Proliferation and transformation of cultured liver fat-storing cells (perisinusoidal lipocytes) under conditions of β- d-xyloside-induced abrogation of proteoglycan synthesis

Abstract

Fat-storing cells (perisinusoidal lipocytes, Ito cells) are the major connective tissue-producing cell type in liver. In areas of necroinflammation the cells proliferate and transform into desmin and smooth muscle α-actin-positive myofibroblast-like cells which synthesize a broad spectrum of significant amounts of collagens, proteoglycans, and matrix glycoproteins. Available data suggest a central role for these cells in the pathogenesis of fibrosis. β- d-Xyloside, an artificial initiation site for galactose-linked glycosaminoglycans, thereby uncoupling the synthesis of core protein and GAG, was used as a probe to study main cellular functions under conditions of abrogated proteoglycan synthesis. The exposure for 48 hr of fat-storing cells to p-nitrophenyl β- d-xyloside (PNP-Xyl) increased dose-dependently the synthesis of [ 35S]sulfate-labeled medium GAG. Maximum stimulation of fivefold above normal was reached at 1.0 m M PNP-Xyl. Higher concentrations of PNP-Xyl progressively decreased the stimulatory effect on GAG synthesis. The relative composition of GAG in medium (60% chondroitin sulfate, 34% dermatan sulfate), at the cell surface, and intracellularly (mainly heparan sulfate) was not changed significantly by PNP-Xyl. The amounts of intracellular and cell surface-bound GAG were reduced by 40 and 30%, respectively, by PNP-Xyl leading to a depletion of heparan sulfate at the cell surface. Pulse-chase experiments revealed that xyloside-initiated GAG were secreted immediately after synthesis into the medium. GAG synthesized in the presence of 1 and 5 m M PNP-Xyl were free of core protein, and the molecular size of the GAG chains was smaller than that of GAG obtained from β-eliminated proteoglycans synthesized in control cultures. At concentrations above 3 m M PNP-Xyl generated a dose-dependent inhibition of cell proliferation, which was at any stage of culture fully reversible upon removal of the drug. Viability and general protein synthesis were not reduced, but fat-storing cell transformation and deposition of matrix glycoproteins were retarded. Only a very small fraction of drug-treated cells (5 m M PNP-Xyl) did express on the 11th culture day smooth muscle iso-α-actin- and desmin-containing cytoskeletal filaments, which are important indicators of transformation into myofibroblast-like cells. Furthermore, the synthesis of hyaluronan and the expression of immunostained fibronectin, laminin, and tenascin were reduced in cultures exposed to 5 m M PNP-Xyl. The described cellular functions were not affected by exposure of fat-storing cells to p-nitrophenyl β- d-galactoside. The data indicate that PNP-Xyl at a concentration of 5 m M interferes with any of the reactions of fat-storing cells, i.e., proliferation, transformation, and matrix glycoprotein deposition, known to be activated in liver fibrogenesis. No evidence for a cytotoxic action of this compound was found. The results support the hypothesis that proteoglycans might play a role in the activation of fat-storing cells, a major event in the initiation of the fibrogenic response of injured liver.