Immortalized Human hTert/KER-CT Keratinocytes a Model System for Research on Desmosomal Adhesion and Pathogenesis of Pemphigus Vulgaris.
ABSTRACT: Pemphigus Vulgaris is an autoimmune disease that results in blister formation in the epidermis and in mucosal tissues due to antibodies recognizing desmosomal cadherins, mainly desmoglein-3 and -1. Studies on the molecular mechanisms of Pemphigus have mainly been carried out using the spontaneously immortalized human keratinocyte cell line HaCaT or in primary keratinocytes. However, both cell systems have suboptimal features, with HaCaT cells exhibiting a large number of chromosomal aberrations and mutated p53 tumor suppressor, whereas primary keratinocytes are short-lived, heterogeneous and not susceptible to genetic modifications due to their restricted life-span. We have here tested the suitability of the commercially available human keratinocyte cell line hTert/KER-CT as a model system for research on epidermal cell adhesion and Pemphigus pathomechanisms. We here show that hTert cells exhibit a calcium dependent expression of desmosomal cadherins and are well suitable for typical assays used for studies on Pemphigus, such as sequential detergent extraction and Dispase-based dissociation assay. Treatment with Pemphigus auto-antibodies results in loss of monolayer integrity and altered localization of desmoglein-3, as well as loss of colocalization with flotillin-2. Our findings demonstrate that hTert cells are well suitable for studies on epidermal cell adhesion and Pemphigus pathomechanisms.
Project description:Pemphigus vulgaris (PV) is an autoimmune disease of the skin and mucous membranes and is characterized by development of autoantibodies against the desmosomal cadherins desmoglein (Dsg) 3 and Dsg1 and formation of intraepidermal suprabasal blisters. Depletion of Dsg3 is a critical mechanism in PV pathogenesis. Because we did not detect reduced Dsg3 levels in keratinocytes cultured for longer periods under high-Ca(2+) conditions, we hypothesized that Dsg depletion depends on Ca(2+)-mediated keratinocyte differentiation. Our data indicate that depletion of Dsg3 occurs specifically in deep epidermal layers both in skin of patients with PV and in an organotypic raft model of human epidermis incubated using IgG fractions from patients with PV. In addition, Dsg3 depletion and loss of Dsg3 staining were prominent in cultured primary keratinocytes and in HaCaT cells incubated in high-Ca(2+) medium for 3 days, but were less pronounced in HaCaT cultures after 8 days. These effects were dependent on protein kinase C signaling because inhibition of protein kinase C blunted both Dsg3 depletion and loss of intercellular adhesion. Moreover, protein kinase C inhibition blocked suprabasal Dsg3 depletion in cultured human epidermis and blister formation in a neonatal mouse model. Considered together, our data indicate a contribution of Dsg depletion to PV pathogenesis dependent on Ca(2+)-induced differentiation. Furthermore, prominent depletion in basal epidermal layers may contribute to the suprabasal cleavage plane observed in PV.
Project description:Desmosomes are adhesion plaques that mediate cell-cell adhesion in many tissues, including the epidermis, and generate mechanical resistance to tissues. The extracellular domains of desmosomal cadherin proteins, desmogleins and desmocollins, are required for the interaction with cadherins of the neighbouring cells, whereas their cytoplasmic tails associate with cytoplasmic proteins which mediate connection to intermediate filaments. Disruption of desmosomal adhesion by mutations, autoantibodies or bacterial toxins results in severe human disorders of e.g. the skin and the heart. Despite the vital role of desmosomes in various tissues, the details of their molecular assembly are not clear. We here show that the two members of the flotillin protein family directly interact with the cytoplasmic tails of desmogleins. Depletion of flotillins in human keratinocytes results in weakened desmosomal adhesion and reduced expression of desmoglein-3, most likely due to a reduction in the desmosomal pool due to increased turnover. In the absence of flotillins, desmoglein-3 shows an altered localisation pattern in the cell-cell junctions of keratinocytes, which is highly similar to the localisation observed upon treatment with pemphigus vulgaris autoantibodies. Thus, our data show that flotillins, which have previously been connected to the classical cadherins, are also of importance for the desmosomal cell adhesion.
Project description:Pemphigus is an autoimmune blistering disease targeting the desmosomal proteins desmoglein (Dsg) 1 and Dsg3. Recently, a genetic variant of the Suppression of tumorigenicity 18 (ST18) promoter was reported to cause ST18 up-regulation, associated with pemphigus vulgaris (PV)-IgG-mediated increase in cytokine secretion and more prominent loss of keratinocyte cohesion. Here we tested the effects of PV-IgG and the pathogenic pemphigus mouse anti-Dsg3 antibody AK23 on cytokine secretion and ERK activity in human keratinocytes dependent on ST18 expression. Without ST18 overexpression, both PV-IgG and AK23 induced loss of keratinocyte cohesion which was accompanied by prominent fragmentation of Dsg3 immunostaining along cell borders. In contrast, release of pro-inflammatory cytokines such as IL-1?, IL-6, TNF?, and IFN-? was not altered significantly in both HaCaT and primary NHEK cells. These experiments indicate that cytokine expression is not strictly required for loss of keratinocyte cohesion. Upon ST18 overexpression, fragmentation of cell monolayers increased significantly in response to autoantibody incubation. Furthermore, production of IL-1? and IL-6 was enhanced in some experiments but not in others whereas release of TNF-? dropped significantly upon PV-IgG application in both EV- and ST18-transfected HaCaT cells. Additionally, in NHEK, application of PV-IgG but not of AK23 significantly increased ERK activity. In contrast, ST18 overexpression in HaCaT cells augmented ERK activation in response to both c-IgG and AK23 but not PV-IgG. Because inhibition of ERK by U0126 abolished PV-IgG- and AK23-induced loss of cell cohesion in ST18-expressing cells, we conclude that autoantibody-induced ERK activation was relevant in this scenario. In summary, similar to the situation in PV patients carrying ST18 polymorphism, overexpression of ST18 enhanced keratinocyte susceptibility to autoantibody-induced loss of cell adhesion, which may be caused in part by enhanced ERK signaling.
Project description:Plakophilin-1 (PKP-1) is an armadillo family protein critical for desmosomal adhesion and epidermal integrity. In the autoimmune skin-blistering disease pemphigus vulgaris (PV), autoantibodies (IgG) target the desmosomal cadherin desmoglein 3 (Dsg3) and compromise keratinocyte cell-cell adhesion. Here, we report that enhanced expression of PKP-1 protects keratinocytes from PV IgG-induced loss of cell-cell adhesion. PKP-1 prevents loss of Dsg3 and other desmosomal proteins from cell-cell borders and prevents alterations in desmosome ultrastructure in keratinocytes treated with PV IgG. Using a series of Dsg3 chimeras and deletion constructs, we find that PKP-1 clusters Dsg3 with the desmosomal plaque protein desmoplakin in a manner dependent on the plakoglobin-binding domain of the Dsg3 tail. Furthermore, PKP-1 expression transforms desmosome adhesion from a calcium-dependent to a calcium-independent and hyperadhesive state. These results demonstrate that manipulating the expression of a single desmosomal plaque protein can block the pathogenic effects of PV IgG on keratinocyte adhesion.
Project description:The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2 hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell-surface Dsg3 but not Dsc3 through early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24 hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.
Project description:Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway.
Project description:OBJECTIVES:? Desmoglein 3 (Dsg3) is a desmosomal adhesion protein expressed in basal and immediate suprabasal layers of skin. Importance of Dsg3 in cell-cell adhesion and maintenance of tissue integrity is illustrated by findings of keratinocyte dissociation in the autoimmune disease, pemphigus vulgaris, where autoantibodies target Dsg3 on keratinocyte surfaces and cause Dsg3 depletion from desmosomes. However, recognition of possible participation of involvement of Dsg3 in cell proliferation remains controversial. Currently, available evidence suggests that Dsg3 may have both anti- and pro-proliferative roles in keratinocytes. The aim of this study was to use RNA interference (RNAi) strategy to investigate effects of silencing Dsg3 in cell-cell adhesion and cell proliferation in two cell lines, HaCaT and MDCK. MATERIALS AND METHODS:? Cells were transfected with siRNA, and knockdown of Dsg3 was assessed by western blotting, fluorescence-activated cell sorting and confocal microscopy. Cell-cell adhesion was analysed using the hanging drop/fragmentation assay, and cell proliferation by colony forming efficiency, BrdU incorporation, cell counts and organotypic culture. RESULTS:? Silencing Dsg3 caused defects in cell-cell adhesion and concomitant reduction in cell proliferation in both HaCaT and MDCK cells. CONCLUSION:? These findings suggest that Dsg3 depletion by RNAi reduces cell proliferation, which is likely to be secondary to a defect in cell-cell adhesion, an essential function required for cell differentiation and morphogenesis.
Project description:Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.
Project description:Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.
Project description:Desmosomes and adherens junctions are cadherin-based protein complexes responsible for cell-cell adhesion of epithelial cells. Type 1 cadherins of adherens junctions show specific homophilic adhesion that plays a major role in developmental tissue segregation. The desmosomal cadherins, desmocollin and desmoglein, occur as several different isoforms with overlapping expression in some tissues where different isoforms are located in the same desmosomes. Although adhesive binding of desmosomal cadherins has been investigated in a variety of ways, their interaction in desmosome-forming epithelial cells has not been studied. Here, using extracellular homobifunctional cross-linking, we provide evidence for homophilic and isoform-specific binding between the Dsc2, Dsc3, Dsg2, and Dsg3 isoforms in HaCaT keratinocytes and show that it represents trans interaction. Furthermore, the cross-linked adducts are present in the detergent-insoluble fraction, and electron microscopy shows that extracellular cross-linking probably occurs in desmosomes. We found no evidence for either heterophilic or cis interaction, but neither can be completely excluded by our data. Mutation of amino acid residues Trp-2 and Ala-80 that are important for trans interaction in classical cadherin adhesive binding abolished Dsc2 binding, indicating that these residues are also involved in desmosomal adhesion. These interactions of desmosomal cadherins may be of key importance for their ordered arrangement within desmosomes that we believe is essential for desmosomal adhesive strength and the maintenance of tissue integrity.