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Flightless I, a contributing factor to skin blistering in Kindler syndrome patients?

Kindler syndrome is the autosomal recessive skin disorder characterized by congenital blistering, photosensitivity and mutations is the FERMT1 gene encoding the focal adhesion protein Kindlin-1. Kindlin-1 is an integrin binding protein which links integrins to the F-actin cytoskeleton and regulates their activity. Loss of Kindlin-1 results in basement membrane splitting, inflammatory response, and development of blistering. This impairs the adhesion of basal keratinocytes to the extracellular matrix leading to modifications of the cortical actin network and increased plasticity of the plasma membrane. Recent studies have shown that Kindlin-1 regulates epidermal growth factor receptor (EGRF) signaling and protects the receptor from lysosomal-mediated degradation with Kindler syndrome patients having reduced EGRF signaling, leading to defective cell migration and wound re-epithelialization. Mechanistic studies using a transgenic mouse lacking Kindlin-1 in the epidermis have identified a role for Kindlin-1 in distinct pathways controlling Wnt and TGF-β availability. Kindlin-1 regulates stem cell proliferation and cancer development independently of β-integrin signaling; however, the mechanism behind its regulation of the cytoskeleton has not been fully elucidated. Here we describe a link between Kindlin-1 and the actin remodeling protein Flightless I (Flii) which may contribute to the excessive blistering observed in Kindler syndrome patients. Flii is a conserved member of the gelsolin family of actin remodeling proteins with unique roles during development, inflammation, and recovery of the skin barrier function following injury. Flii overexpression results in reduced cellular adhesion and migration, impaired integrin signaling, and increased skin fragility. A recent study has described Flii as an inhibitor of epidermal stem cell activation, via its negative effect on Wnt/β-catenin signaling which may explain its adverse effects on wound re-epithelialization. Previous studies have also shown that Flii promotes fibrosis via increasing TGF-β-mediated collagen production as well as impairing cellular adhesion via rac-1 dependent pathway leading to increased skin blistering in epidermolysis bullosa (EB) mouse models. Additionally, Flii is elevated in skin of EB patients, present in blister fluid and found at excessive levels in epidermal keratinocytes, dermal fibroblasts, and inflammatory cells surrounding the blister site of patients with simplex, junctional, and dystrophic EB. While the exact mechanisms of Flii action during blistering in EB are yet to be identified, Flii effects on cellular adhesion, collagen secretion and TGF-β signaling have been linked to disease pathogenesis. Using samples from three Kindler syndrome patients, we show that Flii is increased in this fourth major EB type. Skin biopsies were collected from patients harboring a large genomic deletion mutation in the FERMT1 gene and displaying characteristic symptoms of mild to severe oral involvement, and severe esophageal and anal stenosis (Figure 1A). Mutational analysis and clinical pathology of these patients has previously been published. Collected biopsy samples were analyzed for expression and localization of Flii in the skin using immunohistochemistry. Flii was significantly increased in both keratinocytes and fibroblasts of blistered skin of Kindler syndrome patients when compared to nonblistered healthy controls (Figure 1B,C) suggesting a potential role for Flii in contributing to excessive blistering seen in this EB type. Whether the observed increase in Flii is due to a decrease in Kindlin-1 levels or as a secondary consequence to blistering yet remains to be elucidated. Previous studies have shown that reducing Flii levels improves the healing of blistered EB murine skin therefore reducing the level of Flii may be beneficial for Kindler syndrome patients. Flii increases during development and its overexpression delays the development of a skin barrier, alters tight junctions, and impairs skin barrier recovery following blistering. To understand the effect of Flii expression on Kindlin-1 levels during development, back skin from mice with low (Flii), normal (Flii), or high (Flii) levels was analyzed for Kindlin-1 expression during development. Kindlin-1 levels during murine epidermal development has previously been documented showing strong epidermal expression and co-localization with integrin-mediated adhesion sites in keratinocytes. Here we show that Kindlin-1 is specifically localized to the basolateral keratinocytes in the epidermis and decreasing Flii levels during development resulted in up-regulation of Kindlin-1 at embryonic day 17, suggesting that Kindlin-1 effects on actin cytoskeleton function may be important for more rapid skin barrier development and function, as well as recovery from injury as has been reported in these mice (Figure 2A). No effect of changes in Flii levels on Kindlin-1 expression were observed on embryonic day 19 or in adult skin (Figure 2A). Kindlin-1 plays an essential role in integrin-mediated adhesion and signaling, however unlike Flii, it directly interacts with cytoplasmic tails of β1 and β3 integrins. In addition, both Flii and Kindlin-1 have been shown to regulate focal adhesions via associations with paxillin and governing of the Rho family GTPase signaling. Overexpression of Flii impairs focal adhesion turnover in a Rac-1-dependent manner and Received: 2 September 2019 Revised: 7 October 2019 Accepted: 8 October 2019