Immunohistochemical evaluation of keratins and involucrin in differentiating between palmoplantar pustulosis and pompholyx.

BACKGROUND: Palmoplantar pustulosis (PPP) and pompholyx are chronic diseases characterized by pustules and vesicles on the palms and soles. These disorders often have similar clinicopathological features, which lead to diagnostic difficulties. We aimed to investigate the expression patterns of keratins and involucrin in PPP and pompholyx using immunohistochemical staining. METHODS: Skin biopsies from patients with PPP (n = 40) and pompholyx (n = 22) were immunohistochemically analyzed for Keratin 5, 9, 14, and involucrin expression. RESULTS: K5 expression was higher in PPP than in pompholyx, with diffusely positive expression in the basal, spinous, and granular layers. K14 expression did not differ between groups. K9 expression was observed near the pompholyx vesicle (P = 0.014) and stratum spinosum (P < 0.001) but was almost absent around PPP pustules. Involucrin expression was diffused around the PPP pustules and partially around the pompholyx vesicles, but without statistical significance (P = 0.123). Involucrin expression was elevated in the basal layer of the PPP compared with that in the pompholyx (P = 0.023). CONCLUSION: PPP and pompholyx exhibited distinctive differentiation in the expression of K5, K9, and involucrin.

Understanding and Harnessing Epithelial‒Mesenchymal Interactions in the Development of Palmoplantar Identity.

Palmoplantar skin has several unique characteristics such as increased thickness, high resilience, hypopigmentation, and lack of hair follicles. The establishment of palmoplantar identity occurs through keratinocyte‒fibroblast interactions, with keratin 9 expression and Wnt signaling playing key roles. Understanding how palmoplantar features develop may help efforts to reproduce them at both palmoplantar and nonpalmoplantar body sites.

Cytokeratin 7 and 19 expression in oropharyngeal and oral squamous cell carcinoma.

PURPOSE: The precise etiopathogenesis of human papillomavirus (HPV)-related oropharyngeal squamous cell carcinoma (OPSCC), and reasons for predilection for crypt epithelium, remain uncertain. The purpose of this study is to investigate the interaction between HPV and specific cytokeratins 7 (CK7) and 19 (CK19) in crypt epithelium. METHODS: This is a retrospective cohort study of patients presenting between 1999 and 2015 at a tertiary referral center. CK7 and CK19 positivity and H Scores were determined by immunohistochemistry. Disease-specific and overall survival rates were analyzed. RESULTS: There were 253 patients presenting with OPSCC (134), squamous cell carcinoma (SCC) of unknown primary site (22), and oral tongue SCC (97). Primary tumor CK7 and CK19 positivity and H Scores were significantly higher in HPV-positive OPSCC than HPV-negative OPSCC and oral tongue SCC. Higher CK19 Scores, but not CK7 Scores, were also seen in regional metastases from HPV-positive OPSCC than other sites. No impact on disease-specific or overall survival was identified on multivariate analysis. CONCLUSION: The increased expression of CK7 and CK19 in HPV-positive OPSCC compared to HPV-negative disease supports the theory for a role for these cytokeratins in the etiopathogenesis of HPV-related OPSCC.

Altered keratinocyte differentiation is an early driver of keratin mutation-based palmoplantar keratoderma.

The type I intermediate filament keratin 16 (KRT16 gene; K16 protein) is constitutively expressed in ectoderm-derived appendages and in palmar/plantar epidermis and is robustly induced when the epidermis experiences chemical, mechanical or environmental stress. Missense mutations at the KRT16 locus can cause pachyonychia congenita (PC, OMIM:167200) or focal non-epidermolytic palmoplantar keratoderma (FNEPPK, OMIM:613000), which each entail painful calluses on palmar and plantar skin. Krt16-null mice develop footpad lesions that mimic PC-associated PPK, providing an opportunity to decipher its pathophysiology, and develop therapies. We report on insight gained from a genome-wide analysis of gene expression in PPK-like lesions of Krt16-null mice. Comparison of this data set with publicly available microarray data of PPK lesions from individuals with PC revealed significant synergies in gene expression profiles. Keratin 9 (Krt9/K9), the most robustly expressed gene in differentiating volar keratinocytes, is markedly downregulated in Krt16-null paw skin, well-ahead of lesion onset, and is paralleled by pleiotropic defects in terminal differentiation. Effective prevention of PPK-like lesions in Krt16-null paw skin (via topical delivery of the Nrf2 inducer sulforaphane) involves the stimulation of Krt9 expression. These findings highlight a role for defective terminal differentiation and loss of Krt9/K9 expression as additional drivers of PC-associated PPK and highlight restoration of KRT9 expression as a worthy target for therapy. Further, we report on the novel observation that keratin 16 can localize to the nucleus of epithelial cells, implying a potential nuclear function that may be relevant to PC and FNEPPK.

Drug-induced keratin 9 interaction with Hsp70 in bladder cancer cells.

A pull-down experiment (co-immunoprecipitation) was performed on a T24 human bladder cancer cell lysate treated with the Hsp inhibitor VER155008 using an Hsp70 antibody attached to Dynabeads. Keratin 9, a cytoskeleton intermediate filament protein, was identified by LC MS/MS analysis. This novel finding was confirmed by Western blotting, RT-PCR, and immunocytochemistry. Other members of the keratin family of proteins have been shown to be involved in cancer progression, most recently identified to be associated with cell invasion and metastasis. The specific role of keratin 9 expression in these cells is yet to be determined.

To Control Site-Specific Skin Gene Expression, Autocrine Mimics Paracrine Canonical Wnt Signaling and Is Activated Ectopically in Skin Disease.

Despite similar components, the heterogeneity of skin characteristics across the human body is enormous. It is classically believed that site-specific fibroblasts in the dermis control postnatal skin identity by modulating the behavior of the surface-overlying keratinocytes in the epidermis. To begin testing this hypothesis, we characterized the gene expression differences between volar (ventral; palmoplantar) and nonvolar (dorsal) human skin. We show that KERATIN 9 (KRT9) is the most uniquely enriched transcript in volar skin, consistent with its etiology in genetic diseases of the palms and soles. In addition, ectopic KRT9 expression is selectively activated by volar fibroblasts. However, KRT9 expression occurs in the absence of all fibroblasts, although not to the maximal levels induced by fibroblasts. Through gain-of-function and loss-of-function experiments, we demonstrate that the mechanism is through overlapping paracrine or autocrine canonical WNT-ß-catenin signaling in each respective context. Finally, as an in vivo example of ectopic expression of KRT9 independent of volar fibroblasts, we demonstrate that in the human skin disease lichen simplex chronicus, WNT5a and KRT9 are robustly activated outside of volar sites. These results highlight the complexities of site-specific gene expression and its disruption in skin disease.

Rationalising the role of Keratin 9 as a biomarker for Alzheimer's disease.

Keratin 9 was recently identified as an important component of a biomarker panel which demonstrated a high diagnostic accuracy (87%) for Alzheimer's disease (AD). Understanding how a protein which is predominantly expressed in palmoplantar epidermis is implicated in AD may shed new light on the mechanisms underlying the disease. Here we use immunoassays to examine blood plasma expression patterns of Keratin 9 and its relationship to other AD-associated proteins. We correlate this with the use of an in silico analysis tool VisANT to elucidate possible pathways through which the involvement of Keratin 9 may take place. We identify possible links with Dickkopf-1, a negative regulator of the wnt pathway, and propose that the abnormal expression of Keratin 9 in AD blood and cerebrospinal fluid may be a result of blood brain barrier dysregulation and disruption of the ubiquitin proteasome system. Our findings suggest that dysregulated Keratin 9 expression is a consequence of AD pathology but, as it interacts with a broad range of proteins, it may have other, as yet uncharacterized, downstream effects which could contribute to AD onset and progression.

Keratins K2 and K10 are essential for the epidermal integrity of plantar skin.

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.

The influence of stromal cells on the pigmentation of tissue-engineered dermo-epidermal skin grafts.

It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application.

Expression analysis of KAP9.2 and Hoxc13 genes during different cashmere growth stages by qRT-PCR method.

Keratin-associated protein 9.2 (KAP9.2) and Homeobox C13 (Hoxc13) genes were chosen to study because of their biological functions involving hair formation. KAP9.2 gene belongs to the ultra high sulfur KAPs, which is important for hair formation and may have association with cashmere. Hoxc13 takes part in the formation of cashmere keratin and maintaining the normal structure of follicle. It has been reported that Hoxc13 gene exists binding site of KP and KAP genes at its promoter regions in mouse. So the expression of KAP9.2 and Hoxc13 genes was detected at anagen stage vs telogen stage by qRT-PCR. The data showed that KAP9.2 and Hoxc13 gene had similar expression trend at different stages, which indicated that there was interaction between them. KAP9.2 and Hoxc13 gene had lower expression level in anagen than that of in telogen of cashmere growth. In anagen, KAP9.2 and Hoxc13 expressed lower in high cashmere yield individuals than that of in low cashmere yield ones. In telogen, the result was reverse. The study would provide the evidence of involvement of KAP9.2 and Hoxc13 in hair periodic growth.

[Differential protein expressions in breast cancer between drug sensitive tissues and drug resistant tissues].

OBJECTIVE: To investigate the differential expression of the sensitive and resistant relative proteins in human breast cancer tissue. METHODS: A drug sensitive group and a drug resistant group for chemotherapy in patients with breast cancer were selected through neoadjuvant. The differential protein expression in 2 groups was detected by proteomics techniques, and parts of differential proteins were identified by Western blot. RESULTS: There were 13 differential proteins in the 2 groups, in which the expression of 3 proteins was up-regulated and 10 down-regulated. Seven proteins were identified by Western blot. The expression of keratin type I cytoskeletal 19 (KIC19), thymidine phosphorylase (TYPH) was upregulated, and the expression of heat shock protein 27 (HSP27), keratin type I cytoskeletal 9 (KIC9), collagen alpha-2(VI) (CO6A2), vimentin (VIME), and actin cytoplasmic 1 (ACTB) was down-regulated in the drug resistant group. There was significant difference between the 2 groups (P<0.01). CONCLUSION: The expression of KIC19 and TYPH may be correlated with drug resistance in patients with breast cancer, and HSP27, KIC9, CO6A2, VIME, and ACTB may be correlated with drug sensitivity.

Proteomic analysis of the cerebrospinal fluid in multiple sclerosis and neuromyelitis optica patients.

The present study aimed to compare the 2-dimensional (2D) electrophoresis pattern of the cerebrospinal fluid (CSF) in multiple sclerosis (MS), neuromyelitis optica (NMO) and control individuals, to identify the proteins with differential expression and to examine their significance. CSF samples from the three groups were collected and total protein was isolated and quantified using the Bradford method. 2D electrophoresis of the samples was conducted using equal amounts of CSF. In the CSF 2D gel electrophoresis map, 118 points were obtained from the MS group, 155 points from the NMO group and 350 points from the normal control group. Non-matching proteins appeared in 14 spots in the MS group and in 45 spots in the NMO group, and were also expressed in the 2D electrophoresis pattern of the normal control group. Four differential proteins were identified through the HD-MS/MS and MASCOT network search. Pre-albumin (PA) was found only in the CSF 2D gel electrophoresis map of the MS patients. Keratin 1 was expressed in the normal control group, but not in the MS group. The difference in the expression of keratin 9 in the MS group was twice that in the normal control group. The expression of keratin 1, keratin 9 and transferrin in the NMO group was twice that in the normal control group. The expression of PA was found only in the CSF 2D gel electrophoresis map of the MS patients, and not in the NMO group. Keratin 1 was expressed in the NMO group, but not in the MS group. The expression of a variety of proteins in the 2D electrophoresis pattern of CSF was significantly different in the MS, NMO and control groups. PA, keratin 1, transferrin and keratin 9 are identified as significantly differentially expressed proteins.

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/beta-catenin signaling in keratinocytes.

The epidermis (containing primarily keratinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia-associated transcription factor (MITF) and beta-catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and alpha-Kelch-like ECT2 interacting protein (alphaKLEIP) but down-regulated the expression of beta-catenin, glycogen synthase kinase 3beta, protein kinase C, and proteinase-activated receptor-2 (PAR-2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.

Myeloperoxidase interacts with endothelial cell-surface cytokeratin 1 and modulates bradykinin production by the plasma Kallikrein-Kinin system.

During an inflammatory state, functional myeloperoxidase (MPO) is released into the vessel as a result of intravascular neutrophil degradation. One mechanism of resulting cellular injury involves endothelial internalization of MPO, which causes oxidative damage and impairs endothelial signaling. We report the discovery of a protein that facilitates MPO internalization, cytokeratin 1 (CK1), identified using affinity chromatography and mass spectrometry. CK1 interacts with MPO in vitro, even in the presence of 100% human plasma, thus substantiating biological relevance. Immunofluorescent microscopy confirmed that MPO added to endothelial cells can co-localize with endogenously expressed CK1. CK1 acts as a scaffolding protein for the assembly of the vasoregulatory plasma kallikrein-kinin system; thus we explored whether MPO and high molecular weight kininogen (HK) reside on CK1 together or whether they compete for binding. The data support cooperative binding of MPO and HK on cells such that MPO masked the plasma kallikrein cleavage site on HK, and MPO-generated oxidants caused inactivation of both HK and kallikrein. Collectively, interactions between MPO and the components of the plasma kallikrein-kinin system resulted in decreased bradykinin production. This study identifies CK1 as a facilitator of MPO-mediated vascular responses and thus provides a new paradigm by which MPO affects vasoregulatory systems.

Immunocyto- and histochemical profiling of nucleostemin expression: marker of epidermal stem cells?

BACKGROUND AND OBJECTIVE: Because the nucleolar protein nucleostemin is present in bone marrow and neuronal stem cells and malignancies originating thereof we monitored its expression in frozen sections from normal human epidermis, basal cell carcinomas, cultured keratinocytes and cells of the squamous carcinoma line FaDu. In addition, probing the value of this protein as a marker of epidermal stem cells was an aim of this study. MATERIALS AND METHODS: To further characterize cell features we added analysis of expression of keratins 10 or 19 as markers of terminal differentiation and Ki67 as marker of proliferating cells as well as three adhesion/growth-regulatory galectins. RESULTS: Immunohistochemical monitoring revealed expression of nucleostemin in cells of both Ki67-positive and -negative nuclei regardless of the K10-expression status. Cultured keratinocytes were positive, when they were prepared from hair follicles and cultured in the presence of feeder cells. A small population of these nucleostemin-positive cells also expressed galectin-1 but not galectins-3 and -9 in their nucleoli. Part of these cells also expressed keratin 19. FaDu cells were strongly positive, illustrating expression in malignant cells which require no feeder layer. Of note, the number of galectin-1-positive nucleoli was reduced in the course of culture. CONCLUSION: Nucleostemin positivity cannot be considered as marker for stem cells in skin sections. In cultured cells, nucleostemin is expressed in a distinct population of the epidermal cells from hair follicle kept in the presence of a feeder layer, intimating an association of nucleostemin expression with this type of epithelio-mesenchymal interaction which is not essential during propagation of malignant cells.