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International audience; Basement membranes contain three major components (ie collagen IV, laminin, and the heparan sulfate proteoglycan termed perlecan). Although the distribution and origin of both collagen IV and laminin have been well documented in the liver, perlecan has been poorly investigated, so far. We have studied the distribution and cellular origin of perlecan in rat livers in various conditions as well as in hepatocyte primary culture. By immunolocalization in both adult and 18-day-old fetal liver, perlecan was found in portal spaces, around central veins, and throughout the lobule. Immunoelectron microscopy revealed its presence at the level of basement membranes surrounding bile ducts and blood vessels, and in the space of Disse discontinuously interacting with hepatocyte microvilli. Precursors of perlecan were detected in the rough endoplasmic reticulum of bile duct cells and both vascular and sinusoidal endothelial cells. Both hepatocytes and Ito cells were negative. Northern-blot analysis confirmed the lack of appreciable expression of perlecan in hepatocytes isolated from either fetal or adult livers. In 18-month-diethylnitrosamine-treated rat liver, perlecan was abundant in neoplastic nodules. Electron microscopic investigation revealed an almost continuous layer of perlecan in the space of Disse and intracellular staining in sinusoidal endothelial cells, only. Perlecan mRNAs were detectable in malignant nodules, and absent in hepatocytes from nontumorous areas. Hepatocytes expressed high levels of perlecan mRNAs only when put in culture. This expression was reduced in conditions that allow improvement of hepatocyte survival and function (ie addition of corticoids, dimethylsulfoxide or nicotinamide to the medium, or in coculture with liver epithelial cells from biliary origin). Immunolocalization by light and electron microscopy showed that deposition of the proteoglycan occurred in coculture, in basement membranelike structures located around hepatocyte cords. In vitro attachment assay of hepatocytes on purified perlecan substrate indicated that these cells may interact with the proteoglycan through integrins which belong to the beta 1 family. These data suggest that deposition of perlecan in the space of Disse requires cellular cooperation. This article on perlecan, the third major component of hepatic basement membranes, shows a unique cellular origin in the liver and, as found for both collagen IV and laminin, an expression in adult hepatocytes when place in culture.

Laminin deposition is increased in fetal liver and in a variety of liver diseases, including the development of carcinoma. We investigated the role of the hepatocyte in the synthesis of both laminin and its 32- to 67-kd receptor in normal adult, fetal, and diethylnitrosamine-treated rat livers, and in adult rat hepatocyte primary cultures. Laminin was localized by immunoelectron microscopy in the endoplasmic reticulum of hepatocytes in fetal-derived and in 18-month-old diethylnitrosamine-treated rat livers. Steady-state mRNA levels for the three chains of laminin (A, B1, and B2) and the laminin receptor (LBP-32) were examined. Northern-blot analyses showed that hepatocytes at all stages lacked the A-chain mRNA. B1-chain mRNA was undetectable in normal adult hepatocytes, while significant levels of B1-chain mRNA were found in fetal hepatocytes and in adult hepatocyte primary culture. In hepatocytes from diethylnitrosamine-treated rats, B1-chain mRNAs were abundant and were present mainly in nodular formations rather than in the nontumorous areas. B2-chain mRNAs were barely detectable in either normal adult or fetal hepatocytes. In diethylnitrosamine-treated rats, the steady-state B2-chain mRNA level was higher in nodules than in nontumorous areas. In primary culture, B2-chain mRNAs were present as early as 4 hours after adult hepatocyte seeding, and dramatically increased during the following 2 days. Only low levels of laminin-receptor (LBP-32) mRNAs were present in normal adult hepatocytes, whereas the levels were high in the fetal and in the tumor-containing livers. In diethylnitrosamine-treated rats, LBP-32 mRNAs were more abundant in nodular formations rather than in nontumorous areas. In hepatocyte primary culture, the expression of the LBP-32 mRNA dramatically increased during the first 24 hours. These results show that in hepatocytes, expression of laminin chains and its receptor LBP-32 are not coordinated and depends on the maturation of the cells. In addition, they suggest that the expression of B1 and B2 chains in adult hepatocytes is related to changes of the normal phenotype and/or the pericellular environment.

Cellular and molecular mechanisms involved in the deposition of extracellular matrix components in both normal and fibrotic liver are still poorly understood. We have investigated the influence of cooperation between Ito cells and hepatocytes in matrix deposition in vitro. Immunoprecipitation of radiolabeled proteins from media of 5-day-old Ito cell primary cultures showed that these cells secreted high levels of the major basement membrane components, ie, collagen IV, laminin, and entactin/nidogen. By immunocytochemistry, precursors of basement membrane components were found intracellularly, but only scarce deposits were seen around the cells. When hepatocytes were added to 2-day-old Ito cell primary cultures, they established close contacts with Ito cells in less than 24 hours and expressed ZO-1, a tight junction-associated protein not detectable in standard hepatocyte culture. Cytochemistry analysis revealed an abundant extracellular matrix deposited over hepatocyte cords and between hepatocytes and Ito cells. Immunocytochemistry studies showed that this matrix contained laminin, fibronectin, and collagens proIII and IV. These data indicate that a high level of matrix protein synthesis by liver cells in vitro is not sufficient to induce extracellular matrix deposition, and that cell-cell interactions are strongly involved in this process. Hepatocyte/Ito cell co-culture, which may reflect the actual situation in vivo, represents a useful tool for studying liver fibrogenesis.

Fibroblast growth factor (FGF-2) is a multifunctional growth factor. In cells producing this factor, FGF-2 is synthesized as different molecular weight isoforms lacking the signal peptide sequence for secretion. All forms are highly concentrated in cells. The presence of a nuclearization signal sequence in some isoforms suggests the involvement of these isoforms in cell functions bypassing the cell surface receptors. Our aims were to better define the intracellular localizations of the FGF-2 isoforms by immunocytochemistry and confocal microscopy and to analyze whether these isoforms were involved in the expression of extracellular matrix (ECM) components. We chose the pancreatic acinar cell line AR4-2J since it does not synthesize FGF-2. These cells were retrovirally transfected by point-mutated FGF-2 cDNAs. The cell lines obtained produced either the 18 kDa form (A5 cells) or the 22.5 kDa form (A3 cells). In A5 cells, the 18 kDa form was found in the cytoplasm, on the cell surface reflecting its secretion, and in the nucleoli. Parental AR4-2J cells treated with exogenous FGF-2 exhibited identical localizations, suggesting that in A5 cells the 18 kDa form followed the same translocation pathways than the exogenous FGF-2. By contrast, in A3 cells the 22.5 kDa form was predominantly localized in the nucleoplasm but was undetectable on the cell surface, suggesting its direct translocation to the nucleus. Northern and Western blot analysis showed that cells expressing the high molecular weight form exhibited a decrease of laminin B1 protein level and mRNA stability. In contrast, collagen IV and fibronectin expressions were unmodified either in FGF-2-transfected cells or in parental cells treated by exogenous FGF-2. Thus, these data indicate that: 1) 18 and 22.5 kDa FGF-2 are preferentially localized in different nuclear compartments and 2) the high molecular weight form plays a role on the expression of some ECM components.