Regulation of gene expression in adult rat hepatocytes cultured on a basement membrane matrix

Freshly isolated adult rat hepatocytes, when cultured on type I collagen (commercially available as Vitrogen), assume a polygonal shape, form a stable monolayer within 24 hours, but lose the capacity to express some liver-specific functions over time in culture. We incubated hepatocytes in a serum-free medium on a reconstituted basement membrane gel, "matrigel" (prepared from an extract of extracellular matrix of the murine Engelbreth-Holm-Swarm sarcoma), and observed that the cells adhered firmly, remained rounded as single cells or clusters, and maintained liver-specific gene expression for more than 1 week in vitro. Hepatocytes on matrigel secreted substantially higher amounts of albumin, transferrin, haptoglobin, and hemopexin, Northern blot analyses of extracted cellular RNA, expressed increased amounts of mRNA for the liver-specific protein albumin (as compared with cells on vitrogen). In cultures treated with phenobarbital, cytochrome P-450b, and cytochrome P-450e, mRNAs and proteins were barely detectable in cells on Vitrogen but were induced to levels similar to those in the liver in vivo in matrigel cultures. Likewise, the use of matrigel greatly enhanced the induction of mRNA and protein for P-450c by 3-methylcholanthrene and for P-450p by steroidal and nonsteroidal inducers. However, neither substratum permitted induction of P-450d by 3-methylcholanthrene, suggesting that the effects of matrigel are selective even for expression in liver of members of the superfamily of cytochrome P-450 genes. Within 5 days in cultures on Vitrogen, hepatocytes expressed detectable amounts of fetal liver aldolase activity and also mRNA for vimentin and type I collagen, each considered a phenotypic change reflecting hepatocyte "dedifferentiation." None of these was present in cells on matrigel. Responsiveness to mitogenic stimuli, as judged by incorporation of 3H-thymidine into DNA, was also decreased in hepatocytes cultured on matrigel. Finally, there was a remarkable increase in the levels of both matrices during the first 2 days in culture. However, the continuously cytoskeleton mRNA over time in culture than did the rounded cells on matrigel. We conclude that hepatocytes cultured on matrigel, as opposed to the standard collagen, exhibit remarkably enhanced expression of many liver-specific functions.     

Expression of cell adhesion molecule and albumin genes in primary culture of rat hepatocytes

Changes in the expression of cell adhesion molecule and albumin genes were investigated in primary cultures of rat hepatocytes with and without poly- N-p -vinylbenzyl-D-lactonamide (PVLA) coating of the dishes. In PVLA-coated cultures, hepatocytes aggregated into spheroids and expressed liver cadherin and albumin mRNAs at higher levels. In uncoated cultures, hepatocytes revealed low levels of cadherin and albumin mRNAs, but higher levels of integrin alpha-1 mRNA. The changes in mRNA levels of liver cadherin and integrin alpha-1 coordinated well with those in spheroid and monolayer formation of hepatocytes, respectively. These results suggest that, in the PVLA-coated culture, hepatocytes expressed cadherin at higher levels to promote cell-cell adhesion and further maintain the differentiated function, such as albumin secretion, for prolonged times.     

Modulation of fetal and neonatal rat hepatocyte functional activity by glucocorticoids in co-culture

Fetal and neonatal rat hepatocytes were cultured alone or in association with another liver epithelial cell type, in a medium with or without hydrocortisone. Secretion of albumin and alpha-fetoprotein decreased in pure hepatocyte culture, whereas in co-culture it remained stable for several days. Furthermore, addition of hydrocortisone to the co-culture medium induced a rapid increase in albumin production which was maintained at a high level. In contrast, alpha-fetoprotein production was inhibited. At the same time, an abundant extracellular material was secreted between and around hepatocyte colonies. The results demonstrate that the reciprocal relation between albumin and alpha-fetoprotein production which occurs during in vivo perinatal hepatocyte maturation is also observed in vitro. Both cell-cell contacts and glucocorticoids play a key role in this process. It appears that fetal and neonatal hepatocytes can maturate when maintained in a co-culture system.     

Modulation of alpha-fetoprotein, albumin and transferrin gene expression by cellular interactions and dexamethasone in cocultures of fetal rat hepatocytes

Fetal hepatocytes were cultured alone or in association with primitive biliary cells (RLEC) in the presence or absence of dexamethasone. Cell-cell contacts were established 3 h or five days after hepatocyte seeding and their effects on hepatocyte growth and functional activities were evaluated in the presence or absence of dexamethasone. Establishment of cellular interactions with RLEC in coculture decreased hepatocyte growth, while it stimulated production of alpha-fetoprotein, albumin and transferrin. Addition of dexamethasone to coculture inhibited alpha-fetoprotein secretion and maintained the synthesis rate of albumin and transferrin together with an additional inhibition of DNA synthesis. The levels of mRNAs corresponding to the three proteins were also measured. We observed that the levels of alpha-fetoprotein, albumin and transferrin secretion in cocultures maintained in the presence or absence of dexamethasone were well correlated with the relative amounts of their corresponding mRNAs. Consequently, it may be assumed that the primitive mechanism involved in the increased functional activity of fetal hepatocytes in coculture is of pretranslational origin. Furthermore, the present data provide evidence that heterotypic interactions and dexamethasone act as distinct modulators of growth and maturation of fetal rat hepatocytes.     

Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type

When adult rat hepatocytes were co-cultured with another liver epithelial cell type in a medium supplemented or not with fetal calf serum (FCS), it was found that 1. They survived for more than 2 months 2. Albumin secretion levels remained high over the whole culture period 3. Decreased secretion might be reversed 4. This protein secretion activity appeared to be dependent upon both the presence of cell-cell contacts and the production of an extracellular material. The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatocytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.     

Cell contact but not junctional communication (dye coupling) with biliary epithelial cells is required for hepatocytes to maintain differentiated functions

Specific differentiated gene expression and the morphology of adult rat hepatocytes can be maintained for as long as 8 weeks in vitro only when they are cultured in the presence of biliary epithelial cells; when primary hepatocytes are cultured alone, they lose these functions within 2 to 3 days. We obtained evidence suggesting that contact between hepatocytes and biliary epithelial cells is necessary for maintaining hepatocyte functions. We examined whether junctional communication between and among hepatocytes and biliary epithelial cells is required for long-term maintenance of hepatocyte functions, using a dye-transfer method, in three co-cultures: (1) hepatocytes and biliary epithelial cells prepared from Sprague-Dawley rats; (2) hepatocytes from Sprague-Dawley rats and epithelial cells of the IAR 20 line, originally established from BDVI rats; and (3) hepatocytes from BDVI rats and IAR 20 epithelial cells. The established epithelial cell line (IAR 20) and early-passage cultures of biliary epithelial cells maintained hepatocyte-specific functions in culture for 40 and 70 days, respectively, but the latter induced more stable maintenance of albumin secretion. Hepatocytes cultured alone lost their characteristic morphology within 5 to 8 days, and almost no dye transfer was observed. In co-cultures, the capacity of biliary epithelial cells to communicate among themselves remained relatively high throughout the culture period, whereas hepatocytes showed almost no junctional communication at an early phase of culture and first began to communicate after 2 weeks, communication capacity increasing for at least the next 10 days of culture. The most notable finding was that there was no dye transfer between hepatocytes and biliary epithelial cells in any co-culture system. These results suggest that the maintenance of hepatocyte-specific functions requires intercellular contact but probably not gap-junctional communication between hepatocytes and biliary epithelial cells. This system is useful for studying heterotypic cell-cell interactions and the control of gene expression.     

Chronic iron overload inhibits protein secretion by adult rat hepatocytes maintained in long-term primary culture

Short-term pure cultures and long-term cocultures of adult rat hepatocytes with rat liver epithelial cells, presumably derived from primitive biliary cells, were used to define in vitro models of iron overloaded hepatocytes in order to understand the molecular mechanism responsible for liver damage occurring in patients with hemochromatosis. In vitro iron overload was obtained by daily addition of ferric nitrilotriacetate to the culture medium. A concentration of 20 microM ferric salt induced hepatocyte iron overload with minimal cytotoxicity as evaluated by cell viability, morphological changes of treated cells and cytosolic enzyme leakage into the culture medium. The effects of iron overload on protein biosynthesis and secretion were studied in both short-term pure cultures and long-term cocultures of hepatocytes. The amounts of intracellular and newly synthesized proteins were never modified by the iron treatment. Furthermore, neither the relative amounts of transferrin and albumin mRNAs nor their translational products were altered by iron overload. Moreover, no change in the transferrin isomeric forms were observed in treated cells. In contrast, a prolonged exposure of cocultured hepatocytes to 20 microM ferric salt led to a significant decrease in the amount of proteins secreted in the medium. This decrease included the two major secreted proteins, namely albumin and transferrin, and probably all other secreted proteins. These results demonstrate that iron loading alters neither the total nor the liver specific protein synthesis activity of cultured hepatocytes. They suggest that chronic overload may impede the protein secretion process.     

Functional engineering of hepatocytes via heterocellular presentation of a homoadhesive molecule, E-cadherin.

Engineering functional activity of liver cell cultures requires the modulation of specific cell-cell interactions. We have investigated the quantitative role of systematically varied presentation of the cell-cell adhesion molecule, E-cadherin, on the differentiated function of cocultured parenchymal liver cells, hepatocytes. Specifically, we incorporated different proportions of E-cadherin transfected L-929 chaperone cells and untransfected chaperone cells, within cultures of primary rat hepatocytes on a collagen substrate. By using a strongly adhesive substrate that restricted cadherin-induced variations in cell spreading and growth-arresting chaperone cells, we could carefully isolate the potential role of cell-cell adhesion on cell differentiation. Using immunofluorescence microscopy, we confirmed that cadherins expressed at hepatocyte-hepatocyte contacts as well as hepatocyte-chaperone contacts were crossreactive. However, hepatocytes cocultured with cadherin-presenting chaperone cells had a 55-65% increase in longterm function over hepatocytes cocultured with control, nonpresenting chaperone cells. Notably, the cadherin-induced increase in function occurred over and above the basal, coculture-induced functional elevation. Further, we quantified the stoichiometric importance of cadherin contacts by comparing established markers of hepatocyte functional activity across a graded range of E-cadherin presentation. At low levels of cadherin-mediated contacts, the induction of differentiated function was weak, while high levels of contacts elicited a marked increase in function. Thus, hepatocyte biochemical functions (albumin and urea secretion) were biphasically governed by the degree of cadherin-based contacts presented during culture. Overall, our results demonstrate the unequivocal role of cell-cell adhesion molecules in hepatocyte functional engineering, through the graded use of cadherin presentation from functionally incompetent, heterotypic chaperone cells.     

Cellular density regulation of plasminogen gene expression in mouse hepatocytes

The liver produces a variety of proteins including plasminogen. Plasminogen is pro-enzyme that is converted into plasmin by plasminogen activator. Plasmin has a broad substrate spectrum and participates in several biological processes, such as fibrinolysis, tissue remodeling, cell migration, angiogenesis and embryogenesis. In the present study, the regulation of plasminogen expression in mouse hepatocytes was investigated in the primary culture system. Expression level of plasminogen mRNA in the culture at the low cell density condition (0.2 x 10(5) cells / cm(2)) was compared with that at the high cell density condition (1.0 x 10 (5) cells / cm(2)). In the low cell density culture, the expression level of plasminogen mRNA decreased by a time-dependent manner. However, mRNAs for albumin and alpha(2)-antiplasmin were not influenced by the low cell density culture. On the other hand, in the high cell density culture, plasminongen mRNA expressed constantly as well as albumin and alpha(2)-antiplasmin mRNAs. Thus, the decrease in plasminogen mRNA expression could specifically occur when the density of hepatocytes was low. The down-regulation of plasminogen mRNA in the low cell density culture is not observed in the presence of cycloheximide, suggesting that the de novo protein synthesis is required for the regulatory mechanism. These findings indicate that the expression of plasminogen mRNA from hepatocyte is dependent on the cell density and the stimulation by cell-cell contact may be an important factor for the constitutive expression of plasminogen gene in hepatocytes.     

Phenotype of hepatocyte spheroids in synthetic thermo-reversible extracellular matrix

Aggregates of specific cells are often regarded as a better form in artificial organs and mammalian cell bioreactors in terms of cell-specific functionality. In this study, the morphology and liver-specific functions of freshly harvested primary rat hepatocytes, which were cultivated as spheroids and entrapped in a synthetic thermo-reversible extracellular matrix, were examined and compared to a control (hepatocytes in single cell form). A copolymer of N-isopropylacrylamide (98 mole % in feed) and acrylic acid (poly(NiPAAm-co-AAc)), a thermo-reversible copolymer gel matrix, was used to entrap hepatocytes either in spheroids or single cells. During a 7-day culture period, the spheroids maintained higher viability and produced albumin and urea at a relatively constant rate, while the single cell culture showed a slight increase in cell numbers and a reduction in albumin secretion. Hepatocytes cultured as spheroids present a potentially useful three-dimensional cell culture system for application in a bioartificial liver device.     

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 μM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.