Journal of Hepatology 2ooO; 32: 242-250 Printed in Denmark All rights reserved Munksgaard. Copenhagen zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Co pyright 0 European Association for the Study of the Liver Zoo0 zyxwvutsr Journal of Hepatology ISSN 0168-8278 Fibronectin regulates morphology, cell organization and gen of rat fetal hepatocytes in primary culture Aranzazu S&nchez’, Albert0 M. Alvarez2, Roser Pagan3, CCsar Roncero’, Se&n Vilarb3,Manuel Benito’ and Isabel Fabregat l ‘Departamento de Bioquimica y Biologia Molecular, Centro Mixto CSICIUCM and 2Centro de Citometria de Flujo y Microscopia Confocal UCM, Facultad de Farmacia,Universidad Complutense de Madrid,Madrid, and 3Departament de Biologia Cellular, Fact&at de Biologia, Universitat de Barcelona, Barcelona, Spain Background/Aims: The extracellular matrix regulates hepatic development and regeneration, modulating the maintenance of liver architecture in the differentiated state. The aim of this work was to analyze how differ- ent extracellular matrix molecules modulate fetal he- patocyte morphology, growth and differentiation. Met/‘&s: We cultured fetal hepatocytes either on plastic or on different extracellular matrix proteins, i.e., collagen I, fibronectin or E-C-L (entactin-colla- gen IV-laminin) and we analyzed cell attachment, morphological organization, proliferative response and gene expression. Results: Cell attachment was increased by all the extracellular matrix proteins to a similarextent. However, only fibronectin facilitated the formation of elongated cord-like structures, reminiscent of liver plate organization. Immnnocytochemical analysis of the cellsin these structures revealed highlevels of albumin and cytokeratin 18, phenotypical markers of parenchymal hepatocytes. Fibronectin did not block zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIH I NTERACTIONS between cells and the extracellular ma- trix (ECM) activate a flow of information that acts to regulate many fundamental processes duringdevel- opment. These include regulation of growth and death, migration of the cells and differentiation programs thatcontribute to morphogenesis during organ forma- tion (for review, 1,2). Recent studies indicate that the ECM modulates hepatic development, regeneration Received 14 April: revised 27 Jtdy; accepted 17 August1999 Correspondence: Isabel Fabregat, Dpto. de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain. Tel: 34 91 3941627. Fax: 34 91 3941779. e-mail: isabelf@eucmax.sim.ucm.es the mitogenic stimuli induced by epidermal growth factor in these cells and the elongated structures ap peared either in the absence or in the presence mitogen. Cellscultured on fibronectin, regardless of whether epidermal growth factor was present or not, alsopresented the maximal levels of expression for liver specific genes, such as albumin or alpha-fe tein. This expression was coincident with an increased expression of hepatocyte nuclear factor (HNF)-4 and a higher HNF-MHNF-l/3 ratio, when compared with those cells thatwere cultured on collagen or E-C-L extracellular matrix. Conclusions: Theseresults suggest that fibronectin might play a differential role,as compared to other extracellular matrix proteins, in fetal hepatocyte or- ganization and gene expression. Key words: Extracellular matrix; Fibronectin; He- patocyte; HNFs; Liver differentiation; Proto-onco- gene. and maintenance of the liver architecture in the normal differentiated state (3). The liver forms as an out- growth of the foregut into the septum transversum. In the rat, by day 13.5 of fetal development, hepatoblasts are becoming organized into cell plates. At this early gestational stage, discontinuous deposits of fibronectin, laminin and collagens I and IV (with laminin being the mostprevalent) are present in the perisinusoidal space, a small space between hepatocytes and sinusoidal en- dothelial cells that contains an ECMbut lacks a true basement membrane (4). However, at later gestational dates,fibronectin distribution in the portal triads ap- proaches that of collagen type I, being present in the interstitial connective tissues. Collagen type IV and la- minin are located in vascularand biliary basement 242