Roles of keratins in intestinal health and disease

Intermediate filament keratins (K) play a pivotal role in protein targeting and epithelialcytoprotection from stress as evidenced by keratin mutations predisposing to human liver and skin diseases and possibly inflammatory bowel disease (IBD). The K8-null (K8-/-) mice exhibit colonic phenotype similar to IBD and marked spontaneous colitis, epithelial hyperproliferation, decreased apoptosis, mistargeting of proteins leading to defective ion transport and diarrhea. The K8-heterozygote (K8+/-) mouse colon appears normal but displays a defective sodium (Na+) and chloride (Cl-) transport similar to, but milder than K8-/-. Characterization of K8+/- colon revealed ~50% less keratins (K7, K8, K19, K20) compared to K8 wild type (K8+/+). A similar ~50% decrease was seen in K8+/- mRNA levels as compared to K8+/+, while the mRNA levels for the other keratins were unaltered. K8+/- keratins were arranged in a normal colonic crypt expression pattern, except K7 which was expressed at the top of crypts in contrast to K8+/+. The K8+/- colon showed mild hyperplasia but no signs of inflammation and no resistance to apoptosis. Experimental colitis induced by using different concentrations of dextran sulphate sodium (DSS) showed that K8+/- mice are slightly more sensitive to induced colitis and showed a delayed recovery compared to K8+/+. Hence, the K8+/- mouse with less keratins and without inflammation, provided a novel model to study direct molecular mechanisms of keratins in intestinal homeostasis and ion transport. Different candidate ion transporters for a possible role in altered ion transport seen in the K8-/- and K8+/- mouse colon were evaluated. Besides normal levels of CFTR, PAT-1 and NHE-3, DRA mRNA levels were decreased 3-4-fold and DRA protein nearly entirely lost in K8-/- caecum, distal and proximal colon compared to K8+/+. In K8+/- mice, DRA mRNA levels were unaltered while decreased DRA protein level and patchy distribution was detected particularly in the proximal colon and as compared to K8+/+. DRA was similarly decreased when K8 was knocked-down in Caco-2 cells, confirming that K8 levels modulate DRA levels in an inflammation-independent manner. The dramatic loss of DRA in colon and caecum of K8-/- mice was responsible for the chloride transport defect. The milder ion transport in K8+/- colon might be related to DRA suggesting a role for K8 in regulation of DRA expression and targeting. The current study demonstrates the importance of keratins in stress protection and cell signaling. Furthermore, we have also successfully developed a novel, simple, fast, cost effective, non-invasive in vivo imaging method for the early diagnosis of murine colitis with specificity for both genetic and experimental colitis. The said modality provides continuous measurements of reactive oxygen and nitrogen species (RONS) and minimizes the use of an increased number of experimental animals by using a luminal derivative chemiluminescent probe, L-012 which provides a cost-effective tool to study the level and longitudinal progression of colitis.

Immunoexpression of keratins in the calcifying cystic odontogenic tumor epithelium

Ameloblastomatous epithelium containing clusters of ghost cells is the typical histopathology of calcifying cystic odontogenic tumor (CCOT). This paper aimed to assess keratins AE1-AE3, K7, K10/13, K14, K18, K19, vimentin, laminin, and collagen IV in 08 CCOTs to discuss their histopathogenesis. Similarity to the immunoprofile of the stratified squamous epithelium was seen in the with the basal layer expressing K14 and the upper cells expressing K10/13. When compared to the immunoprofile of the normal odontogenic epithelium, of odontogenic tumor epithelia and of the ghost cells described in the literature, it was possible to suggest that the CCOT epithelium differentiates towards squamous type.

Interaction of Plectin with Keratins 5 and 14: Dependence on Several Plectin Domains and Keratin Quaternary Structure.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein-binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.Journal of Investigative Dermatology advance online publication, 10 July 2014; doi:10.1038/jid.2014.255.

Formation of a Normal Epidermis Supported by Increased Stability of Keratins 5 and 14 in Keratin 10 Null Mice

The expression of distinct keratin pairs during epidermal differentiation is assumed to fulfill specific and essential cytoskeletal functions. This is supported by a great variety of genodermatoses exhibiting tissue fragility because of keratin mutations. Here, we show that the loss of K10, the most prominent epidermal protein, allowed the formation of a normal epidermis in neonatal mice without signs of fragility or wound-healing response. However, there were profound changes in the composition of suprabasal keratin filaments. K5/14 persisted suprabasally at elevated protein levels, whereas their mRNAs remained restricted to the basal keratinocytes. This indicated a novel mechanism regulating keratin turnover. Moreover, the amount of K1 was reduced. In the absence of its natural partner we observed the formation of a minor amount of novel K1/14/15 filaments as revealed by immunogold electron microscopy. We suggest that these changes maintained epidermal integrity. Furthermore, suprabasal keratinocytes contained larger keratohyalin granules similar to our previous K10T mice. A comparison of profilaggrin processing in K10T and K10−/− mice revealed an accumulation of filaggrin precursors in the former but not in the latter, suggesting a requirement of intact keratin filaments for the processing. The mild phenotype of K10−/− mice suggests that there is a considerable redundancy in the keratin gene family.

Hydrolyse des keratins aus horn und wolle sowie des ichtylepidi...

Thesis (doctoral)--

A novel mutation in the L12 domain of keratin 5 in the Kobner variant of epidermolysis bullosa simplex

We have identified a novel mutation within the linker L12 region of keratin 5 (K5) in a family with the Kobner variant of epidermolysis bullosa simplex. The pattern of inheritance of the disorder in this family is consistent with an autosomal dominant mode of transmission. Affected individuals develop extensive and generalized blistering at birth or early infancy but in later years clinical manifestations are largely confined to palmo-plantar surfaces. Direct sequencing of polymerase chain reaction products revealed a T to C transition within codon 323 of K5 in affected individuals, resulting in a valine to alanine substitution of the seventh residue within the L12 linker domain. This mutation was not observed in unaffected family members or in 100 K5 alleles of unrelated individuals with normal skin. The other critical regions of K5 and K14 were unremarkable in this family except for common polymorphisms that have been previously described. The valine at position 7 of the L12 domain is absolutely conserved in all type II keratins, and in other intermediate filament subunits as well, which suggests that this residue makes an important contribution to filament integrity. Secondary structure analysis revealed that alanine at this position markedly reduces both the hydrophobicity and the beta-sheet nature of the L12 domain. This is the first report of a mutation at this position in an intermediate filament subunit and reinforces the importance of this region to filament biology.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

In vitro biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate composite using cultures of human periodontal ligament fibroblasts and keratinocytes

The mm of this work was to evaluate the biocompatibility of poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane to be used in guided tissue regeneration (GTR) Fibroblasts from human periodontal ligament (hPDLF) and keratinocytes (SCC9) were plated on P(VDF-TrFE)/BT and polytetrafluorethylene membranes at a cell density of 20.000 cells well(-1) and Cultured for up to 21 days Cell morphology, adhesion and proliferation were evaluated in hPDLF and keratinocytes, while total protein content and alkaline phosphatase (ALP) activity were assayed only for hPDLF Using a higher cell density. real-time polymerase chain reaction (PCR) was performed to assess the expression of typical genes of hPDLF, such as periostin, PDLs17, S100A4 and fibromodulin, and key phenotypic markers of keratinocytes, including involucrin, keratins 1. 10 and 14 Expression of the apoptotic genes bax, bcl-2 and Survivin was evaluated for both cultures hPDLF adhered and spread more oil P(VDF-TrFE)/BT, whereas keratinocytes showed a round shape on both membranes. hPDLF adhesion was greater oil P(VDF-TrFE)/BT at 2 and 4 h, while keratinocyte adhesion was similar for both membranes. Whereas proliferation was significantly higher for hPDLF on P(VDF-TrFE)/BT at days 1 and 7. no signs of keratinocyte proliferation could be noticed for both membranes Total protein content was greater on P(VDF-TrFE)/BT at 7, 14 and 21 days, and higher levels of ALP activity were observed oil P(VDF-TrFE)/BT at 21 days. Real-time PCR revealed higher expression of phenotypic markers of hPDLF and keratinocytes as well as greater expression of apoptotic genes in cultures grown on P(VDF-TrFE)/BT. These results indicate that, by favoring hPDLF adhesion. spreading. proliferation and typical mRNA expression, P(VDF-TrFE)/BT membrane should be considered an advantageous alternative for GTR (C) 2009 Acta Materialia Inc Published by Elsevier Ltd All rights reserved

Enzymatic phosphorylation of hair keratin enhances fast adsorption of cationic moieties

The current study describes the in vitro phosphorylation of a human hair keratin, using protein kinase for the first time. Phosphorylation of keratin was demonstrated by 31P NMR (Nuclear Magnetic Resonance) and Diffuse Reflectance Infrared Fourier Transform (DRIFT) techniques. Phosphorylation induced a 2.5 fold increase of adsorption capacity in the first 10 minutes for cationic moiety like Methylene Blue (MB). Thorough description of MB adsorption process was performed by several isothermal models. Reconstructed fluorescent microscopy images depict distinct amounts of dye bound to the differently treated hair. The results of this work suggest that the enzymatic phosphorylation of keratins might have significant implications in hair shampooing and conditioning, where short application times of cationic components are of prime importance.

Mutation in the gene for connexin 30.3 in a family with erythrokeratodermia variabilis.

Erythrokeratodermia variabilis (EKV) is an autosomal dominant keratinization disorder characterized by migratory erythematous lesions and fixed keratotic plaques. All families with EKV show mapping to chromosome 1p34-p35, and mutations in the gene for connexin 31 (Cx31) have been reported in some but not all families. We studied eight affected and three healthy subjects in an Israeli family, of Kurdish origin, with EKV. After having mapped the disorder to chromosome 1p34-p35, we found no mutations in the genes for Cx31, Cx31.1, and Cx37. Further investigation revealed a heterozygous T-->C transition leading to the missense mutation (F137L) in the human gene for Cx30.3 that colocalizes on chromosome 1p34-p35. This nucleotide change cosegregated with the disease and was not found in 200 alleles from normal individuals. This mutation concerns a highly conserved phenylalanine, in the third transmembrane region of the Cx30.3 molecule, known to be implicated in the wall formation of the gap-junction pore. Our results show that mutations in the gene for Cx30.3 can be causally involved in EKV and point to genetic heterogeneity of this disorder. Furthermore, we suggest that our family presents a new type of EKV because of the hitherto unreported association with erythema gyratum repens.

EIU in the rat promotes the potential of syngeneic retinal cells injected into the vitreous cavity to induce PVR.

PURPOSE: To determine whether syngeneic retinal cells injected in the vitreous cavity of the rat are able to initiate a proliferative process and whether the ocular inflammation induced in rats by lipopolysaccharide (LPS) promotes this proliferative vitreoretinopathy (PVR). METHODS: Primary cultured differentiated retinal Müller glial (RMG) and retinal pigmented epithelial (RPE) cells isolated from 8 to 12 postnatal Lewis rats were injected into the vitreous cavity of 8- to 10-week-old Lewis rats (10(5) cells/eye in 2 microlieter sterile saline), with or without the systemic injection of 150 microgram LPS to cause endotoxin-induced uveitis (EIU). Control groups received an intravitreal injection of 2 microliter saline. At 5, 15, and 28 days after cell injections, PVR was clinically quantified, and immunohistochemistry for OX42, ED1, vimentin (VIM), glial fibrillary acidic protein (GFAP), and cytokeratin was performed. RESULTS: The injection of RMG cells, alone or in combination with RPE cells, induced the preretinal proliferation of a GFAP-positive tissue, that was enhanced by the systemic injection of LPS. Indeed, when EIU was induced at the time of RMG cell injection into the vitreous cavity, the proliferation led to retinal folds and localized tractional detachments. In contrast, PVR enhanced the infiltration of inflammatory cells in the anterior segment of the eye. CONCLUSIONS: In the rat, syngeneic retinal cells of glial origin induce PVR that is enhanced by the coinduction of EIU. In return, vitreoretinal glial proliferation enhanced the intensity and duration of EIU.

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium.

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.

High frequencies of functionally impaired cytokeratin 18-specific CD8+ T cells in healthy HLA-A2+ donors.

Combining cell surface phenotyping with functional analysis, human CD8+ T cells have been divided into several subsets which are being studied extensively in diverse physiological situations, such as viral infection, cancer and ageing. In particular, so-called terminally differentiated effector cells possess a CD45RA+ CCR7- CD27- CD28- phenotype, contain perforin and, in different models, have been shown to exert direct ex vivo killing and to release interleukins upon both antigen-nonspecific and -specific stimulation. Using HLA class I multimers, we have identified a high frequency of peripheral CD8+ T cells that recognize a peptide derived from the self protein cytokeratin 18 presented by the HLA-A*0201 molecule. These cells can be detected in approximately 15% of the HLA-A2-positive healthy donors tested. A detailed analysis revealed that they must have divided extensively in vivo, have an effector cell phenotype and express various natural killer cell-associated receptors. Interestingly, however, they remained unresponsive to antigen-specific stimulation in vitro in terms of cytotoxicity and cytokine secretion. Thus, cytokeratin 18-specific cells constitute a frequently encountered, new CD8+ T lymphocyte subpopulation without classical effector status and with so far unknown function.