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The cytoskeletal protein Flightless (Flii) is a negative regulator of wound healing. Upregulation of Flii is associated with impaired migration, proliferation and adhesion of both fibroblasts and keratinocytes. Importantly, Flii translocates from the cytoplasm to the nucleus in response to wounding in fibroblasts but not keratinocytes. This cell-specific nuclear translocation of Flii suggests that Flii may directly regulate gene expression in fibroblasts, providing one potential mechanism of action for Flii in the wound healing response. To determine whether the tissue-specific upregulation of Flii in fibroblasts was important for the observed inhibitory effects of Flii on wound healing, an inducible fibroblast-specific Flii overexpressing mouse model was generated. The inducible ROSA26 system allowed the overexpression of Flii in a temporal and tissue-specific manner in response to tamoxifen treatment. Wound healing in the inducible mice was impaired, with wounds at day 7 postwounding significantly larger than those from non-inducible controls. There was also reduced collagen maturation, increased myofibroblast infiltration and elevated inflammation. The impaired healing response was similar in magnitude to that observed in mice with non-tissue-specific upregulation of Flii suggesting that fibroblast-derived Flii may have an important role in the wound healing response., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Aims/hypothesis: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound., Methods: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response., Results: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation., Conclusions/interpretation: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.

Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF- κ B production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.

Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent-ColVII-hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post-induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii(+/-) blistered skin showed reduced α-SMA, TGF-β1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii-deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF-β1 restored collagen contraction, suggesting an interplay between Flii and TGF-β1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

The gelsolin related actin binding protein, Flii, is able to regulate wound healing; mice with decreased Flii expression show improved wound healing whereas mice with elevated Flii expression exhibit impaired wound healing. In both mice and humans Flii expression increases with age and amelioration of FLII activity represents a possible therapeutic strategy for improved wound healing in humans. Despite analysis of Flii function in a variety of organisms we know little of the molecular mechanisms underlying Flii action. Two new murine alleles of Flii have been produced to drive constitutive or tissue-specific expression of Flii. Each strain is able to rescue the embryonic lethality associated with a Flii null allele and to impair wound healing. These strains provide valuable resources for ongoing investigation of Flii function in a variety of biological processes., (Copyright © 2011 Wiley-Liss, Inc.)

Flightless I (Flii) is an actin-remodeling protein that influences diverse processes including cell migration and gene transcription and links signal transduction with cytoskeletal regulation. Here, we show that Flii modulation of focal adhesions and filamentous actin stress fibers is Rac1-dependent. Using primary skin fibroblasts from Flii overexpressing (Flii(Tg/Tg)), wild-type, and Flii deficient (Flii(+/-)) mice, we show that elevated expression of Flii increases stress fiber formation by impaired focal adhesion turnover and enhanced formation of fibrillar adhesions. Conversely, Flii knockdown increases the percentage of focal complex positive cells. We further show that a functional effect of Flii at both the cellular level and in in vivo mouse wounds is through inhibiting paxillin tyrosine phosphorylation and suppression of signaling proteins Src and p130Cas, both of which regulate adhesion signaling pathways. Flii is upregulated in response to wounding, and overexpression of Flii inhibits paxillin activity and reduces adhesion signaling by modulating the activity of the Rho family GTPases. Overexpression of constitutively active Rac1 GTPase restores the spreading ability of Flii(Tg/Tg) fibroblasts and may explain the reduced adhesion, migration, and proliferation observed in Flii(Tg/Tg) mice and their impaired wound healing, a process dependent on effective cellular motility and adhesion.

Regeneration of cells, tissues, and organs has long captured the attention of researchers for its obvious potential benefits in biomedical applications. Although mammals are notoriously poor at regeneration compared with many lower-order species, the hair follicle, paradoxically a defining characteristic of mammals, is capable of regeneration following partial amputation. To investigate the role of a negative regulator of wound healing, flightless I (Flii), on hair follicle regeneration, the bulbar region of vibrissae from rats as well as strains of mice expressing low (Flii(+/-)), normal (Flii(+/+)), and high (FLII(Tg/Tg)) levels of Flii were surgically amputated, and then allowed to regenerate in vivo. Macroscopic and histological assessment of the regeneration process revealed impaired or delayed regenerative potential in Flii(+/-) follicles. Regenerated follicles expressing high levels of Flii (FLII(Tg/Tg)) produced significantly longer terminal hair fibers. Immunohistochemical analysis was used to characterize the pattern of expression of Flii, as well as markers of hair follicle development and wound healing-associated factors during hair follicle regeneration. These studies confirmed that Flii appears to have a positive role in the regeneration of hair follicles, contrary to its negative influence on wound healing in skin.

Flightless I (Flii), a highly conserved member of the gelsolin family of actin-remodelling proteins associates with actin structures and is involved in cellular motility and adhesion. Our previous studies have shown that Flii is an important negative regulator of wound repair. Here, we show that Flii affects hemidesmosome formation and integrin-mediated keratinocyte adhesion and migration. Impaired hemidesmosome formation and sparse arrangements of keratin cytoskeleton tonofilaments and actin cytoskeleton anchoring fibrils were observed in Flii(Tg/+) and Flii(Tg/Tg) mice with their skin being significantly more fragile than Flii(+/-) and WT mice. Flii(+/-) primary keratinocytes showed increased adhesion on laminin and collagen I than WT and Flii(Tg/Tg) primary keratinocytes. Decreased expression of CD151 and laminin-binding integrins alpha3, beta1, alpha6 and beta4 were observed in Flii overexpressing wounds, which could contribute to the impaired wound re-epithelialization observed in these mice. Flii interacts with proteins directly linked to the cytoplasmic domain of integrin receptors suggesting that it may be a mechanical link between ligand-bound integrin receptors and the actin cytoskeleton driving adhesion-signaling pathways. Therefore Flii may regulate wound repair through its effect on hemidesmosome formation and integrin-mediated cellular adhesion and migration.