Epidermal growth factor-induced selective phosphorylation of cultured rat hepatocyte 55-kD cytokeratin before filament reorganization and DNA synthesis.

We have reported previously that the addition of dexamethasone to cultured quiescent suckling rat hepatocytes in the presence of insulin, a culture condition which does not cause growth activation, induces a selective increase in the synthesis of the 49-kD/55-kD cytokeratin (CK49/CK55) pair over a 24-h period. This increased synthesis coincides with the formation of dense filament networks reminiscent of those observed in situ at the cell periphery (Marceau, N., H. Baribault, and I. Leroux-Nicollet. 1985. Can. J. Biochem. Cell Biol. 63:448-457). We show here for the first time that when EGF is added 48 h after insulin and dexamethasone, there is an early preferential phosphorylation of the CK55 of the CK49/CK55 pair, an induced filament rearrangement from the cell periphery to the cytoplasm, and a subsequent entry into S phase and mitosis after a lag period of 8 h. Indirect immunofluorescence microscopy with monoclonal antibodies to CK49 and CK55 indicate that, while before EGF treatment the cytokeratin filaments were mainly distributed near the cell periphery, the addition of EGF resulted in their reorganization to a predominantly cytoplasmic localization within less than 3 h. Antitubulin and anti-actin antibodies showed no detectable alteration in the distribution of microtubules and microfilaments. Pulse-chase measurements with [35S]methionine showed no apparent change in the turnover of either CK49 or CK55 during the period that precedes the initiation of DNA synthesis. 32P-labeling in vivo followed by SDS-PAGE demonstrated that CK55 was phosphorylated at a much higher level than CK49 in nonstimulated hepatocytes, and that the addition of EGF resulted in a selective stimulation of 32P-CK55 labeling within less than 30 min. Comparative analyses by two-dimensional PAGE of [35S]methionine and 32P-labeled cytokeratins at various times after EGF stimulation demonstrated a rapid increase in a first phosphorylated form of CK55 and the appearance of a second phosphorylated form at 30 min poststimulation. The changes in the relative proportion of nonphosphorylated and phosphorylated forms were confirmed by immunoblotting with the anti-CK55 monoclonal antibody. Determinations of the 32P-labeled phosphoamino acids of CK55 extracted from the gels demonstrated that the radioactivity was mostly in serine residues. Labeling of Triton-permeabilized hepatocytes with gamma 32P-ATP after treatment with EGF for 30 min to 3 h at 37 degrees C, also demonstrated a phosphorylation of CK55 and CK49 as well, implying that the EGF-responsive serine protein kinase is detergent insoluble and probably part of the surface membrane skeleton.(ABSTRACT TRUNCATED AT 400 WORDS)

Simple epithelium keratins 8 and 18 provide resistance to Fas-mediated apoptosis. The protection occurs through a receptor-targeting modulation.

Keratins 8 and 18 belong to the keratin family of intermediate filament (IF) proteins and constitute a hallmark for all simple epithelia, including the liver. Hepatocyte IFs are made solely of keratins 8 and 18 (K8/K18). In these cells, the loss of one partner via a targeted null mutation in the germline results in hepatocytes lacking K8/K18 IFs, thus providing a model of choice for examining the function(s) of simple epithelium keratins. Here, we report that K8-null mouse hepatocytes in primary culture and in vivo are three- to fourfold more sensitive than wild-type (WT) mouse hepatocytes to Fas-mediated apoptosis after stimulation with Jo2, an agonistic antibody of Fas ligand. This increased sensitivity is associated with a higher and more rapid caspase-3 activation and DNA fragmentation. In contrast, no difference in apoptosis is observed between cultured K8-null and WT hepatocytes after addition of the Fas-related death-factors tumor necrosis factor (TNF) alpha or TNF-related apoptosis-inducing ligand. Analyses of the Fas distribution in K8-null and WT hepatocytes in culture and in situ demonstrate a more prominent targeting of the receptor to the surface membrane of K8-null hepatocytes. Moreover, altering Fas trafficking by disrupting microtubules with colchicine reduces by twofold the protection generated against Jo2-induced lethal action in K8-null versus WT hepatocytes. Together, the results strongly suggest that simple epithelium K8/K18 provide resistance to Fas-mediated apoptosis and that this protection occurs through a modulation of Fas targeting to the cell surface.

Spheroidal aggregate culture of rat liver cells: histotypic reorganization, biomatrix deposition, and maintenance of functional activities.

Liver cells isolated from newborn rats and seeded on a non-adherent plastic substratum were found to spontaneously re-aggregate and to form, within a few days, spheroidal aggregates that eventually reached a plateaued diameter of 150-175 micron. Analyses on frozen sections from these spheroids by immunofluorescence microscopy using antibodies to various cytoskeletal elements and extracellular matrix components revealed a sorting out and a histotypic reorganization of three major cell types. A first type consisted of cells that segregated out on the aggregate surface forming a monolayer cell lining; a second type was identified as hepatocytes that regrouped in small islands often defining a central lumen; and a third group of cells reorganized into bile duct-like structures. This intercellular organization in the aggregates was paralleled by the accumulation of extracellular matrix components (laminin, fibronectin, and collagen) and their deposition following a specific pattern around each cell population structure. Determinations of albumin secretion and tyrosine aminotransferase induction by dexamethasone and glucagon at various times after the initiation of the cultures revealed a maintenance of the hepatocyte-differentiated functions for at least up to 2 mo at the levels measured at 3-5 d. It is concluded that cells dispersed as single cells from newborn rat liver conserve in part the necessary information to reconstruct a proper three-dimensional cyto-architecture and that the microenvironment so generated most likely represents a basic requirement for the optimal functioning of these differentiated cells.

Cell growth recovery after treatments at various supraoptimal temperatures.

The growth recovery kinetics of HeLa cells was investigated after treatments at intermediate (43-45 degrees) or high (49-55 degrees) supraoptimal temperatures for various periods of time (2 to 300 min) or after irradiations with nanosecond infrared CO2-laser pulses at energy densities equivalent to very high temperatures rises. After treatments at intermediate temperatures, single cells developed into colonies smaller than those obtained from nonheated control cells. Daily incorporations of pulsed [3H]thymidine in whole populations and microscopic observations of individual cell proliferation revealed a complex growth recovery for both survivors and dying cells. In contrast, colonies arising from cells treated at temperatures above 49 degrees do not differ in size from those developed from controls, and growth evaluations by [3H]thymidine and microscopy demonstrated that survivors resume normal proliferation immediately after treatments. The results which are supported by other studies on the effects of dose fractionation and metabolism status on cell survival are further discussed in relation to the "multistep" kinetic model for the cell response to hyperthermia.

Differential cytokeratin and alpha-fetoprotein expression in morphologically distinct epithelial cells emerging at the early stage of rat hepatocarcinogenesis.

The various liver cell populations emerging during the transitory reappearance of alpha-fetoprotein (AFP) in serum of 3'-methyl-4-dimethylaminoazobenzene-ingesting rats were analyzed in situ and in vitro on isolated cell preparations in terms of their cytokeratin and AFP expression using single and double indirect immunofluorescence microscopy. A polyclonal guinea pig antibody raised against cow hoof prekeratin, which recognized a Mr 52,000 cytokeratin, was found to react with bile ductular epithelial cells and oval cells but not with hepatocytes. A monoclonal antibody against a Mr 55,000 cytokeratin reacted not only with bile ductular and oval cells but also with hepatocytes. In contrast, a polyclonal antibody against porcine eye lens vimentin reacted with sinusoidal cells and stroma cells. To assess further the heterogeneity of the emerging cell populations, liver cells were isolated after 4 weeks of treatment and fractionated according to cell size and ploidy level into 4 fractions (I to IV) by velocity sedimentation at 1 X g. A cell-type analysis using AFP and albumin as functional markers revealed the presence of AFP-producing cells in Fraction IV at a mean velocity equivalent to that of newborn diploid rat hepatocytes, whereas most of the albumin-producing cells were distributed in Fractions I to III at velocities similar to those of adult tetraploid rat hepatocytes. A similar analysis based on the differential expression of Mr 52,000 and Mr 55,000 cytokeratins and vimentin in bile ductular and other diploid epithelial cells, hepatocytes, and mesenchymal cells showed that large cells in Fractions I to III were tetraploid hepatocytes, whereas viable cells present in Fraction IV were diploid epithelial cells and mesenchymal cells in proportion of 62 and 38%, respectively. These cell populations could be resolved further by changing the sedimentation time. A subsequent examination of the Mr 55,000 cytokeratin-containing diploid epithelial cells in Fraction IV using double immunofluorescence microscopy resolved three cell populations with respect to Mr 52,000 cytokeratin and AFP expression, namely, two cell populations expressing either protein marker and a third one containing both markers. These results suggest a ductular origin of oval cells and a possible relation to immature hepatocytes.

Promotion of growth and differentiation of rat ductular oval cells in primary culture.

Oval cells emerging in rat liver at the early period of 3-methyl-4-dimethylaminoazobenzene treatment constitute a mixed epithelial cell compartment with respect to alpha-fetoprotein (AFP) and cytokeratin differential expression, and include a subpopulation which exhibits a phenotype intermediate between ductular cells and hepatocytes (Germain et al., Cancer Res., 45:673-681, 1985). In the present study we have examined the developmental potential of ductular oval cells in primary culture and after in vivo transfer. The use of monoclonal and polyclonal antibodies directed against cytokeratins of Mr 39,000 (CK39), 52,000 (CK52), and 55,000 (CK55) and vimentin, and also monoclonal antibodies against exposed surface components of oval cells (BDS7) and normal hepatocytes (HES6) allowed us to establish the ductular phenotype of the oval cells. A highly enriched preparation of oval cells was obtained by perfusion/digestion of the liver with collagenase, treatment of the cell suspension with trypsin and DNase, selective removal of hepatocytes by panning using the anti-HES6 antibody, and cell separation by isopyknic centrifugation in a Percoll gradient. The procedure yielded about 8 x 10(7) cells, of which 95% expressed CK39, CK52, and BDS7, 84% gamma-glutamyl transpeptidase, and 5% albumin and AFP. The primary response of cultured oval cells to various combinations of growth and differentiation promoting factors was evaluated with respect to their capacity to initiate DNA synthesis as measured by [3H]thymidine labeling from day 1 to 3, and/or to produce albumin and AFP and express tyrosine aminotransferase. Culture in the presence of either serum or clot blood extract resulted in a low proliferative activity with less than 5% of the nuclei being labeled. Over a 5-day period, fusion of a large portion of the oval cells led to multinucleated cells. When the cells were cultured in the presence of an elaborate combination of supplements [minimum essential medium containing 1 mM pyruvate, 0.2 mM aspartate, 0.2 mM serine, 1 mM tyrosine, 1 mM proline, 1 mM phenylalanine and supplemented with 20% clot blood extract, 10 ng/ml oxidized bile acids, 17 microM bilirubin, 10 ng/ml cholera toxin, 1 microM dexamethasone, 2.5 micrograms/ml insulin, 50 mM beta-mercaptoethanol, and 5 micrograms/ml transferrin (medium MX)], the labeling index increased to around 30% and the level of cell fusion greatly decreased. The addition of dimethyl sulfoxide further enhanced the initiation of DNA synthesis, while sodium butyrate acted as an inhibitor.(ABSTRACT TRUNCATED AT 400 WORDS)

Biliary epithelial and hepatocytic cell lineage relationships in embryonic rat liver as determined by the differential expression of cytokeratins, alpha-fetoprotein, albumin, and cell surface-exposed components.

The differentiation patterns of epithelial cells in fetal rat liver were analyzed in situ and in primary culture by indirect immunofluorescence microscopy using polyvalent and monoclonal antibodies directed against cytokeratins with molecular weights of 55,000 (CK55), 52,000 (CK52), and 39,000 (CK39) and against vimentin, albumin, alpha-fetoprotein, and surface-exposed components of bile ductular cells (BDS7) and hepatocytes (HES6). The anti-CK52 antibody, which reacted with biliary ductal cells in the liver of adult rats (Germain et al., Cancer Res., 45:673, 1985; Germain et al., Cancer Res., 48: 368-378, 1988), stained essentially all of the epithelial cells of embryonic day 12 (E12) rat liver. The anti-BDS7 antibody reacted with a few cell foci, which enlarged and became more numerous at later developmental ages. At E12 essentially all of the cells were positive for albumin and alpha-fetoprotein but did not express HES6. In fact HES6 was not detected until E15 in cells with the morphology of immature hepatocytes. By E18 staining with anti-HES6 reached the level of that observed on adult rat hepatocytes. Liver cells isolated from E12 rats were seeded on fibronectin-treated dishes and their response to various combinations of growth- and differentiation-promoting factors was evaluated with respect to their capacity to express either the hepatocytic or the bile ductular phenotype. In medium supplemented with serum, insulin, dexamethasone, and dimethyl sulfoxide, the E12 cells were capable of differentiating in culture to mimic over a 6-day period the sequential phenotypic changes which occur in vivo during normal hepatoontogeny, namely the loss of CK52 and the appearance of HES6. In contrast, the addition of sodium butyrate to the above supplement mixture resulted in the massive expression of BDS7. To further assess the developmental potential of fetal rat liver cells toward the biliary epithelial cell lineage, the in vitro assay was performed using cells isolated from livers of E18 rats and also from 2-day-old (P2) and P14 rats. While a slight expression of BDS7 was induced in cell culture from E18 liver, essentially no expression was observed in cells from postnatal livers. These findings strongly suggest that the emerging hepatic tissue in rat embryo is composed of bipotential progenitor epithelial cells that are capable of differentiating along either the hepatocytic or biliary epithelial cell lineage. These observations constitute a clear demonstration of the plasticity of liver differentiation and also provide a striking example of environmental influences on liver progenitor cell differentiation.

Synthesis and degradation of heat shock proteins during development and decay of thermotolerance.

Morris hepatoma 7777 cells, heat conditioned at 43 degrees for 0.5 hr, become gradually thermoresistant during an incubation at 37 degrees as judged by their ability to form colonies following a second heat challenge. Pulse incorporation of [35S]methionine into proteins at various times after the conditioning treatment and subsequent fractionation of the proteins by polyacrylamide gel electrophoresis indicate that the gradual putative modifications occurring at the cellular level and leading to the thermotolerance state are accompanied by an elevated synthesis above the normal level of a small set of polypeptides with apparent molecular weights of 27,000, 65,000, 68,000, 70,000, 89,000, and 107,000. Both thermotolerance development and protein induction are completed after a 6- to 8-hr period. At the end of this period, thermotolerance is at its maximum level and heat shock protein synthesis is returned to normal. This acquired thermal resistance eventually disappears between 60 and 80 hr following conditioning treatment. In a parallel manner, the heat shock-induced proteins synthesized during the first 4 hr following the conditioning treatment are maintained in the cells at a high level for several hr but become undetectable by 82 hr. The results provide strong circumstantial evidence that heat shock proteins are involved in the acquisition, maintenance, and decay of thermotolerance.

The relative rates of degradation of the plasma membrane glycoproteins from normal rat liver.

Rat liver plasma membranes, as fractionated by sodium dodecylsulfate/polyacrylamide gel electrophoresis, have been examined for the incorporation in their subunits of radioactive leucine, glucosamine and fucose. Specific spectra were obtained. In contrast to leucine, where the activity is distributed in many peaks all over the fractions, the glucosamine and fucose activities are found principally in the high molecular weight region. The relative rates of degradation of the glycoprotein components of the plasma membrane have been measured in normal liver using the double isotope technique. A marked heterogeneity of degradation was observed among the different subunits and a correlation between the rate of degradation and the size of the labelled subunits was found with glucosamine and fucose as well with leucine. This suggests a similar mode for the degradation of these membrane components.

Differential phosphorylation of CK8 and CK18 by 12-O-tetradecanoyl-phorbol-13-acetate in primary cultures of mouse hepatocytes.

The phosphorylation of cytokeratin was investigated in primary cultures of hepatocytes. The two hepatocyte cytokeratins CK8 and CK18 (55,000 and 49,000 M(r) respectively) were phosphorylated, CK8 being more phosphorylated than CK18. Treatment of the hepatocytes with 150 nM 12-O-tetradecanoyl-phorbol-13-acetate (TPA) an activator of protein kinase C induced a transient increase in the level of phosphorylation of CK8 but not CK18. This effect was maximal after 15 min of TPA treatment and was maintained for up to 3 h. After 22 h of treatment with TPA, which down-regulates protein kinase C, CK8 phosphorylation was returned to the basal level. Further addition of TPA to the 22-h treated cells did not cause an increase in CK8 phosphorylation. Indirect immunofluorescence microscopy with a monoclonal antibody to CK8 indicated that while the addition of TPA induced the formation of granular cytokeratin aggregates in some hepatocytes, in most hepatocytes no major changes in the intermediate filament network were observed. Staining for actin showed that actin microfilaments were rapidly reorganized after the treatment and a loss of stress fibres were observed. We propose that CK8 is an in vivo substrate for protein kinase C and that the specific phosphorylation of CK8 plays a role in protein kinase C signal transduction.

Thermotolerance and heat shock proteins induced by hyperthermia in rat liver cells.

Hepatic epithelial cells become thermotolerant when conditioned with a 30 minute heat-treatment at 43 degrees C. The effect reaches a full amplitude after a 4-8 hour period at 37 degrees C and lasts for more than one day at a level corresponding to a 50-fold increase in cellular thermoresistance. During the development period, electrophoretic patterns of proteins from cells incubated in presence of 35S-methionine reveal an increased synthesis of a small set of proteins with molecular weights of 107, 89, 70, 68 and 27KD. The maximal synthesis of the induced proteins occurs concomitantly with the maximal increase of cell thermotolerance and has returned to normal when thermotolerance levels off. The induction of specific protein synthesis is also observed in other liver epithelial cells of normal and cancerous origins and in freshly isolated hepatocytes. It is suggested that the accumulation of these proteins in the cells plays a role in the process leading to a thermotolerant state.

Immunocytochemical localization of actin in the pancreatic exocrine cell.

Immunoreactive actin molecules have been localized in pancreatic acinar cells by the protein A-gold technique. The labeling was found at the level of the filamentous cell web, and in close association with the Golgi cisternae, condensing vacuoles, and zymogen granules delimiting membranes as well as with the plasma membrane. A weak labeling was also present over the dense content of the zymogen granules. The association of actin with different membranes implicates that contractile proteins might constitute structural membrane proteins and, thus, might play an important role in protein secretion.

Gap junctional intercellular communication and connexin expression in normal and SV 40-transformed human liver cells in vitro.

The gap junction intercellular communication (GJIC) capacity of two normal human liver-derived epithelial cell strains and their SV40 large T oncogene-transformed counterparts was examined. In homologous cultures the GJIC capacity of the transformed cells was considerably less than the parental cells. In heterologous cultures, transformed cells appeared to be able to form GJIC channels with normal cells. Only non-transformed cells expressed connexin (cx) 43 gene and cx 26 or cx 32 transcripts were not detectable in any cell strains tested. When GJIC was assayed in the presence of the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13- acetate (TPA), all four strains showed a marked sensitivity to TPA in inhibitory activity at 1-10 ng/ml. In contrast to a rat liver epithelial cell line, this effect of TPA did not appear to become refractory even after 24 h exposure. These studies demonstrate that GJIC of human liver cells in culture can be decreased by viral oncogene and tumour promoter action. Such disturbance may be an important component of the carcinogenic activity of these agents.

Expression of organ-specific structures and functions in long-term cultures of aggregates from adult rat liver cells.

Freshly isolated hepatocytes, endothelial cells, Kupffer and fat-storing cells from adult rats were found to be able to form spheroidal aggregates within 24 hr when cultured under serum-free and rotatory conditions. The ultrastructure, including intracellular organization and extracellular matrix deposition was investigated in 7-day aggregates by scanning and electron microscopy. The different cell types expressed a histotypic organization and reconstructed their extracellular matrix (fibronectin and laminin) as well as junctional complexes (desmosomes, tight junctions and gap junctions). The aggregates preserved, over a period of at least 7 days, high albumin expression (mRNA) and secretion as well as high secretion of the acute phase protein T-kininogen. Dexamethasone (10(-7) M) increased the tyrosine aminotransferase activity fourfold after a 24-hr exposure. Aggregates cultured in the presence of 10(-7) M dexamethasone showed an increased expression and secretion of albumin concomitantly with a decrease in T-kininogen secretion. Induction of 7-ethoxycoumarin O-deethylase (ECOD) was effective after exposure to 3-methylcholanthrene or Aroclor for 48 hr. Up to a 13-fold increase in (ECOD) activity was found. The results demonstrate that spheroidal cultures of liver cell aggregates obtained under rotatory conditions provide a useful model for studies on the combined cellular interactions between non-parenchymal liver cells and hepatocytes from adult rats.