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

Differentiation (1 992) 52 :45-54 Onrogeny. Vpoplasiu and Diflrrenrlshoo Therapy

8 Springer-Verlag 1992

Cytokeratin 14 expression in rat liver cells in culture and localization in vivo Richard Blouin, Marie-Jose Blouin, Isabelle Royal, AndrCe Grenier, Dennis R. Roop, Anne Loranger, and Normand Marceau Laval University Cancer Research Center, L‘HBtel-Dieu de Qutbec. 11 C6te du Palais Qutbec. Canada G1R 256. and Departments

of Cell Biology and Dermatology (DRR), Baylor College of Medicine, Houston, Texas 77030, USA Accepted in revised form September 21, 1992

Abstract. 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’-MeDAB) 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 (HESZ cells) and was expressed in some biliary epithelial (BDST cells). CK14-positive cells were also found in the Glisson’s capsule. However, CKlCpositive 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. Correspondance to: N. Marceau, Centre de recherche en canctrologie de I’Universitt Laval; L‘H6teCDieu de Quebec, 11 c8te du Palais, Qutbec, Canada G1 R 216

Introduction Cytokeratins (CKs) form the intermediate filaments (IFs) of both keratinized and non-keratinized epithelial cells. They represent the largest and most diversified group in the I F family and their genes are expressed in a tissue, differentiation and developmental specific fashion in most eukaryotic cells [16, 371. So far, some 20 different CKs have been identified in various types of simple, stratified and pseudostratified epithelial cells [32, 33, 481. They can be subdivided into two classes, type I and type 11, which differ in their molecular weights, isoelectric points and sequences [16, 321, and their assembly into IFs requires the participation of type I and type I1 CK pairs [25, 491. CK IFs form an extensive network in the cytoplasm of non-keratinized epithelial cells and make connections with nuclei, various cytoplasmic organelles and desmosomes at the surface [21, 221. Although the actual function(s) of IFs is unclear [20, 21, 261, the differential expression of CK genes can still be used to advantage to distinguish different epithelial cell types and lineages. Previous work on the CK composition of mammalian (including rat) liver has clearly established that the hepatocytes contain CK8 and CK18 [2,28, 29, 301 the typical C K pair of simple epithelium [32], whereas biliary epithelial cells contain CK7, CK8, CK18 and CK19 [lo, 11, 19, 291. While hepatocytes are defined as parenchymal cells on the basis that they express diverse metabolic activities, biliary epithelial cells belong to a mixed compartment of non-parenchymal cells [ 5 . 12, 23, 29, 30, 31,46,47]. The evidence for the presence of other epithelial cell types in the compartment comes from two lines of evidence: (1) Atypical epithelial cells, designated as oval cells appear during the early phase of rat liver chernical hepatocarcinogenesis [9, 12, 19, 42, 431. Oval cells express few metabolic features of rat liver fetal cells and it has been proposed that they could correspond to progenitor (stem) cells in adult liver [9,42, 531. (2) Hepatocytes isolated from normal or carcinogen-fed rats exhibit a low survival in primary culture and they are replaced by epithelial cells that bear no morphological resem-

46

blance to hepatocytes during the first 2 weeks post-seeding (for review, see [23]). These non-parenchymal cells, named liver epithelial cells (LECs), emerge as rapidly growing colonies and can be easily cloned and established as cell lines [4, 6, 17, 23, 24, 29, 55-58]. While the actual CK composition of oval cells is unclear 1301, we reported recently some evidence indicating that the LEC-derived cell line T51B contains a CK of 55 kDa (CK55) equivalent to human CK8 and a CK of 52 kDa (CK52) equivalent to human CK14 [3, 291. The in vivo cells of origin of oval cells and LECs are unknown and, therefore, their respective cell lineage affiliation remains unsettled. In the work reported here, we first analyzed the CK14 gene expression in post-natal rat hepatocytes in primary culture, cells from a secondary LEC line isolated from embryonic rat liver, LECs freshly isolated from 4-day old rats or a few hours after seeding, ductular oval cells in primary culture isolated from 3’-Me-DAB rat liver and, finally, in T5lB cells and rat skin. In the light of the data on the usual pairing of CK5 and CK14 [16, 32,491, the analyses also included the use of CK5 cDNA and specific antibody. The results demonstrate that the CK14 gene is selectively expressed in LECs, without CK5 gene expression. We then used immunofluorescence laser scanning confocal microscopy to localize CK14 positive LECs in the liver of 4-day old rats. The results show that LECs are distributed at sites different from hepatocytes, but their location coincides with biliary epithelial cells and cells of the Glisson’s capsule.

Methods Materials. a-minimal essential medium (a-MEM) was purchased from Gibco. Burlington, Ontario, Canada. Fetal calf serum (FCS) was purchased from Dextran Products, Scarborough, Ontario, Canada. Tissue Tek O.C.T. compound was obtained from Canlab, Ste-Foy, Quebec, Canada. 3-Methyl-4-dimethylaminoazobenzene (3’-Me-DAB) was purchased from American Tokyo, Kasei, Portland, Oregon, USA, enzymes and HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonicacid) from Boehringer Mannheim, Montreal, Quebec, Canada. Methionine 35S-label, 32P-dCTP, phenylmethylsulfonylfluoride (PMSF) and urea were from ICN Biochemicals Canada, Montreal, Quebec, Canada. Products for gel electrophoresis were obtained from Bio-Rad, Mississauga, Ontario, Canada. All others chemicals were obtained from BDH Chemicals, Montreal, Quebec, Canada. The cDNA probes used were the following: a 350 bp Pstl fragment from a RECXVI cDNA clone encompassing most of the tail region and part of the 3’ non-coding region which is very specific for CK8 [44]; a 1168 bp Hind 111 fragment containing the head region and most of the central rod domain of mouse CK18 cDNA, PUC9B7 [45]; a 450 bp Hind 111EcoR 1 fragment corresponding to the 3’ non-coding region of mouse CK14 cDNA cloned in pGEM3 [39]; a 350 bp HindIIIEcoR I corresponding to the 3’ non-coding region of CK5 cDNA cloned in pGEM3 [37] and a 550 bp Psr I-Barn HI fragment containing the C-terminal half of the complete 1.1 kb human vimentin cDNA clone hp4fl [13]. The antibodies were the following: mouse monoclonal antibodies generated against surface-exposed components of hepatocytes (HES,) and biliary epithelial cells [18, 191; mouse monoclonal antibodies against rat CK8, CK18 [29] and vimentin [29] ; rabbit antibodies produced against specific peptides of mouse CK5 and CK14 carboxy termini [38, 481. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse Ig antibodies

were purchased from Rego Industries, Quebec, Quebec, Canada. Biotin conjugated donkey anti-rabbit Ig antibody and Texas Redtagged streptavidin were purchased from Amersham, Oakville, Ontario, Canada. Rat h e r epithelial cells in culture. The procedures used to obtained LECs as primary and secondary cell cultures and ako as cell lines from newborn and adult rat livers have been described in detail before [29]. LECs can also be derived in a similar manner from embryonic day 12 rat liver (see below). In contrast to newborn and adult rat hepatocytes and even embryonic rat bipotential progenitor cells, LECs exhibit a high proliferative activity in culture. Therefore, LECs can selectively be derived as proliferating colonies of morphologically distinct epithelial cells emerging rapidly in primary cultures of hepatocytes or bipotential progenitor cells and then be established as cell lines. Previous studies from our [29] and other [6] laboratories have shown that LECs can also be obtained as enriched cell populations from newborn rat liver cell preparations by applying selective proteolysis with trypsin to damage hepatocytes and then to remove them. In the present study. the following LEC models were used: the established cell line T51 B derived from adult rat liver, a low passage LEC cell line derived from embryonic day 12 rat liver and LECs obtained as an enriched cell population from a newborn rat liver. Hepatocytes isolated from a 14-day-old rat were used as controls. Finally, we used 3‘-Me-DAB oval cells set up in primary culture. TSIB. This is an established cell line used here as a typical LEC line derived from an adult Fischer rat liver [29]. It was kindly provided by Dr. Sabine Swierenga from Health and Welfare, Ottawa, Canada [52]. The present work was performed on T51B cells cultured in a-MEM supplemented with 10% heat-inactivated FCS at passages 12 to 15. Embryonic rat LEC.9. They were derived as growing colonies emerging in the cultures of embryonic rat day-12 bipotential progenitor cells. The procedures used for the isolation and primary culture of the progenitor cells have been described in detail [2]. Briefly, the minced livers were incubated in a-MEM supplemented with 10% FCS, 0.05% collagenase, 0.48% Dispase I and 0.075% hyaluronidase. The procedure was repeated once, using fresh enzyme solution. The two tissue digests were pooled, subjected to gentle trituration and then centrifuged. The cell pellet was resuspended at appropriate dilutions in a-MEM containing 10% FCS. Cells were seeded on fibronectin coated culture plastic dishes and then maintained in primary cultures. LEC-emerging colonies were recognized on the basis of cell morphology and then collected by trypsin treatment using stainless steel cloning rings. LECs were used at passages 3 to 5. Newborn rat LECs. The procedure to obtain enriched preparations of LECs from 4-day-old Fischer rats has been reported before [6, 29). It involves successive digestions of minced rat livers with dispase I and trypsin. LECs were plated on fibronectin-coated 100 mm dishes or on fibronectin-coated glass coverslips in 24-well plates in a-MEM medium. These cell preparations were used here to examine the expression of CK14 at the time of LEC seeding and then at 17 and 96 h of primary culture in the presence or absence of 10% FCS. Hepatocytes. Hepatocytes were isolated from 14-day old suckling rats by the two-step collagenase perfusion method described before [8, 29, 411. Cells were plated on fibronectin-coated 100 mm dishes or on fibronectin-coated glass coverslips in 24-well plates and cultured in serum-free a-MEM medium containing 150 ng/ml insulin and 1 pM dexamethasone [29]. The CK gene expression was examined at 48 h post-seeding. Biliary-like ooal cells. Oval cells were isolated from a 4-week 3‘-MeDAB treated Fischer rat, as described before [19]. The liver was perfused by the two-step collagenase method, minced in a collagen-

47 ase solution and then incubated in a shaking water bath. The procedure was repeated once. The tissue was centrifuged and digested with trypsin. The resulting cell suspension was filtered and then centrifuged. Highly enriched preparations of oval cells were recovered after removal of hepatocytes by panning using the anti-HES, antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. Cell typing analyses based on the expression of BDS7 and gamma glutamyl-transpeptidase (GGT) have shown that they were mostly related to biliary epithelial cells [19]. The cells were plated on fibronectin-coated 100 mm dishes and cultured in medium M X [19] for 2 days.

USA). Photographs were taken with Kodak Tri-X films. Other observations were made with a Bio-Rad confocal scanning laser microscope. In this case, an argon ion laser beam was used for double-fluorescence emission, with excitation at 514 nm and a filter-separator for fluorescein and Texas Red emissions; two photomultipliers were used in parallel. Confocal sections were obtained using a high numerical aperture (1.4 NA, 60x1 oil-immersion lens and zoom factors. Images obtained after contrast enhancement with an MRC-600 processor were photographed directly from the video screen using Kodak T-Max 100 ASA film. Single immunofluorescence was performed by using the respective staining procedures.

Intermediate filament isolation and CK14 idenrification. The Triton X-lOO/KCl insoluble fraction of T51B cell line was obtained as described [14]. The cytoskeletal fraction of adult rat skin was obtained by modifications of procedures described before [14, 151. Briefly, we used the buffers described by Fey et a]. [14] with the technique of Franke et al. [15]. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was performed as described [36], except that CHAPS was used instead of NP-40.Ampholines of pH 5-7:3-10:3-5 (7:2: 1) were mixed to yield a pH gradient that ranged from 4.5 to 7.0. In some cases, proteins were electrophoretia l l y transferred to nitrocellulose sheets [29, 541. The nitrocellulose papers were incubated with the CK14 antibody and the bound antibody was detected by HRP-conjugated donkey anti-rabbit Ig using the ECL Western blotting detection system (Amersham, Oakville, Ontario, Canada).

R N A preparation and Northern analyses. Total RNA was isolated by the acid guanidinium thiocyanate-phenol-chloroformextraction procedure [7]. The RNA was fractionated by electrophoresis in a 1.5% agarose gel containing formaldehyde, and transferred to a nylon membrane by blotting [40]. Prehybridization and hybridization were performed in 50% formamide, 0.5 M Na,HPO, (pH 7.2). 1 mM EDTA, 1% bovine serum albumin (BSA) and 5% sodium dodecyl sulfate (SDS) at 42" C for 4 h and 18 h, respectively. Filters were probed with specific cDNAs labeled to 1-10 x lo* cpm/ pg DNA by random priming [40]. After hybridization, filters were washed twice for 15 min in 2 x SSC (1 x SSC is 150 mM NaCI. 15 mM sodium citrate, pH 7.0), 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 0.2xSSC, 0.1% SDS at 55" C. Filters were then exposed to Kodak X-Omat AR film at -70" C with intensifying screens.

Indirect immunojluorescencc microscopy. Analyses were performed in both cultured LECs and 4-day-old rat liver. LECs cultured on coverslips were fixed in 100 percent acetone for 10 min at -20" C, as described before [19, 291. Small pieces of rat liver embedded in O.C.T. and frozen in liquid nitrogen were cut with a cryostat to provide 6-10 sections which were then laid out on glass slides and air-dried [ 18, 191. Double indirect immunofluorescence staining was done with a modified version of a published procedure [18. 19, 291. Briefly, one reaction was performed with a mouse monoclonal antibody generated against HES,, BDS7, CK8, CK18 or vimentin, and detected with a FITC-tagged goat anti-mouse Ig. The other reaction involved the successive use of rabbit antibody against the mouse CK5 or CK14 peptide, biotin-tagged donkey anti-rabbit Ig and then Texas Red conjugated streptavidin. Some observations were made with a conventional Leitz Ortholux I1 microscope equipped with epifluorescence illumination and a special emission filter (535DF45, Omega Optical, Brattleboro, Utah,

CK14 is a constituent of LECs Figure 1 shows a Northern blot of total RNA extracted from T51B cells and cultured rat hepatocytes probed with CK8, CK14, CK18 and vimentin cDNAs. A single mRNA species of 1.65 kb was detected with the CK8 cDNA clone in both cell types (Fig. IA). The CK14 cDNA probes detected a mRNA of 1.6 kb only in RNA from T5lB cells, whereas the CK18 cDNA probe detected a mRNA of 1.5 kb only in hepatocytes (Fig. 1 B D

C

B

A

Results

Fig. 1. Northern blot analysis of cytokeratin (CK) and vimentin mRNAs in T51B cells and rat hepatocytes. Total RNA (10 pg) isolated from T51 B cells (lane 1 ) and rat hepatocytes (lane 2) was fractionated by formaldehydeagarose gel electrophoresis, transferred to a nylon membrane by blotting and hybridized to 32P-labeled CK8 (A), CK14 (B), CK18 (C) and vimentin (D) cDNA probes. The exposure time was 24 h for A, B and D and 6 h for C

18s -

1

2

1

2

1

2

1

2

48

A

B

-

C

D

E

28s

18s

Fig. 2. Northern blot analysis of CK14 mRNAs in liver epithelial cells (LECs). Total RNA (10 pg) isolated from T51B cells ( A ) , rat hepatocytes (B), 12-day fetal (C) or 4-day newborn rat LEC (D)and ductular oval cells ( E ) was fractionated by formaldehydeagarose gel electrophoresis, transferred to a nylon membrane by blotting and hybridized to a 32P-labeled CK14 cDNA probe. Thc exposure time was 24 h

The probe, CK14 cDNA, recognized a mRNA of 1.6 kb in T51B cells and LECs (Fig. 2, lanes A, C and D). The ductular oval cells contained an mRNA shorter than that in RNA from typical LEC lines (Fig. 2, lane E). There was no detectable reaction with the probe in RNA from rat hepatocytes (Fig. 2, lane B). No CK5 mRNAs were detected in the various cell types (data not shown). Previous work has established that the amino acid sequence KVVSTHEQVLRTKN is common to the carboxy terminus of the mouse and human CK14 polypeptides [38, 501. Moreover, CK14 is normally found in the epidermis as well as other stratified epithelia [32]. To determine whether rat CK52 was equivalent to CK14, Triton-resistant cytoskeletons were prepared from T51B cells and from newborn rat epidermis and their proteins were fractionated by 2D-PAGE and analyzed by Western blotting. The protein recognized by the anti-CK14 antibody in the epidermis had a molecular weight and an isoelectric point identical to those of T51 B cell CK52 (Fig. 3).

CK14 is a component of the liver epithelial cell and 1 C). The vimentin cDNA probe demonstrated the presence of a hybridization signal in T51B cells only (Fig. 1 D). Figure 2 shows a Northern blot of total RNA preparations isolated from T51 B cells, rat hepatocytes in primary culture, LECs from 4-day-old rat in primary culture, LECs from day 12 embryos obtained as a cell line and ductular oval cells isolated from the liver of 3’-MeDAB treated rats maintained for 48 h in primary culture.

intermediate filuments

T51B cells and cultured rat hepatocytes were examined by indirect immunofluorescence microscopy with antiCK14, anti-CK8, anti-CK18 and anti-CK5 antibodies (Fig. 4). Anti-CK14 and anti-CK8 antibodies decorated a well-organized filamentous matrix present in the cytoplasmic space and in the perinuclear region (Fig. 4a and c). No staining was obtained with either anti-CK18

PI

I

5.4

53

5.4

53

7 r57

0

CK 8

143 B

*.

57

r43

C

-D

Fig. 3. Western blot analysis of CK14 in rat skin and T51B cells. Triton-resistant cytoskeletons from adult rat skin (A, C) and T51B cells (B, D) were fractionated by two-dimensional gel electrophoresis and stained with Coomassie blue (A, B) or transferred to nitrocellulose membranes for the immunoblot analysis (C, D). Blots were incubated with the rabbit polyclonal antibody against CK14 and the bound antibody was detected by using the enhanced chemiluminescence (ECL) system (see Methods). Arronheads denote the position of CK14; ac, actin; D, vimentin

49

Fig. 4. Irnmunofluorescence microscopy of CKS, CK8, CK14 and CK18 in TS1B cells and cultured rat hepatocytes. TSIB cells (a, c, e, g) and hepatocytes (b, d, f, h) were stained first with an anti-CK14 (a, b), anti-CK8 (e, d), anti-CK18 (e, f) or anti-CKS (8, h) antibody and then with the appropriate fluorescein isothiocyanate (F1TC)-tagged Ig. Bur equals 5 Pm

(Fig. 4e) or anti-CK5 (Fig. 4g) antibody. Hepatocytes contained CK8 and CK18 (Fig. 4d and f) but not CK14 and CK5 (Fig. 4 b and h). The same analysis was performed on LECs obtained as cells freshly isolated from 4-day-old rat liver or seeded on a fibronectin-coated plastic substratum. While some definitive staining was detected in LECs at the time of seeding (Fig. 5A), the presence of CK14 IFs was best seen at 2 h as the cells spread on the culture substratum (Fig. 5B). LECs derived from the 4-day-old rat liver were set up in primary culture in the presence or absence of 10% fetal calf serum in order to determine the influence of cell growth on CK14 IF composition and distribution. While the addition of serum actively stimulated LEC proliferation, as revealed by the emergence of rapidly enlarging colonies, typical CK14 IFs were distrib-

uted in the cytoplasm even in the absence of serum (Fig. 5C-F). At 96 h the staining was inore intense in LECs cultured in serum-free medium, suggesting a modulation in the CK content with the cell microenvironment. LEC lines established from such cultures in the presence of serum exhibited a morphology and a CK network similar to those of LECs at 96 h and also to that of T51 B cells (data not shown).

CK14 is a marker of liver epithelial cells in vivo Immunofluorescence analyses of serial sections throughout the lobes of a 4-day-old rat liver established that CK14-positive cells were present in all portions of the liver, but restricted mainly to the portal area and the

50

Similar analyses performed on embryonic rat liver showed very few CKlCpositive cells (data not shown); supporting the observations made here in primary culture showing that LECs can only be detected as few emerging colonies morphologically distinct from the islands of bipotential progenitor cells (as reported above).

2H

OH

Discussion

17 H

96 H

SERUM

W

Fig. 5. Immunofluorescence microscopy of various LEC prepara-

tions containing CK14. The staining was done with the rabbit anti-CK14 antibody plus a biotin-tagged anti-rabbit Ig and Texas Red conjugated streptavidin on enriched LECs obtained from a 4-day-old rat liver at the time of cell isolation (A), after 2 h on a fibronectin-coated substratum (B), and after 17 h (C, D) and 96 h (E, F) in primary culture in the absence (C, E) or presence (D, F) of serum

Glisson’s capsule (Fig. 6). Interestingly, some of the CKlCpositive cells in the portal area were parts of biliary ductules (Fig. 6A and B), whereas others were dispersed in small regions that were close to the biliary epithelial cell structures (Fig. 6C and D). Not all of the cells in the Glisson’s capsule were CK14-positive (Fig. 6E-H). A more precise identification of the CK14-positive cells was obtained by double immunolocalization using confocal microscopy with monoclonal antibodies generated against a surface marker of hepatocytes (HE&), a surface marker of biliary epithelial cells (BDS7), CK8 or vimentin (Fig. 7). Not all of the BDS,-biliary epithelial cells were CK14-positive (Fig. 7A). None of the CK14-positive cells in the portal area were vimentinpositive (Fig. 7 B). Hepatocytes (HE&-positive cells) were always CK14-negative (data not shown). This finding was confirmed by double labeling of CK8 and CK14 (Fig. 7C). Indeed, CK8 was present in all hepatocytes as well as in cells of the Glisson’s capsule, whereas the CK14 staining was restricted to those of the capsule. Finally, the CKlCpositive cells of this structure were also vimentin-positive (Fig. 7 D).

The results of the present study demonstrate that a CK equivalent to a human CK14 is selectively expressed in the absence of its usual partner CK5 and form typical IFs with CK8 in LECs obtained as freshly isolated rat liver cell preparations or as cell lines. We also present here, for the first time, the localization of CKlCpositive cells in normal intact newborn rat liver. The results show that LECs are distinct from hepatocytes but co-localize with some cells of the biliary ductules and Glisson’s capsule. Previous work from our laboratory on CK differential expression in T51 B cells and other LEC lines derived from newborn and adult rat livers provided the first experimental evidence that a distinct population of liver epithelial cells could express in vitro a CK, known then as CK52, not present in other differentiated hepatic epithelial cells [29]. CK52 was first identified as a CK by its resistance to Triton extraction and its reaction with a guinea pig antiserum raised against cow hoof CKs [29]. Our conclusion that CK52 is the equivalent of human CK14 and that it can be used as a specific marker of LECs in culture comes from the following: our preliminary data from Northern blot analysis and immunofluorescence microscopy performed recently on T51 B cells [3] ; the present data from immunoblottings performed on Triton-resistant cytoskeletons from T51 B cells and adult rat skin; the use of a monospecific rabbit antibody raised against a peptide common to mouse and human skin CK14 [38,48]; and the present results from Northern blotting and immunofluorescence microscopy performed on various isolated LEC preparations, obtained from fetal and postnatal rats. CK IFs present in various epithelial cells usually contain matched pairs of type I and type I1 CKs [32, 391. For example, CK8 is matched with CK18 whereas CK5 is the partner of CK14 [32, 351. Experiments on CK reconstitution into 1Fs in vitro have shown that many combinations of mismatched type I and type I1 CKs can assemble to form IFs [25]. Other work using gene transfer of intact and deleted cDNAs for CK7, CK8, CK14, CK18 and CK19 to examine the molecular mechanisms underlying the formation of CK IFs in mouse 3T3 cells, has led to the conclusion that the matched CK pairs, such as CK8/CK18 form typical filament networks whereas mismatched (type I-type 11) pairs such as CK8/CK14 yield a perturbed IF network 1271. In fact, the present data on CK IF organization in LECs clearly show that a CK8/CK14 pair can form typical, stable CK IFs. The biological significance of such CK

51

Fig. 6. Phase contrast and immunofluorescence microscopy of CK14-positive cclls in 4-day-old rat liver. Tissue sections were obtained from the portal area (a-d) and the Glisson’s capsule (eh), and stained first with the rabbit antibody against CK14 and then with a biotin conjugated donkey anti-rabbit Ig antibody and Texas Red-tagged streptavidin. CK 14positive cells were found in biliary ductules (a, b) and surrounding areas (c, a). They were also distributed in discontinous manner in the capsule (eh). Burs equal 20 pm (a, c, e, g) and 10 pm (b, d, f, h). bd denotes biliary ductules

pairing will be better understood, once the actual role(s) of IFs in cell function is established. Attempts to define the differentiation status and to assign the proper lineage to LECs have been largely based on cell morphology and liver-specific metabolic activities [I,23, 34, 561. At low passages under standard culture conditions, LEC lines derived from either newborn or adult rat liver contain virtually none of the metabolic markers present in hepatocytes and biliary epithelial cells [23]. However, under some culture conditions, LECs can be forced to express a few hepatocytic traits such as albumin production [34,56], as well as to exhibit the activity of enzymes (e.g. gamma-glutamyl transpeptidase, GGT) usually restricted to biliary epithelial cells in postnatal rat liver [57]. This sort of plasticity has

led to the proposal that LECs may constitute a population of cells derived from a multipotential ‘stem’ cell compartment in postnatal rat liver [l]. However, by combining our recent findings on the differentiation potential of emerging embryonic rat liver cells [I81 with the data accumulated here on the differentiation status of LECs, it is now possible to propose a new hypothesis for the origin of LEC lineage. On the basis of the differential expression of BDS,, HE&, distinct CK sets, AFP and albumin, embryonic day 12 rat liver cells can be classified as a population of CKlCnegative bipotential progenitor cells capable of differentiating along the hepatocytic or biliary epithelial cell lineage [18, 28, 301. On the other hand, the present data show that a minor CKI 4-positive cell population lacking AFP and albumin

52

Fig. 7. Confocal immunofluorescence microscopy of 4-day-old rat liver cells containing CK14, BDS,, CK8 and/or vimentin. The four microscopic fields presented in Fig. 6b. d, f and h were examined by double immunofluorescence with a laser scanning confocal system. The first reaction involved the use of anti-BDS, (a), anti-CKI (c) and anti-vimentin (b, d) mouse monoclonal antibodies and their

detection with a FITC-tagged goat anti-mouse Ig. The second reaction for the detection CK14 was described in Fig. 6. The yellow/ orange color corresponded to cells that expressed two markers. Note that not all BDS,-positive cells are CKlCpositive. The same is true for cells of the Glisson’s capsule. Bur denotes 20 pm

emerges from a primary culture containing mostly CKlCnegative progenitor cells. This suggests, indeed, that LECs of the portal area constitute a population of partially differentiated cells that belong to a third cell lineage derived from progenitor cells. In the light of the observation that CKlCpositive LECs are BDS7positive, it could well be that they are derived from differentiating biliary epithelial cells. This possibility fits with our recently proposed model for liver epithelial cell differentiation, where differentiating progenitor cells progress through a ‘differentiation window’ that we defined as a short period during which the cells can switch reversibly from the hepatocytic to the biliary epithelial cell lineage [28]. In this context, the apparent high plasticity of biliary LECs suggests that they remain close to the differentiation window. There have been several attempts to establish the actual relationship between LECs and oval cells. The latter have been described as atypical epithelial cells that emerge in the liver of rats fed toxic doses of hepatocarcinogens [12, 19, 511. Although oval cells have originally been defined as an homogenous cell population [9, 421, recent data from studies performed on freshly isolated or cultured oval cells from the livers of rats fed various hepatocarcinogens indicate that they constitute a mixed cell type compartment containing sub-poulations of cells equivalent to immature hepatocytes or biliary epithelial

cells, as well as cells with phenotypic features of LEC lines derived from normal non-treated rat liver [12, 30, 421. For example, oval cells isolated from 3’-Me-DAB treated rats show an extremely low proliferative activity in primary culture [19], but within 2 weeks after seeding, CKlCpositive cells exhibiting the LEC morphological phenotype progressively emerge as a few colonies [19]. Yet, at seeding time, more than 97% of the oval cells express BDS, and GGT, two standard markers of biliary epithelial cells [19]. Therefore, the fact that biliary-like oval cells in primary culture express CK14 mRNAs (Fig. 2) and can be forced to produce albumin and to express tyrosine aminotransferase activity [ 191 would mean that they can partially switch to either the LEC or hepatocytic cell lineage. Until now, the location of LECs in postnatal rat liver has been uncertain [17]. It has been proposed that LECs are derived from terminal bile ductules, at a site in the periportal area that may contain a population of ‘hepatocytic facultative stem cells’ [23]. However, our immunolocalization analyses of 4-day-old rat liver demonstrate for the first time the presence of CK14-positive cells not only in the portal area but also in the Glisson’s capsule, thus suggesting the presence of two distinct CK14-containing cell populations in vivo. This conclusion is supported by the observation that CK14-positive cells in the portal region are vimentin-negative whereas

~

53

those in the Glisson’s capsule are vimentin-positive. Since the LEC lines that are usually derived are vimentin-positive, one would be tempted to conclude that they come from the Glisson’s capsule. However, it could well be that LECs derived from the portal area start to produce vimentin when put in culture. The next step then is to examine the biological significance of the presence of CK 14-positive cells in both biliary ductal structures and the Glisson’s capsule. Acknowledgements. We thank Dr. W.I. Waithe for the critical reading of the manuscript, M. Noel for her expert technical assistance, E. Lemay for her excellent secretarial assistance and G. Langlois and P. Paquin for their competent photography assistance. R. Blouin was recipient of a studentship from the Medical Research Council of Canada, and A. Loranger of a post-doctoral fellowship from the “Fonds de la Recherche en Sante du Quebec”, whereas M.-J. Blouin and I. Royal both held studentships from the Cancer Research Society Inc. This work was supported by the National Cancer Institute and the Medical Research Council of Canada.

References 1. Bisgaard HC, Thorgeirsson S S (1991) Evidence for a common cell of origin for primitive epithelial cells isolated from rat liver and pancreas. J Cell Physiol 147 :333-343 2. Blouin MJ, Marceau N (1991) Primary culture of fetal rat liver bipotential progenitor cells. J Tiss Cult Meth 13:117-120 3. Blouin R, Swierenga SHH, 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 4. Boynton AL, Whitfield JF, Klein LP (1983) Ca*+/phospholipid-depend protein kinase activity correlates to the ability of transformed liver cells to proliferate in Ca2’-deficient medium. Biochem Biophys Res Commun 1 15 :383-390 5. Bucher NLR, McGowan JA (1979) Regulatory mechanisms in liver regeneration. In: Wright R, Alberti GGMM, Karsan S, Millward-Sadler H (eds) Liver and biliary disease: a pathophysiological approach. W.B. Saunders, Philadelphia, pp 210227 6. Chessebeuf M, Olsson A, Bourret P, Desgres J, Guignet M, Maune B, Erissel B, Padieu P (1974) Long-term culture of rat liver epithelial cells retaining some hepatic functions. Biochimie 56: 1365-1379 7. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159 8. Deschenes J, Valet JP, Marceau N (1980) Hepatocytes from newborn and weanling rats in monolayer culture: isolation by perfusion, fibronectin-mediated adhesion, spreading, and functional activities. In Vitro 16: 722-730 9. Evarts RP, Nagy P, Nakatsukasa H. Marsden E, Thorgeirsson S S (1989) In vivo differentiation of rat liver oval cells into hepatocytes. Cancer Res 49: 1541-1 547 10. Eyken P van, Sciot R, Callea F, Van der Steen K, Moerman P, Desmet VJ (1988) The development of the intrahepatic bile ducts in man : a keratin-immunohistochemical study. Hepatology 8:15861595 11. Eyken P van, Sciot R, Desmet V (1988) Intrahepatic bile duct development in the rat : a cytokeratin-immunohistochemical study. Lab Invest 59: 52-59 12.Fausto N, Thompson NL, Braun L (1987) Purification and culture of oval cells from rat liver. In: Pretlow TGH 11, Pretlow TP (eds) Cell separation: methods and selected applications, vol4, Academic Press, Orlando, pp 45-77 13. Ferrari S, Battini R, Kaczmarek L, Rittling S, Calabretta B, Kim de Riel J, Philiponis V, Wei JF, Baserga R (1986) Coding

sequence and growth regulation of the human vimentin gene. Mol Cell Biol 6:361&3620 14. Fey EG, Wan KM, Penman S (1984) Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold : three-dimensional organization and protein composition. J Cell Biol98: 1973-1984 15. Franke WW, Schiller DL, Moll R, Winter S. Schmid E, Engelbrecht I, Denk H, Krepler R, Platzer B (1981) Diversity of cytokeratins. Differentiation specific expression of cytokeratin polypeptides in epithelial cells and tissues. J Mol Biol 153:933959 16. Fuchs E, Tyner AG, Guidice GJ, Marchuk D, RayChaudhury A, Rosenberg M (1987) The human keratin genes and their differential expression. Curr Top Dev Biol 22: 5 3 4 17. Gebhardt R, Schafer-Degenhart I (1988) Monoclonal antibodies directed against rat liver epithelial ccll lines selectively recognize bile duct epithelium in livers of adult rats. Cell Biol Toxicol4: 379-392 18. Germain L, Blouin MJ, 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 :4909-491 8 19. Germain L, Noel M, Gourdeau H, Marceau N (1988) Promotion of growth and differentiation of rat ductular oval cells in primary culture. Cancer Res 48 :368-378 20. Goldman R, Goldman A, Green K, Jones J , Lieska N, Yang HY (1985) Intermediate filaments: possible functions a cytoskeletal connection links between the nucleus and the cell surface. Ann NY Acad Sci 455: 1-17 21. Goldman RD, Zackroff RV, Steinert PM (1990) Intermediate filaments. An overview. In: Goldman RD, Steinert PM (eds) Cellular and molecular biology of intermediate filaments. Plenum Press, New York, pp 3-17 22. Green KJ, Jones JCR (1990) Interaction of intermediate filaments with the cell surface. In: Goldman RD, Steinert PM (ed) Cellular and molecular biology of intermediate filaments. Plenum Press, New York, pp 147-171 23. Grisham JW (1983) Cell types in rat liver cultures: their identification and isolation. Mol Cell Biochem 53/54: 23-33 24. Guguen-Guillouzo C (1986) Role of homotypic and heterotypic cell interactions in expression of specific functions by cultured hepatocytes. In: Guillouzo A, Guguen-Guillouzo C (eds) Research in isolated and cultured hepatocytes. John Libbey Eurotext Ltd/INSERM, pp 259-284 25. Hatzfeld M, Franke WW (1985) Pair formation and promiscuity of cytokeratins: formation in vitro of heterotypic complexes and intermediate-sized filaments by homologous and heterologous recombinations of purified polypeptides. J Cell Biol 101:1826-1841 26. Lazarides E (1980) Intermediate filaments as mechanical integrators of cellular space. Nature 238:249-256 27. Lu X, Lane EB (1990) Retrovirus-mediated transgenic keratin expression in cultured fibroblasts: specific domain functions in keratin stabilization and filament formation. Cell 62:681-696 28. Marceau N (1990) Cell lineages and differentiation programs in epidermal, urothelial and hepatic tissues and their neoplasms. Lab Invest 63 :4-20 29. Marceau N, Germain L, Goyette R, Noel M, Gourdeau H (1986) Cell origin of distinct cultured rat liver epithelial cells, as typed by cytokeratin and surface component selective cxpression. Biochem Cell Biol64: 788-802 30. Marceau N, Blouin MJ, Germain L, Noel M (1989) Role of different liver epithelial cell types in liver ontogenesis, regeneration and neoplasia. In Vitro Cell Dev Biol25:336-341 31. Michalopoulos GK,Strom SC, Jirtle RL (1986) Use of hepatocytes for studies of mutagenesis and carcinoge~~esis. In : Guillouzo A, Guguen-Guillouzo C (eds) Research in isolated and cultured hepatocytes. John Libbey Eurotext Ltd/INSERM, pp 333-352

54 32. Moll R, Franke WW, Schiller DL, Geiger B, Krepler R (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors, and cultured cells. Cell 31 :11-24 33. Moll R, Schiller DL, Franke WW (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 34. Nagy P, Evarts R, McMahon JB, Thorgeirsson SS (1989) Role of TGFP in normal differentiation and oncogenesis in rat liver. Mol Carcinog 2: 345-354 35. Nelson W, Sun TT (1983) The 50- and 58-Kdalton keratin classes as molecular markers for stratified squamous epithelia: cell culture studies. J Cell Biol 97:244251 36. O’Farrell PH (1 975) High-resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:40074021 37. Roop DR (1987) Regulation of keratin gene expression during differentiation of epidermal and vaginal epithelial cells. Curr Top Dev Biol22: 195-207 38. Roop DR. Cheng CK, Titterington L, Meyers CA, Stanley JR, Steinert PM, Yuspa SH (1984) Synthetic peptides corresponding to keratin subunits elicit highly specific antibodies. J Biol Chem 259: 8037-8040 39. Roop DR, Krieg TM, Mehrel T, Cheng CK, Yuspa SH (1988) Transcriptional control of high molecular weight keratin gene expression in multistage mouse skin carcinogenesis. Cancer Res 48: 3245-3252 40. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. 2nd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 41. Seglen PO (1987) Preparation of isolated rat liver cells. In: Prescott DM (ed) Methods in cell biology. Academic Press, New York, pp 29-83 42. SellS(1990)Is therealiverstemcell?CancerRes50:3811-3815 43. Sell S, Salman J (1984) Light- and electron-microscopic autoradiographic analysis of proliferating cells during the early stages of chemical hepatocarcinogenesis in the rat induced by feeding N-2-fluorenylacetamide in a choline-deficient diet. Am J Pathol 114:287-300 44.Sknat A, Vasseur M, Maillet L, Briilet P, Darmon M (1988) Sequence analysis of murine cytokeratin endoA (no8) cDNA. Evidence for mRNA species initiated upstream of the normal 5’ end in PCC4 cells. Differentiation 37:40-46 45. Singer PA, Trevor K, Oshima RG (1986) Molecular cloning and characterization of the EndoB cytokeratin expressed in preimplantation mouse embryos. J Biol Chem 261 :538-547 46. Sirica AE, Mathis GA, Sano N, Elmore LW (1990) Isolation,

culture, and transplantation of intrahepatic biliary epithelial cells and oval cells. Pathobiology 58:44-64 47. Sirica AE, Elmore LW, Sano N (1991) Characterization of rat hyperplastic bile ductular epithelial cells in culture and in vjvo. Dig Dis Sci 36:494-501 48. Smith GH, Mehrel T, Roop DR (1990) Differential keratin gene expression in developing, differentiating, preneoplastic, and neoplastic mouse mammary epithelium. Cell Growth Differ 1:161-170 49. Steinert PM, Roop DR (1988) Molecular and cellular biology of intermediate filaments. Annu Rev Biochem 57: 593-625 50. Stoler A, Kopan R, Duvic M, Fuchs E (1988) Use of monospecific antisera and cRNA probes to localize the major changes in keratin expression during normal and abnormal epidermal differentiation. J Cell Biol 107:427-446 51. Sun TT, Tseng SCG, Huang AJW, Cooper D, Schermer A, Lynch MH, Weiss R, Eichner R (1985) Monoclonal antibody studies of mammalian epithelial keratins: a review. Ann NY Acad Sci 455: 307-329 52. Swierenga SHH (1985) Use of low calcium medium in carcinogenicity testing - studies with rat liver cells. In: Weber NM (ed) In vitro models for cancer research. CRC Press, Boca Raton, pp 61-89 53.Tournier I, Legres L, Schoevaert D, Feldmann G. Bernuau D (1988) Cellular analysis of a-fetoprotein gene activation during carbon tetrachloride and D-galactosamine-induced acute liver injury in rats. Lab Invest 59:657-665 54. Towbin H, Stachelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:1350-1354 55. Tsao MS, Grisham JW (1987) Hepatocarcinomas, cholangiocarcinomas, and hepatoblastomas produced by chemically transformed cultured rat liver epithelial cells: a light- and electron-microscopic analysis. Am J Pathol 127: 168-181 56. Tsao MS, Smith JD, Nelson KG, Grisham JW (1984) A diploid epithelial cell line from normal adult rat liver with phenotypic properties of “oval” cells. Exp Cell Res 154:38-52 57. Tsao MS. Grisham JW, Cou BB, Smith J D (1985) Clonal isolation of populations of gamma-glutamyl-transpeptidase-positive and -negative cells from rat liver epithelial cells chemically transformed in vitro. Cancer Res 45:51345138 58. Tsao MS, Grisham JW, Nelson KG (1985) Clonal analysis of tumorigenicity and paratumorigenic phenotypes in rat liver epithelial cells chemically transformed in vitro. Cancer Res 45:5139-5144