Evidence for post-transcriptional regulation of cytokeratin gene expression in a rat liver epithelial cell line RICHARD BLOUIN Centre de recherche en cancPrologie de I'Universite Laval, L 'HGteI-Dieude Quebec, Quebec (Quebec), Canada GlR 2 56 SABINE H. H. SWIERENGA Drugs Directorate, Health and Welfare Canada, Ottawa, Canada KIA OL2 AND NORMAND MARCEAU Centre de recherche en cancerologie de IYUniversitP Laval, L'H6tel-Dieu de Quebec, 11 c6te du Palais, Quebec (Quebec), Canada GlR 2 56 Received June 24, 1991 BLOUIN, R., SWIERENGA, S. H. H., and ~~ARCEAU, N. 1992. 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. How- ever, 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. BLOUIN, R., SWIERENGA, S. H. H., et MARCEAU, N. 1992. Evidence for post-transcriptional regulation of cytokeratin 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 fa~on post-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 pseudo- stratified) (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. '~uthor to whom correspondence should be addressed. 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 regula- tion of CK gene expression occurs primarily at the transcrip- tional 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, Printed in Canada / Imprim6 au Canada Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by 216.208.156.69 on 06/05/13 For personal use only.