Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line RICHARDBLOUIN Centre de recherche en cancPrologie de I'Universite Laval, L 'HGteI-Dieude Quebec, Quebec (Quebec), Canada G l R 2 56
SABINE H. H. SWIERENGA Drugs Directorate, Health and Welfare Canada, Ottawa, Canada KIA OL2
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NORMANDMARCEAU Centre de recherche en cancerologie de IYUniversitPLaval, L'H6tel-Dieu de Quebec, 11 c6te du Palais, Quebec (Quebec), Canada G l R 2 56 Received June 24, 1991 BLOUIN,R., SWIERENGA, S. H. H., and ~ ~ A R C E A N.U1992. , Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line. Biochem. Cell Biol. 70: 1-9. T51B, a cell line of the rat liver nonparenchymal cell compartment, contains a cytokeratin (CK) pair composed of CK8, a CK typical of simple epithelium, and CK14, a CK usually present in proliferative stratified epithelium. T5lB-Ni, a subclone selected by prolonged exposure of the parental clone to nickel subsulfide contains CK8 and CK18 (its usual partner in simple epithelium), as well as CK14, at a lower level. The two clones have comparable levels of vimentin. Northern blot analyses of cytoplasmic mRNA demonstrated that the differences in the steady state mRNA levels of each CK paralleled those observed at the protein level, thus showing that the regulatory events occurred prior to translation. The most prominent difference was a 30-fold higher level of CK18 mRNA in T51B-Ni cells. Run-off assays of isolated nuclei revealed that the level of CK8, CK14, and vimentin was regulated primarily at the transcriptional level. However, the large increase in CK18 mRNA levels in T51B-Ni cells did not result from a corresponding increase in the relative level of CK18 gene transcription nor from a change in cytoplasmic CK18 mRNA stability. Comparative Northern blot analyses of nuclear and cytoplasmic mRNAs further suggested that the control of CK18 gene expression in T5lB cells is post-transcriptionally mediated by nuclear events. Key words: cytokeratins, gene regulation, T5lB cells, intermediate filaments, liver. S. H. H., et MARCEAU, N. 1992. Evidence for post-transcriptional regulation of cytokeratin BLOUIN,R., SWIERENGA, gene expression in a rat liver epithelial cell line. Biochem. Cell Biol. 70 : 1-9. La lignte T51B, dtrivte du compartiment cellulaire non-parenchymateux du foie de rat, contient une paire de cytoktratines (CKs) comprenant la CK8, une CK typique de l'tpithtlium simple, et la CK14, une CK habituellement prtsente dans l'tpithtlium stratifit en proliftration. T51B-Ni, un sous-clone stlectionnt par un traitement prolong6 du clone parental avec du subsulfure de nickel, contient la CK8 et la CK18 (son partenaire habitue1 dans l'tpithtlium simple) ainsi que la CK14, mais B un niveau plus bas. Les niveaux de vimentine sont comparables. Ces changements de la composition en CKs se reflktent dans le niveau d'ARNm correspondant 21 chacune d'elles. Des essais de transcription nucltaire rtalists A partir de noyaux isolts de cellules T51B et T51B-Ni rhtlent que l'expression des gtnes de la CK8, de la CK14 et de la vimentine est principalement contralte au niveau transcriptionnel. La situation difftre pour le gtne de la CK18 puisque I'augmentation importante du niveau d'ARNm observte dans le sous-clone, T51B-Ni, ne peut &tre associte a des changements comparables de l'activitt transcriptionnelle ni A des variations dans la stabilitt des transcrits. L'examen comparatif du contenu en ARN nuclbire et cytoplasmiquedes cellules T5lB et T5 1B-Ni suggtre que l'expression du gtne de la CK18 est contralte de f a ~ o npost-transcriptionnelle par des tvtnements se dtroulant dans le noyau. Mots cles : cytoktratines, rtgulation gtnique, cellules TSlB, filaments intermtdiaires, foie.
Introduction Intermediate filaments (IFs) constitute a complex family of polypeptides present in a tissue, differentiation, and developmental specificfashion in most eukaryotic cells (Moll et al. 1982). Cytokeratins (CKs) are unique in that they constitute a family of at least 20 polypeptides present in various types of epithelia (simple, stratified, and pseudostratified) (Moll et al. 1982, 1990). They can be subdivided into two distinct classes, type I (CK9 to CK20) and type I1 (CKl to CK8), which differ in terms of molecular weight, isoelectric point, and gene sequence (Sun et al. 1985; Fuchs ABBREVIATIONS: CK(s), cytokeratin(s); CX, cycloheximide; IF(s), intermediate filament(s); LEC, liver epithelial cells; NM-IFs, nuclear matrix-intermediate filaments; PBS, phosphate-buffered saline. ' ~ u t h o rto whom correspondence should be addressed. Printed in Canada / Imprim6 au Canada
et al. 1987). At least one member of each type is necessary for IF formation (Hatzfeld and Franke 1985; Eichner et al. 1986; Steinert and Roop 1988). Type I and type I1 CKs are usually present as specific pairs, and distinct pairs are present in different cell lineages and differentiation states (Quinlan et al. 1985). While there is some indication that the regulation of CK gene expression occurs primarily at the transcriptional level in stratified epithelial cells (Jorcano et al. 1984; Fuchs et al. 1987), very little is known about the regulatory mechanisms responsible for the appearance of the different CK pairs in simple epithelial cells. Among the CKs normally present in simple (e.g., liver) and stratified (e.g., epidermis) epithelial cells are the CK8-CK18 and CK5-CK14 pairs, respectively (Moll et al. 1982; Nelson and Sun 1983; Quinlan et al. 1984). However, recent work from our laboratory has demonstrated that while rat hepatocytes indeed contain only CK8 and CK18,
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basic
PI
acidic
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FIG. 1. Analysis of CK composition of T5 1B and T5 1B-Ni cells. Subconfluent T5 1B (a) and T5 1B-Ni (b) cells were labeled overnight with [35~]methionine, and the Triton X-100 insoluble fractions, which were enriched in intermediate filaments, were analyzed by twodimensional gel electrophoresis. Each gel was loaded with 100 000 cpm. Spots corresponding to actin (Ac), vimentin (V), and CK8, CK14, and CK18 are identified.
other liver cell types known as liver epithelial cells (LECs, Marceau et a/. 1986), which readily emerge in enriched preparations of hepatocytes in culture, contain CK14 without CK5 (R. Blouin, M.J. Blouin, I. Royal et a/., in preparation). LECs can be established as cell lines (Grisham 1980; Marceau et a/. 1986), and one of them is T51B (Swierenga 1985), a LEC clone that exhibits the additional feature of containing CK8 and CK14 in the almost complete absence of CK18 (Marceau et a/. 1986). But we have recently selected, as a result of nickel exposure, a subclone designated as T5lB-Ni which contains CK8, CK18, and a decreased amount of CK14 (Swierenga et a/. 1989). In the present study these two T5lB clones were used to determine the level(s) at which CK and vimentin gene expression can be regulated in LECs. The results show that while the expression of CK8, CK14, and vimentin genes is regulated mainly at the transcriptional level, that of CK18 seems to be regulated by a post-transcriptional mechanism that involves nuclear RNA processing events.
previously (Fey et al. 1984) and analyzed by two-dimensional gel electrophoresis (O'Farrell 1975).
In vitro transcription assay T5 1B and T5 1B-Ni cells were rinsed with phosphate-buffered saline (PBS), removed from plates by scraping in the same buffer, pelleted (500 g, 5 min), resuspended at lo7 cells/mL in lysis buffer (10 mM Tris-HC1 (pH 7.4). 10 mM NaCl, 5 mM MgCl,, 1 mM dithiothreitol, 100 U RNasin/mL) containing 0.5% Nonidet P-40. The cells were incubated on ice for 5 min. Nuclei were then pelleted by centrifugation (1000 g, 4 min) and kept on ice. Postnuclear supernatants were saved for preparation of cytoplasmic RNA by a guanidine isothiocyanate technique (Chomczynski and Sacchi 1987). Nuclear pellets were rinsed gently in ice-cold lysis buffer alone, repelleted and suspended at a concentration of 5 x 10' nuclei/mL in nuclear freezing buffer (50 mM Tris-HC1 (pH 8.0), 5 mM MgCI,, 0.1 mM EDTA, 40% glycerol, 1 mM dithiothreitol, 100 U RNasin/mL). Nuclei were frozen and stored at - 70°C until used. The transcription assay was carried out with 5 x lo7 nuclei. One hundred microlitres of 2 x transcription buffer (10 mM Tris-HC1 (pH 8.0), 5 mM MgCl,, 300 mM KCI, 1 mM ATP, 1 mM CTP, 1 mM GTP, 1 U RNasin/pL) containing 250 pCi of [a-'*P]UTP (3000 Ci/mmol) was added to each Methods sample, and transcription was allowed to proceed for 30 min at Cell culture 30°C. The nuclei were then pelleted and resuspended in 0.5 M Normal T51B cells (Swierenga 1985) and nickel subsulfide treated NaCI, 10 mM Tris-HCI (pH 7.4), 50 mM MgCI,, 100 U RQ1 TSlB cells (referred to as T5lB-Ni in this study and clone 4 in RNase-free DNase/mL (Promega), 0.4 mg proteinase K/mL, Swierenga et al. 1989) were grown in minimal essential medium 10 mM EDTA, 0.5% SDS, and 10 mM vanadyl ribonucleoside (alpha-modification) containing 10% (v/v) heat-inactivated fetal complex for 30 min at 37°C. Labeled RNA was isolated by phenol bovine serum, 100 U penicillin/mL, and 100 pg streptomycin/mL. extraction and ethanol precipitation. The resulting RNA pellets were then dissolved in 10 mM Tris-HC1 (pH 7.5), 0.3 M NaCI, C K extraction and analysis T51B and TSlB-Ni cells were radiolabeled with [35~]methionine 1 mM EDTA, and 1% SDS and passed through Bio-Spin 30 columns (Bio-Rad Laboratories) to eliminate unincorporated (10 pCi/mL; 1 Ci = 37 GBq) for 18 h and the nuclear matrixnucleotides. Usually, 10 x lo6 to 20 x lo6 cpm were hybridized, intermediate filaments (NM-IFs) were prepared as described
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FIG. 2. Double immunofluorescent microscopy analysis of CK14 and CK8 distribution in T5lB and T51B-Ni cells. Subconfluent T5lB (A and B) and T51B-Ni (C and D) cells grown on glass coverslips were fixed and stained for CK14 (A and C) and CK8 (B and D) using highly specific polyclonal and monoclonal antibodies. in 1 mL, to filter-bound linear plasmid DNAs. DNAs were immobilized on nitrocellulose using a BRL dot blot apparatus after denaturation in 0.2 M NH40H - 2 M NaCl at 100°C for 5 min and quick-cooling on ice. Plasmid DNAs were dissolved directly into 0.2 M NH40H - 2 M NaCl to bring the DNA concentration to 50 pg/mL, and 100 pL (5 pg) of each plasmid was applied to each sample well. Nitrocellulose membranes were baked at 80°C for 2 h. Prehybridization was done at 42°C for 16 h in 50% formamide, 6 x SSC (1 x SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)), 0.5% SDS, 10 mM Na2HP04(pH 6.8), 1 mM EDTA, 0.1 mM UTP, 5 x Denhardt's, 100 pg herring sperm DNA/mL, and 1 pg poly(A)/mL. Hybridization reactions were
done in the same buffer with 3Z~-labeled RNA for 72 h at 42"C, in triplicate. After hybridization, filters were washed twice for 15 min in 2 x SSC, 0.1% SDS at room temperature, once for 15 min in 0.2 x SSC, 0.1% SDS at room temperature, and twice for 15 min in 0.2 x SSC, 0.1% SDS at 55°C. Filters were then exposed to Kodak X-Omat AR film at - 70°C with intensifying screens. Plasmids used contained specific cDNAs: CK18, mouse CK18 full-length cDNA (Singer et al. 1986); CK8, mouse CK8 cDNA (Bralet and Jacob 1982); CK14, mouse CK14 cDNA (Roop et al. 1989); vimentin, human vimentin cDNA (Ferrari et al. 1986); and actin, chicken P-actin cDNA (Kost et al. 1983). pBR322 was used as a control.
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pg DNA by random priming (Feinberg and Vogelstein 1983) CK18; CK8; CK14; vimentin; and actin. A mouse c-myc gene fragment (Land et al. 1983) was also used as a control in the mRNA stability assay. Conditions for washing and autoradiography were the same as for the in vitro transcription assay.
Zmmunofluorescence microscopy Cells grown on glass coverslips were washed three times at room temperature with PBS, treated in cold methanol (100%) for 10 min at - 20°C. and then rinsed again with phosphate-buffered saline. Indirect immunofluorescence microscopy was performed as described previously (Germain et al. 1988), using a mouse monoclonal antibody to rat CK8 (Marceau et al. 1986), and a rabbit polyclonal antibody to mouse CK14 (Smith et al. 1990). Fluorescein-conjugated goat anti-mouse Ig (Rego Lab, Qutbec), and Texas-Red-conjugated donkey anti-rabbit Ig were used as second antibodies (Jackson Immunoresearch Laboratory, West Grove, PA). Cells were observed with a Leitz microscope equipped with epifluorescence illumination and a special emission filter (535 DF45, Omega Optical, Inc., Brathleboro, VT, U.S.A.).
Results CK composition T51B and T51B-Ni cells were labeled with [35~]methionine, Triton-resistant cytoskeletons were prepared, and the IF proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (PAGE). The parental T5 1B contained comparable amounts of CK8 and CK14, very little CK18, and a high level of vimentin (Fig. la). T51B-Ni cells contained the same pattern of IF proteins but had much more CK18 and slightly less CK14 (Fig. 16). Double indirect immunofluorescence analyses of T51B cells using anti-CK8 and anti-CK14 antibodies showed that both CKs formed typical IFs in all cells (Fig. 2). The same analyses performed in T5 1B-Ni cells revealed that while all cells contained CK8, a small proportion of them were CK14 negative.
FIG. 3. Analysis of CK mRNA levels in T51B and T51B-Ni cells. Cytoplasmic RNA (20 pg) isolated from TSlB (lane 1) and T5 1B-Ni (lane 2) cells was resolved by formaldehyde - agarose gel electrophoresis, transferred to a nylon membrane by blotting, and hybridized to "P-labeled CK18, CK14, CK8, vimentin, and P-actin cDNAs.
Cytoplasmic and nuclear RNA analysis Cells were lysed and nuclei isolated as described for the in vitro transcription assay. After centrifugation in lysis buffer containing Nonidet P-40, the cytosol was saved and centrifuged once more; the nuclei were washed once more with lysis buffer alone and centrifuged. Three volumes of 4 M guanidine isothiocyanate solution were added to the cytosol; the nuclear pellet was resuspended directly in the same solution and the RNA of each fraction was isolated as described by Chomczynski and Sacchi (1987). The RNA was fractionated by electrophoresis in a 1.5% agarose gel containing formaldehyde, and transferred to a nylon filter. Prehybridization and hybridization were performed in 50% formamide, 0.5 M Na2HP0, (pH 7.2), 1 mM EDTA, 1% BSA, and 5% SDS at 42OC for 4 and 18 h, respectively. Filters were probed with specific cDNAs labeled to 1 x 10' to 10 X 10' cpm/
CK mRNA levels As a first step to determine the level at which the changes in CK composition are determined, the steady-state levels of CK8, CK14, and CK18 mRNAs were measured by Northern blotting (using appropriate cDNA probes) and compared with those of vimentin and actin mRNAs (Fig. 3). The main finding was a 30-fold greater level of CK18 mRNA in T5lBNi cells as compared with T51B cells (Figs. 3a and 5). Moreover, T51B-Ni cells showed a slightly reduced CK14 mRNA level (Fig. 36) but a higher level CK8 mRNA (Figs. 3c and 5). This down-regulation of CK14 mRNA expression in T51B-Ni is probably due to the inhibition of CK14 protein synthesis in a subset of these cells (Fig. 2). Finally, vimentin and actin mRNA levels of the two cell types were similar (Figs. 3d and 3e). The differences in CK composition were thus reflected at the mRNA level, which implies that the mechanism responsible for the observed differences occurred prior to translation. CK gene transcription Since variations in mRNA steady-state levels may be due to transcriptional or post-transcriptional regulatory events, run-off assays were performed on nuclei isolated from T5 lB and T5lB-Ni cells to monitor transcription of CK8, CK14, CK18, vimentin, and actin genes (Fig. 4). Despite the negligible steady-state level of CK18 mRNA in T51B cells, the CK18 gene was transcriptionally active. Furthermore, although there was at least a 30-fold difference in the CK18
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mRNA STEADY-STATE L EVEL
CK18
'
CK14
'
CK8
' V I M E N T I N ACTlN .
Vimentin TRANSCRIPTION LEVEL 1.47
FIG. 4. Nuclear transcription analysis of CK genes in T5lB and T5 1B-Nicells. Nuclei isolated from T5 1B (a) and T5 1B-Ni (b) cells were incubated in the presence of [ a - ' 2 ~ under ] ~ ~conditions ~ that favored elongation of previously initiated RNA transcripts. Equivalent counts of labeled nuclear RNA were hybridized to denatured, nitrocellulose-immobilized plasmid DNAs containing CK18, CK14, CK8, vimentin, and P-actin cDNA sequences. pBR322 was used as a control for nonspecific background hybridization. steady-state mRNA level between T51B and T51B-Ni cells, there was only a 4-fold increase in the transcriptional level of the corresponding gene (Figs. 4 and 5). The relative levels of transcription of the CK8, CK14, and vimentin genes in T5 1B-Ni cells were 5, 1.6, and 1.5-fold higher, respectively, than in T51B cells. By comparing these differences with those of the respective mRNA steady-state levels, the regulation of CK8, CK14, and vimentin gene expression seems to be mainly at the transcriptional level. Thus, the differences in transcription levels alone in T51B and T5 1B-Ni cells cannot account for the much greater CK18 mRNA level measured in T5lB-Ni cells (Fig. 5). CKI8 mRNA stability RNA synthesis was blocked with actinomycin D and the rate of CK18 mRNA degradation in T5 1B and T5 1B-Ni cells was determined in the presence or absence of cycloheximide (CX) (Fig. 6). The same analyses were performed for vimentin, actin, and myc mRNAs (the latter was used as an internal reference). The stability of CK18 mRNA, as well as that of vimentin and actin mRNAs, was the same in T51B and T51B-Ni cells (Figs. 6a, 6c, and 6d).In fact, no degradation could be detected in the 3-h period examined even in
CK18
CK14
CK8
VlMENTlN ACTlN
FIG. 5. Quantitative analysis of the relative mRNA steady-state levels and transcription levels of CK18, CK14, CK8, and vimentin. The data were obtained by densitometric scanning of the autoradiographsfrom the Northern blots shown in Fig. 3, and from the nuclear transcription assay shown in Fig. 4. The values given in the ordinate are optical densities normalized to P-actin mRNA and transcription levels. The data are representative of three separate experiments. the absence of CX in actinomycin D treated cells. As expected, the half-life of c-myc mRNA was relatively short (30 min) and CX greatly prolonged it (Fig. 6b). Thus the differences observed in the steady-state levels of CK18 cytoplasmic mRNA in T5 1B and T5 1B-Ni cells are not determined by their relative rates of decay. Nuclear and cytoplasmic CK RNA levels To follow early steps of transcript processing, total nuclear and cytoplasmic RNA fractions were isolated from T5 1B and T5 1B-Ni cells and examined by Northern blotting (Fig. 7). Although there was a large amount of CK18 RNA present in the nuclei and cytoplasm of T51B-Ni cells, there were no detectable CK18 transcripts in the nuclei or cytoplasm of T51B cells (Fig. 7a). Very little unprocessed
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FIG. 6. Analysis of CK18 mRNA stability in TSlB and TSlB-Ni cells. TSlB (lanes 1-10) and TSlB-Ni (lanes 11-20) cells were treated for 60 min with 10 pg CX/mL. After that time, some of the plates were washed 3 times with Hepes buffer and incubated further with or-MEM containing 10% heat-inactivated FCS, 5 pg actinomycin D/rnL, but without CX. Other plates were washed and incubated under identical conditions, but with medium containing 10 pg CX/mL. Cytoplasmic RNA was prepared at 0, 30, 60, 120, and 180 min after actinomycin D addition. Equal amounts of cytoplasmic RNA 20 pg were fractionated by formaldehyde - agarose gel electrophoresis, transferred to nylon membrane by blotting, and hybridized to P-labeled CK18, c-myc, vimentin, and O-actin cDNAs or gene fragments.
6
CK18 RNA was present in the nuclei from T5 1B and T5lBNi cells, indicating a very rapid processing or degradation of precursor transcripts. Nevertheless, it is clear that in spite of transcription of the CK18 gene, a large pool of CK18 transcripts did not accumulate in the nuclei of T51B cells. The difference in the level of mature CK14 mRNAs in the nucleus of T5 1B and T51B-Ni was reflected in the cytoplasm (Fig. 7b). Similar levels of CK8 RNAs were detected in the nuclei of T51B and T5 1B-Ni cells but not in the cytoplasm, perhaps suggesting a slight difference in processing (Fig. 7c). The analysis of the amount of nuclear and cytoplasmic vimentin RNA revealed no difference in the steady-state vimentin RNA levels between the two cellular compartments of T5lB and Tl5B-Ni cells (Fig. 7d). Among the various explanations for the lack of detectable nuclear CK18 mRNA in T5lB cells are differences that might exist in the degradation or maturation of nascent transcripts. To examine this, we compared the accumulation of nuclear and cytoplasmic CKs RNAs in T5lB and T51B-Ni cells treated with CX for prolonged periods (Figs. 7e-7h). CX treatment resulted in the appearance of precursor and mature CK18 mRNAs, particularly in T5lB-Ni cells. However, CK18 mRNA transcripts were also present in the cytoplasmic fraction of T5 1B cells, but at a lower level than that in T5lB-Ni cells (Fig. 7e). In spite of the fact that little contamination of cytoplasmic RNA with nuclear RNA occurred in T51B and T5 1B-Ni cells, precursors were present
)
at comparable levels in the cytoplasmic fraction of both cell types. This result was in contrast to the observations made for mature CK18 mRNA. CX treatment also increased the steady-state levels of precursor and mature mRNAs for CK14, CK8, and vimentin in both cell types, the most prominent increase being for the CK14 RNA (Figs. 7f-7h).
Discussion The present results demonstrate that in T5lB LEC expressing a CK8-CK14 pair, the expression of CK18, the usual partner of CK8 in simple epithelium, is largely regulated by a post-transcriptional mechanism taking place at the level of nuclear RNA processing. In contrast, the expression of CK8 and CK14 is transcriptionally regulated. The expression of CK18 in T5lB-Ni cells somehow is affected in a stable and heritable manner. Previous studies performed on the mode of expression of several CK genes in stratified epithelial cells indicated that the regulating events occur mainly at the transcriptional level (Jorcano et al. 1984; Tyner and Fuchs 1986; Fuchs et al. 1987; Roop et al. 1987, 1988). The present data on mRNA and transcription levels of CKs and vimentin suggest that the regulation of the IF genes expressed in T51B cells differ widely depending on the gene. For example, it is clear that the regulation of vimentin gene expression takes place only at the transcriptional level. The changes in CK8 mRNA and transcription levels imply that regulation in this case can
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FIG. 7. Analysis of nuclear and cytoplasmic CK RNA content in TSlB and T51B-Ni cells. T51B (lanes 1 and 3) and TSlB-Ni (lanes 2 and 4) cells were cultured without (a-d) or with (e-h) CX (5 pg/mL) for 5 h. After this treatment, nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) RNA was isolated from both cell types. Equal amounts of these RNA fractions (20 pg) were electrophoresed on a formaldehyde - agarose gel, transferred to a nylon membrane by blotting, and hybridized to 32~-labeled CK18 (a and e), CK14 ( b and f), CK8 (c and g), and vimentin (d and h) cDNAs.
occur at both transcriptional and post-transcriptional levels. The CK14 gene, on the other hand, showed transcriptional regulation. The data on the expression of the CK18 gene can be best interpreted as a regulation occurring primarily at the post-transcriptional level. The decay rate of steadystate CK18 mRNA in the presence of actinomycin D suggests that the stability of CK18 mRNA in the cytoplasm cannot account for the much larger differences in mRNA accumulation observed in T51B-Ni cells. These results suggest therefore that post-transcriptional regulation can occur via changes in nuclear mRNA stability and (or) the rate of RNA processing. The change between the two cell types may represent an increase in the efficiency with which CK18 hnRNA is converted to CK18 mRNA. A greater proportion of the poly(A)+ CK18 hnRNA is converted into poly(A)+ CK18 mRNA in T51B-Ni. The analyses of the CK18 nuclear RNA levels support this interpretation. There are data from work performed on other genes suggesting that regulation at the level of nuclear mRNA stability and (or) rate of RNA processing (Leys et al. 1984; Vannice et al. 1984) depends on the presence of a labile protein (Wilkinson and MacLeod 1988). However, since treatment with cycloheximide did not significantly alter the accu-
mulation of mature CK18 transcripts in the nucleus, it seems unlikely that the nuclear processing of CK18 mRNA in T51B cells is due to the action of such a labile protein. T51B, which contains CK8 and CK14, is a nontransformed cell line derived from a culture of adult rat liver cells (San et al. 1979). This line has been used extensively to study the role of c a 2 + in cell growth regulation and to screen for potential carcinogens (Swierenga 1985; Swierenga and McClean 1985). The finding that a prolonged exposure of T51B cells to nickel subsulfide led to the appearance of a subclone (T51B-Ni) that contains CK8 and CK18, but a lesser amount of CK14, is certainly intriguing. While there are data indicating that nickel compounds can influence the interactions between proteins and DNA (Sunderman 1984; Wedrychowski et al. 1986), the actual cellular mechanisms underlying this selective effect are unknown. Much interest has been focused on the regulation of CK gene expression in stratified epithelial cells, particularly as the progenitor cells differentiate (Roop et al. 1988). In contrast, very little has been done on this aspect in simple epithelial cells. While the cell of origin of LECs in vivo is still unsettled, the experimental evidence accumulated so far suggests that they are derived from distinct portions of bile
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ductules (Marceau et al. 1986; R. Blouin, M.J. Blouin, A. Grenier et al., t o be published). T h e finding that LECs contain CK14 without CK5 is in line with recent d a t a obtained in mouse mammary glands, where a few CK14 positive cells are detected within a field of CK14 negative cells (Smith et al. 1990). Although the physiological significance of this selective C K gene expression by a few cells is unclear, the present d a t a show that simple epithelial cells can use distinct molecular mechanisms to regulate the appearance o f different C K pairs.
Acknowledgements We thank Drs. William I. Waithe and Alan Anderson f o r reviewing the manuscript, AndrCe Grenier f o r her expert technical assistance, Michde Gagnon a n d Elisabeth Lemay f o r their secretarial assistance, a n d Pierre Paquin a n d G u y Langlois f o r photographic assistance. Richard Blouin was recipient of a studentship from t h e Medical Research Council of Canada. This work was supported by the Medical Research Council of Canada.
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Chomczynski, P., and Sacchi, N. 1987. Single-stepmethod of RNA isolation by guanidium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159. Eichner, R., Sun, T.-T., and Aebi, U. 1986. The role of keratin subfamilies and keratin pairs in the formation of human epidermal intermediate filaments. J. Cell Biol. 102: 1767-1777. Fausto, N., and Mead, J.E. 1989. Regulation of liver growth: protooncogenes and transforming growth factors. Lab. Invest. 60: 4-13. Feinberg, A.P., and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. Ferrari, S., Battini, R., Kaczmarek, L., et al. 1986. Coding sequence and growth regulation of the human vimentin gene. Mol. Cell. Biol. 6: 3614-3620. Fey, E.G., Wan, K.M., and Penman, S. 1984. Epithelial cytoskeletal framework and nuclear matrix-intermediate fiiament scaffold: three-dimensional organization and protein composition. J. Cell Biol. 98: 1973-1984. Fuchs, E., Tyner, A.G., Guidice, G.J., et al. 1987. The human keratin genes and their differential expression. Curr. Topics Dev. Biol. 22: 5-34. Germain, L., Blouin, M.-J., and Marceau, N. 1988. Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, a-fetoprotein, albumin, and cell surface-exposed components. Cancer Res. 48: 4909-4918. Grisham, J.W. 1980. Cell types in long term propagable cultures of rat liver. Ann. N.Y. Acad. Sci. 349: 128-137. Hatzfeld, M., and Franke, W.W. 1985. Pair formation and promiscuity of cytokeratin: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J. Cell Biol. 101: 1826-1841. Jorcano, J.L., Magin, T.M., and Franke, W.W. 1984. Cell typespecific expression of bovine keratin genes as demonstrated by the use of complementary DNA clones. J. Mol. Biol. 176: 21-37. Kost, T.A., Theodorakis, N., and Hughes, S.H. 1983. The nucleotide sequence of the chick cytoplasmic &actin gene. Nucleic Acids Res. 11: 8287-8301.
Land, H., Parada, L.F., and Weinberg, R.A. 1983. Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature (London), 304: 596-602. Leyes, E.J., Crouse, G.F., and Kellems, R.E. 1984. Dihydrofolate reductase gene expression in cultured mouse cells is regulated by transcript stabilization in the nucleus. J. Cell Biol. 99: 180-187.
Marceau, N., Germain, L., Goyette, R., et al. 1986. Cell of origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective expression. Biochem. Cell Biol. 64: 788-802. Moll, R., Franke, W.W., Schiller, D.L., et al. 1982. The catalog of human cytokeratin polypeptides patterns of expression of specific cytokeratins in normal epithelia, tumors and cultured cells. Cell, 31: 11-24. Moll, R., Schiller, D.L., and Franke, W.W. 1990. Identification of protein IT of the intestinal cytoskeleton as a novel type I cytokeratin with unusual properties and expression patterns. J. Cell Biol. 111: 567-580. Nelson, W., and Sun, T.-T. 1983. The 50- and 58-kd keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J. Cell Biol. 97: 244-251. O'Farrell, P.H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: 4007-4021. Quinlan, R.A., Cohlberg, J.A., Schiller, D.L., et al. 1984. Heterotypic tetramer (A2D3 complexes of non-epiderm keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells. J. Mol. Biol. 178: 365-388. Quinlan, R.A., Schiller, D.L., Hatzfeld, M., et al. 1985. Pattern of expression and organization of cytokeratin intermediate filaments. Ann. N.Y. Acad. Sci. 455: 282-306. Roop, D.R. 1987. Regulation of keratin gene expression during differentiation of epiderma and vaginal epithelial cells. Curr. Topics Dev. Biol. 22: 195-207. Roop, D.R., Kreig, T.M., Mehrel, T., et al. 1988. Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res. 48: 3248-3252. Roop, D.R., Mehrel, T., Kreig, T.M., et al. 1989. Keratin expression in mouse epidermal tumors. In Skin tumors experimental and clinical aspects. Edited by C.J. Conti, T.J. Slager, and A.J.P. Klein-Szanto. Raven Press, New York. pp. 257-271. San, R.H.C., Laspia M.F., Soiefer, A.I., et al. 1979. A survey of growth in soft agar and cell surface properties as markers for transformation in adult rat liver epithelial-like cell culture. Cancer Res. 39: 1026-1034. Singer, P.A., Trevor, K., and Oshima, R.G. 1986. Molecular cloning and characterization of the Endo B cytokeratin expressed in preimplantation mouse embryos. J. Biol. Chem. 261: 538-547. Smith, G.H., Mehrel, T., and Roop, D.R. 1990. Differential keratin gene expression in developing, differentiating, preneoplastic, and neoplastic mouse mammary epithelium. Cell Growth & Differ. 1: 161-170. Steinert, P.M., and Roop, D.R. 1988. Molecular and cellular biology of intermediate filaments. Annu. Rev. Biochem. 57: 593-625.
Sun, T.-T., Tseng, S.C.G., Huang, A.J.-W., et al. 1985. Monoclonal antibody studies of mammalian epithelial keratins: a review. Ann. N.Y. Acad. Sci. 455: 307-329. Sunderman. F.W., Jr. 1984. Recent progress in nickel carcinogenesis. Toxicol. Environ. Chem. 8: 235-252. Swierenga, S.H.H. 1985. Use of low calcium medium in carcinogenicity testing-studies with rat liver cells. In In vitro models for cancer research. Vol. 2. Edited by M.M. Weber. CRC Press, Boca Raton. pp. 61-89. Swierenga, S.H.H., and McClean, J.R. 1985. Further insights into mechanisms of nickel-induced DNA damage: studies with cultured rat liver cells. Progr. Nickel Toxicol. Proc. Int. Conf. Nickel Metab. Toxicol. 3nd, S4-7. pp. 101-104. Swierenga, S.H.H., Marceau, N., Katsuma, Y., et al. 1989. Altered
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cytokeratin expression and differentiation induction during neoplastic transformation of cultured rat liver cells by nickel subsulfide. Cell Biol. Toxicol. 5: 271-286. Tyner, A.L., and Fuchs, E. 1986. Evidence for posttranscriptional regulation of the keratins expressed during hyperproliferation and malignant transformation in human epidermis. J. Cell Biol. 103: 1945-1955. Vannice, J.L., Taylor, J.M., and Ringold, G.M. 1984. Glucocorticoid-mediated induction of a,-acid glycoprotein:
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evidence for hormone-regulated RNA processing. Proc. Natl. Acad. Sci. U.S.A. 81: 4241-4245. Wedrychowski, A., Schmidt, W.N., Ward, W.S., and Hrilica, L.S. 1986. Cross-linking of cytokeratinsto DNA in vivo by chromium salt and cis-diamine dichloroplatinum (11). Biochemistry, 25: 1-9. Wilkinson, M.F., and MacLeod, C.L. 1988. Induction of T-cell receptor-a and @ mRNA in SL12 cells can occur by transcriptional and post-transcriptional mechanisms. EMBO J. 7: 101-109.
SABINE H. H. SWIERENGA Drugs Directorate, Health and Welfare Canada, Ottawa, Canada KIA OL2
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AND
NORMANDMARCEAU Centre de recherche en cancerologie de IYUniversitPLaval, L'H6tel-Dieu de Quebec, 11 c6te du Palais, Quebec (Quebec), Canada G l R 2 56 Received June 24, 1991 BLOUIN,R., SWIERENGA, S. H. H., and ~ ~ A R C E A N.U1992. , Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line. Biochem. Cell Biol. 70: 1-9. T51B, a cell line of the rat liver nonparenchymal cell compartment, contains a cytokeratin (CK) pair composed of CK8, a CK typical of simple epithelium, and CK14, a CK usually present in proliferative stratified epithelium. T5lB-Ni, a subclone selected by prolonged exposure of the parental clone to nickel subsulfide contains CK8 and CK18 (its usual partner in simple epithelium), as well as CK14, at a lower level. The two clones have comparable levels of vimentin. Northern blot analyses of cytoplasmic mRNA demonstrated that the differences in the steady state mRNA levels of each CK paralleled those observed at the protein level, thus showing that the regulatory events occurred prior to translation. The most prominent difference was a 30-fold higher level of CK18 mRNA in T51B-Ni cells. Run-off assays of isolated nuclei revealed that the level of CK8, CK14, and vimentin was regulated primarily at the transcriptional level. However, the large increase in CK18 mRNA levels in T51B-Ni cells did not result from a corresponding increase in the relative level of CK18 gene transcription nor from a change in cytoplasmic CK18 mRNA stability. Comparative Northern blot analyses of nuclear and cytoplasmic mRNAs further suggested that the control of CK18 gene expression in T5lB cells is post-transcriptionally mediated by nuclear events. Key words: cytokeratins, gene regulation, T5lB cells, intermediate filaments, liver. S. H. H., et MARCEAU, N. 1992. Evidence for post-transcriptional regulation of cytokeratin BLOUIN,R., SWIERENGA, gene expression in a rat liver epithelial cell line. Biochem. Cell Biol. 70 : 1-9. La lignte T51B, dtrivte du compartiment cellulaire non-parenchymateux du foie de rat, contient une paire de cytoktratines (CKs) comprenant la CK8, une CK typique de l'tpithtlium simple, et la CK14, une CK habituellement prtsente dans l'tpithtlium stratifit en proliftration. T51B-Ni, un sous-clone stlectionnt par un traitement prolong6 du clone parental avec du subsulfure de nickel, contient la CK8 et la CK18 (son partenaire habitue1 dans l'tpithtlium simple) ainsi que la CK14, mais B un niveau plus bas. Les niveaux de vimentine sont comparables. Ces changements de la composition en CKs se reflktent dans le niveau d'ARNm correspondant 21 chacune d'elles. Des essais de transcription nucltaire rtalists A partir de noyaux isolts de cellules T51B et T51B-Ni rhtlent que l'expression des gtnes de la CK8, de la CK14 et de la vimentine est principalement contralte au niveau transcriptionnel. La situation difftre pour le gtne de la CK18 puisque I'augmentation importante du niveau d'ARNm observte dans le sous-clone, T51B-Ni, ne peut &tre associte a des changements comparables de l'activitt transcriptionnelle ni A des variations dans la stabilitt des transcrits. L'examen comparatif du contenu en ARN nuclbire et cytoplasmiquedes cellules T5lB et T5 1B-Ni suggtre que l'expression du gtne de la CK18 est contralte de f a ~ o npost-transcriptionnelle par des tvtnements se dtroulant dans le noyau. Mots cles : cytoktratines, rtgulation gtnique, cellules TSlB, filaments intermtdiaires, foie.
Introduction Intermediate filaments (IFs) constitute a complex family of polypeptides present in a tissue, differentiation, and developmental specificfashion in most eukaryotic cells (Moll et al. 1982). Cytokeratins (CKs) are unique in that they constitute a family of at least 20 polypeptides present in various types of epithelia (simple, stratified, and pseudostratified) (Moll et al. 1982, 1990). They can be subdivided into two distinct classes, type I (CK9 to CK20) and type I1 (CKl to CK8), which differ in terms of molecular weight, isoelectric point, and gene sequence (Sun et al. 1985; Fuchs ABBREVIATIONS: CK(s), cytokeratin(s); CX, cycloheximide; IF(s), intermediate filament(s); LEC, liver epithelial cells; NM-IFs, nuclear matrix-intermediate filaments; PBS, phosphate-buffered saline. ' ~ u t h o rto whom correspondence should be addressed. Printed in Canada / Imprim6 au Canada
et al. 1987). At least one member of each type is necessary for IF formation (Hatzfeld and Franke 1985; Eichner et al. 1986; Steinert and Roop 1988). Type I and type I1 CKs are usually present as specific pairs, and distinct pairs are present in different cell lineages and differentiation states (Quinlan et al. 1985). While there is some indication that the regulation of CK gene expression occurs primarily at the transcriptional level in stratified epithelial cells (Jorcano et al. 1984; Fuchs et al. 1987), very little is known about the regulatory mechanisms responsible for the appearance of the different CK pairs in simple epithelial cells. Among the CKs normally present in simple (e.g., liver) and stratified (e.g., epidermis) epithelial cells are the CK8-CK18 and CK5-CK14 pairs, respectively (Moll et al. 1982; Nelson and Sun 1983; Quinlan et al. 1984). However, recent work from our laboratory has demonstrated that while rat hepatocytes indeed contain only CK8 and CK18,
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basic
PI
acidic
basic
PI
acidic
FIG. 1. Analysis of CK composition of T5 1B and T5 1B-Ni cells. Subconfluent T5 1B (a) and T5 1B-Ni (b) cells were labeled overnight with [35~]methionine, and the Triton X-100 insoluble fractions, which were enriched in intermediate filaments, were analyzed by twodimensional gel electrophoresis. Each gel was loaded with 100 000 cpm. Spots corresponding to actin (Ac), vimentin (V), and CK8, CK14, and CK18 are identified.
other liver cell types known as liver epithelial cells (LECs, Marceau et a/. 1986), which readily emerge in enriched preparations of hepatocytes in culture, contain CK14 without CK5 (R. Blouin, M.J. Blouin, I. Royal et a/., in preparation). LECs can be established as cell lines (Grisham 1980; Marceau et a/. 1986), and one of them is T51B (Swierenga 1985), a LEC clone that exhibits the additional feature of containing CK8 and CK14 in the almost complete absence of CK18 (Marceau et a/. 1986). But we have recently selected, as a result of nickel exposure, a subclone designated as T5lB-Ni which contains CK8, CK18, and a decreased amount of CK14 (Swierenga et a/. 1989). In the present study these two T5lB clones were used to determine the level(s) at which CK and vimentin gene expression can be regulated in LECs. The results show that while the expression of CK8, CK14, and vimentin genes is regulated mainly at the transcriptional level, that of CK18 seems to be regulated by a post-transcriptional mechanism that involves nuclear RNA processing events.
previously (Fey et al. 1984) and analyzed by two-dimensional gel electrophoresis (O'Farrell 1975).
In vitro transcription assay T5 1B and T5 1B-Ni cells were rinsed with phosphate-buffered saline (PBS), removed from plates by scraping in the same buffer, pelleted (500 g, 5 min), resuspended at lo7 cells/mL in lysis buffer (10 mM Tris-HC1 (pH 7.4). 10 mM NaCl, 5 mM MgCl,, 1 mM dithiothreitol, 100 U RNasin/mL) containing 0.5% Nonidet P-40. The cells were incubated on ice for 5 min. Nuclei were then pelleted by centrifugation (1000 g, 4 min) and kept on ice. Postnuclear supernatants were saved for preparation of cytoplasmic RNA by a guanidine isothiocyanate technique (Chomczynski and Sacchi 1987). Nuclear pellets were rinsed gently in ice-cold lysis buffer alone, repelleted and suspended at a concentration of 5 x 10' nuclei/mL in nuclear freezing buffer (50 mM Tris-HC1 (pH 8.0), 5 mM MgCI,, 0.1 mM EDTA, 40% glycerol, 1 mM dithiothreitol, 100 U RNasin/mL). Nuclei were frozen and stored at - 70°C until used. The transcription assay was carried out with 5 x lo7 nuclei. One hundred microlitres of 2 x transcription buffer (10 mM Tris-HC1 (pH 8.0), 5 mM MgCl,, 300 mM KCI, 1 mM ATP, 1 mM CTP, 1 mM GTP, 1 U RNasin/pL) containing 250 pCi of [a-'*P]UTP (3000 Ci/mmol) was added to each Methods sample, and transcription was allowed to proceed for 30 min at Cell culture 30°C. The nuclei were then pelleted and resuspended in 0.5 M Normal T51B cells (Swierenga 1985) and nickel subsulfide treated NaCI, 10 mM Tris-HCI (pH 7.4), 50 mM MgCI,, 100 U RQ1 TSlB cells (referred to as T5lB-Ni in this study and clone 4 in RNase-free DNase/mL (Promega), 0.4 mg proteinase K/mL, Swierenga et al. 1989) were grown in minimal essential medium 10 mM EDTA, 0.5% SDS, and 10 mM vanadyl ribonucleoside (alpha-modification) containing 10% (v/v) heat-inactivated fetal complex for 30 min at 37°C. Labeled RNA was isolated by phenol bovine serum, 100 U penicillin/mL, and 100 pg streptomycin/mL. extraction and ethanol precipitation. The resulting RNA pellets were then dissolved in 10 mM Tris-HC1 (pH 7.5), 0.3 M NaCI, C K extraction and analysis T51B and TSlB-Ni cells were radiolabeled with [35~]methionine 1 mM EDTA, and 1% SDS and passed through Bio-Spin 30 columns (Bio-Rad Laboratories) to eliminate unincorporated (10 pCi/mL; 1 Ci = 37 GBq) for 18 h and the nuclear matrixnucleotides. Usually, 10 x lo6 to 20 x lo6 cpm were hybridized, intermediate filaments (NM-IFs) were prepared as described
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FIG. 2. Double immunofluorescent microscopy analysis of CK14 and CK8 distribution in T5lB and T51B-Ni cells. Subconfluent T5lB (A and B) and T51B-Ni (C and D) cells grown on glass coverslips were fixed and stained for CK14 (A and C) and CK8 (B and D) using highly specific polyclonal and monoclonal antibodies. in 1 mL, to filter-bound linear plasmid DNAs. DNAs were immobilized on nitrocellulose using a BRL dot blot apparatus after denaturation in 0.2 M NH40H - 2 M NaCl at 100°C for 5 min and quick-cooling on ice. Plasmid DNAs were dissolved directly into 0.2 M NH40H - 2 M NaCl to bring the DNA concentration to 50 pg/mL, and 100 pL (5 pg) of each plasmid was applied to each sample well. Nitrocellulose membranes were baked at 80°C for 2 h. Prehybridization was done at 42°C for 16 h in 50% formamide, 6 x SSC (1 x SSC is 150 mM NaCl, 15 mM sodium citrate (pH 7.0)), 0.5% SDS, 10 mM Na2HP04(pH 6.8), 1 mM EDTA, 0.1 mM UTP, 5 x Denhardt's, 100 pg herring sperm DNA/mL, and 1 pg poly(A)/mL. Hybridization reactions were
done in the same buffer with 3Z~-labeled RNA for 72 h at 42"C, in triplicate. After hybridization, filters were washed twice for 15 min in 2 x SSC, 0.1% SDS at room temperature, once for 15 min in 0.2 x SSC, 0.1% SDS at room temperature, and twice for 15 min in 0.2 x SSC, 0.1% SDS at 55°C. Filters were then exposed to Kodak X-Omat AR film at - 70°C with intensifying screens. Plasmids used contained specific cDNAs: CK18, mouse CK18 full-length cDNA (Singer et al. 1986); CK8, mouse CK8 cDNA (Bralet and Jacob 1982); CK14, mouse CK14 cDNA (Roop et al. 1989); vimentin, human vimentin cDNA (Ferrari et al. 1986); and actin, chicken P-actin cDNA (Kost et al. 1983). pBR322 was used as a control.
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pg DNA by random priming (Feinberg and Vogelstein 1983) CK18; CK8; CK14; vimentin; and actin. A mouse c-myc gene fragment (Land et al. 1983) was also used as a control in the mRNA stability assay. Conditions for washing and autoradiography were the same as for the in vitro transcription assay.
Zmmunofluorescence microscopy Cells grown on glass coverslips were washed three times at room temperature with PBS, treated in cold methanol (100%) for 10 min at - 20°C. and then rinsed again with phosphate-buffered saline. Indirect immunofluorescence microscopy was performed as described previously (Germain et al. 1988), using a mouse monoclonal antibody to rat CK8 (Marceau et al. 1986), and a rabbit polyclonal antibody to mouse CK14 (Smith et al. 1990). Fluorescein-conjugated goat anti-mouse Ig (Rego Lab, Qutbec), and Texas-Red-conjugated donkey anti-rabbit Ig were used as second antibodies (Jackson Immunoresearch Laboratory, West Grove, PA). Cells were observed with a Leitz microscope equipped with epifluorescence illumination and a special emission filter (535 DF45, Omega Optical, Inc., Brathleboro, VT, U.S.A.).
Results CK composition T51B and T51B-Ni cells were labeled with [35~]methionine, Triton-resistant cytoskeletons were prepared, and the IF proteins were analysed by two-dimensional polyacrylamide gel electrophoresis (PAGE). The parental T5 1B contained comparable amounts of CK8 and CK14, very little CK18, and a high level of vimentin (Fig. la). T51B-Ni cells contained the same pattern of IF proteins but had much more CK18 and slightly less CK14 (Fig. 16). Double indirect immunofluorescence analyses of T51B cells using anti-CK8 and anti-CK14 antibodies showed that both CKs formed typical IFs in all cells (Fig. 2). The same analyses performed in T5 1B-Ni cells revealed that while all cells contained CK8, a small proportion of them were CK14 negative.
FIG. 3. Analysis of CK mRNA levels in T51B and T51B-Ni cells. Cytoplasmic RNA (20 pg) isolated from TSlB (lane 1) and T5 1B-Ni (lane 2) cells was resolved by formaldehyde - agarose gel electrophoresis, transferred to a nylon membrane by blotting, and hybridized to "P-labeled CK18, CK14, CK8, vimentin, and P-actin cDNAs.
Cytoplasmic and nuclear RNA analysis Cells were lysed and nuclei isolated as described for the in vitro transcription assay. After centrifugation in lysis buffer containing Nonidet P-40, the cytosol was saved and centrifuged once more; the nuclei were washed once more with lysis buffer alone and centrifuged. Three volumes of 4 M guanidine isothiocyanate solution were added to the cytosol; the nuclear pellet was resuspended directly in the same solution and the RNA of each fraction was isolated as described by Chomczynski and Sacchi (1987). The RNA was fractionated by electrophoresis in a 1.5% agarose gel containing formaldehyde, and transferred to a nylon filter. Prehybridization and hybridization were performed in 50% formamide, 0.5 M Na2HP0, (pH 7.2), 1 mM EDTA, 1% BSA, and 5% SDS at 42OC for 4 and 18 h, respectively. Filters were probed with specific cDNAs labeled to 1 x 10' to 10 X 10' cpm/
CK mRNA levels As a first step to determine the level at which the changes in CK composition are determined, the steady-state levels of CK8, CK14, and CK18 mRNAs were measured by Northern blotting (using appropriate cDNA probes) and compared with those of vimentin and actin mRNAs (Fig. 3). The main finding was a 30-fold greater level of CK18 mRNA in T5lBNi cells as compared with T51B cells (Figs. 3a and 5). Moreover, T51B-Ni cells showed a slightly reduced CK14 mRNA level (Fig. 36) but a higher level CK8 mRNA (Figs. 3c and 5). This down-regulation of CK14 mRNA expression in T51B-Ni is probably due to the inhibition of CK14 protein synthesis in a subset of these cells (Fig. 2). Finally, vimentin and actin mRNA levels of the two cell types were similar (Figs. 3d and 3e). The differences in CK composition were thus reflected at the mRNA level, which implies that the mechanism responsible for the observed differences occurred prior to translation. CK gene transcription Since variations in mRNA steady-state levels may be due to transcriptional or post-transcriptional regulatory events, run-off assays were performed on nuclei isolated from T5 lB and T5lB-Ni cells to monitor transcription of CK8, CK14, CK18, vimentin, and actin genes (Fig. 4). Despite the negligible steady-state level of CK18 mRNA in T51B cells, the CK18 gene was transcriptionally active. Furthermore, although there was at least a 30-fold difference in the CK18
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mRNA STEADY-STATE L EVEL
CK18
'
CK14
'
CK8
' V I M E N T I N ACTlN .
Vimentin TRANSCRIPTION LEVEL 1.47
FIG. 4. Nuclear transcription analysis of CK genes in T5lB and T5 1B-Nicells. Nuclei isolated from T5 1B (a) and T5 1B-Ni (b) cells were incubated in the presence of [ a - ' 2 ~ under ] ~ ~conditions ~ that favored elongation of previously initiated RNA transcripts. Equivalent counts of labeled nuclear RNA were hybridized to denatured, nitrocellulose-immobilized plasmid DNAs containing CK18, CK14, CK8, vimentin, and P-actin cDNA sequences. pBR322 was used as a control for nonspecific background hybridization. steady-state mRNA level between T51B and T51B-Ni cells, there was only a 4-fold increase in the transcriptional level of the corresponding gene (Figs. 4 and 5). The relative levels of transcription of the CK8, CK14, and vimentin genes in T5 1B-Ni cells were 5, 1.6, and 1.5-fold higher, respectively, than in T51B cells. By comparing these differences with those of the respective mRNA steady-state levels, the regulation of CK8, CK14, and vimentin gene expression seems to be mainly at the transcriptional level. Thus, the differences in transcription levels alone in T51B and T5 1B-Ni cells cannot account for the much greater CK18 mRNA level measured in T5lB-Ni cells (Fig. 5). CKI8 mRNA stability RNA synthesis was blocked with actinomycin D and the rate of CK18 mRNA degradation in T5 1B and T5 1B-Ni cells was determined in the presence or absence of cycloheximide (CX) (Fig. 6). The same analyses were performed for vimentin, actin, and myc mRNAs (the latter was used as an internal reference). The stability of CK18 mRNA, as well as that of vimentin and actin mRNAs, was the same in T51B and T51B-Ni cells (Figs. 6a, 6c, and 6d).In fact, no degradation could be detected in the 3-h period examined even in
CK18
CK14
CK8
VlMENTlN ACTlN
FIG. 5. Quantitative analysis of the relative mRNA steady-state levels and transcription levels of CK18, CK14, CK8, and vimentin. The data were obtained by densitometric scanning of the autoradiographsfrom the Northern blots shown in Fig. 3, and from the nuclear transcription assay shown in Fig. 4. The values given in the ordinate are optical densities normalized to P-actin mRNA and transcription levels. The data are representative of three separate experiments. the absence of CX in actinomycin D treated cells. As expected, the half-life of c-myc mRNA was relatively short (30 min) and CX greatly prolonged it (Fig. 6b). Thus the differences observed in the steady-state levels of CK18 cytoplasmic mRNA in T5 1B and T5 1B-Ni cells are not determined by their relative rates of decay. Nuclear and cytoplasmic CK RNA levels To follow early steps of transcript processing, total nuclear and cytoplasmic RNA fractions were isolated from T5 1B and T5 1B-Ni cells and examined by Northern blotting (Fig. 7). Although there was a large amount of CK18 RNA present in the nuclei and cytoplasm of T51B-Ni cells, there were no detectable CK18 transcripts in the nuclei or cytoplasm of T51B cells (Fig. 7a). Very little unprocessed
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FIG. 6. Analysis of CK18 mRNA stability in TSlB and TSlB-Ni cells. TSlB (lanes 1-10) and TSlB-Ni (lanes 11-20) cells were treated for 60 min with 10 pg CX/mL. After that time, some of the plates were washed 3 times with Hepes buffer and incubated further with or-MEM containing 10% heat-inactivated FCS, 5 pg actinomycin D/rnL, but without CX. Other plates were washed and incubated under identical conditions, but with medium containing 10 pg CX/mL. Cytoplasmic RNA was prepared at 0, 30, 60, 120, and 180 min after actinomycin D addition. Equal amounts of cytoplasmic RNA 20 pg were fractionated by formaldehyde - agarose gel electrophoresis, transferred to nylon membrane by blotting, and hybridized to P-labeled CK18, c-myc, vimentin, and O-actin cDNAs or gene fragments.
6
CK18 RNA was present in the nuclei from T5 1B and T5lBNi cells, indicating a very rapid processing or degradation of precursor transcripts. Nevertheless, it is clear that in spite of transcription of the CK18 gene, a large pool of CK18 transcripts did not accumulate in the nuclei of T51B cells. The difference in the level of mature CK14 mRNAs in the nucleus of T5 1B and T51B-Ni was reflected in the cytoplasm (Fig. 7b). Similar levels of CK8 RNAs were detected in the nuclei of T51B and T5 1B-Ni cells but not in the cytoplasm, perhaps suggesting a slight difference in processing (Fig. 7c). The analysis of the amount of nuclear and cytoplasmic vimentin RNA revealed no difference in the steady-state vimentin RNA levels between the two cellular compartments of T5lB and Tl5B-Ni cells (Fig. 7d). Among the various explanations for the lack of detectable nuclear CK18 mRNA in T5lB cells are differences that might exist in the degradation or maturation of nascent transcripts. To examine this, we compared the accumulation of nuclear and cytoplasmic CKs RNAs in T5lB and T51B-Ni cells treated with CX for prolonged periods (Figs. 7e-7h). CX treatment resulted in the appearance of precursor and mature CK18 mRNAs, particularly in T5lB-Ni cells. However, CK18 mRNA transcripts were also present in the cytoplasmic fraction of T5 1B cells, but at a lower level than that in T5lB-Ni cells (Fig. 7e). In spite of the fact that little contamination of cytoplasmic RNA with nuclear RNA occurred in T51B and T5 1B-Ni cells, precursors were present
)
at comparable levels in the cytoplasmic fraction of both cell types. This result was in contrast to the observations made for mature CK18 mRNA. CX treatment also increased the steady-state levels of precursor and mature mRNAs for CK14, CK8, and vimentin in both cell types, the most prominent increase being for the CK14 RNA (Figs. 7f-7h).
Discussion The present results demonstrate that in T5lB LEC expressing a CK8-CK14 pair, the expression of CK18, the usual partner of CK8 in simple epithelium, is largely regulated by a post-transcriptional mechanism taking place at the level of nuclear RNA processing. In contrast, the expression of CK8 and CK14 is transcriptionally regulated. The expression of CK18 in T5lB-Ni cells somehow is affected in a stable and heritable manner. Previous studies performed on the mode of expression of several CK genes in stratified epithelial cells indicated that the regulating events occur mainly at the transcriptional level (Jorcano et al. 1984; Tyner and Fuchs 1986; Fuchs et al. 1987; Roop et al. 1987, 1988). The present data on mRNA and transcription levels of CKs and vimentin suggest that the regulation of the IF genes expressed in T51B cells differ widely depending on the gene. For example, it is clear that the regulation of vimentin gene expression takes place only at the transcriptional level. The changes in CK8 mRNA and transcription levels imply that regulation in this case can
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BLOUIN ET At.
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FIG. 7. Analysis of nuclear and cytoplasmic CK RNA content in TSlB and T51B-Ni cells. T51B (lanes 1 and 3) and TSlB-Ni (lanes 2 and 4) cells were cultured without (a-d) or with (e-h) CX (5 pg/mL) for 5 h. After this treatment, nuclear (lanes 1 and 2) and cytoplasmic (lanes 3 and 4) RNA was isolated from both cell types. Equal amounts of these RNA fractions (20 pg) were electrophoresed on a formaldehyde - agarose gel, transferred to a nylon membrane by blotting, and hybridized to 32~-labeled CK18 (a and e), CK14 ( b and f), CK8 (c and g), and vimentin (d and h) cDNAs.
occur at both transcriptional and post-transcriptional levels. The CK14 gene, on the other hand, showed transcriptional regulation. The data on the expression of the CK18 gene can be best interpreted as a regulation occurring primarily at the post-transcriptional level. The decay rate of steadystate CK18 mRNA in the presence of actinomycin D suggests that the stability of CK18 mRNA in the cytoplasm cannot account for the much larger differences in mRNA accumulation observed in T51B-Ni cells. These results suggest therefore that post-transcriptional regulation can occur via changes in nuclear mRNA stability and (or) the rate of RNA processing. The change between the two cell types may represent an increase in the efficiency with which CK18 hnRNA is converted to CK18 mRNA. A greater proportion of the poly(A)+ CK18 hnRNA is converted into poly(A)+ CK18 mRNA in T51B-Ni. The analyses of the CK18 nuclear RNA levels support this interpretation. There are data from work performed on other genes suggesting that regulation at the level of nuclear mRNA stability and (or) rate of RNA processing (Leys et al. 1984; Vannice et al. 1984) depends on the presence of a labile protein (Wilkinson and MacLeod 1988). However, since treatment with cycloheximide did not significantly alter the accu-
mulation of mature CK18 transcripts in the nucleus, it seems unlikely that the nuclear processing of CK18 mRNA in T51B cells is due to the action of such a labile protein. T51B, which contains CK8 and CK14, is a nontransformed cell line derived from a culture of adult rat liver cells (San et al. 1979). This line has been used extensively to study the role of c a 2 + in cell growth regulation and to screen for potential carcinogens (Swierenga 1985; Swierenga and McClean 1985). The finding that a prolonged exposure of T51B cells to nickel subsulfide led to the appearance of a subclone (T51B-Ni) that contains CK8 and CK18, but a lesser amount of CK14, is certainly intriguing. While there are data indicating that nickel compounds can influence the interactions between proteins and DNA (Sunderman 1984; Wedrychowski et al. 1986), the actual cellular mechanisms underlying this selective effect are unknown. Much interest has been focused on the regulation of CK gene expression in stratified epithelial cells, particularly as the progenitor cells differentiate (Roop et al. 1988). In contrast, very little has been done on this aspect in simple epithelial cells. While the cell of origin of LECs in vivo is still unsettled, the experimental evidence accumulated so far suggests that they are derived from distinct portions of bile
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BIOCHEM. CELL BIOL. VOL. 70, 1992
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ductules (Marceau et al. 1986; R. Blouin, M.J. Blouin, A. Grenier et al., t o be published). T h e finding that LECs contain CK14 without CK5 is in line with recent d a t a obtained in mouse mammary glands, where a few CK14 positive cells are detected within a field of CK14 negative cells (Smith et al. 1990). Although the physiological significance of this selective C K gene expression by a few cells is unclear, the present d a t a show that simple epithelial cells can use distinct molecular mechanisms to regulate the appearance o f different C K pairs.
Acknowledgements We thank Drs. William I. Waithe and Alan Anderson f o r reviewing the manuscript, AndrCe Grenier f o r her expert technical assistance, Michde Gagnon a n d Elisabeth Lemay f o r their secretarial assistance, a n d Pierre Paquin a n d G u y Langlois f o r photographic assistance. Richard Blouin was recipient of a studentship from t h e Medical Research Council of Canada. This work was supported by the Medical Research Council of Canada.
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