Abstract
We reported previously that the potency of heparin-binding fibroblast growth factor-1 (FGF-1) as a mitogen for rat hepatocytes in primary culture is as high as that of epidermal growth factor (EGF) and hepatocyte growth factor. To gain insight into the pathophysiological significance of FGF-1 in hepatocyte growth, we analyzed the cooperative mitogenicity of FGF-1 and EGF. Results from a nuclear labeling assay using [3H]thymidine suggest that most hepatocytes in primary culture consist of two cell populations that differ in response to FGF-1; one is an FGF-1-responsive cell population, and the other is an EGF-responsive (but not FGF-1-responsive) cell population. On the other hand, autoradiographic analysis of 125I-FGF-1 binding demonstrated that high affinity FGF receptors were homogeneously distributed on the surface of all hepatocytes. Cross-linking 125I-FGF-1 to the nonstimulated hepatocyte surface indicated that the high affinity FGF receptors comprise two FGF receptors that differ in molecular mass (128 and 79 kDa). Furthermore, the 79-kDa receptor was preferentially down-regulated when the hepatocytes were stimulated with EGF or hepatocyte growth factor. These data suggest that the abundant expression of the 79-kDa FGF receptor on some populations of hepatocytes is involved in their lack of response to FGF-1. The 128- and 79-kDa FGF receptors were assigned as FGFR2 using an antibody specific to the ectodomain of FGFR2, whereas the 79-kDa receptor was not reactive to the antibody against the carboxyl terminus of FGFR2. This 79-kDa FGF receptor was not tyrosine-phosphorylated in response to FGF-1 stimulation, while the 128-kDa FGF receptor was recognized by anti-phosphotyrosine antibody under the same conditions. Also, the heterodimer of 79- and 128-kDa FGF receptors was less tyrosine-phosphorylated than the homodimer of 128-kDa FGF receptors. These data suggest that the 79-kDa FGF receptor inhibits the function of the 128-kDa FGF receptor through their heterodimerization. Thus, we surmise that the difference in response to FGF-1 between the cell populations of normal rat hepatocytes was caused by the different levels of the 79-kDa FGF receptor in each cell population.
Highlights
Various growth factors, including transforming growth factor-␣ (TGF-␣),1 epidermal growth factor (EGF), and hepatocyte growth factor (HGF), have been identified as potent hepatotrophic factors in primary-cultured hepatocytes [1,2,3]
To clarify the significance of these two growth factors on hepatocyte growth, we examined the combinational effects of EGF and fibroblast growth factor-1 (FGF-1) by measuring the time course of DNA synthesis by rat hepatocytes
We demonstrated that two major hepatocyte mitogens, FGF-1 and EGF, additively stimulated DNA synthesis in rat hepatocytes as follows. (i) Each growth factor had a saturating level of stimulating DNA synthesis in hepatocytes. (ii) Even when the DNA synthesis was stimulated up to the maximal level by one of these growth factors, the stimulation of the other growth factor was additive. (iii) This stimulation was additive throughout the first S phase. (iv) The fractions of hepatocytes that responded to FGF-1, EGF, and both growth factors were about 55, 55, and 80%, respectively
Summary
Primary Culture of Rat Hepatocytes— Adult rat (7–9-week-old female Wistar) hepatocytes were isolated by perfusing the liver in situ with 0.05% (w/v) collagenase as reported [16], and morphologically identified hepatocytes constituted Ͼ95% of the preparation. Dimerization of FGF Receptors on Rat Hepatocytes— Rat hepatocytes were incubated in Williams’ E medium supplemented with 10% FBS and 10Ϫ7 M dexamethasone in 6-cm culture dishes for 2 h; sodium orthovanadate (50 M) was added, and the cells were further cultured for 3 h. The 125I-FGF-1-cross-linked hepatocytes were lysed in RIPA buffer (10 mM Tris-Cl, pH 7.4, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 1 mM EDTA, 1 g/ml aprotinin, and 10 M E-64) supplemented with phosphatase inhibitors at 4 °C for 30 min, and the lysates were obtained by centrifugation at 10,000 ϫ g at 4 °C for 30 min. Immunoprecipitation of FGF Receptor by Anti-FGF Receptor Antibodies—Rat hepatocytes were plated in collagen-coated 6-cm culture dishes, and the medium was replaced with methionine-free Williams’ E medium supplemented with 10% FBS, 10Ϫ7 M insulin, and 10Ϫ7 M dexamethasone 5 h after cell seeding.
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