization in vivo. PHDi-treatment accelerated wound closure and migration within 12 hours, associated with increased integrin-α2 and α6 protein,but not α3. Functional blockade of integrins-α2 and α6 inhibited PHDi-mediated accelerated wound closure. HIF-1 bound directly to the integrin-α6 promoter. PHDi treatment accelerated mucosal healing, which was associated with increased α6 immunohistochemical staining in wound-associated epithelium and wound-adjacent tissue. PHDi-treatment increased α6 protein levels in colonocytes of TNBS mice and induced α6 staining in regenerating crypts and re-epithelialized inflammatory lesions. Together these data demonstrate a role for HIF-1 in regulating both integrin-α2 and α6 responses during intestinal epithelial healing.

中文翻译：

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药理学上的HIF-1稳定通过调节α整合素的表达和功能促进肠道上皮的愈合

肠上皮对于维持胃肠稳态至关重要。上皮屏障损伤，引起炎症和血管损伤，导致炎症性缺氧，因此在氧气受限的环境中发生愈合。转录因子低氧诱导因子（HIF）-1调节细胞存活和修复基因重要的，包括细胞粘附蛋白β ₁-整联蛋白。整联蛋白起αβ-二聚体的作用，α-整联蛋白-基质结合对于细胞迁移至关重要。我们假设HIF-1稳定通过整合素依赖性途径加速上皮迁移。我们旨在检查在HIF-1介导的肠上皮愈合过程中α-整合素的功能和翻译后活性。在有/没有脯氨酰羟化酶抑制剂（PHDi）（GB-004）稳定HIF-1的情况下，在24小时内评估T84单层的伤口愈合情况。通过RT-PCR和免疫印迹测量基因和蛋白质表达，通过免疫荧光评估α-整联蛋白定位。通过抗体介导的阻滞评估α-整联蛋白功能，并通过HIF-1α染色质免疫沉淀法确定整联蛋白-α6调节。粘膜伤口和TNBS诱导的结肠炎模型用于检查整联蛋白的表达和体内定位。PHDi治疗可在12小时内加速伤口闭合和迁移，并增加整联蛋白-α2和α6蛋白，但不是α3。整合素α2和α6的功能性阻断抑制了PHDi介导的加速伤口闭合。HIF-1直接与整联蛋白-α6启动子结合。PHDi治疗可加速粘膜愈合，这与伤口相关上皮和伤口附近组织中α6免疫组化染色的增加有关。PHDi处理可增加TNBS小鼠结肠细胞中α6蛋白的水平，并在新生隐窝和上皮炎性病变中诱导α6染色。这些数据共同证明了HIF-1在肠上皮愈合过程中在调节整联蛋白-α2和α6反应中的作用。