RESUMO
Mesoderm arises at gastrulation and contributes to both the mouse embryo proper and its extra-embryonic membranes. Two-photon live imaging of embryos bearing a keratin reporter allowed recording filament nucleation and elongation in the extra-embryonic region. Upon separation of amniotic and exocoelomic cavities, keratin 8 formed apical cables co-aligned across multiple cells in the amnion, allantois, and blood islands. An influence of substrate rigidity and composition on cell behavior and keratin content was observed in mesoderm explants. Embryos lacking all keratin filaments displayed a deflated extra-embryonic cavity, a narrow thick amnion, and a short allantois. Single-cell RNA sequencing of sorted mesoderm cells and micro-dissected amnion, chorion, and allantois, provided an atlas of transcriptomes with germ layer and regional information. It defined the cytoskeleton and adhesion expression profile of mesoderm-derived keratin 8-enriched cells lining the exocoelomic cavity. Those findings indicate a novel role for keratin filaments in the expansion of extra-embryonic structures and suggest mechanisms of mesoderm adaptation to the environment.
Assuntos
Gastrulação , Mesoderma , Animais , Embrião de Mamíferos , Membranas Extraembrionárias , Queratinas/genética , Queratinas/metabolismo , Mesoderma/metabolismo , CamundongosRESUMO
Desmosomes are intercellular junctions which mediate cohesion and communication in tissues exposed to mechanical strain by tethering the intermediate filament cytoskeleton to the plasma membrane. While mature desmosomes are characterized by a hyperadhesive, Ca2+-independent state, they transiently loose this state during wound healing, pathogenesis and tissue regeneration. The mechanisms controlling the hyperadhesive state remain incompletely understood. Here, we show that upon Ca2+-induced keratinocyte differentiation, expression of keratin 17 (K17) prevents the formation of stable and hyperadhesive desmosomes, accompanied by a significant reduction of desmoplakin (DP), plakophilin-1 (PKP1), desmoglein-1 (Dsg1) and -3 (Dsg3) at intercellular cell borders. Atomic force microscopy revealed that both increased binding strength of desmoglein-3 molecules and amount of desmoglein-3 oligomers, known hallmarks of hyperadhesion, were reduced in K17- compared to K14-expressing cells. Importantly, overexpression of Dsg3 or DPII enhanced their localization at intercellular cell borders and increased the formation of Dsg3 oligomers, resulting in stable, hyperadhesive desmosomes despite the presence of K17. Notably, PKP1 was enriched in these desmosomes. Quantitative image analysis revealed that DPII overexpression contributed to desmosome hyperadhesion by increasing the abundance of K5/K17-positive keratin filaments in the proximity of desmosomes enriched in desmoglein-3. Thus, our data show that hyperadhesion can result from recruitment of keratin isotypes K5/K17 to desmosomes or from enhanced expression of DP and Dsg3 irrespective of keratin composition. The notion that hyperadhesive desmosomes failed to form in the absence of keratins underscores the essential role of keratins and suggest bidirectional control mechanisms at several levels.
Assuntos
Desmossomos , Queratinas , Adesão Celular , Citoesqueleto/metabolismo , Desmogleínas/metabolismo , Desmossomos/metabolismo , Queratinócitos/metabolismo , Queratinas/metabolismoRESUMO
Keratin intermediate filament (IF) proteins constitute the major cytoskeletal components in epithelial cells. Missense mutations in keratin 5 (K5; also known as KRT5) or keratin 14 (K14; also known as KRT14), highly expressed in the basal epidermis, cause the severe skin blistering disease epidermolysis bullosa simplex (EBS). EBS-associated mutations disrupt keratin networks and change keratinocyte mechanics; however, molecular mechanisms by which mutations shape EBS pathology remain incompletely understood. Here, we demonstrate that, in contrast to keratin-deficient keratinocytes, cells expressing K14R125C, a mutation that causes severe EBS, generate lower traction forces, accompanied by immature focal adhesions with an altered cellular distribution. Furthermore, mutant keratinocytes display reduced directionality during collective migration. Notably, RhoA activity is downregulated in human EBS keratinocytes, and Rho activation rescues stiffness-dependent cell-extracellular matrix (ECM) adhesion formation of EBS keratinocytes. Collectively, our results strongly suggest that intact keratin IF networks regulate mechanotransduction through a Rho signaling pathway upstream of cell-ECM adhesion formation and organized cell migration. Our findings provide insights into the underlying pathophysiology of EBS.This article has an associated First Person interview with the first author of the paper.
Assuntos
Epidermólise Bolhosa Simples , Queratinas , Citoesqueleto/metabolismo , Epidermólise Bolhosa Simples/genética , Humanos , Queratinas/genética , Queratinas/metabolismo , Mecanotransdução Celular , Mutação/genética , TraçãoRESUMO
The isotype-specific composition of the keratin cytoskeleton is important for strong adhesion, force resilience, and barrier function of the epidermis. However, the mechanisms by which keratins regulate these functions are still incompletely understood. In this study, the role and significance of the keratin network for mechanical integrity, force transmission, and barrier formation were analyzed in murine keratinocytes. Following the time-course of single-cell wound closure, wild-type (WT) cells slowly closed the gap in a collective fashion involving tightly connected neighboring cells. In contrast, the mechanical response of neighboring cells was compromised in keratin-deficient cells, causing an increased wound area initially and an inefficient overall wound closure. Furthermore, the loss of the keratin network led to impaired, fragmented cell-cell junctions, and triggered a profound change in the overall cellular actomyosin architecture. Electric cell-substrate impedance sensing of cell junctions revealed a dysfunctional barrier in knockout (Kty-/-) cells compared to WT cells. These findings demonstrate that Kty-/- cells display a novel phenotype characterized by loss of mechanocoupling and failure to form a functional barrier. Re-expression of K5/K14 rescued the barrier defect to a significant extent and reestablished the mechanocoupling with remaining discrepancies likely due to the low abundance of keratins in that setting. Our study reveals the major role of the keratin network for mechanical homeostasis and barrier functionality in keratinocyte layers.
Assuntos
Queratinócitos/citologia , Queratinas/metabolismo , Animais , Fenômenos Biomecânicos , Linhagem Celular , Epiderme/metabolismo , Epiderme/ultraestrutura , Deleção de Genes , Junções Intercelulares/genética , Junções Intercelulares/metabolismo , Junções Intercelulares/ultraestrutura , Queratinócitos/metabolismo , Queratinas/genética , Queratinas/ultraestrutura , Camundongos , CicatrizaçãoRESUMO
The human interosseous membrane (IOM) is a fundamental stabilizer during forearm rotation. To investigate the dynamic aspects of forearm stability, we analyzed sensory nerve endings in the IOM. The distal oblique bundle (DOB), the distal accessory band (DAB), the central band (CB), the proximal accessory band (PAB), the dorsal oblique accessory cord (DOAC) and the proximal oblique cord (POC) were dissected from 11 human cadaver forearms. Sensory nerve endings were analyzed at two levels per specimen as total cell amount/mm2 after immunofluorescence staining with low-affinity neurotrophin receptor p75, protein gene product 9.5, S-100 protein and 4',6-diamidino-2-phenylindole on an Apotome microscope, according to Freeman and Wyke's classification. Sensory nerve endings were significantly more commonly found to be equally distributed throughout the structures, rather than being epifascicular, interstitial, or close to the insertion into bone (P ≤ 0.001, respectively). Free nerve endings were the predominant mechanoreceptor in all six structures, with highest density in the DOB, followed by the POC (P ≤ 0.0001, respectively). The DOB had the highest density of Pacini corpuscles. The DOAC and CB had the lowest amounts of sensory innervation. The high density of sensory corpuscles in the DOB, PAB and POC indicate that proprioceptive control of the compressive and directional muscular forces acting on the distal and proximal radioulnar joints is monitored by the DOB, PAB and POC, respectively, due to their closed proximity to both joints, whereas the central parts of the IOM act as structures of passive restraint.
Assuntos
Antebraço/inervação , Membrana Interóssea/metabolismo , Células Receptoras Sensoriais/metabolismo , Idoso , Fenômenos Biomecânicos/fisiologia , Feminino , Imunofluorescência , Humanos , Masculino , Propriocepção/fisiologiaRESUMO
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.
Assuntos
Citoesqueleto/fisiologia , Receptor de Insulina/fisiologia , Fenômenos Fisiológicos da Pele , Pele/fisiopatologia , Animais , Humanos , Queratinas/genética , CamundongosRESUMO
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.
Assuntos
Azetidinas/farmacologia , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Piperidinas/farmacologia , Proteínas Proto-Oncogênicas c-raf/genética , Animais , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/fisiologia , Avaliação Pré-Clínica de Medicamentos , Regulação da Expressão Gênica no Desenvolvimento , Genes Letais , Intestinos/enzimologia , Larva , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Especificidade de Órgãos , Fenótipo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/fisiologia , Proteínas Proto-Oncogênicas c-raf/biossíntese , Proteínas Proto-Oncogênicas c-raf/deficiência , Proteínas Proto-Oncogênicas c-raf/fisiologia , Vemurafenib/farmacologiaRESUMO
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.
Assuntos
Ectima Contagioso/complicações , Fibroblastos/virologia , Molécula 1 de Adesão Intercelular/metabolismo , Queratinócitos/virologia , Vírus do Orf , Morte Celular , Humanos , Sistema Imunitário , Inflamação , Interferon gama/metabolismo , Interleucina-6/metabolismo , Microscopia de Contraste de Fase , Pele/citologia , Regulação para Cima , Replicação ViralRESUMO
Cytoplasmic intermediate filaments (IFs) represent a major cytoskeletal network contributing to cell shape, adhesion and migration as well as to tissue resilience and renewal in numerous bilaterians, including mammals. The observation that IFs are dispensable in cultured mammalian cells, but cause tissue-specific, life-threatening disorders, has pushed the need to investigate their function in vivo. In keeping with human disease, the deletion or mutation of murine IF genes resulted in highly specific pathologies. Epidermal keratins, together with desmin, are essential to protect corresponding tissues against mechanical force but also participate in stabilizing cell adhesion and in inflammatory signalling. Surprisingly, other IF proteins contribute to tissue integrity to a much lesser extent than anticipated, pointing towards their role in stress situations. In support, the overexpression of small chaperones or the interference with inflammatory signalling in several settings has been shown to rescue severe tissue pathologies that resulted from the expression of mutant IF proteins. It stills remains an open issue whether the wide range of IF disorders share similar pathomechanisms. Moreover, we lack an understanding how IF proteins participate in signalling processes. Now, with a large number of mouse models in hand, the next challenge will be to develop organotypic cell culture models to dissect pathomechanisms at the molecular level, to employ Crispr/Cas-mediated genome engineering to optimize models and, finally, to combine available animal models with medicinal chemistry for the development of molecular therapies.
Assuntos
Proteínas de Filamentos Intermediários , Animais , Doenças do Tecido Conjuntivo , Citoplasma , Modelos Animais de Doenças , HumanosRESUMO
Cell motility and cell shape adaptations are crucial during wound healing, inflammation, and malignant progression. These processes require the remodeling of the keratin cytoskeleton to facilitate cell-cell and cell-matrix adhesion. However, the role of keratins for biomechanical properties and invasion of epithelial cells is only partially understood. In this study, we address this issue in murine keratinocytes lacking all keratins on genome engineering. In contrast to predictions, keratin-free cells show about 60% higher cell deformability even for small deformations. This response is compared with the less pronounced softening effects for actin depolymerization induced via latrunculin A. To relate these findings with functional consequences, we use invasion and 3D growth assays. These experiments reveal higher invasiveness of keratin-free cells. Reexpression of a small amount of the keratin pair K5/K14 in keratin-free cells reverses the above phenotype for the invasion but does not with respect to cell deformability. Our data show a unique role of keratins as major players of cell stiffness, influencing invasion with implications for epidermal homeostasis and pathogenesis. This study supports the view that down-regulation of keratins observed during epithelial-mesenchymal transition directly contributes to the migratory and invasive behavior of tumor cells.
Assuntos
Movimento Celular/fisiologia , Forma Celular/fisiologia , Queratinas/metabolismo , Invasividade Neoplásica/fisiopatologia , Pele/citologia , Animais , Fenômenos Biomecânicos , Ensaio de Unidades Formadoras de Colônias , Transição Epitelial-Mesenquimal/fisiologia , Imunofluorescência , Engenharia Genética/métodos , Indóis , Queratinas/genética , Camundongos , VinculinaRESUMO
Keratins are major components of the epithelial cytoskeleton and are believed to play a vital role for mechanical integrity at the cellular and tissue level. Keratinocytes as the main cell type of the epidermis express a differentiation-specific set of type I and type II keratins forming a stable network and are major contributors of keratinocyte mechanical properties. However, owing to compensatory keratin expression, the overall contribution of keratins to cell mechanics was difficult to examine in vivo on deletion of single keratin genes. To overcome this problem, we used keratinocytes lacking all keratins. The mechanical properties of these cells were analyzed by atomic force microscopy (AFM) and magnetic tweezers experiments. We found a strong and highly significant softening of keratin-deficient keratinocytes when analyzed by AFM on the cell body and above the nucleus. Magnetic tweezers experiments fully confirmed these results showing, in addition, high viscous contributions to magnetic bead displacement in keratin-lacking cells. Keratin loss neither affected actin or microtubule networks nor their overall protein concentration. Furthermore, depolymerization of actin preserves cell softening in the absence of keratin. On reexpression of the sole basal epidermal keratin pair K5/14, the keratin filament network was reestablished, and mechanical properties were restored almost to WT levels in both experimental setups. The data presented here demonstrate the importance of keratin filaments for mechanical resilience of keratinocytes and indicate that expression of a single keratin pair is sufficient for almost complete reconstitution of their mechanical properties.
Assuntos
Forma Celular/fisiologia , Queratinócitos/citologia , Queratinas/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Fenômenos Biomecânicos/fisiologia , Western Blotting , Cruzamentos Genéticos , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde , Imuno-Histoquímica , Queratina-14/metabolismo , Queratinócitos/metabolismo , Queratinas/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Micromanipulação , Microscopia de Força Atômica , Estatísticas não ParamétricasAssuntos
Anfirregulina/imunologia , Receptores ErbB/imunologia , Queratinas/imunologia , Prurido/imunologia , Transdução de Sinais/imunologia , Anfirregulina/genética , Animais , Citocinas , Receptores ErbB/genética , Humanos , Queratinas/genética , Camundongos , Camundongos Knockout , Prurido/genética , Prurido/patologia , Transdução de Sinais/genética , Linfopoietina do Estroma do TimoRESUMO
Keratin 1 (KRT1) and its heterodimer partner keratin 10 (KRT10) are major constituents of the intermediate filament cytoskeleton in suprabasal epidermis. KRT1 mutations cause epidermolytic ichthyosis in humans, characterized by loss of barrier integrity and recurrent erythema. In search of the largely unknown pathomechanisms and the role of keratins in barrier formation and inflammation control, we show here that Krt1 is crucial for maintenance of skin integrity and participates in an inflammatory network in murine keratinocytes. Absence of Krt1 caused a prenatal increase in interleukin-18 (IL-18) and the S100A8 and S100A9 proteins, accompanied by a barrier defect and perinatal lethality. Depletion of IL-18 partially rescued Krt1(-/-) mice. IL-18 release was keratinocyte-autonomous, KRT1 and caspase-1 dependent, supporting an upstream role of KRT1 in the pathology. Finally, transcriptome profiling revealed a Krt1-mediated gene expression signature similar to atopic eczema and psoriasis, but different from Krt5 deficiency and epidermolysis bullosa simplex. Our data suggest a functional link between KRT1 and human inflammatory skin diseases.
Assuntos
Inflamação/patologia , Interleucina-18/metabolismo , Queratina-1/metabolismo , Pele/metabolismo , Pele/patologia , Animais , Caspase 1/metabolismo , Diferenciação Celular , Desmossomos/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Deleção de Genes , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Inflamação/metabolismo , Queratinócitos/metabolismo , Queratinócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Estrutura Quaternária de Proteína , Proteínas S100/metabolismo , Pele/imunologia , Regulação para CimaRESUMO
BACKGROUND: CFTR function is tightly regulated by many interacting proteins. RESULTS: Intermediate filament protein keratin 18 increases the cell surface expression of CFTR by interacting with the C-terminal hydrophobic patch of CFTR. CONCLUSION: K18 controls the function of CFTR. SIGNIFICANCE: These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis. Malfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) leads to cystic fibrosis, but the regulation of CFTR is not fully understood. Here, we identified the intermediate filament protein keratin K18 (K18) as a CFTR-binding protein by various approaches. We mapped a highly conserved "hydrophobic patch" ((1413)FLVI(1416)) in the CFTR C-terminus, known to determine plasmalemmal CFTR stability, as the K18-binding site. On the other hand, the C-terminal tail of K18 was found to be a critical determinant for binding CFTR. Overexpression of K18 in cells robustly increased the surface expression of wild-type CFTR, whereas depletion of K18 through RNA interference specifically diminished it. K18 binding increased the surface expression of CFTR by accelerating its apical recycling rate without altering CFTR biosynthesis, maturation, or internalization. Importantly, CFTR surface expression was markedly reduced in duodenal and gallbladder epithelia of K18(-/-) mice. Taken together, our results suggest that K18 increases the cell surface expression of CFTR by interacting with the CFTR C-terminal hydrophobic patch. These findings offer novel insights into the regulation of CFTR and suggest that K18 and its dimerization partner, K8, may be modifier genes in cystic fibrosis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Queratina-18/metabolismo , Animais , Linhagem Celular , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Queratina-18/química , Queratina-18/genética , Camundongos , Camundongos Knockout , Ligação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
A new study reports that the extracellular matrix component laminin-111 shields the nucleus from actin-mediated forces by engaging the keratin cytoskeleton. Thus, matrix composition represents a means by which tissues can protect cell nuclei from mechanical damage.
Assuntos
Actinas , Citoesqueleto , Fenômenos Fisiológicos Celulares , Microtúbulos , Biofísica , Núcleo Celular , Matriz Extracelular , Estresse MecânicoRESUMO
We suggest that the human body can be viewed as of textile nature whose fabric consists of interconnected fiber systems. These fiber systems form highly dynamic scaffolds, which respond to environmental changes at different temporal and spatial scales. This is especially relevant at sites where epithelia border on connective tissue regions that are exposed to dynamic microenvironments. We propose that the enormous heterogeneity and adaptability of epithelia are based on a "keratin code", which results from the cell-specific expression and posttranslational modification of keratin isotypes. It thereby defines unique cytoskeletal intermediate filament networks that are coupled across cells and to the correspondingly heterogeneous fibers of the underlying extracellular matrix. The resulting fabric confers unique local properties.
Assuntos
Citoesqueleto , Queratinas , Humanos , Queratinas/metabolismo , Citoesqueleto/metabolismo , Epitélio/metabolismo , Filamentos Intermediários/metabolismo , TêxteisRESUMO
The keratin cytoskeleton protects epithelia against mechanical, nonmechanical, and physical stresses, and participates in multiple signaling pathways that regulate cell integrity and resilience. Keratin gene mutations cause multiple rare monoallelic epithelial diseases termed keratinopathies, including the skin diseases Epidermolysis Bullosa Simplex (EBS) and Pachyonychia Congenita (PC), with limited available therapies. The disease-related keratin mutations trigger posttranslational modifications (PTMs) in keratins and their associated proteins that can aggravate the disease. Recent findings of drug high-throughput screening have led to the identification of compounds that may be repurposed, since they are used for other human diseases, to treat keratinopathies. These drugs target unique PTM pathways and sites, including phosphorylation and acetylation of keratins and their associated proteins, and have shed insights into keratin regulation and interactions. They also offer the prospect of testing the use of drug mixtures, with the long view of possible beneficial human use coupled with increased efficacy and lower side effects.
Assuntos
Epidermólise Bolhosa Simples , Queratinas , Humanos , Queratinas/genética , Queratinas/metabolismo , Citoesqueleto/metabolismo , Epidermólise Bolhosa Simples/genética , Epidermólise Bolhosa Simples/metabolismo , Mutação , Processamento de Proteína Pós-TraducionalRESUMO
Keratin (K) and other intermediate filament (IF) protein mutations at conserved arginines disrupt keratin filaments into aggregates and cause human epidermolysis bullosa simplex (EBS; K14-R125C) or predispose to mouse liver injury (K18-R90C). The challenge for more than 70 IF-associated diseases is the lack of clinically utilized IF-targeted therapies. We used high-throughput drug screening to identify compounds that normalized mutation-triggered keratin filament disruption. Parthenolide, a plant sesquiterpene lactone, dramatically reversed keratin filament disruption and protected cells and mice expressing K18-R90C from apoptosis. K18-R90C became hyperacetylated compared with K18-WT and treatment with parthenolide normalized K18 acetylation. Parthenolide upregulated the NAD-dependent SIRT2, and increased SIRT2-keratin association. SIRT2 knockdown or pharmacologic inhibition blocked the parthenolide effect, while site-specific Lys-to-Arg mutation of keratin acetylation sites normalized K18-R90C filaments. Treatment of K18-R90C-expressing cells and mice with nicotinamide mononucleotide had a parthenolide-like protective effect. In 2 human K18 variants that associate with human fatal drug-induced liver injury, parthenolide protected K18-D89H- but not K8-K393R-induced filament disruption and cell death. Importantly, parthenolide normalized K14-R125C-mediated filament disruption in keratinocytes and inhibited dispase-triggered keratinocyte sheet fragmentation and Fas-mediated apoptosis. Therefore, keratin acetylation may provide a novel therapeutic target for some keratin-associated diseases.
Assuntos
Queratinas , Sirtuína 2 , Animais , Humanos , Camundongos , Proteínas de Filamentos Intermediários , Queratinas/genética , Queratinas/metabolismo , Mutação , Sirtuína 2/genéticaRESUMO
In the original publication [...].
RESUMO
Keratin 8 (K8) and keratin 18 (K18) form the major hepatocyte cytoskeleton. We investigated the impact of genetic loss of either K8 or K18 on liver homeostasis under toxic stress with the hypothesis that K8 and K18 exert different functions. krt8â»/â» and krt18â»/â» mice crossed into the same 129-ola genetic background were treated by acute and chronic administration of 3,5-diethoxy-carbonyl-1,4-dihydrocollidine (DDC). In acutely DDC-intoxicated mice, macrovesicular steatosis was more pronounced in krt8â»/â» and krt18â»/â» compared with wild-type (wt) animals. Mallory-Denk bodies (MDBs) appeared in krt18â»/â» mice already at an early stage of intoxication in contrast to krt8â»/â» mice that did not display MDB formation when fed with DDC. Keratin-deficient mice displayed significantly lower numbers of apoptotic hepatocytes than wt animals. krt8â»/â», krt18â»/â» and control mice displayed comparable cell proliferation rates. Chronically DDC-intoxicated krt18â»/â» and wt mice showed a similarly increased degree of steatohepatitis with hepatocyte ballooning and MDB formation. In krt8â»/â» mice, steatosis was less, ballooning, and MDBs were absent. krt18â»/â» mice developed MDBs whereas krt8â»/â» mice on the same genetic background did not, highlighting the significance of different structural properties of keratins. They are independent of the genetic background as an intrinsic factor. By contrast, toxicity effects may depend on the genetic background. krt8â»/â» and krt18â»/â» mice on the same genetic background show similar sensitivity to DDC intoxication and almost resemble wt animals regarding survival, degree of porphyria, liver-to-body weight ratio, serum bilirubin and liver enzyme levels. This stands in contrast to previous work where krt8â»/â» and krt18â»/â» mice on different genetic backgrounds were investigated.