Hypoxia Inducible Factor (HIF)‐1 accelerates mucosal wound healing through regulation and trafficking of integrin‐α5β1
Authors: Goggins Bridie Jane , Minahan Kyra , Sherwin Simonne , Liu Gang , Walker Marjorie , Horvat Jay , Knight Darryl , Keely Simon 
Published: April 2020

The intestinal epithelium functions as a selective barrier, critical for mucosal protection. Inflammatory tissue damage leads to barrier destruction and epithelial healing is necessary to restore barrier integrity and gut homeostasis. Mucosal healing occurs in an hypoxic environment due to vascular damage and increased metabolic demands. The transcription factor Hypoxia Inducible Factor (HIF)‐1 is a key co‐ordinator of mucosal healing during hypoxia. HIF‐1 mediates adaptive mechanisms for cell survival and repair including transmembrane integrins. Our initial studies identified HIF‐1‐mediated induction of the α5β1 integrin at the mucosa is associated with accelerated healing. However, integrin transport and recycling mechanisms are also important for integrin function but little is known of the role of HIF‐1 in these processes. We hypothesised that HIF‐1α stabilisation accelerates epithelial restitution through both transcriptional and integrin transport pathways. The aim of this study was to examine the functional, transcriptional and post‐translational activity, as well as trafficking mechanisms of integrin‐α5, during HIF‐1‐mediated wound healing. Functional scratch assays were performed over 24 hours on T84 monolayers treated with PHDi or vehicle with/without functional integrin‐α5 antibodies. Protein expression of integrin‐α5 and KIF1C mictrotubule motor protein, a known α5 transporter was analysed via Western blot. Monolayers were stained by immunofluorescence to examine α5‐integrin and KIF1C cellular localisation. Biopsy‐wound models and TNBS colitis models were used to examine the role of HIF‐mediated integrin response during mucosal healing as well as early (day 2) and late (day 6) healing responses during inflammatory hypoxia (PHDi ‐ 5mg/kg). PHDi‐mediated accelerated wound closure was inhibited by functional blockade of integrin‐α5, against controls (‐14.03%, p<0.0001). Immunocytochemistry staining of α5 showed increased cytoplasmic trafficking following PHDi treatments, concurrent with KIF1C induction. Integrin‐α5 and KIF1C cytoplasmic protein expression was increased (α5 5.25‐fold, p<0.0001; KIF1C 1.19‐fold, p<0.0001) following PHDi treatment. In vivo, PHDi treatments accelerated mucosal wound closure in biopsy wounds (+0.0964mm2, p<0.0012), which was associated with increased expression of α5 (17.02, p=0.0343) and KIF1C (13.16, p=0.0140) in wound granulation tissue (IHC). Integrin‐α5 protein from TNBS colonocytes was increased on day 2 in vehicle (0.90‐fold, p=0.0004) and PHDi (0.84‐fold, p=0.0007) mice compared to vehicle/ctrl, as well as increased KIF1C in TNBS/PHDi mice (7.44‐fold, p=0.0189). There was no difference in α5 protein at day 6, however there was sustained increase in KIF1C in TNBS/PHDi against ctrl/PHDi (1.16‐fold, p=0.0012 ) and TNBS/PHDi (0.76‐fold, p=0.0366). This may suggest that HIF‐1 stabilisation increases integrin‐α5 in early stages of healing and promotes α5 transport in late stages through up‐regulation of KIF1C. This data indicates that KIF1C may be hypoxia responsive and plays a role in integrin‐α5 transport to promote mucosal healing in both early and late stages.