Cytokeratin 14 expression in rat liver cells in culture and localization in vivo

Rat liver epithelial cells (LECs) are non-parenchymal proliferating cells that readily emerge in primary culture and can be established as cell lines, but their in vivo cell(s) of origin is unclear. We reported recently some evidence indicating that the LEC line, T51B, contains two cytokeratins (CKs) equivalent to human CK8 and CK14 respectively. T51B cells also contain vimentin assembled as a network of intermediate filaments distinct from that of the CKs. In the present study, we examined the expression of CK14 gene in various LEC preparations and a Triton-resistant rat skin cytoskeletal fraction, and then assessed its usefulness as an LEC specific marker in the liver. Northern and Western blot analyses with cDNAs and antibodies for CK8, CK14, CK18 and vimentin confirmed that rat hepatocytes express CK8 and CK18 genes only, whereas T51B cells express CK8, CK14 and vimentin genes in the absence of CK18. CK14 was also present in LECs derived as primary from embryonic-day 12 rat liver and secondary cultures from 4-day-old rat liver. Primary cultures of oval cells isolated from 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) treated rat liver (an enriched source of biliary epithelial cells) contained CK14 mRNAs which were slightly shorter than those in LECs. The analyses of CK5 (the usual partner of CK14) gene expression using specific cDNA and antibody clearly demonstrated its absence in LECs. In situ double immunolocalization analyses by laser scanning confocal microscopy showed that CK14 was not present in hepatocytes (HES6+ cells) and was expressed in some biliary epithelial (BDS7+ cells). CK14-positive cells were also found in the Glisson's capsule. However, CK14-positive cells of the portal region were vimentin negative, whereas those of the Glisson's capsule were vimentin positive. Our results suggest that CK14 gene expression is part of the differentiation program of two types of LECs and that this differential CK14 gene expression can be used as a new means to type LECs in culture and in vivo.     

Association of mRNA and eIF-2 alpha with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2 alpha to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2 alpha shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

Association of mRNA and eIF-2α with the cytoskeleton in cells lacking vimentin

The human bladder carcinoma cell lines RT4 and T24 and the human breast adenocarcinoma cell line MCF-7 were found to be negative for vimentin when studied by means of immunofluorescence and immunoblotting. Northern blot analysis revealed that these cells lacked detectable levels of vimentin mRNA with the exception of T24, which contains trace amounts of vimentin mRNA compared to the RNA level in vimentin-containing HeLa cells. CAT assays performed on these cells showed that a hamster vimentin promoter is inactive in RT4 and MCF-7 cells. In the vimentin-lacking cells, the binding of polyribosomes, specific mRNAs, and translation factor eIF-2α to the cytoskeletal fraction was examined. Our results indicate that the presence of a vimentin network is not crucial for the association of the translation machinery with the cytoskeleton. Furthermore, in these vimentin-negative cell lines the immunofluorescence staining pattern of eIF-2α shows a fibro-granular structure that has no resemblance to the cytokeratin or actin cytoskeleton present in these cells.     

Prostaglandin endoperoxide synthase (cyclooxygenase) mRNA and protein production in mouse myoblasts and a differentiation-defective variant.

Northern blot analysis revealed that a differentiation-defective variant (DD-1) of MM14 mouse myoblasts has seven times the prostaglandin endoperoxide synthase mRNA than the parental MM14 myoblasts. There was an even greater increase in the level of prostaglandin endoperoxide synthase protein in the DD-1 cells as compared to that in the MM14 myoblasts. In fact, prostaglandin endoperoxide synthase was not detectable by Western blot analysis of extracts from MM14 myoblasts. Since prostaglandin endoperoxide synthase has been reported to be a gene whose expression is induced transiently, i.e., growth-regulated, upon mitogen stimulation of quiescent cells, the RNA abundance of other growth-regulated genes was examined including: KC, JE, c-myc, 1B6, and vimentin. Northern blot analysis revealed that the mRNA abundance of JE, KC, and c-myc is 12-, 17-, and 2-fold higher, respectively, in growing DD-1 cells than in growing MM14 myoblasts. In contrast, there was little difference in the mRNA abundance of 1B6 and vimentin. These results are consistent with the hypothesis that increases in the levels of expression of prostaglandin endoperoxide synthase and some growth-regulated genes are integral to the expression of the differentiation-defective phenotype and may in fact contribute to this phenotype.     

Down-regulation of cytokeratin 14 mRNA in polyoma virus middle T-transformed rat liver epithelial cells.

We have recently shown that rat liver nonparenchymal epithelial cells, such as T51B cells, selectively express cytokeratin (CK) 14 as a partner of CK8 in their intermediate filaments, and we proposed CK14 as a unique cell lineage marker of the liver epithelial cell population (R. Blouin, M-J. Blouin, I. Royal, A. Grenier, A. Loranger, D. R. Roop, and N. Marceau, Differentiation, submitted for publication, 1992). In the present study, T51B-261A (spontaneously transformed) and T51B-261B (aflatoxin B1-treated) clones and clones derived from T51B cells transfected with SV40 large T (LT) and polyoma virus middle T (MT) were used to investigate CK gene expression in nontransformed and transformed liver epithelial cells. T51B-261A, T51B-261B, MT-T51B, and LT/MT-T51B clones all grew in calcium-deficient medium and formed colonies in soft agar, whereas LT-T51B clones did not grow at all in either one of these assays. T51B-261A and T51B-261B clones formed small, slow growing tumors when injected into newborn syngenic rats, whereas the MT-T51B and LT/MT-T51B clones produced rapidly forming, large tumors. There was no effect of cell transformation on CK expression, except in the clones expressing MT, where the CK intermediate filaments were completely lost. Analyses of [35S]methionine incorporation into the Triton-resistant cytoskeleton and of total proteins confirmed that CKs were absent. In contrast, vimentin intermediate filaments remained unaffected in all of the clones.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spontaneous losses of control of cytokeratin gene expression in transformed, non-epithelial human cells occurring at different levels of regulation

Intermediate filaments (IFs) of the cytokeratin (CK) type are cytoskeletal elements typical for epithelial differentiation. However, in diverse transformed culture lines of non-epithelial origin, rare cells emerge spontaneously, which synthesize, in addition to their vimentin IFs, CKs 8 and 18. We enriched such cells by cloning and studied the level(s) of regulation at which these changes occur. We found that in SV40-transformed fibroblasts the CK 18 gene is constitutively transcribed into translatable mRNA but that the protein is rapidly degraded in the absence of its complex partner, CK 8. In contrast, cells immunocytochemically positive for CK IFs contained both CKs 8 and 18, which apparently stabilized in heterotypic complexes. These findings and related observations of active genes for CKs 8 and/or 18 in several other transformed non-epithelial cell lines indicate that the genes for CKs 18 and, less frequently, 8 can be active in diverse different non-epithelial cell lines; synthesis of type I and type II CK pair partners can be uncoupled; control of CK IF formation can take place at different levels. We suggest that the intrinsic instability of the inactive state of these genes is responsible for the occurrence of CKs 8 and 18 in certain non-epithelial tissues and tumors, a caveat in tumor diagnosis.     

Heat-resistant variants of the Chinese hamster ovary cell: alteration of cellular structure and expression of vimentin.

Three heat-resistant mutant cell lines (78-1, 78-2, 78-3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat-resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two-dimensional gel electrophoresis and Western blot showed a 2.3-2.9-fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin-containing intermediate filaments are correlated with the heat-resistant phenotypes.     

Immuno- and lectin histochemistry of epithelial subtypes and their changes in a radiation-induced lung fibrosis model of the mini pig

Cell types of lung epithelia of mini pigs have been studied using a panel of monoclonal and polyclonal antibodies against cytokeratins (CKs) and vimentin and three lectins before and after radiation-induced fibrosis. In normal tissues, CK18 specific antibodies reacted above all with type II alveolar epithelial cells, while CK7 and pan CK-specific antibodies stained the whole alveolar epithelium. In bronchial epithelial cells, CKs 7, 8, 18 and focally CKs 4 and 13 as well as vimentin were found. Cell specificity of the CK pattern was confirmed by double label immunofluorescence using type II cell-specific Maclura pomifera (MPA) lectin, type I cell specific Lycopersicon esculentum (LEA) lectin and capillary endothelium-binding Dolichos biflorus (DBA) lectin. In experimental pulmonary fibrosis, enhanced coexpression of CK and vimentin was observed in bronchial epithelium. Subtypes of alveolar epithelial cells were no longer easily distinguishable. CK18 was found to be expressed in the entire alveolar epithelium. The gradual loss of the normal alveolar epithelial marker, as seen by the binding of MPA to type I-like cells, of LEA to type II-like cells and the partial loss of MPA-binding to type II cells, was paralleled by the appearance of CK4, typical for squamous epithelia, and the occurrence of DBA-binding in epithelial cells. Implications of these results for general concepts of intermediate filament protein expression and lectin binding in the fibrotic process are discussed.     

Expression of cytokeratin-mRNAs in squamous-cell carcinoma and balloon-cell formation of human oesophageal epithelium

Summary Using digoxigenin-labelled cRNA probes, relationships between morphological characteristics and in situ hybridization for cytokeratin (CK)-mRNAs were analysed in cases of squamous-cell carcinoma of variable differentiation and in balloon-cell formation within the oesophageal mucosa. The present results were correlated to our previous findings on normal oesophageal epithelium. Our results from in situ hybridization study on oesophageal squamous-cell carcinoma provide strong evidence that changes in CK expression occur with differences in malignant potential. Cells of poorly differentiated carcinoma lose an ability to produce CK-mRNAs characteristic of their normal progenitor cells. Moderately differentiated and, still more pronounced, well differentiated carcinoma cells retain an ability to produce CKs characteristic of their tissue of origin (CK 6, CK 14, CK 15 and CK 19). Furthermore, well differentiated carcinoma cells may also gain an ability to synthesize new types of CKs that are not characteristic of the normal oesophageal epithelium (CK 8 and CK 18 characteristic of most simple epithelia, and CK 10 characteristic of keratinizing epithelia). Moreover, some oesophageal CK-genes are expressed in an obviously higher amount (CK 6, CK 14, and CK 19), but the expression of genes coding for the oesophageal differentiation-related CKs (CK 4 and CK 13) is obviously decreased or apparently lost. At the interface zone, observed in sections of well differentiated carcinomas, CK 8 and CK 18 mRNA were expressed in intermediate cell layers, and the centrally located cell layers were found positive for CK 10 mRNA. These findings largely extend the existing results from immunoblotting and immunohistochemical studies. The reduced or non-detectable expression of oesophageal differentiation-related CK-mRNAs (CK 4 and CK 13) on the appearance of balloon cells, suggests molecular changes that may be a marker for pathological progression. In addition, the abundant expression of CK 6 and CK 14 mRNA within areas of balloon-cell formation showing basal hyperplasia, and the higher expression of CK 19 in comparison with normal epithelium, points rather to de-differentiation than to normal vertical differentiation of the oesophageal epithelium. Whether CK-mRNAs can be used as biomarkers for evaluation of oesophageal pathologies remains to be further elucidated.     

Patterns of cytokeratin and vimentin expression in the human eye

We studied the expression of the various cytokeratin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaic-like staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.     

Sequence and expression of testis-expressed gene 14 (Tex14): a gene encoding a protein kinase preferentially expressed during spermatogenesis

To discover germ cell-specific genes, we used in silico subtraction and identified testis expressed gene 14 (Tex14). Mouse Tex14 contains an open reading frame encoding a 1450-amino-acid protein, which shares 64% amino acid identity with the predicted human TEX14 protein. The predicted TEX14 amino acid sequence consists of three ankyrin repeats, a protein kinase domain, and a leucine zipper dimerization motif. Northern blot analysis and in situ hybridization show that Tex14 mRNA is expressed specifically in the testis, with highest levels observed in pachytene, diplotene, and meiotically dividing spermatocytes. Two 5' splice variants of mouse Tex14 were discovered by sequencing 5'-RACE polymerase chain reaction products. TEX14 is predicted to be localized to the nucleus, suggesting that it may play a key role in regulating gene expression or modulating nuclear events during mammalian spermatogenesis.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Patterns of cytokeratin and vimentin expression in the human eye

Summary We studied the expression of the various cytokeralin (CK) polypeptides and vimentin in tissues of the human eye by applying immunocytochemical procedures using a panel of monoclonal antibodies as well as by performing biochemical analyses of microdissected tissues. Adult corneal epithelium was found to contain significant amounts of the cornea-specific CKs nos. 3 and 12 as well as CK no. 5, and several additional minor CK components. Among these last CKs, no. 19 was found to exhibit an irregular mosaiclike staining pattern in the peripheral zone of the corneal epithelium, while having a predominantly basal distribution in the limbal epithelium. Both the fetal corneal epithelium and the conjunctival epithelium were uniformly positive for CK no. 19. In the ciliary epithelium, co-expression of CKs nos. 8 and 18 and vimentin was detected, whereas in the retinal pigment epithelium, CKs nos. 8 and 18 were dominant. The present data illustrate the remarkable diversity and complexity of CK-polypeptide expression in the human eye, whose significance with respect to histogenetic and functional aspects is, as yet, only partially clear. The unusual distribution of CK no. 19 in different zones of the corneal epithelium may be related to the specific topography of corneal stem cells. The occurrence of the expression of simple-epithelium CKs in the ciliary and pigment epithelium demonstrates that, despite their neuroectodermal derivation, these are true epithelia.Supported by a grant from the Deutsche Forschungsgemeinschaft (Mo 345-3).