Identification of trichoplein, a novel keratin filament-binding protein.
(1/198)
Keratins 8 and 18 (K8/18) are major components of the intermediate filaments (IFs) of simple epithelia. We report here the identification of a novel protein termed trichoplein. This protein shows a low degree of sequence similarity to trichohyalin, plectin and myosin heavy chain, and is a K8/18-binding protein. Among interactions between trichoplein and various IF proteins that we tested using two-hybrid methods, trichoplein interacted significantly with K16 and K18, and to some extent with K5, K6a, K8 and K14. In in vitro co-sedimentation assays, trichoplein directly binds to K8/18, but not with vimentin, desmin, actin filaments or microtubules. An antibody raised against trichoplein specifically recognized a polypeptide with a relative molecular mass of 61 kDa in cell lysates. Trichoplein was immunoprecipitated using this antibody in a complex with K8/18 and immunostaining revealed that trichoplein colocalized with K8/18 filaments in HeLa cells. In polarized Caco-2 cells, trichoplein colocalized not only with K8/18 filaments in the apical region but also with desmoplakin, a constituent of desmosomes. In the absorptive cells of the small intestine, trichoplein colocalized with K8/18 filaments at the apical cortical region, and was also concentrated at desmosomes. Taken together, these results suggest that trichoplein is a keratin-binding protein that may be involved in the organization of the apical network of keratin filaments and desmosomes in simple epithelial cells. (+info)
Use of adenoviral E1A protein to analyze K18 promoter deregulation in colon carcinoma cells discloses a role for CtBP1 and BRCA1.
(2/198)
BACKGROUND: The promoter of the keratin 18 (K18) gene is 5- to 10-fold more active in tumorigenic (T-type) cell clones derived from the SW613-S human colon carcinoma cell line than in non-tumorigenic (NT-type) clones. We have reported previously that the mechanism responsible for this differential activity is acting on the minimal K18 promoter (TATA box and initiation site). This mechanism does not require the binding of a factor to a specific site on the DNA but involves the acetylation of a non-histone substrate. To get further insight into this mechanism, we investigated the effect of the adenovirus E1A protein on the activity of the K18 promoter, both in T and NT cells. RESULTS: Wild type adenovirus E1A protein and C-terminal deletion mutants inhibit the K18 promoter, specifically in T-type cells. The domain responsible for this inhibitory effect is located in the 12-25 region of the viral protein. E1A mutants that have lost this region but retain the PLDLS motif (the C-terminal binding site for CtBP1) stimulate the K18 promoter, specifically in NT cells. The inhibitory or stimulatory effects of the different E1A mutants are not dependent on a particular sequence of the promoter. An E1A N-terminal deletion mutant carrying point mutations in the PLDLS motif cannot stimulate the K18 promoter. CtBP1 interacts with CtIP, which is a known partner of BRCA1, itself a component of the RNA polymerase II holoenzyme. The stimulatory effect of two BRCA1 mutants, specifically in NT cells, implicates a tripartite BRCA1-CtIP-CtBP1 complex in the regulation of the K18 promoter. CONCLUSION: Since we have shown previously that the K18 promoter is stimulated by deacetylase inhibitors, specifically in NT cells, we conclude that the activity of the promoter is repressed in NT cells by a mechanism involving the recruitment, by a BRCA1/CtIP complex, of CtBP1 and associated deacetylases to the preinitiation complex. We propose a model depicting the mechanism responsible for the differential activity of the K18 promoter between T and NT cells of the SW613-S cell line. (+info)
Transfection of keratin 18 gene in human breast cancer cells causes induction of adhesion proteins and dramatic regression of malignancy in vitro and in vivo.
(3/198)
This study shows that high keratin 18 (K18) expression in tumor cells is associated with reduced invasiveness in vitro and lack of tumorigenicity in nude mice. We previously showed that high K18 expression correlated with a good prognosis and that reducing K18 expression increased the aggressiveness of established breast cancer cell lines. To confirm these observations, we transfected the human K18 gene into the human breast cancer cell line MDA-MB-231 and isolated a stable overexpressing clone. The forced K18 expression was associated with a complete loss of the previously strong vimentin expression in the parent cell line, induction of the K18 dimerization partner K8, and up-regulation of adhesion proteins. These changes were accompanied by a dramatic reduction in the aggressiveness of the K18 transfectants in vitro and in vivo. We conclude that forced reexpression of K18 causes at least partial redifferentiation of the tumor cell, followed by a corresponding regression of malignant phenotype. (+info)
Keratin 8 overexpression promotes mouse Mallory body formation.
(4/198)
Keratins 8 and 18 (K8/18) are major constituents of Mallory bodies (MBs), which are hepatocyte cytoplasmic inclusions seen in several liver diseases. K18-null but not K8-null or heterozygous mice form MBs, which indicates that K8 is important for MB formation. Early stages in MB genesis include K8/18 hyperphosphorylation and overexpression. We used transgenic mice that overexpress K8, K18, or K8/18 to test the importance of K8 and/or K18 in MB formation. MBs were induced by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). Livers of young K8 or K8/K18 overexpressors had no histological abnormalities despite increased keratin protein and phosphorylation. In aging mice, only K8-overexpressing livers spontaneously developed small "pre-MB" aggregates. Only K8-overexpressing young mice are highly susceptible to MB formation after short-term DDC feeding. Thus, the K8 to K18 ratio, rather than K8/18 overexpression by itself, plays an essential role in MB formation. K8 overexpression is sufficient to form pre-MB and primes animals to accumulate MBs upon DDC challenge, which may help explain MB formation in human liver diseases. (+info)
Differential sensitivity of mouse epithelial tissues to the polyomavirus middle T oncogene.
(5/198)
To determine how different epithelial cell types respond to the same oncogenic stimulation, we have used a modified human keratin 18 gene to conditionally express the polyomavirus middle T antigen (PyMT) oncogene in simple epithelial tissues of transgenic mice. Activation of PyMT expression by transgenic Cre recombinase in mammary epithelial cells resulted in carcinomas in all bitransgenic females. PyMT expression induced by K18-driven Cre in internal epithelial organs resulted in pancreatic acinar metaplasia and ductal dysplasia with remarkable desmoplastic stromal responses in all 25 bitransgenic mice. Hepatoma formation with altered lipid metabolism and gastric adenocarcinoma occurred in 96 and 54% of these mice, respectively. Elevated PyMT RNA expression also correlated with intraepithelial neoplasia in the prostate. Activated Erk2 was found in mammary tumors, pancreatic tissues, and affected livers. Hes1 RNA, a target of Notch signaling that has been implicated downstream of Ras pathway activation, was elevated in pancreatic and liver lesions. The variety of responses of different epithelia to PyMT demonstrates the importance of the differentiated state in interpreting oncogenic signals. (+info)
Rescue of DeltaF508-CFTR (cystic fibrosis transmembrane conductance regulator) by curcumin: involvement of the keratin 18 network.
(6/198)
The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, DeltaF508, causes retention of DeltaF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl(-) channels in the plasma membrane. DeltaF508-CFTR retains some Cl(-) channel activity so increased expression of DeltaF508-CFTR in the plasma membrane can restore Cl(-) secretion deficiency. Recently, curcumin was shown to rescue DeltaF508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in DeltaF508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional DeltaF508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of DeltaF508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or DeltaF508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in DeltaF508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated DeltaF508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic DeltaF508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments. With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis. (+info)
Focal adhesions are hotspots for keratin filament precursor formation.
(7/198)
Recent studies showed that keratin filament (KF) formation originates primarily from sites close to the actin-rich cell cortex. To further characterize these sites, we performed multicolor fluorescence imaging of living cells and found drastically increased KF assembly in regions of elevated actin turnover, i.e., in lamellipodia. Abundant KF precursors (KFPs) appeared within these areas at the distal tips of actin stress fibers, moving alongside the stress fibers until their integration into the peripheral KF network. The earliest KFPs were detected next to actin-anchoring focal adhesions (FAs) and were only seen after the establishment of FAs in emerging lamellipodia. Tight spatiotemporal coupling of FAs and KFP formation were not restricted to epithelial cells, but also occurred in nonepithelial cells and cells producing mutant keratins. Finally, interference with FA formation by talin short hairpin RNA led to KFP depletion. Collectively, our results support a major regulatory function of FAs for KF assembly, thereby providing the basis for coordinated shaping of the entire cytoskeleton during cell relocation and rearrangement. (+info)
Method validation and preliminary qualification of pharmacodynamic biomarkers employed to evaluate the clinical efficacy of an antisense compound (AEG35156) targeted to the X-linked inhibitor of apoptosis protein XIAP.
(8/198)
Data are presented on pharmacodynamic (PD) method validation and preliminary clinical qualification of three PD biomarker assays. M65 Elisa, which quantitates different forms of circulating cytokeratin 18 (CK18) as putative surrogate markers of both apoptotic and nonapoptotic tumour cell death, was shown to be highly reproducible: calibration curve linearity r2 = 0.996, mean accuracy > 91% and mean precision < 3%, n = 27. Employing recombinant (r) CK18 and caspase cleaved CK18 (CK18 Asp396 neo-epitope) as external standards, kit to kit reproducibly was < 6% (n = 19). rCK18 was stable in plasma for 4 months at -20 degrees C and -80 degrees C, for 4 weeks at 4 degrees C and had a half-life of 2.3 days at 37 degrees C. Cytokeratin 18 Asp396 NE, the M30 Apoptosense Elisa assay antigen, was stable in plasma for 6 months at -20 degrees C and -80 degrees C, for 3 months at 4 degrees C, while its half-life at 37 degrees C was 3.8 days. Within-day variations in endogenous plasma concentrations of the M30 and M65 antigens were assessed in two predose blood samples collected from a cohort of 15 ovarian cancer patients receiving carboplatin chemotherapy and were shown to be no greater than the variability associated with methods themselves. Between-day fluctuations in circulating levels of the M30 and M65 antigens and in XIAP mRNA levels measured in peripheral blood mononuclear cells by quantitative (q) RT-PCR were evaluated in two predose blood samples collected with a 5- to 7-day gap from 23 patients with advanced cancer enrolled in a phase I trial. The mean variation between the two pretreatment values ranged from 13 to 14 to 25%, respectively, for M65, M30 and qRT-PCR. These data suggest that the M30 and M65 Elisa's and qRT-PCR as PD biomarker assays have favourable performance characteristics for further investigation in clinical trials of anticancer agents which induce tumour apoptosis/necrosis or knockdown of the anti-apoptotic protein XIAP. (+info)