Defining molecular mechanisms of hypoxia-induced migration during would healing

About This Grant

PROJECT SUMMARY To date, most efforts to address scarring have focused on treatment at the tissue level rather than the cellular level. Therefore, identifying the molecular events involved in scar formation provides an opportunity to develop new drugs and treatment strategies to reduce scar formation. In the early stages of wound healing, hypoxia (low oxygen) stimulates fibroblasts to migrate into the wound bed where they synthesize the granulation tissue needed for healing. In successful wound healing that does not leave a scar, this extracellular matrix (ECM) is processed by proteolytic cleavage, the wound is revascularized, and oxygen is restored. When wound healing is not optimal, the new tissue suffers from poor re-vascularization and hypoxia does not resolve after healing is complete. Chronic hypoxia increases the migration of fibroblasts into the healed tissue, which is a rate limiting step for scar formation. Based on this concept, blocking the ability of fibroblasts to migrate into scar tissue will prevent and/or reduce scar formation. The objectives of this proposal are to determine how hypoxia rewires cytoskeletal dynamics to increase fibroblast migration. Cell migration involves the formation of focal adhesions (FA; mediated by integrin binding to ECM) and protrusion at the leading edge (mediated by actin polymerization). To date, the study of how hypoxia stimulates cell migration has focused almost exclusively on hypoxia-inducible factor-1 (HIF-1), which has limited progress in the field because HIF-1 amplifies well known actin regulatory pathways. As a result, the discovery of new mechanisms and therapeutic targets to address hypoxia in scar formation is lacking. PIM1 is a unique target to oppose hypoxia because it is stabilized in hypoxia at the protein level, independent of HIF-1. My lab recently discovered that PIM1 kinase is essential for hypoxia-induced migration. Mechanistically, we showed that PIM1 phosphorylates Abl interactor 2 (Abi2), a member of the WAVE regulatory complex (WRC), which enhances protrusive activity. Preliminary data from an unbiased phospho- proteomic screen identified kindlin 2 (K2) as a novel PIM1 substrate in hypoxia. K2 is an adapter protein that drives integrin clustering and focal adhesion (FA) maturation. Our central hypothesis is that unresolved hypoxia in hypertrophic scar tissue increases PIM1, which enhances fibroblast migration and worsens scar formation. In our model, PIM1 accumulates at the cell membrane in hypoxia, where it phosphorylates Abi2 (stabilizing the WRC and increasing protrusion) and K2 (activating K2 and increasing FA). Therefore, blocking PIM1 represents a new, HIF-independent, strategy to prevent fibroblast infiltration and restrain scar formation. The knowledge gained from these studies will inform future efforts to control cell migration as a strategy to treat scars and other diseases associated with hypoxia (i.e., cancer, stroke). My lab has a heavy investment in developing PIM kinase inhibitors, so we are in a strong position to lead the translation of PIM inhibitors as anti-fibrotic agents.