The control of DNA synthesis in primary cultures of hepatocytes from adult and young rats: interactions of extracellular matrix components, epidermal growth factor, and the cell cycle

Hepatocytes from adult and 4-week-old rats cultured on one of several extracellular matrix components were stimulated to replicate by epidermal growth factor (EGF). DNA synthesis was increased at 44-48 hr in adult hepatocytes and at 24, 48, and 72 hr in hepatocytes from young rats when EGF was added 2 hr after explantation. When EGF was added at 24 hr, maximal DNA synthesis of adult hepatocytes was observed at 48 hr, whereas that of 4-week-old hepatocytes was seen at 48 and 72 hr. Ten ng EGF per ml was the optimal concentration for maximal DNA synthesis in both adult and young cells. DNA synthesis decreased with increasing cell density, but this effect was less in hepatocytes from young than in those from adults. When hepatocytes were cultured on substrata consisting of individual extracellular matrix components, neither the time that adult cells needed to respond to EGF nor the time from stimulation by EGF to the peak of maximal DNA synthesis was altered in either adult or young cells. The optimal EGF concentration for maximal DNA synthesis and the cell density control of replication were also not altered by the substrata used. Substrata made from each of the extracellular matrix components studied enhanced DNA synthesis of adult and young hepatocytes stimulated by EGF in the following decreasing order: fibronectin, type IV collagen, type I collagen, and laminin. In both adult and young hepatocytes the enhancement of DNA synthesis was greatest when cultured on fibronectin. Thus the initiation and magnitude of DNA synthesis in primary cultures of rat hepatocytes were altered both by the age of the donor and the substratum on which the cells were explanted.     

Integrin alpha5-induced EGFR activation by prothrombin triggers hepatocyte apoptosis via the JNK signaling pathway

We have previously shown that prothrombin, a blood coagulation factor, can cause an inhibition of DNA synthesis in normal rat hepatocytes. To explore the mechanisms of this prothrombin action, we examined its effects on the activation of fibronectin receptor integrin alpha5, since fibronectin was found to be degraded by prothrombin actions in primary hepatocyte cultures. We found that prothrombin treatment of rat hepatocytes without addition of any growth factor induced tyrosine phosphorylation of integrin alpha5 and interaction of integrin alpha5 with epidermal growth factor receptor (EGFR), leading to EGFR tyrosine phosphorylation at tyrosine residues Tyr-845 and Tyr-1173. EGFR tyrosine phosphorylation triggered phosphorylation of its down-stream target Shc and the activation of the c-Jun N-terminal kinase (JNK) pathway. Prothrombin also induced hepatocyte apoptosis, a change in cell shape and activation of caspase 3 pathway. The JNK pathway is most likely involved in prothrombin-induced hepatocyte apoptosis, because pre-treatment of hepatocytes with JNK kinase inhibitor II (SP600125) antagonized these prothrombin actions. The data suggest that integrin-related EGFR activation by prothrombin can induce cell growth inhibition and apoptosis via an EGFR-JNK signaling pathway.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.     

Regulation of cell adhesion signaling by synthetic glycopolymer matrix in primary cultured hepatocyte

Control of cell-matrix interactions is a central principle for the design of biomaterial in tissue engineering. In this study, we evaluated a synthetic glycopolymer, which is recognized by the asialoglycoprotein receptor (ASGPR) expressed on the surface of hepatocytes, as an artificial matrix to regulate integrin-mediated signaling. The phosphorylation of focal adhesion kinase was restricted in hepatocytes cultured on the glycopolymer compared with fibronectin. In addition, there was no reorganization of cytoskeleton-related proteins such as actin filaments, microtubules, and vinculin in hepatocytes cultured on the glycopolymer. DNA synthesis and cyclin D1 expression were suppressed in hepatocytes grown on the glycopolymer as compared with those grown on fibronectin and collagen. The data suggest that the glycopolymer will be a good artificial matrix to regulate integrin-mediated signaling and cell growth through the unique ASGPR-carbohydrate interaction.     

Modulation of protein synthesis and secretion by substratum in primary cultures of rat hepatocytes

Hepatocytes isolated by perfusion of adult rat liver and cultured on substrata consisting of one or more of the major components of the liver biomatrix (fibronectin, laminin, type IV collagen) have been examined for the synthesis of defined proteins. Under these conditions, tyrosine amino transferase, a marker of hepatocyte function, is maintained at similar levels in response to dexamethasone over 5 days in culture on each substratum, and total cellular protein synthesis remains constant. By contrast, there is a rapid decrease in synthesis and secretion of albumin and a 3-7-fold increase in synthesis and secretion of alpha-fetoprotein which are most marked on a laminin substratum, but least evident on type IV collagen, and an increased synthesis of fibronectin and type IV collagen. The newly synthesized matrix proteins are present in the cell layer as well as in cell secretions. The enhanced synthesis of fibronectin is less in cells seeded onto a fibronectin substratum than on laminin or type IV collagen substrata, and its synthesis by hepatocytes seeded onto a mixed substratum of laminin and fibronectin is down-regulated by fibronectin in a dose-related manner. Similarly, type IV collagen synthesis is less when the cells are seeded on the homologous matrix protein substratum than on heterologous substrata. These results indicate that hepatocytes cultured in serum-free medium on substrata composed of components of the liver biomatrix maintain certain functions of the differentiated state (tyrosine amino transferase), lose others (albumin secretion) and switch to increased synthesis of matrix components as well as fetal markers such as alpha-fetoprotein. The magnitude of these effects depends on the substratum on which the hepatocytes are cultured.     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells

Cytoskeletal elements, enriched in intermediate-sized filaments and insoluble in buffers of high salt concentrations and Triton X-100, were isolated from various cultures of rat hepatocytes and hepatoma cells, and their proteins were studied by one- and two-dimensional gel electrophoresis and immunofluorescence microscopy. The cells examined included several permanent cell lines (MH₁C₁, HTC, hepatoma 72/22, clone 12 from Gunn rat hepatocytes, and cell clones from normal rat hepatocytes), as well as freshly dissociated hepatocytes that were cultured and allowed to attach to substratum for increasing periods of time, beginning at 24 h after removal of the liver from the animal. Filaments containing vimentin, which were not found in hepatocytes grown in liver tissue, were detected in most of the cultured hepatocytes and hepatoma cells, except in MH₁C₁ cells, and were shown to be newly synthesized during the first days of primary culture. Maintenance of expression of filaments containing proteins immunologically related to epidermal prekeratin (‘cytokeratins’) was observed in all cells examined but HTC cells. Detailed comparison of the cytokeratin polypeptides present in various hepatocyte and hepatoma cell cultures showed that, in some of the cultured epithelial liver cells, cytokeratins are expressed which are identical with, or similar to, those of normal hepatocytes grown in the liver. On the other hand, differences in cytokeratin polypeptides were also found among different hepatocyte-derived cell cultures. Changes of expression of cytoskeletal proteins were found to occur even in cloned cell populations, and cells positive for certain cytokeratins could be seen next to other cells that were negative.The results demonstrate that profound changes of cytoskeletal composition, especially concerning intermediate filament protein patterns, can occur during culturing in vitro. Moreover, we show that different intermediate filament proteins can be expressed in different hepatocyte-derived cell cultures and that changes of cytoskeletal composition can occur in a given cell population, without obvious effects on cell growth rate and cell morphology. During culturing of hepatocytes and hepatoma cells, there seems to be a general tendency to induce the production of vimentin filaments as well as to maintain the production of cytokeratins similar to the hepatocyte-specific cytokeratins in liver tissue. However, the demonstrated exceptions speak against a role of these filament proteins as prerequisites for the growth of an epithelial cell in vitro. Rather, the presence of filaments containing certain cytokeratins and of desmosomes in epithelial cells growing in vitro seems to reflect the synthesis of specific differentiation markers which may be lost, independently, in some cells during culturing.     

Stimulation of integrin-mediated cell contractility by fibronectin polymerization

Ligation of integrins with extracellular matrix molecules induces the clustering of actin and actin-binding proteins to focal adhesions, which serves to mechanically couple the matrix with the cytoskeleton. During wound healing and development, matrix deposition and remodeling may impart additional tensile forces that modulate integrin-mediated cell functions, including cell migration and proliferation. We have utilized the ability of cells to contract floating collagen gels to determine the effect of fibronectin polymerization on mechanical tension generation by cells. Our data indicate that fibronectin polymerization promotes cell spreading in collagen gels and stimulates cell contractility by a Rho-dependent mechanism. Fibronectin-stimulated contractility was dependent on integrin ligation; however, integrin ligation by fibronectin fragments was not sufficient to induce either tension generation or cell spreading. Furthermore, treatment of cells with polyvalent RGD peptides or pre-polymerized fibronectin did not stimulate cell contractility. Fibronectin-induced contractility was blocked by agents that inhibit fibronectin polymerization, suggesting that the process of fibronectin polymerization is critical in triggering cytoskeletal tension generation. These data indicate that Rho-mediated cell contractility is regulated by the process of fibronectin polymerization and suggest a novel mechanism by which extracellular matrix fibronectin regulates cytoskeletal organization and cell function.     

The role of collagen structure in mitogen stimulation of ERK,cyclin D1 expression,and G1-S progression in rat hepatocytes

Adhesion to type 1 collagen can elicit different cellular responses dependent upon whether the collagen is in a fibrillar form (gel) or monomeric form (film). Hepatocytes adherent to collagen film spread extensively, express cyclin D1, and increase DNA synthesis in response to epidermal growth factor, whereas hepatocytes adherent to collagen gel have increased differentiated function, but lower DNA synthesis. The signaling mechanisms by which different forms of type I collagen modulate cell cycle progression are unknown. When ERK MAP kinase activation was analyzed in hepatocytes attached to collagen film, two peaks of ERK activity were demonstrated. Only the second peak, which correlated with an increase of cyclin D1, was required for G1-S progression. Notably, this second peak of ERK activity was absent in cells adherent to collagen gel, but not required in the presence of exogenous cyclin D1. Expression of activated mutants of the Ras/Raf/MEK signaling pathway in cells adherent to collagen gel restored ERK phosphorylation and DNA synthesis, but differentially affected cell shape. Although Ras, Raf, and MEK all increased expression of cyclin D1 on collagen film, only Ras and Raf significantly up-regulated cyclin D1 levels on collagen gel. These results demonstrate that adhesion to polymerized collagen induces growth arrest by inhibiting the Ras/ERK-signaling pathway to cyclin D1 required in late G1.     

Type I collagen preparations inhibit DNA synthesis in glial cells of the peripheral nervous system

The mechanisms underlying cessation of glial proliferation in the developing peripheral nervous system are obscure. One possibility, as yet little explored, is that mitotic inhibitory signals play a part in regulating glial cell numbers. In this study we demonstrate that type I collagen preparations from several different sources can inhibit the rate of DNA synthesis in purified populations of enteric glia and both short-term and long-term secondary Schwann cells in dissociated cell cultures. When these cells are grown on gelled or dried type I collagen substrata, they proliferate at substantially lower rates than on polylysine substrata. In contrast, type III or V collagen preparations do not inhibit glial DNA synthesis and laminin, fibronectin, type IV collagen, and secreted matrix from bovine corneal endothelial cells all stimulate thymidine incorporation. The inhibitory effect is not observed with heat denatured type I collagen preparations, but is seen equally in serum-containing medium, in medium containing fibronectin-free serum, or in serum-free medium, suggesting that the interaction of collagen with the cells requires structurally intact collagen molecules and does not occur via intermediary linkage to fibronectin. The inhibition on collagen is accompanied by a shape change from a more flattened morphology to a narrow spindle form. The labeling index of a rat Schwannoma cell line, 33B, is not inhibited on type I collagen substrata. These results demonstrate that type I collagen preparations inhibit the DNA synthesis levels of early postnatal peripheral glial cells in vitro. It remains to be determined whether this effect occurs via direct collagen-cell membrane interactions or whether it depends on accessory molecules, perhaps present in the collagen preparations themselves, since these are not purified to absolute homogeneity.     

Stimulation of growth of primary cultured adult rat hepatocytes without growth factors by coculture with nonparenchymal liver cells

DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.     

Rat chromosome 5 (q22–23) contains elements that control cell morphology and interactions with the extracellular matrix: A study of normal fibroblast X malignant hepatoma cell hybrids

Cell interactions with the extracellular matrix are consistently modified in neoplasia. Malignant transformation has been correlated with modifications in the synthesis and distribution of matrix components and with alterations of cell adhesive properties to these components. A particular class of genes, able to suppress the transformed phenotype in normal cells, may be involved in those phenotypic changes. By studying somatic cell hybrids between mouse hepatoma (BWTG3) cells and normal rat skin fibroblasts (RSF), Islam and co-workers were able to localize a gene or a group of genes controlling anchorage dependence and cell growth in vitro. This (or these) gene(s) was (were) assigned to the q22-23 fragment of rat chromosome 5. In the present study, we compare the morphology and the interactions with the extracellular matrix proteins (laminin, fibronectin, and collagen IV) and the synthesis of these proteins by RSF X BWTG3 hybrid cells that had either retained (BS181p10) or lost (BS181a5) the q22-23 region of rat chromosome 5. Our results suggest that the rat 5q22-23 fragment controls a part of the cell differentiation program including morphology, attachment to extracellular matrix, and synthesis of some matrix proteins, particularly alpha 1 and alpha 2 chains of collagen IV.     

Extracellular matrix- and cytoskeleton-dependent changes in cell shape and stiffness

Cell spreading is correlated with changes in important cell functions including DNA synthesis, motility, and differentiation. Spreading is accompanied by a complex reorganization of the cytoskeleton that can be related to changes in cell stiffness. While cytoskeletal organization and the resulting cell stiffness have been studied in motile cells such as fibroblasts, less is known of these events in nonmigratory, epithelial cells. Hence, we examined the relationship between cell function, spreading, and stiffness, as measured by atomic force microscopy. Cell stiffness increased with spreading on a high density of fibronectin (1000 ng/cm(2)) but remained low in cells that stayed rounded on a low fibronectin density (1 ng/cm(2)). Disrupting actin or myosin had the same effect of inhibiting spreading, but had different effects on stiffness. Disrupting f-actin assembly lowered both stiffness and spreading, while inhibiting myosin light chain kinase inhibited spreading but increased cell stiffness. However, disrupting either actin or myosin inhibited DNA synthesis. These results demonstrate the relationship between cell stiffness and spreading in hepatocytes. They specifically show that normal actin and myosin function is required for hepatocyte spreading and DNA synthesis and demonstrate opposing effects on cell stiffness upon disruption of actin and myosin.     

Cell shape, cytoskeletal mechanics, and cell cycle control in angiogenesis

Capillary endothelial cells can be switched between growth and differentiation by altering cell-extracellular matrix interactions and thereby, modulating cell shape. Studies were carried out to determine when cell shape exerts its growth-regulatory influence during cell cycle progression and to explore the role of cytoskeletal structure and mechanics in this control mechanism. When G₀-synchronized cells were cultured in basic fibroblast growth factor (FGF)-containing defined medium on dishes coated with increasing densities of fibronectin or a synthelic integrin ligand (RGD-containing peptide), cell spreading, nuclear extention, and DNA synthesis all increased in parallel. To determine the minimum time cells must be adherent and spread on extracellular matrix (ECM) to gain entry into S phase, cells were removed with trypsin or induced to retract using cytochalasin D at different times after plating. Both approaches revealed that cells must remain extended for approximately 12–15 h and hence, most of G₁, in order to enter S phase. After this restriction point was passed, normally ‘anchorage-dependent’ endothelial cells turned on DNA synthesis even when round and in suspension. The importance of actin-containing microfilaments in shape-dependent growth control was confirmed by culturing cells in the presence of cytochalasin D (25–1000 ng ml⁻¹): dose-dependent inhibition of cell spreading, nuclear extension, and DNA synthesis resulted. In contrast, induction of microtubule disassembly using nocodazole had little effect on cell or nuclear spreading and only partially inhibited DNA synthesis. Interestingly, combination of nocodazole with a suboptimal dose of cytochalasin D (100 ng ml⁻¹) resulted in potent inhibition of both spreading and growth, suggesting that microtubules are redundant structural elements which can provide critical load-bearing functions when microfilaments are partially compromised. Similar synergism between nocodazole and cytochalasin D was observed when cytoskeletal stiffness was measured directly in living cells using magnetic twisting cytometry. These results emphasize the importance of matrix-dependent changes in cell and nuclear shape as well as higher order structural interactions between different cytoskeletal filament systems for control of capillary cell growth during angiogenesis.     

Gab2 tyrosine phosphorylation by a pleckstrin homology domain-independent mechanism: role in epidermal growth factor-induced mitogenesis

In primary rat hepatocyte cultures, activation of phosphatidylinositol 3-kinase is both necessary and sufficient to account for epidermal growth factor (EGF)-induced DNA synthesis. In these cells, three major p85-containing complexes were formed after EGF treatment: ErbB3-p85, Shc-p85, and a multimeric Gab2-Grb2-SHP2-p85, which accounted for more than 80% of total EGF-induced PI3K activity (Kong, M., C. Mounier, J. Wu, and B. I. Posner, J Biol Chem, 2000, 275:36035-36042). More recently, we found that EGF-dependent tyrosine phosphorylation of endogenous Gab2 is essential for EGF-induced DNA synthesis in rat hepatocytes. Here we show that, after EGF treatment, ErbB3-p85 and Shc-p85 complexes were localized to plasma membrane and endosomes, whereas the multimeric Gab2-Grb2-SHP2-p85 complex was formed rapidly (peak at 30 sec) and exclusively in cytosol. Western blotting of subcellular fractions from intact liver and immunofluorescence analyses in cultured hepatocytes demonstrated that EGF did not promote the association of cytosolic Gab2 with cell membranes. These observations prompted us to evaluate the role of the PH domain of Gab2 in regulating its function. Overexpression of the PH domain of Gab2 did not affect EGF-induced Gab2 phosphorylation, PI3K activation, and DNA synthesis. Overexpressed Gab2 lacking the PH domain (DeltaPHGab2) was comparable to wild-type Gab2 in respect to EGF-induced tyrosine phosphorylation, recruitment of p85, and DNA synthesis. In summary, after EGF stimulation, ErbB3, Shc, and Gab2 are differentially compartmentalized in rat liver, where they associate with and activate PI3K. Our data demonstrate that Gab2 mediates EGF-induced PI3K activation and DNA synthesis in a PH domain-independent manner.     

Hyaluronic acid inhibits adhesion of hepatic stellate cells in spite of its stimulation of DNA synthesis

Unbalanced accumulation of fibers in extracellular matrix (ECM) results from attachment and activation of hepatic stellate cells (HSCs) during chronic liver diseases, in which the content of hyaluronic acid (HA), a glycosaminoglycan, in ECM changes. No information is available on the effect of HA on adhesion and activation of HSCs although that of collagen (Col) on HSCs was extensively studied. This study investigated the effects of HA with or without Col on adhesion of HSCs or the rate of DNA synthesis. Attachment of primary cultured HSCs was microscopically monitored in the plate simultaneously coated with HA or other ECM components. HA inhibited adhesion of quiescent HSCs at least up to 7 days after seeding, whereas HSCs were adherent to plastic or type I collagen (Col-I), type III collagen (Col-III), type IV collagen (Col-IV) or fibronectin. Both microscopy and alpha-smooth muscle actin immunocytochemistry revealed that the number of HSCs, which had been re-seeded after 15 days of culture, attached to HA-coated area was remarkably lower compared to that of HSCs on Col-I or plastic. Incorporation of HA into Col-I prevented adhesion of activated HSCs to matrix film. The number of HSCs adherent to HA at early times after seeding was minimal and significantly lower than that of the cells adherent to plastic. In contrast, either Col-I or Col-IV increased the number of adherent cells. Attachment of HSCs to plastic was inhibited by soluble HA in culture medium. CD44, the cell surface receptor to which HA binds, was immunochemically detected in HSCs. Adhesion of HSCs to plastic, HA or Col-I was not changed by anti-CD44 antibody. Either HA or Col increased the basal or platelet-derived growth factor-inducible rate of thymidine incorporation into DNA in HSCs. In conclusion, HA inhibits adhesion of quiescent or activated HSCs in spite of its stimulation of DNA synthesis, whereas Col increases HSC attachment and DNA synthesis, and inhibition of HSC adhesion by HA does not involve CD44.     

TGF-β1 stimulates cultured human fibroblasts to proliferate and produce tissue-like fibroplasia: A fibronectin matrix-dependent event

During wound repair, fibroblasts accumulate in the injured area until any defect is filled with stratified layers of cells and matrix. Such fibroplasia also occurs in many fibrotic disorders. Transforming growth factor-β (TGF-β), a promotor of granulation tissue in vivo and extracellular matrix production in vitro, is expressed during the active fibroplasia of wound healing and fibroproliferative diseases. Under usual tissue culture conditions, normal fibroblasts grow to confluence and then cease proliferation. In this study, culture conditions with TGF-β1 have been delineated that promote human fibroblasts to grow in stratified layers mimicking in vivo fibroplasia. When medium supplemented with serum, ascorbate, proline, and TGF-β was added thrice weekly to normal human dermal fibroblasts, the cells proliferated and stratified up to 16 cell layers thick within the culture dish, producing a tissue-like fibroplasia. TGF-β stimulated both DNA synthesis as measured by 1H-thymidine uptake and cell proliferation as measured by a Hoechst dye DNA assay in these postconfluent cultures. The stratification was dependent on fibronectin assembly, as demonstrated by anti-fibronectin antibodies which inhibited both basal and TGF-β-stimulated cell proliferation and stratification. Suppression of collagen matrix assembly in cell layers with β-amino-proprionitrile (BAPN) did not inhibit basal or TGF-β stimulated in vitro fibroplasia. BAPN did not interfere with fibronectin matrix assembly as judged by immunofluorescence microscopy. Thus, in concert with serum factors, TGF-β stimulates postconfluent, fibronectin matrix-dependent, fibroblast growth creating a fibroplasia-like tissue in vitro. J Cell Physiol 170:69–80, 1997 © 1997 Wiley-Liss, Inc.     

Primary cultured murine hepatocytes but not hepatoma cells regulate the cell number through density-dependent cell death

The mechanisms by which hepatocytes regulate their cell numbers in culture have been examined. We found that when murine hepatocytes were cultured at an overconfluent stage, the number of viable cells were reduced to that of the confluent stage 48 h later by cell death. Cell death was accompanied by LDH release, and it was observed only in primary cultured hepatocytes but not in hepatoma cells. Genomic DNA analysis using electrophoresis showed that DNA fragmentation, a biochemical hallmark of apoptosis, was induced in superconfluent cultures of hepatocytes in a cell-density-dependent fashion, but not in pre-confluent cells. DNA fragmentation was rapidly induced 2 h after the beginning of the in vitro culture and continued up to 24 h later. Flow cytometry analysis demonstrated that the nuclei from the hepatocytes in a high density culture were condensed and that the DNA content was reduced. These data suggest that the mechanism of cell death is apoptosis. The DNA fragmentation seen in the high density hepatocyte culture was not observed in hepatoma cell lines. Moreover, apoptosis was induced in hepatocytes of MRL/lpr mice, suggesting that the Fas antigen was not involved in the apoptotic process. Apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, and by a calmodulin antagonist, W-7. Taken together, the results indicate that high density culture of murine hepatocytes though not hepatoma cells regulate their cell numbers by an apoptotic mechanism. The apoptosis is dependent on de novo protein synthesis and intracellular calcium metabolism.     

Changes in TGF-β receptors of rat hepatocytes during primary culture and liver regeneration: Increased expression of TGF-β receptors associated with increased sensitivity to TGF-β-mediated growth inhibition

To clarify the role of transforming growth factor-β (TGF-β) and its receptors in hepatocyte growth, we studied the expression of TGF-β1 and its receptors and the sensitivity to growth inhibition by TGF-β1 protein in rat hepatocytes derived from resting and regenerating livers. In hepatocytes derived from resting livers, mRNAs for TGF-β type II receptor (TβR-II), insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M-6-PR), and TGF-β1 increased with time in primary culture. The cell surface TGF-β receptor proteins (TβR-I, II, and III), examined by the receptor affinity-labeling assay using ¹²⁵I-TGF-β1, also increased, especially after 48 hr of culture. Hepatocytes were more sensitive to inhibition of DNA synthesis, when the TGF-β1 protein was added at later times in culture, corresponding to the presence of increased TGF-β receptors. In hepatocytes from regenerating livers after a partial hepatectomy (PH), an increase of TβR-I, TβR-II, TβR-III, IGF-II/M-6-PR, and TGF-β1 mRNAs was found, compared with hepatocytes from resting livers. Similarly, using TGF-β receptor affinity-labeling assay, hepatocytes from PH livers were found to have an increase in TβR-I, II, and III proteins, with a peak at 4 days post-PH, compared with hepatocytes from resting livers. When TGF-β1 protein was added for a short period (6 or 24 hr) after cell attachment to hepatocyte cultures, it inhibited DNA synthesis more effectively in hepatocytes from regenerating compared with resting livers. Our results show that hepatocyte TGF-β receptors and sensitivity to growth inhibition by TGF-β1 protein change together and are modulated during liver regeneration, as well as during the conditions of primary culture. J. Cell. Physiol. 176:612–623, 1998. © 1998 Wiley-Liss, Inc.