Yinghong He

Integrin α3 mutations with kidney, lung, and skin disease.

Integrin α(3) is a transmembrane integrin receptor subunit that mediates signals between the cells and their microenvironment. We identified three patients with homozygous mutations in the integrin α(3) gene that were associated with disrupted basement-membrane structures and compromised barrier functions in kidney, lung, and skin. The patients had a multiorgan disorder that included congenital nephrotic syndrome, interstitial lung disease, and epidermolysis bullosa. The renal and respiratory features predominated, and the lung involvement accounted for the lethal course of the disease. Although skin fragility was mild, it provided clues to the diagnosis.

Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes.

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Lack of plakoglobin leads to lethal congenital epidermolysis bullosa: a novel clinico-genetic entity

Epidermal integrity is essential for skin functions. It is maintained by adhesive structures between keratinocytes, mainly the desmosomes and adherens junctions, which provide resistance against mechanical stress and regulate the formation of the skin barrier. As a constituent of both types of intercellular junctions, plakoglobin has multiple interaction partners and mutations in its gene [junction plakoglobin (JUP)] have been associated with mild cutaneous disease, palmoplantar keratoderma and arrhythmogenic heart disease. Here we report a novel lethal phenotype caused by a homozygous nonsense JUP mutation, c.1615C>T, p.Q539X, which is very different from any human or murine JUP phenotype described so far. The patient suffered from severe congenital skin fragility with generalized epidermolysis and massive transcutaneous fluid loss, but apparently no cardiac dysfunction. In contrast to previously reported JUP mutations where truncated proteins were still present, in this case there was complete loss of plakoglobin in the patients skin, as demonstrated by immunofluorescence and immunoblot analysis. As a consequence, only very few abnormal desmosomes were formed and no adhesion structures between keratinocytes were recognizable. The expression and distribution of desmosomal components was severely affected, suggesting an essential role for plakoglobin in desmosomal assembly. Adherens junction proteins were localized to keratinocyte plasma membrane, but did not provide proper cell-cell adhesion. This lethal congenital epidermolysis bullosa highlights the fundamental role of plakoglobin in epidermal cohesion.

Loss of desmoglein 1 associated with palmoplantar keratoderma, dermatitis and multiple allergies

Monoallelic desmoglein 1 mutations have been known for many years to cause striate palmoplantar keratoderma, but only recently, biallelic loss‐of‐function mutations were associated with a new disorder, designated as SAM syndrome (comprising severe dermatitis, multiple allergies and metabolic wasting) in two consanguineous families. We report on a new case from a third independent family with the homozygous nonsense mutation, c.2659C>T, p.R887* in exon 15 of DSG1 (desmoglein 1 gene). This mutation led to mRNA decay and loss of expression of desmoglein 1. The clinical phenotype consisted of severe palmoplantar keratoderma, dermatitis and multiple allergies. In contrast to the previous cases, malabsorption, hypoalbuminaemia, developmental delay, hypotrichosis or severe recurrent infections were not observed.

Kindlin-1 and -2 Have Overlapping Functions in Epithelial Cells: Implications for Phenotype Modification

Kindlins are a novel family of intracellular adaptor proteins in integrin-containing focal adhesions. Kindlin-1 and -2 are expressed in the skin, but whether and how they cooperate in adult epithelial cells have remained elusive. We uncovered the overlapping roles of kindlin-1 and -2 in maintaining epithelial integrity and show that the phenotype of kindlin-1-deficient cells can be modulated by regulating kindlin-2 gene expression and vice versa. The experimental evidence is provided by use of human keratinocyte cell lines that express both kindlins, just kindlin-1 or kindlin-2, or none of them. Double deficiency of kindlin-1 and -2 had significant negative effects on focal adhesion formation and actin cytoskeleton organization, cell adhesion, survival, directional migration, and activation of β(1) integrin, whereas deficiency of one kindlin only showed variable perturbation of these functions. Cell motility and formation of cell-cell contacts were particularly affected by lack of kindlin-2. These results predict that kindlin-1 and -2 can functionally compensate for each other, at least in part. The high physiologic and pathologic significance of the compensation was emphasized by the discovery of environmental regulation of kindlin-2 expression. UV-B irradiation induced loss of kindlin-2 in keratinocytes. This first example of environmental regulation of kindlin expression has implications for phenotype modulation in Kindler syndrome, a skin disorder caused by kindlin-1 deficiency.

Revertant mosaicism in a human skin fragility disorder results from slipped mispairing and mitotic recombination

Spontaneous gene repair, also called revertant mosaicism, has been documented in several genetic disorders involving organs that undergo self-regeneration, including the skin. Genetic reversion may occur through different mechanisms, and in a single individual, the mutation can be repaired in various ways. Here we describe a disseminated pattern of revertant mosaicism observed in 6 patients with Kindler syndrome (KS), a genodermatosis caused by loss of kindlin-1 (encoded by FERMT1) and clinically characterized by patchy skin pigmentation and atrophy. All patients presented duplication mutations (c.456dupA and c.676dupC) in FERMT1, and slipped mispairing in direct nucleotide repeats was identified as the reversion mechanism in all investigated revertant skin spots. The sequence around the mutations demonstrated high propensity to mutations, favoring both microinsertions and microdeletions. Additionally, in some revertant patches, mitotic recombination generated areas with homozygous normal keratinocytes. Restoration of kindlin-1 expression led to clinically and structurally normal skin. Since loss of kindlin-1 severely impairs keratinocyte proliferation, we predict that revertant cells have a selective advantage that allows their clonal expansion and, consequently, the improvement of the skin condition.

Role of Kindlin-2 in Fibroblast Functions: Implications for Wound Healing

Kindlins are a protein family that regulates cell-matrix interactions. Although kindlin-2 is known to be essential for the early developmental processes, its role in the homeostasis of adult tissues has remained elusive. In this study, we used new domain-specific antibodies and small interfering RNA technology to uncover physiological functions of kindlin-2 in human dermal fibroblasts in vitro and in adult skin in situ. In primary dermal fibroblasts, kindlin-2 is essential for the integrin clustering and activation, and it regulates cell adhesion and directed migration by guiding formation and maturation of focal adhesions (FAs) and organization of the cytoskeleton. These functions are linked to the transmission of mechanical cues between cells and their microenvironment, typically in processes wherein fibroblasts differentiate to myofibroblasts under mechanical tension. In concert, kindlin-2 was shown to be required for the stabilization and maturation of FAs and stress fibers in activated fibroblasts and myofibroblasts. These findings implicated that in physiological wound closure and tissue repair, the prediction was validated by strong upregulation of kindlin-2 in contractile myofibroblasts during middle stages of wound healing in human skin. Taken together, the data reveal a physiological role for kindlin-2 in skin fibroblasts under normal steady-state conditions and during tissue regeneration.

Crucial role of posttranslational modifications of integrin α3 in interstitial lung disease and nephrotic syndrome

Interstitial lung disease, nephrotic syndrome and junctional epidermolysis bullosa is an autosomal recessive multiorgan disorder caused by mutations in the gene for the integrin α3 subunit (ITGA3). The full spectrum of manifestations and genotype-phenotype correlations is still poorly characterized. Here, we uncovered the disease-causing role and the molecular mechanisms underlying a homozygous ITGA3 mutation leading to the single amino acid substitution, p.R463W. The patient suffered from respiratory distress and episodes of cyanosis with onset in the first week of life and had a nephrotic syndrome. Although there was no clinical evidence for cutaneous fragility, the analysis of a skin sample and of skin epithelial cells enabled the direct assessment of the authentic mutant protein. We show that the mutation altered the conformation of the extracellular β-propeller domain of the integrin α3 subunit preventing correct processing of N-linked oligosaccharides, heterodimerization with β1 integrin and maturation through cleavage into heavy and light chains in the Golgi. Confocal microscopy demonstrated that the mutant protein accumulated intracellularly, but it was not present in focal adhesions or on the cell membrane as shown by flow cytometry. These findings highlight that single amino acid changes in the integrin α3 subunit may crucially alter the structure and complex processing of this integrin, completely preventing its functionality. The present report also underscores that ITGA3 mutations may account for atypical cases solely with early onset respiratory and renal involvement.

Induction of phenotype modifying cytokines by FERMT1 mutations

Kindler syndrome (KS) is a progressive skin disorder caused by FERMT1 mutations. Early in life, KS manifests as a mechanobullous disease reflecting diminished cell adhesion, but the mechanisms of its later phenotypic features, progressive poikiloderma, and mucocutaneous fibrosis, remain elusive. The FERMT1 gene product and KS protein, kindlin‐1, is an epithelial‐specific phosphoprotein involved in integrin beta‐1 activation, without an obvious link to dermal connective tissue. Here we show how lack of intracellular kindlin‐1 in epidermal keratinocytes leads to profound changes in another skin compartment, the dermis. Kindlin‐1‐deficient keratinocytes respond to cell stress by upregulating the expression of cytokines such as IL‐20, IL‐24, TGF‐β2, IL1F5, PDGFB, and CTGF. These launch—via paracrine communication—an inflammatory response in the dermis, accompanied by the presence of TGF‐β, IL‐6, and CTGF, activation of fibroblasts and their differentiation to myofibroblasts, which secrete and deposit increased amounts of extracellular matrix proteins. These data are concordant with a model wherein repeated cycles of epidermal cell stress, cytokine secretion, dermal inflammation, and profibrotic processes underlie mucocutaneous fibrosis in KS.

RhoA activation by CNFy restores cell-cell adhesion in kindlin-2-deficient keratinocytes.

Kindlins are a family of integrin adapter and cell–matrix adhesion proteins causally linked to human genetic disorders. Kindlin‐2 is a ubiquitously expressed protein with manifold functions and interactions. The contribution of kindlin‐2 to integrin‐based cell–matrix adhesions has been extensively explored, while other integrin‐independent roles emerge. Because of the early involvement of kindlin‐2 in development, no viable animal models with its constitutional knockout are available to study its physiological functions in adult skin. Here, we uncovered a critical physiological role of kindlin‐2 in the epidermis by using a skin‐equivalent model with shRNA‐mediated knock‐down of kindlin‐2 in keratinocytes. Kindlin‐2‐deficient keratinocytes built stratified epidermal layers, but displayed impaired dermal–epidermal and intra‐epidermal adhesion and barrier function. Co‐immunoprecipitation studies demonstrated that kindlin‐2 interacts with both integrin‐ and cadherin‐based adhesions. In kindlin‐2‐deficient keratinocytes, reduced cell–cell adhesion was associated with abnormal cytoplasmic distribution of adherens junctions and desmosomal proteins, which was dependent on RhoA activation. Direct activation of RhoA with recombinant bacterial cytotoxic necrotizing factor y (CNFy) reverted the abnormal phenotype and barrier function of kindlin‐2‐deficient keratinocytes and skin equivalents. These findings have physiological and pathological significance, since kindlin‐2 expression modulates the phenotype in Kindler syndrome, a skin fragility disorder caused by kindlin‐1 deficiency. Our results suggest that pharmacological regulation of RhoGTPase activity may represent a therapeutic option for skin fragility. Copyright