ABSTRACT
BACKGROUND: K1 and K2 are the main type II keratins in the suprabasal epidermis where each of them heterodimerizes with the type I keratin K10 to form intermediate filaments. In regions of the ears, tail, and soles of the mouse, only K2 is co-expressed with K10, suggesting that these keratins suffice to form a mechanically resilient cytoskeleton. OBJECTIVE: To determine the effects of the suppression of both main keratins, K2 and K10, in the suprabasal plantar epidermis of the mouse. METHODS: Krt2(-/-) Krt10(-/-) mice were generated by crossing Krt2(-/-) and Krt10(-/-) mice. Epidermal morphology of soles of hind-paws was examined macroscopically and histologically. Immunofluorescence analysis and quantitative PCR analysis were performed to analyze the expression of keratins in sole skin of wildtype and Krt2(-/-) Krt10(-/-) mice. Highly abundant proteins of the sole stratum corneum were determined by electrophoretic and chromatographic separation and subsequent mass spectrometry. RESULTS: K2 and K10 are the most prominent suprabasal keratins in normal mouse soles with the exception of the footpads where K1, K9 and K10 predominate. Mice lacking both K2 and K10 were viable and developed epidermal acanthosis and hyperkeratosis in inter-footpad epidermis of the soles. The expression of keratins K1, K9 and K16 was massively increased at the RNA and protein levels in the soles of Krt2(-/-) Krt10(-/-) mice. CONCLUSIONS: This study demonstrates that the loss of the main cytoskeletal components of plantar epidermis, i.e. K2 and K10, can be only partly compensated by the upregulation of other keratins. The thickening of the epidermis in the soles of Krt2(-/-) Krt10(-/-) mice may serve as a model for pathomechanistic aspects of palmoplantar keratoderma.
Subject(s)
Epidermis/physiology , Keratin-10/physiology , Keratin-2/physiology , Animals , Biomechanical Phenomena , Disease Models, Animal , Epidermis/anatomy & histology , Extremities , Humans , Keratin-1/genetics , Keratin-1/metabolism , Keratin-10/genetics , Keratin-16/genetics , Keratin-16/metabolism , Keratin-2/deficiency , Keratin-2/genetics , Keratin-9/genetics , Keratin-9/metabolism , Keratoderma, Palmoplantar/genetics , Keratoderma, Palmoplantar/metabolism , Keratoderma, Palmoplantar/pathology , Keratosis/genetics , Keratosis/metabolism , Keratosis/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Up-RegulationABSTRACT
Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.
Subject(s)
Arsenites/pharmacology , Bone Morphogenetic Proteins/antagonists & inhibitors , Keratinocytes/drug effects , Bone Morphogenetic Protein 6/antagonists & inhibitors , Bone Morphogenetic Protein 6/physiology , Bone Morphogenetic Proteins/physiology , Cells, Cultured , Forkhead Transcription Factors/antagonists & inhibitors , Humans , Keratin-1/physiology , Keratin-10/physiology , Keratinocytes/physiology , Real-Time Polymerase Chain Reaction , Receptors, Notch/physiology , Signal Transduction/drug effectsABSTRACT
PURPOSE: Cytokeratin 10 (CK10) was found to be expressed differently in human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials in our previous research. The aim of this study was to assess the value of CK10 alone or in combination with cytokeratin 19 (CK19) in predicting tumor recurrence after curative resection in HCC patients. EXPERIMENTAL DESIGN: CK10 expression in stepwise metastatic HCC cell lines and tumor tissues from 50 HCC patients was investigated using immunofluorescence assay, quantitative real-time reverse transcription-PCR, and Western blot analyses. Tumor tissue microarrays of 300 HCC patients who underwent curative resection between 1997 and 2000 were used to detect the expressions of CK10 and CK19. Clinicopathologic data for these patients were evaluated. The prognostic significance was assessed using Kaplan-Meier survival estimates and log-rank tests. RESULTS: CK10 was overexpressed in the high metastatic HCC cell line and in tumor tissues of recurrent patients. Both univariate and multivariate analyses revealed that CK10 was a significant predictor for overall survival (OS) and disease-free survival, and that CK19 was a significant predictor for OS. CK10 expression was correlated with poor prognosis regardless of alpha-fetoprotein, tumor-node-metastasis stage, and vascular invasion. The 7-year OS and disease-free survival rates in CK10+ and/or CK19+ patients were 30.0% and 37.6%, respectively, which were significantly lower than that of CK10-/CK19- patients (56.1% and 60.0%, respectively; P < 0.001). CONCLUSION: CK10 is associated with HCC invasiveness. CK10 alone, or in combination with CK19, can be a novel predictor for poor prognosis of HCC patients after curative resection.
Subject(s)
Biomarkers, Tumor/physiology , Carcinoma, Hepatocellular/diagnosis , Carcinoma, Hepatocellular/surgery , Keratin-10/physiology , Keratin-19/physiology , Liver Neoplasms/diagnosis , Liver Neoplasms/surgery , Adult , Aged , Aged, 80 and over , Biomarkers, Tumor/metabolism , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/mortality , Cell Line, Tumor , Cohort Studies , Female , Follow-Up Studies , Humans , Keratin-10/metabolism , Keratin-19/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/mortality , Male , Middle Aged , Postoperative Period , Prognosis , Survival Analysis , Tissue Array AnalysisABSTRACT
BACKGROUND: Staphylococcus aureus permanently colonizes the vestibulum nasi of one-fifth of the human population, which is a risk factor for autoinfection. The precise mechanisms whereby S. aureus colonizes the nose are still unknown. The staphylococcal cell-wall protein clumping factor B (ClfB) promotes adhesion to squamous epithelial cells in vitro and might be a physiologically relevant colonization factor. METHODS AND FINDINGS: We define the role of the staphylococcal cytokeratin-binding protein ClfB in the colonization process by artificial inoculation of human volunteers with a wild-type strain and its single locus ClfB knock-out mutant. The wild-type strain adhered to immobilized recombinant human cytokeratin 10 (CK10) in a dose-dependent manner, whereas the ClfB(-) mutant did not. The wild-type strain, when grown to the stationary phase in a poor growth medium, adhered better to CK10, than when the same strain was grown in a nutrient-rich environment. Nasal cultures show that the mutant strain is eliminated from the nares significantly faster than the wild-type strain, with a median of 3 +/- 1 d versus 7 +/- 4 d (p = 0.006). Furthermore, the wild-type strain was still present in the nares of 3/16 volunteers at the end of follow-up, and the mutant strain was not. CONCLUSIONS: The human colonization model, in combination with in vitro data, shows that the ClfB protein is a major determinant of nasal-persistent S. aureus carriage and is a candidate target molecule for decolonization strategies.
Subject(s)
Carrier State/microbiology , Coagulase/physiology , Nasal Cavity/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/pathogenicity , Administration, Intranasal , Adult , Bacterial Adhesion , Coagulase/biosynthesis , Coagulase/deficiency , Coagulase/genetics , Culture Media/pharmacology , Double-Blind Method , Enzyme-Linked Immunosorbent Assay , Female , Gene Expression Regulation, Bacterial/drug effects , Humans , Keratin-10/genetics , Keratin-10/physiology , Male , Middle Aged , Recombinant Fusion Proteins/physiology , Staphylococcus aureus/drug effects , Staphylococcus aureus/geneticsSubject(s)
Keratin-10/physiology , Skin Neoplasms/metabolism , 9,10-Dimethyl-1,2-benzanthracene/toxicity , Animals , Apoptosis , Binding Sites , Carcinogens/toxicity , Cell Proliferation , Cells, Cultured , Disease Susceptibility/metabolism , Disease Susceptibility/physiopathology , Epidermal Cells , Intermediate Filaments/metabolism , Keratin-10/genetics , Keratin-10/metabolism , Keratin-14/chemistry , Keratin-14/genetics , Keratin-14/metabolism , Keratin-14/physiology , Keratin-5/genetics , Keratin-5/metabolism , Keratin-5/physiology , Keratinocytes/cytology , Keratinocytes/metabolism , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Knockout , Mice, Transgenic , Protein Binding , Protein Structure, Tertiary , Skin Neoplasms/etiology , Skin Neoplasms/genetics , Skin Neoplasms/physiopathology , Tetradecanoylphorbol Acetate/toxicityABSTRACT
Keratin 10 (K10) is a type I keratin that is expressed in post-mitotic suprabasal keratinocytes of the skin. Based on cell culture experiments and transgenic mouse studies, it has been proposed that K10 suppresses cell proliferation and tumor formation in the skin. Furthermore, the ability of K10 to suppress cell proliferation was mapped to its unique N- and C-terminal protein domains. In the present study, we modified the endogenous keratin 14 (K14) gene of mice using a knock-in approach to encode a chimeric keratin that consists of the K14 rod domain fused to the K10 head and tail domains (K1014chim). This transgene was expressed in the basal layer of the epidermis and the outer root sheath of hair follicles. Unexpectedly, we found that the K10 end domains had no effect on basal keratinocyte proliferation in vivo. Moreover, when subjected to a chemical skin carcinogenesis protocol, papilloma formation in mutant mice was accelerated instead of being inhibited. Our data suggest that the increased tumor susceptibility of K1014chim mice is in part due to a suppression of apoptosis in mutant keratinocytes. Our results support the notion that intermediate filaments, in addition to their function as cytoskeletal components, affect tumor susceptibility of epithelial cells.
