Hepatocyte growth factor (HGF) is the most potent mitogen for mature parenchymal hepatocytes in primary culture, and seems to be a hepatotrophic factor that acts as a trigger for liver regeneration after partial hepatectomy and liver injury. The partial purification and characterization of HGF have been reported. We have demonstrated that pure HGF from rat platelets is a new growth factor effective at concentrations as low as 1 ng ml-1. The effects of HGF and epidermal growth factor (EGF) are additive. The activity of HGF is not species-specific, although it does not stimulate growth in Swiss 3T3 fibroblasts. HGF has a relative molecular mass (Mr) of 82,000 and is a heterodimer composed of a large alpha-subunit of Mr 69,000 and a small beta-subunit of Mr 34,000. Here we report the amino-acid sequence of human HGF determined by complementary DNA cloning and the expression of biologically active human HGF from COS-1 cells transfected with cloned cDNA. The nucleotide sequence of the human HGF cDNA reveals that both alpha- and beta-chains are contained in a single open reading frame coding for a pre-pro precursor protein of 728 amino acids.

The primary structure of rat hepatocyte growth factor (HGF) was elucidated by determining the base sequence of the complementary DNA (cDNA) of HGF. The cDNA for rat HGF was isolated by screening a liver cDNA library with oligonucleotides based on the partial N-terminal amino acid sequence ofthe P subunit ofpurified rat HGF. HGF is encoded in an mRNA of about 6 kilobases. Both a and 13 subunits of HGF are specified in a single open reading frame for a 728-amino acid protein with a calculated molecular weight of 82,904. The N-terminal part of HGF has a signal sequence and a prosequence with 30 and 25 amino acid residues, respectively. The mature heterodimer structure is derived proteolytically from this single pre-pro precursor polypeptide. The calculated molecular weights of the a and (3 subunits are 50,664 and 25,883, respectively, and each subunit has two potential N-lked glycosylation sites. The amino acid sequence of HGF is 38% identical with that of plasminogen. The a subunit of HGF contains four "Ikringle" structures, and the (3 subunit has 37% amino acid identity with the serine protease domain of plasmin. Northern blot analysis revealed that HGF mRNA was expressed in rat various tissues, including the liver, kidney, lung, and brain.were shown to be markedly elevated in the blood plasma of rats with hepatitis induced by administration ofa hepatotoxin such as CC14 or D-galactosamine (18). In humans, similar findings were observed in the sera of patients after partial hepatectomy (19) and with fulminant hepatitis (33).HGF has been isolated as a homogeneous material from rat platelets and shown to be a heat-labile protein of -82 kDa, composed of a 69-kDa a subunit and a 34-kDa (3 subunit (20,21). HGF at 1 ng/ml markedly stimulated DNA synthesis in adult rat hepatocytes and its effect was additive or synergistic with those of insulin and epidermal growth factor. HGF has no species specificity; for example, rat HGF also stimulated DNA synthesis in human, canine, and porcine hepatocytes in primary culture. In contrast, HGF did not stimulate DNA synthesis in established cell lines such as Swiss mouse 3T3 fibroblasts and BRL (buffalo rat liver) epithelial cells.Here we report the complete amino acid sequence of rat HGF obtained by cloning and sequence analysis of its cDNA. § We have found that HGF mRNA is expressed in various rat tissues including the kidney, heart, lung, and brain, as well as in injured liver.Liver regenerates actively after partial hepatectomy and liver injury. Early investigations (1-4) showed that hepatotrophic factors existed in the blood of partially hepatectomized animals. However, no one succeeded in purifying and characterizing such a humoral factor until recently, largely because no simple, reproducible, and sensitive assay for the factor has been available.Although many in vitro experimental methods using established liver and hepatoma cell lines have been employed in attempts to identify the hepatotrophic factor, these assays are unsuitable because these cell lines have lost al...

A full-length cDNA clone encoding a purified augmenter of liver regeneration (ALR) factor prepared from the cytosol of weanling rat livers was isolated. The 1.2-kb cDNA included a 299-bp 5' untranslated region, a 375-bp coding region, and a 550-bp 3' untranslated region. It encoded a protein consisting of 125 amino acids. The molecular weight of ALR calculated from the cDNA was 15,081, which is consistent with the size estimated by SDS/PAGE under reducing conditions. The molecular weight of the purified native ALR estimated by SDS/PAGE under nonreducing conditions was -30,000; thus ALR apparently has a homodimeric structure. The recombinant ALR produced by expression of the cDNA in COS cells was tested in vivo in the canine Eck fistula model and found to have potency equivalent to the purified native ALR. The 125-aa sequence deduced from the rat ALR cDNA shows 50% homology to the amino acid sequence of the gene for oxidative phosphorylation and vegetative growth in the yeast Saccharomyces cerevisiae.

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Background-Prostacyclin (PGI 2 ) is a short-lived endogenous inhibitor of platelet aggregation and a potent vasodilator and regulator of the growth of vascular smooth muscle cells. To study the role of PGI 2 in the vascular system in vivo, PGI 2 -deficient (PGID) mice were established by genetic disruption of the PGI 2 synthase gene. Methods and Results-PGI 2 synthase-null mice were generated by replacing the exons of PGI 2 synthase gene that encodes for the catalytic site of the enzyme with a neomycin resistance gene. In these mice, PGI 2 levels in the plasma, kidneys, and lungs were reduced, whereas thromboxane and prostaglandin E 2 levels became elevated. Blood pressure and the amounts of urea nitrogen and creatinine in plasma of the PGID mice were significantly higher than those of wild-type mice (PϽ0.05). They developed progressive morphological abnormalities in the kidneys, accompanied by atrophy, surface irregularity, fibrosis, cyst, arterial sclerosis, and hypertrophy of vessel walls. Thickening of the thoracic aortic media and adventitia were observed in aged PGID mice. Importantly, these phenotypes have not been reported in PGI 2 receptor-deficient mice. Conclusions-PGI 2 deficiency resulted in the development of vascular disorders with the thickening of vascular walls and interstitial fibrosis, especially in mouse kidneys. The findings demonstrated in vivo that PGI 2 is important in the homeostasis of blood vessels. Our established PGID mice are useful for studies on the initiation and development of vascular diseases, such as ischemic renal disorders with arterial sclerosis and infarction, and also for studies on the novel signaling pathway of PGI 2 .