epidermal-growth-factor and ciprofibrate

The purpose of this study was to determine whether the extracellular matrix used in hepatocyte culture would alter gene expression induced by the peroxisome proliferator ciprofibrate (CIP). We compared the activities and mRNA levels of two enzymes associated with peroxisome proliferation-fatty acyl-CoA oxidase (FAO), the first enzyme in the peroxisomal beta-oxidation pathway, and lauric acid hydroxylase (LAH), which represents the activity of the cytochrome P450 4A subfamily-in rat hepatocytes cultured on different plates: plastic, collagen-coated, thin and thick Matrigel, and collagen gel plates. CIP increased FAO activity about fivefold in collagen gel plates and sixfold in thick Matrigel plates compared to a fourfold increase in other plates; LAH was increased about threefold in thin Matrigel plates and fourfold in thick Matrigel and collagen gel plates compared to only a twofold increase in plastic and collagen-coated plates. The mRNA level for FAO was highest in hepatocytes cultured on collagen gel and Matrigel plates compared to those cultured on plastic and collagen-coated plates. The P-450 4A1 mRNA expression, however, was highest in the collagen gel plates, with lower expression in the thick Matrigel, collagen-coated and plastic plates. DNA synthesis and the DNA binding activity of the transcription factor AP-1 were also examined in response to epidermal growth factor (EGF) and CIP. Without the addition of EGF, DNA synthesis was significantly higher on collagen-coated plates than on collagen gel plates. The DNA binding activity of AP-1 was also induced after 24 hr culture in collagen-coated plates, whereas it was not detected in collagen gel plates. After the addition of EGF, the DNA binding activity of AP-1 was increased in both collagen-coated plates and collagen gel plates. CIP did not increase the DNA binding activity of AP-1 in either plate. These results demonstrate that components of the extracellular matrix influence the induction of peroxisome proliferator-induced enzyme activities and mRNA levels by CIP, with the highest induction seen in collagen gel and thick Matrigel plates. Furthermore, the induction of cell proliferation and AP-1 DNA binding activity are influenced by the extracellular matrix.

Topics: Acyl-CoA Oxidase; Animals; Blotting, Northern; Cells, Cultured; Clofibric Acid; Culture Media; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; DNA; Epidermal Growth Factor; Extracellular Matrix; Fibric Acids; Hypolipidemic Agents; Liver; Male; Mixed Function Oxygenases; Nuclear Proteins; Oxidoreductases; Peroxisome Proliferators; Peroxisomes; Rats; Rats, Sprague-Dawley; RNA; Transcription Factor AP-1

Several hypolipidemic drugs and environmental contaminants induce hepatic peroxisome proliferation and hepatic tumors when administered to rodents. These chemicals increase the expression of the peroxisomal beta-oxidation pathway and the cytochrome P-450 4A family, which metabolize lipids, including eicosanoids and their precursor fatty acids. We previously found that the peroxisome proliferator ciprofibrate decreases the level of eicosanoids in the liver and in cultured hepatocytes. In this study, we examined the effect of prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha), leukotriene C4 (LTC4) and the peroxisome proliferator ciprofibrate on DNA synthesis in cultured hepatocytes. Primary rat hepatocytes were cultured on collagen gels in serum-free L-15 medium with varying concentrations of eicosanoids and ciprofibrate, and the absence or presence of growth factors. Ciprofibrate lowered hepatocyte eicosanoid concentrations; the addition of eicosanoids restored their levels. After a 48-h exposure with [3H]-thymidine, DNA synthesis was determined by measuring [3H]-thymidine incorporation into DNA. The addition of PGE2, PGF2 alpha, and LTC4 to cultures along with ciprofibrate increased DNA synthesis, whereas treatment with ciprofibrate or eicosanoids alone resulted in a much smaller increase. The addition of epidermal growth factor (EGF) to the eicosanoid-ciprofibrate combination increased DNA synthesis more than EGF or the eicosanoid-ciprofibrate combination alone. The PGF2 alpha-ciprofibrate combination also was comitogenic with transforming growth factor-alpha and hepatocyte growth factor. The addition of both ciprofibrate and prostaglandins also blocked the growth inhibitory effect of transforming growth factor-beta on DNA synthesis induced by EGF. These results show that the eicosanoids PGE2, PGF2 alpha, and LTC4 are comitogenic with the peroxisome proliferator ciprofibrate in cultured rat hepatocytes.

Topics: Animals; Cells, Cultured; Clofibric Acid; Dinoprost; Dinoprostone; DNA Replication; Eicosanoids; Epidermal Growth Factor; Fibric Acids; Hypolipidemic Agents; Leukotriene C4; Liver; Male; Microbodies; Mitogens; Norepinephrine; Rats; Rats, Sprague-Dawley

The response of promoter-exposed hepatocytes to the complete hepatic mitogens hepatocyte growth factor (HGF), epidermal growth factor (EGF) and acidic fibroblast growth factor (aFGF) was studied. Male Fisher 344 rats were administered phenobarbital or ciprofibrate. Hepatocytes were isolated at various time points and cultured in type I collagen gels, a 3-dimensional culture system that allows stable long-term hepatocyte differentiation. DNA synthetic activity in response to addition of HGF, aFGF or EGF to the cultures was assayed by incorporation of [3H]thymidine. Administration of ciprofibrate to rats caused an immediate decreased growth response in hepatocytes to all three growth factors. Phenobarbital administration caused a gradual decrease in responsiveness to the growth factors: after 10 days, hepatocytes became insensitive to the mitogenic effects of HGF, EGF and aFGF. However, an early increase in responsiveness to HGF and aFGF occurred in phenobarbital-exposed hepatocytes. The results indicate that phenobarbital and ciprofibrate have similar inhibitory effects on hepatocyte DNA synthesis in culture after long-term in vivo exposure. However, their early effects on hepatocyte growth response differ considerably, suggesting that their effects on cellular proliferation occur via different mechanisms.

Topics: Animals; Cell Division; Cells, Cultured; Clofibric Acid; Collagen; DNA; Drug Interactions; Epidermal Growth Factor; Fibric Acids; Fibroblast Growth Factor 1; Hepatocyte Growth Factor; Liver; Male; Phenobarbital; Rats; Rats, Inbred F344