Interleukin-6 induces keratin expression in intestinal epithelial cells: potential role of keratin-8 in interleukin-6-induced barrier function alterations.
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Keratin 8 (K8) and keratin-18 (K18) are the major intermediate filament proteins in the intestinal epithelia. The regulation and function of keratin in the intestinal epithelia is largely unknown. In this study we addressed the role and regulation of K8 and K18 expression by interleukin 6 (IL-6). Caco2-BBE cell line and IL-6 null mice were used to study the effect of IL-6 on keratin expression. Keratin expression was studied by Northern blot, Western blot, and confocal microscopy. Paracellular permeability was assessed by apical-to-basal transport of a fluorescein isothiocyanate dextran probe (FD-4). K8 was silenced using the small interfering RNA approach. IL-6 significantly up-regulated mRNA and protein levels of K8 and K18. Confocal microscopy showed a reticular pattern of intracellular keratin localized to the subapical region after IL-6 treatment. IL-6 also induced serine phosphorylation of K8. IL-6 decreased paracellular flux of FD-4 compared with vehicle-treated monolayers. K8 silencing abolished the decrease in paracellular permeability induced by IL-6. Administration of dextran sodium sulfate (DSS) significantly increased intestinal permeability in IL-6-/- mice compared with wild type mice given DSS. Collectively, our data demonstrate that IL-6 regulates the colonic expression of K8 and K18, and K8/K18 mediates barrier protection by IL-6 under conditions where intestinal barrier is compromised. Thus, our data uncover a novel function of these abundant cytoskeletal proteins, which may have implications in intestinal disorders such as inflammatory bowel disease wherein barrier dysfunction underlies the inflammatory response. (+info)
The possible role of cytokeratin 8 in cadmium-induced adaptation and carcinogenesis.
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Chronic exposures to cadmium compounds are carcinogenic. It was hypothesized that the development of resistance to cadmium may drive carcinogenesis. This is achieved by selection of resistant cells in which the apoptotic response is significantly attenuated. The induction of cadmium resistance in rat lung epithelial cells (LEC) was used to explore the mechanisms of cadmium-induced adaptation and carcinogenesis. Our previous results showed that LECs developed resistance to apoptosis during cadmium adaptation possibly due to perturbation of the c-Jun NH(2)-terminal kinase pathway. Here, we further study these cells by comparative proteomics. Interestingly, we showed that two intermediate filament proteins, cytokeratin 8 (CK8) and cytokeratin 14 (CK14), were increased significantly and stably maintained only in the adapted cells but not in cadmium-treated parental cells. It has been documented that CK8/cytokeratin 18 provided resistance to tumor necrosis factor (TNF)-induced apoptosis and CK14 may function as an inhibitor of TNF-TNF receptor 1 (TNFR1) signaling through an association with TNFR1-associated death domain protein, suggesting that up-regulation of CK8 and CK14 may be responsible for apoptotic resistance. Finally, we showed that small interfering RNA-specific knockdown of CK8 in cadmium-adapted cells attenuated the cadmium resistance, indicating the potential role of CK8 in cadmium resistance. This acquired self-resistance to apoptosis could account for cadmium-induced carcinogenesis, as this promotes neoplastic cell survival as well as subsequent clonal expansion and then progression of tumor development. Thus, increased expression of these cytokeratins represents an adaptive survival mechanism that resists cadmium-induced apoptosis and it is unprecedented that cells respond to long-term cadmium exposure by modulating keratin dynamics. (+info)
Severe defect in thymic development in an insertional mutant mouse model.
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Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function. (+info)
Immunohistochemical studies on cytokeratin 8 and 18 expressions in canine cutaneous adnexus and their tumors.
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The expressions of cytokeratin 8 and 18 (CK8 and CK18) in the normal canine skin (2 cases) and cutaneous adnexal tumors (127 cases) were investigated immunohistochemically. In the normal skin, co-expression of CK8/18 was found in the glandular epithelium of apocrine sweat glands, and single CK8-immunoreactivity was detected occasionally in the external root sheath at the isthmus and suprabulbar regions of the hair follicles. Neoplastic glandular epithelial cells in all apocrine gland tumors (21/21 cases, 100%) had co-expression of CK8/18. In trichoblastomas (27/28 cases, 96%), most neoplastic cells were diffusely positive for CK8, but those were negative for CK18. Single CK8-expression was also observed in basaloid neoplastic cells in several cases of trichoepitheliomas (7/19 cases, 37%) and pilomatricoma (1/7 cases, 14%). In several cases of trichoblastomas (4/28 cases, 14%) and trichoepitheliomas (2/19 cases, 11%), tumor cells forming glandular structures had co-expression of CK8/18. There were no positive reactions for both CK8 and 18 in infundibular keratinizing acanthomas, and sebaceous and hepatoid gland tumors. The present findings indicate that co-expression of CK8/18 is a specific feature of apocrine sweat glands and single CK8-expression represents the natures of external root sheath or pluripotential stem cells. Thus, the combination of CK8- and 18-immunostainings may have the utility to confirm the directions of differentiation in canine cutaneous adnexal tumors providing a reliable hallmark for histopathological diagnoses. (+info)
The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues.
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The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)-based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein. (+info)
Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.
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To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues. (+info)
The sentinel node procedure in colon carcinoma: a multi-centre study in The Netherlands.
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BACKGROUND: Lymph node status is the most important predictive factor in colorectal carcinoma. Recurrences occur in 20% of the patients without lymph node metastases. The sentinel lymph node (SLN) biopsy is a tool to facilitate identification of micrometastatic disease and aberrant lymphatic drainage. We studied the feasibility of in vivo SLN detection in a multi-centre setting and evaluated nodal micro-staging using immunohistochemistry (IHC). MATERIALS AND METHODS: Sub-serosal injection with Patent Blue dye was used in the SLN procedure in 69 patients operated for localized colon cancer in six Dutch hospitals. Each SLN was examined with routine haematoxylin-eosin staining. In tumour-negative SLNs, we performed CK7/8 or 18 IHC. RESULTS: The procedure was successful in 67 of 69 patients (97%). The SLN was negative in 43 patients. In three cases, it was false negative, resulting in a negative predictive value of 93% and an accuracy of 96%. In 24 of 27 patients with lymph node metastases in a successful SLN procedure, the SLN was positive (sensitivity 89%). In 15 patients, the SLN was the only positive node (21%). In nine patients, we only found micrometastases or isolated tumour cells, resulting in 18% upstaging. Aberrant lymphatic drainage was seen in three patients (4%). CONCLUSION: The SLN procedure in localized colon carcinoma is reliable in a multi-centre setting. It is helpful to identify patients who would be classified as stage II with conventional staging (18%) and who might benefit from adjuvant treatment. (+info)
Transdifferentiation of peripheral blood mononuclear cells into epithelial-like cells.
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Bone marrow-derived stem cells have the potential to transdifferentiate into unexpected peripheral cells. We hypothesize that circulating bone marrow-derived stem cells might have the capacity to transdifferentiate into epithelial-like cells and release matrix metalloproteinase-1-modulating factors such as 14-3-3varsigma for dermal fibroblasts. We have characterized a subset of peripheral blood mononuclear cells (PBMCs) that develops an epithelial-like profile. Our findings show that these cells develop epithelial-like morphology and express 14-3-3varsigma and keratin-5, -8 as early as day 7 and day 21, respectively. When compared with control, conditioned media collected from PBMCs in advanced epithelial-like differentiation (cultures on days 28, 35, and 42) increased the matrix metalloproteinase-1 expression in dermal fibroblasts (P +info)