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It was determined whether human hepatocyte growth factor (hHGF)-producing ability would change in the human embryonic lung fibroblast cell strains (MRC-5 and IMR-90) until the cells senesced in culture. The effects of phorbol 12-myristate 13-acetate (PMA), dexamethasone, and transforming growth factor-beta 1 (TGF-beta 1) on hHGF production were also studied in these cell strains. For stimulation of DNA synthesis of adult rat hepatocytes in primary culture, hHGF secreted by MRC-5 cells at 39.9 and 69.8 population doubling levels (PDLs) showed almost the same activity as recombinant hHGF. Secretion of hHGF by MRC-5 cells increased about threefold between 37.3 and 67.8 PDLs. IMR-90 cells also showed about a threefold increase in hHGF secretion with increased passage from 37.8 to 66.0 PDL. Both cell strains showed almost the same ratio of hHGF amount in the cell extracts to that secreted into the medium around 40 and 70 PDLs. Northern blot analysis showed that the transcriptional level of the hHGF gene in MRC-5 cells increased about three-fold from 42.0 to 73.6 PDL in culture. These findings indicated that hHGF production increased in both cell strains with aging in culture. Production of hHGF in both cell strains was remarkably stimulated by treatment with 10 nM PMA. On the other hand, hHGF production in both cell strains was slightly suppressed by treatment with 1 microM dexamethasone. TGF-beta at a concentration of 5 ng/ml prominently inhibited hHGF production in both cell strains. The response of both cell strains to these regulators for hHGF production was almost the same around 40 and 70 PDLs in culture.

Hepatocyte growth factor (HGF) is a multifunctional cytokine with mitogenic, motogenic, morphogenic, and tumor-suppressing activities. Despite the broad spectrum of its biological activities, HGF is most likely the physiological hepatotrophic factor that triggers or modulates liver regeneration. Regulatory mechanisms for HGF production are crucial for understanding the control of liver regeneration. We previously reported that HGF production by human skin fibroblasts is stimulated by a protein kinase C (PKC)-mediated pathway. We determined here whether gene expression and production of HGF in human skin fibroblasts can be induced via activation of a cAMP-mediated pathway. HGF secretion by the cells was markedly stimulated by the cAMP-elevating agents, forskolin, cholera toxin, prostaglandin E2 (PGE2), and 3-isobutyl-1-methylxanthine, as well as by the membrane-permeable cAMP analogues, 8-bromo-cAMP and dibutyryl cAMP. The dose-response curves of induction of HGF secretion by cholera toxin and forskolin were nearly parallel with those of the intracellular cAMP levels. HGF mRNA levels did not significantly increase at 5 and 10 h, but increased considerably 15 h or more after the addition of cholera toxin. Forskolin, 8-bromo-cAMP, and PGE2 also caused appreciable up-regulation of HGF gene expression with a similar time course. Although human skin fibroblasts of various origins secreted variable amounts of HGF, the cAMP-elevating agents and the cAMP analogues caused a very marked increase in HGF production in all of them. The agents also enhanced highly active HGF secretion by MRC-5 human embryonic lung fibroblasts. Dexamethasone and transforming growth factor-beta 1, which inhibit PKC-mediated HGF secretion, down-regulated HGF mRNA expression and HGF production in the cells treated with the cAMP-elevating agents and the cAMP analogues. These results indicate that HGF expression in human skin fibroblasts is stimulated by activation of a cAMP-mediated pathway.

The effects of three of the most widely used histamine H2-receptor antagonists, cimetidine, ranitidine and famotidine, on liver cell growth were studied in vitro using adult rat hepatocytes in primary culture, because these antagonists are commonly given to patients with hepatic cirrhosis or fulminant hepatic failure for protection against peptic ulcers and gastrointestinal hemorrhage. At their clinically effective concentrations in the blood (0.5-5 micrograms/ml cimetidine, 0.25-2.5 micrograms/ml ranitidine and 0.05-0.5 microgram/ml famotidine), these three antagonists did not have any effect on replicative DNA synthesis either in the presence or absence of insulin plus epidermal growth factor (EGF). However, unexpectedly DNA synthesis stimulated by insulin and EGF was found to be enhanced by 0.05-0.5 mg/ml cimetidine, although it was unaffected or inhibited by ranitidine and famotidine at the concentrations tested. Cimetidine caused maximal enhancement of 1.5-2 times the control level of DNA synthesis at a concentration of 0.25 mg/ml. Cimetidine also had an enhancing effect at submaximal concentrations of insulin and EGF, but neither cimetidine nor the other antagonists had any stimulatory effect on DNA synthesis in the absence of insulin plus EGF. This enhancement of DNA synthesis by cimetidine resulted in significant increase in the total DNA content of the hepatocytes in culture. Under the conditions used, cimetidine had the lowest toxicity of these three antagonists and ranitidine the highest, as judged from data on DNA synthesis and the total protein content of cultured hepatocytes, leakage of aminotransferases from the cells and morphological observations.

Plasma from patients with fulminant hepatic failure obtained during plasma exchange therapy, like their serum, demonstrated marked stimulatory activity on DNA synthesis in cultured rat hepatocytes. Heat treatment at 56 degrees C for 30 min did not affect this activity of the plasma, but reduced that of the serum. This growth-promoting activity was confirmed by showing that the patients' serum and plasma increased the labeling index with [3H]thymidine and the total number of nuclei in hepatocyte cultures. The activity of pooled active fractions obtained by gel filtration of the heated plasma was lost completely on heat treatment at 80 degrees C for 10 min or on treatment with trypsin or chymotrypsin, which suggests that it was due to a protein. The human hepatocyte growth factor was purified about 600-fold from heated plasma of a patient by ammonium sulfate precipitation and chromatographies on Affi-Gel Blue and hydroxylapatite. The maximum effect of this partially purified factor on DNA synthesis in cultured hepatocytes was greater than that of epidermal growth factor. The molecular weight of the hepatocyte growth factor was about 85,000 as determined by SDS-PAGE.