Kinase Inhibition by PKC412 Prevents Epithelial Sheet Damage in Autosomal Dominant Epidermolysis Bullosa Simplex through Keratin and Cell Contact Stabilization.

Epidermolysis bullosa simplex (EBS) is a severe and potentially life-threatening disorder for which no adequate therapy exists. Most cases are caused by dominant sequence variations in keratin genes K5 or K14, leading to the formation of cytoplasmic keratin aggregates, profound keratinocyte fragility, and cytolysis. We hypothesized that pharmacological reduction of keratin aggregates, which compromise keratinocyte integrity, represents a viable strategy for the treatment of EBS. In this study, we show that the multikinase inhibitor PKC412, which is currently in clinical use for acute myeloid leukemia and advanced systemic mastocytosis, reduced keratin aggregation by 40% in patient-derived K14.R125C EBS-associated keratinocytes. Using a combination of epithelial shear stress assay and real-time impedance spectroscopy, we show that PKC412 restored intercellular adhesion. Molecularly, global phosphoproteomic analysis together with immunoblots using phosphoepitope-specific antibodies revealed that PKC412 treatment altered phosphorylated sites on keratins and desmoplakin. Thus, our data provide a proof of concept to repurpose existing drugs for the targeted treatment of EBS and showcase how one broad-range kinase inhibitor reduced keratin filament aggregation in patient-derived EBS keratinocytes and the fragility of EBS cell monolayers. Our study paves the way for a clinical trial using PKC412 for systemic or local application in patients with EBS.

Structural heterogeneity of cellular K5/K14 filaments as revealed by cryo-electron microscopy.

Keratin intermediate filaments are an essential and major component of the cytoskeleton in epithelial cells. They form a stable yet dynamic filamentous network extending from the nucleus to the cell periphery, which provides resistance to mechanical stresses. Mutations in keratin genes are related to a variety of epithelial tissue diseases. Despite their importance, the molecular structure of keratin filaments remains largely unknown. In this study, we analyzed the structure of keratin 5/keratin 14 filaments within ghost mouse keratinocytes by cryo-electron microscopy and cryo-electron tomography. By averaging a large number of keratin segments, we have gained insights into the helical architecture of the filaments. Two-dimensional classification revealed profound variations in the diameter of keratin filaments and their subunit organization. Computational reconstitution of filaments of substantial length uncovered a high degree of internal heterogeneity along single filaments, which can contain regions of helical symmetry, regions with less symmetry and regions with significant diameter fluctuations. Cross-section views of filaments revealed that keratins form hollow cylinders consisting of multiple protofilaments, with an electron dense core located in the center of the filament. These findings shed light on the complex and remarkable heterogenic architecture of keratin filaments, suggesting that they are highly flexible, dynamic cytoskeletal structures.

Epidermolysis bullosa.

Epidermolysis bullosa (EB) is an inherited, heterogeneous group of rare genetic dermatoses characterized by mucocutaneous fragility and blister formation, inducible by often minimal trauma. A broad phenotypic spectrum has been described, with potentially severe extracutaneous manifestations, morbidity and mortality. Over 30 subtypes are recognized, grouped into four major categories, based predominantly on the plane of cleavage within the skin and reflecting the underlying molecular abnormality: EB simplex, junctional EB, dystrophic EB and Kindler EB. The study of EB has led to seminal advances in our understanding of cutaneous biology. To date, pathogenetic mutations in 16 distinct genes have been implicated in EB, encoding proteins influencing cellular integrity and adhesion. Precise diagnosis is reliant on correlating clinical, electron microscopic and immunohistological features with mutational analyses. In the absence of curative treatment, multidisciplinary care is targeted towards minimizing the risk of blister formation, wound care, symptom relief and specific complications, the most feared of which - and also the leading cause of mortality - is squamous cell carcinoma. Preclinical advances in cell-based, protein replacement and gene therapies are paving the way for clinical successes with gene correction, raising hopes amongst patients and clinicians worldwide.

A dominant vimentin variant causes a rare syndrome with premature aging.

Progeroid syndromes are a group of rare genetic disorders, which mimic natural aging. Unraveling the molecular defects in such conditions could impact our understanding of age-related syndromes such as Alzheimer's or cardiovascular diseases. Here we report a de novo heterozygous missense variant in the intermediate filament vimentin (c.1160 T > C; p.(Leu387Pro)) causing a multisystem disorder associated with frontonasal dysostosis and premature aging in a 39-year-old individual. Human vimentin p.(Leu387Pro) expression in zebrafish perturbed body fat distribution, and craniofacial and peripheral nervous system development. In addition, studies in patient-derived and transfected cells revealed that the variant affects vimentin turnover and its ability to form filaments in the absence of wild-type vimentin. Vimentin p.(Leu387Pro) expression diminished the amount of peripilin and reduced lipid accumulation in differentiating adipocytes, recapitulating key patient's features in vivo and in vitro. Our data highlight the function of vimentin during development and suggest its contribution to natural aging.

Cross-Talk between Hemidesmosomes and Focal Adhesions: A Primer for Wound Healing, Blistering Skin Disease, and Skin Aging.

Hemidesmosomes and focal adhesions attach keratinocytes to the dermis and act as bidirectional signaling centers to control epidermal renewal. Pora and colleagues (Pora et al., 2019) demonstrate that in migrating primary human keratinocytes, hemidesmosomes cluster as ordered arrays consisting of multiple chevrons, flanked by actin-associated focal adhesions. These and related findings have implications for wound healing, cancer invasion, blistering skin diseases, and skin aging.

MEK inhibitor cobimetinib rescues a dRaf mutant lethal phenotype in Drosophila melanogaster.

Since Drosophila melanogaster has proven to be a useful model system to study phenotypes of oncogenic mutations and to identify new anti-cancer drugs, we generated human BRAFV600E homologous dRaf mutant (dRafA572E ) Drosophila melanogaster strains to use these for characterisation of mutant phenotypes and exploit these phenotypes for drug testing. For mutant gene expression, the GAL4/UAS expression system was used. dRafA572E was expressed tissue-specific in the eye, epidermis, heart, wings, secretory glands and in the whole animal. Expression of dRaf A572E under the control of an eye-specific driver led to semi-lethality and a rough eye phenotype. The vast majority of other tissue-specific and ubiquitous drivers led to a lethal phenotype only. The rough eye phenotype was used to test BRAF inhibitor vemurafenib and MEK1/2 inhibitor cobimetinib. There was no phenotype rescue by this treatment. However, a significant rescue of the lethal phenotype was observed under a gut-specific driver. Here, MEK1/2 inhibitor cobimetinib rescued Drosophila larvae to reach pupal stage in 37% of cases as compared to 1% in control experiments. Taken together, the BRAFV600E homolog dRaf A572E exerts mostly lethal effects in Drosophila. Gut-specific dRaf A572E expression might in future be developed further for drug testing.

Treatment of keratinocytes with 4-phenylbutyrate in epidermolysis bullosa: Lessons for therapies in keratin disorders.

BACKGROUND: Missense mutations in keratin 5 and 14 genes cause the severe skin fragility disorder epidermolysis bullosa simplex (EBS) by collapsing of the keratin cytoskeleton into cytoplasmic protein aggregates. Despite intense efforts, no molecular therapies are available, mostly due to the complex phenotype of EBS, comprising cell fragility, diminished adhesion, skin inflammation and itch. METHODS: We extensively characterized KRT5 and KRT14 mutant keratinocytes from patients with severe generalized EBS following exposure to the chemical chaperone 4-phenylbutyrate (4-PBA). FINDINGS: 4-PBA diminished keratin aggregates within EBS cells and ameliorated their inflammatory phenotype. Chemoproteomics of 4-PBA-treated and untreated EBS cells revealed reduced IL1ß expression- but also showed activation of Wnt/ß-catenin and NF-kB pathways. The abundance of extracellular matrix and cytoskeletal proteins was significantly altered, coinciding with diminished keratinocyte adhesion and migration in a 4-PBA dose-dependent manner. INTERPRETATION: Together, our study reveals a complex interplay of benefits and disadvantages that challenge the use of 4-PBA in skin fragility disorders.

Insulin receptor plays a central role in skin carcinogenesis by regulating cytoskeleton assembly.

Diabetes mellitus prevalence is increasing rapidly and is a major cause of mortality and morbidity worldwide. In addition to the known severe complications associated with the disease, in recent years diabetes has been recognized as a major risk factor for cancer. Patients with diabetes experience significantly higher incidence of and higher mortality rates from many types of cancer. However, to date there are no conclusive data on the pathophysiology underlying the association between these two diseases. We previously reported that insulin regulates skin proliferation and differentiation, while IGF1 had different sometimes contrasting effects to those of insulin, suggesting direct involvement of insulin in transformation. To this end, we developed an epidermal skin-specific insulin receptor knockout (SIRKO) mouse, in which the insulin receptor (IR) is inactivated only in skin, with no other metabolic consequences. We found that IR inactivation by itself resulted in a marked decrease in skin tumorigenesis. In the control group 100% of the mice developed tumors, but in the SIRKO group tumor incidence was over 60% lower, and 25% of the SIRKO mice did not develop tumors at all, and the tumors that did develop were smaller and benign in their appearance. Furthermore, IR inactivation in vitro not only prevented cell transformation but also reversed the keratinocyte-transformed phenotype. We found that IR inactivation led to a striking abnormality in the major keratin cytoskeleton filaments structure in both in vivo and in vitro, a change that we were able to link to the decreased transformation potential in IR-null cells. In summary, we identified a unique pathway in which IR regulates cytoskeletal assembly, thus affecting skin transformation, opening a new potential target for cancer treatment and prevention.-Weingarten, G., Ben Yaakov, A., Dror, E., Russ, J., Magin, T. M., Kahn, C. R., Wertheimer, E. Insulin receptor plays a central role in skin carcinogenesis by regulating cytoskeleton assembly.

High Keratin 8/18 Ratio Predicts Aggressive Hepatocellular Cancer Phenotype.

BACKGROUND & AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termed Mallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: We analyzed livers of aged Krt18-/- mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18-/- mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18-/- mice with 26 human hepatoma cell lines and with data sets of >300 patients with HCC, where Krt18-/- gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC.

Orf virus infection of human keratinocytes and dermal fibroblasts: Limited virus detection and interference with intercellular adhesion molecule-1 up-regulation.

Orf virus (Parapoxvirus ovis, ORFV) is a dermatotropic virus causing pustular dermatitis in small ruminants and humans. We analysed isolated human primary keratinocytes (KC) and dermal fibroblasts (FB) for cell death and virus replication by infection with a patient-derived ORFV isolate. ORFV infection was associated with rapid induction of cell death in KC allowing for considerable virus removal. Upon infection with ORFV, KC and FB harboured intracytoplasmic ORFV and showed viral protein presence; however, missing virus spread indicated an abortive infection. Upon ORFV exposure, KC but not FB secreted the pro-inflammatory cytokine interleukin (IL)-6. ORFV infection enhanced the frequency of KC expressing intercellular adhesion molecule (ICAM)-1 which was independent of IL-6. Interestingly, ORFV inhibited ICAM-1 up-regulation on infected but not on non-infected KC. Even interferon-γ, a potent inducer of ICAM-1, up-regulated ICAM-1 only on non-infected KC. Transfer of ORFV-free supernatant from infected to non-infected KC induced ICAM-1 on non-infected KC pointing to the involvement of soluble mediator(s). Similarly as in KC, in FB interference with ICAM-1 up-regulation by ORFV infection was also observed. In conclusion, we shed light on epidermal and dermal defense mechanisms to ORFV infection and point to a novel ICAM-1-related immune evasion mechanism of ORFV in human skin.

Human papilloma virus E7 oncoprotein abrogates the p53-p21-DREAM pathway.

High risk human papilloma viruses cause several types of cancer. The HPV oncoproteins E6 and E7 are essential for oncogenic cell transformation. E6 mediates the degradation of the tumor suppressor p53, and E7 can form complexes with the retinoblastoma pRB tumor suppressor. Recently, it has been shown that HPV E7 can also interfere with the function of the DREAM transcriptional repressor complex. Disruption of DREAM-dependent transcriptional repression leads to untimely early expression of central cell cycle regulators. The p53-p21-DREAM pathway represents one important means of cell cycle checkpoint activation by p53. By activating this pathway, p53 can downregulate transcription of genes controlled by DREAM. Here, we present a genome-wide ranked list of genes deregulated by HPV E7 expression and relate it to datasets of cell cycle genes and DREAM targets. We find that DREAM targets are generally deregulated after E7 expression. Furthermore, our analysis shows that p53-dependent downregulation of DREAM targets is abrogated when HPV E7 is expressed. Thus, p53 checkpoint control is impaired by HPV E7 independently of E6. In summary, our analysis reveals that disruption of DREAM through the HPV E7 oncoprotein upregulates most, if not all, cell cycle genes and impairs p53's control of cell cycle checkpoints.

Growth Retardation, Loss of Desmosomal Adhesion, and Impaired Tight Junction Function Identify a Unique Role of Plakophilin 1 In Vivo.

Desmosomes mediate strong intercellular adhesion through desmosomal cadherins that interact with intracellular linker proteins including plakophilins (PKPs) 1-3 to anchor the intermediate filaments. PKPs show overlapping but distinct expression patterns in the epidermis. So far, the contribution of individual PKPs in differentially regulating desmosome function is incompletely understood. To resolve the role of PKP1 we ablated the PKP1 gene. Here, we report that PKP1(-/-) mice were born at the expected mendelian ratio with reduced birth weight, but they otherwise appeared normal immediately after birth. However, their condition rapidly declined, and the mice died within 24 hours, developing fragile skin with lesions in the absence of obvious mechanical trauma. This was accompanied by sparse and small desmosomes. Newborn PKP1(-/-) mice showed disturbed tight junctions with an impaired inside-out barrier, whereas the outside-in barrier was unaffected. Keratinocytes isolated from these mice showed strongly reduced intercellular cohesion, delayed tight junction formation, and reduced transepithelial resistance and reduced proliferation rates. Our study shows a nonredundant and essential role of PKP1 in desmosome and tight junction function and supports a role of PKP1 in growth control, a function that is crucial in wound healing and epidermal carcinogenesis.

Using Drosophila for Studies of Intermediate Filaments.

Drosophila melanogaster is a useful organism for determining protein function and modeling human disease. Drosophila offers a rapid generation time and an abundance of genomic resources and genetic tools. Conservation in protein structure, signaling pathways, and developmental processes make studies performed in Drosophila relevant to other species, including humans. Drosophila models have been generated for neurodegenerative diseases, muscular dystrophy, cancer, and many other disorders. Recently, intermediate filament protein diseases have been modeled in Drosophila. These models have revealed novel mechanisms of pathology, illuminated potential new routes of therapy, and make whole organism compound screens feasible. The goal of this chapter is to outline steps to study intermediate filament function and model intermediate filament-associated diseases in Drosophila. The steps are general and can be applied to study the function of almost any protein. The protocols outlined here are for both the novice and experienced Drosophila researcher, allowing the rich developmental and cell biology that Drosophila offers to be applied to studies of intermediate filaments.

Distinct Impact of Two Keratin Mutations Causing Epidermolysis Bullosa Simplex on Keratinocyte Adhesion and Stiffness.

Keratin filaments constitute the major component of the epidermal cytoskeleton from heterodimers of type I and type II keratin subunits. Missense mutations in keratin 5 or keratin 14, highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS) in humans by rendering the keratin cytoskeleton sensitive to mechanical stress; yet, the mechanisms by which individual mutations cause cell fragility are incompletely understood. Here, we compared the K14p.Arg125Pro with the K5p.Glu477Asp mutation, both giving rise to severe generalized EBS, by stable expression in keratin-free keratinocytes. This revealed distinctly different effects on keratin cytoskeletal organization, in agreement with in vivo observations, thus validating the cell system. Although the K14p.Arg125Pro mutation led to impaired desmosomes, downregulation of desmosomal proteins, and weakened epithelial sheet integrity upon shear stress, the K5p.Glu477Asp mutation did not impair these functions, although causing EBS with squamous cell carcinoma in vivo. Atomic force microscopy demonstrated that K14 mutant cells were even less resistant against deformation compared with keratin-free keratinocytes. Thus, a keratin mutation causing EBS compromises cell stiffness to a greater extent than the lack of keratins. Finally, re-expression of K14 in K14 mutant cells did not rescue the above defects. Collectively, our findings have implications for EBS therapy approaches.

Keratins Stabilize Hemidesmosomes through Regulation of ß4-Integrin Turnover.

Epidermal integrity and wound healing depend on remodeling of cell-matrix contacts including hemidesmosomes. Mutations in ß4-integrin and plectin lead to severe epidermolysis bullosa (EB). Whether mutations in keratins K5 or K14, which cause EB simplex, also compromise cell-matrix adhesion through altering hemidesmosomal components is not well investigated. In particular, the dependence of ß4-integrin endocytosis and turnover on keratins remains incompletely understood. Here, we show that the absence of keratins causes loss of plectin-ß4-integrin interaction and elevated ß4-integrin phosphorylation at Ser1354 and Ser1362. This triggered a caveolin-dependent endocytosis of ß4-integrin but not of other integrins through Rab5 and Rab11 compartments in keratinocytes. Expressing a phospho-deficient ß4-integrin mutant reduces ß4-integrin endocytosis and rescues plectin localization in keratin-free cells. ß4-integrin phosphorylation in the absence of keratins resulted from elevated Erk1/2 activity downstream of increased EGFR and PKCα signaling. Further, increased Erk1/2 phosphorylation and altered plectin localization occur in keratin-deficient mouse epidermis in vivo. Strikingly, expression of the K14-R125P EBS mutant also resulted in plectin mislocalization and elevated ß4-integrin turnover, suggesting disease relevance. Our data underscore a major role of keratins in controlling ß4-integrin endocytosis involving a plectin-Erk1/2-dependent mechanism relevant for epidermal differentiation and pathogenesis.

Regulation of keratin network organization.

Keratins form the major intermediate filament cytoskeleton of epithelia and are assembled from heterodimers of 28 type I and 26 type II keratins in cell- and differentiation-dependent patterns. By virtue of their primary sequence composition, interactions with cell adhesion complexes and components of major signaling cascades, keratins act as targets and effectors of mechanical force and chemical signals to determine cell mechanics, epithelial cohesion and modulate signaling in keratin isotype-specific manners. Therefore, cell-specific keratin expression and organization impact on cell growth, migration and invasion. Here, we review the recent literature, focusing on the question how keratin networks are regulated and how the interplay of keratins with adhesion complexes affects these processes and provides a framework to understand keratins contribution to blistering and inflammatory disorders and to tumor metastasis.

Genomewide RNAi screen identifies protein kinase Cb and new members of mitogen-activated protein kinase pathway as regulators of melanoma cell growth and metastasis.

A large-scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole-genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen-activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C ß (PKCß) as candidate genes. Knockdown of PKCß most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCß showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCß-specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCß-shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCß seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.