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Hepatocyte growth factor—How do I know thee? Let me count the ways
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are told that these patients tended to have normalcaliber esophagi, suggesting that their stretch receptors in the esophageal wall had not been deactivated by chronic luminal distention. However, this cannot be the entire explanation, because other patients with nondilated esophagi still showed UES failure of relaxation after air instillation. Do the observations of Massey et al. explain the unusual achalasia in patients who develop a dilated esophagus with tracheal compression? Clearly, failure of UES relaxation would explain how the gas would be kept from leaving the esophagus, but how did the gas reach the esophagus in the first place? A study by Holloway et al.’ has shown that patients with achalasia in whom gas is placed in the stomach do not show transient lower esophageal sphincter relaxation and common cavity events as do control subjects in whom gas is instilled into the stomach.7 Therefore, the few unfortunate esophageal bloaters must either relax the lower esophageal sphincter in a manner that their achalasic colleagues do not or inject the air into the esophagus with the pharyngeal muscles during swallowing and then find themselves unable to vent the air. What questions remain? The authors mention that two of their patients had undergone lower esophageal myotomy. Did they have more trouble than the others because intragastric gas could reflux easily into the esophagus, or did they have less difficulty because a distal escape route for gas was now provided? Do patients with tumors infiltrating the lower esophageal sphincter zone producing a pseudoachalasia have a similar defect in UES function? We should be grateful to Massey et al. for emphasizing the clinical point that patients with achalasia are unable to belch. It certainly is an easy question to add to the diagnostic evaluation of swallowing difficulties. The implications of their study show the wonder of the control mechanisms of the esophagus. Who would think that such a simple-looking tube would manifest such complicated behavior? These investigators have extended the concept of achalasia (failure of relaxation) to the UES as well as to the
Unlike the lower esophageal lower sphincter. sphincter in achalasia, which does not relax for any stimulus, the fact that deglutition relaxes the UES but esophageal body distention does not relax the sphincter suggests that the defect in UES function in achalasia does not reside in the efferent limb of the neural control mechanism. Installation of air has certainly given food for thought! CHARLES E. POPE II, M.D.
VA Medical Center and University of Washington Seattle, Washington
References 1.
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4. 5.
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Massey BT, Hogan WJ, Dodds WJ, Dantas RO. Alteration of the upper esophageal belch reflex in patients with achalasia. Gastroenterology 1992;103:1574-1579. Smith D, King NA, Waldron B, Cullen PT, Millar B, Fenwick M, Campbell FC. Study of belching ability in antireflux surgery patients and normal volunteers. Br J Surg 1991;78:32-35. McNally EF, Kelly JE, Ingelfinger FJ. Mechanism of belching: effects of gastric distension with air. Gastroenterology 1964;46:254-259. Dent J. A new technique for continuous sphincter pressure measurement. Gastroenterology 1976;71:263-267. Kahrilas PJ, Dent J, Dodds WJ, Hogan WJ, Arndorfer RC. A method of continuous monitoring of upper esophageal sphincter pressure. Dig Dis Sci 1987;32:121-128. Kahrilas PJ, Dodds WJ, Dent J, Wyman JB, Hogan WJ, Arndorfer RC. Upper esophageal sphincter function during belching. Gastroenterology 1986;91:133-140. Wyman JB, Dent J, Heddle R, Dodds WJ, Toouli J, Downton J. Control of belching by the lower esophageal sphincter. Gut 1990;31:639-646. Holloway RH, Wyman JB, Dent J. Failure of transient lower oesophageal sphincter relaxation in response to gastric distention in patients with achalasia: evidence for neural mediation of transient lower oesophageal sphincter relaxations. Gut 1989;30:762-767.
Address requests for reprints to: Charles E. Pope II, M.D., Gastroenterology Section (111 GI), Seattle VA Medical Center, 1660 South Columbian Way, Seattle, Washington 98108. This is a U.S. government work. There are no restrictions on its use.
Hepatocyte Growth Factor-How Thee? Let Me Count the Ways The existence of a platelet-derived growth factor for hepatocytes was first suggested by Strain et al. in 1982.l One year later, three laboratories simultaneously reported the presence of factors that stimulated DNA synthesis in cultured hepatocytes: Mi-
School of Medicine
Do I Know
chalopoulos et al.’ described hepatopoietin A in rabbit serum, Nakamura et a1.3 reported on hepatotrophin in regenerating rat serum, and Russell et al. expanded their report on hepatocyte growth factor in rat platelets.4 A similar activity was subsequently
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found by Gohda et al.’ in the plasma of patients with fulminant hepatic failure. Work in three different laboratories resulted in the purification of a molecule with similar characteristics from rat platelets,6 the plasma of a patient with fulminant hepatic failure,7 and rabbit serum.’ By 1989, a complete sequence from a human placental complementary DNA library,g a partial rabbit amino acid sequence,” and an entire human liver sequence, including a description of the molecule’s secondary structure,” had been published. The sequences were virtually identical. Because of its powerful mitogenic effect on hepatocytes in culture, the molecule was christened hepatocyte growth factor (HGF).12*13 Characterization
of HGF
HGF has an unusual structure for a growth factor. It is a large heterodimeric molecule made up of a heavy a chain of -69 kilodaltons and a light p chain of -34 kilodaltons. It is synthesized as a single prepro molecule of 728 amino acids and is cleaved to produce the mature heterodimer. HGF has a 38% sequence homology to plasminogen. The heavy chain has four kringle domains reminiscent of the five kringle domains in plasminogen and those in several other fibrinolytic and coagulation-related proteins. They are thought to play an important role in protein-protein interactions. The p chain contains the consensus sequence for serine proteases and has a 37% homology with the p chain of plasmin. However, two of the three amino acids in the catalytic site have been replaced in HGF, and it has no proteolytic activity. In contrast, neither plasminogen nor plasmin is mitogenic to hepatocytes.12*‘3 The gene for human HGF has recently been localized to chromosome 7.15-17 HGF is the single most potent in vitro mitogen for hepatocytes yet described, being loo-fold more active than transforming growth factor a (TGF-a) or epidermal growth factor (EGF).12*13*1* It is species indiscriminate. Increased levels of HGF in the rat following partial hepatectomy (15-Ii’-fold within 1-2 hours’? or carbon tetrachloride-induced liver dam1Q*20 and in patients with acute hepatitis or cirrhoage sis “J as well as its marked increase in patients with fuiminant hepatitis,23 strongly suggest that it plays a key role in liver regeneration. Its ability to stimulate cultured liver cells to proliferate in a completely defined medium containing no serum or growth factors, and its increase in vivo within 1 hour of partial hepatectomy long before the initial increase in DNA synthesis at 16-18 hours, suggests that it might be the long-sought “initiating factor” that triggers liver regeneration.“*13 Concerns about this logical interpretation of the data began to develop with the availability of molecu-
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lar probes and specific antibodies for HGF. The first surprise was the widespread distribution of HGF in vivo. HGF messenger RNA (mRNA) was found in human placenta’ and rat lung, kidney, brain, liver, and thymus.14 HGF protein was not found in liver parenchymal cells, but it was found (along with HGF mRNA) in the liver Ito ce11.24,25HGF protein was also found histochemically in the exocrine pancreas, throughout the gastrointestinal tract mucosa, and in all squamous epithelia and lining glandular epithelia as well as the ovum. It is not clear whether all of these cells produce HGF or whether its presence indicates passive uptake, because not all of them were found to contain HGF mRNA.26 If HGF is a liver-specific growth factor, why is it so universally distributed? HGF was identified during a specific search for the elusive “humoral factors” that had been implicated in liver regeneration since the early cross-circulation experiments of Moolten and Bucherz7 and the reverse-flow and autotransplantation studies of Grisham et aI.” By their very design, these studies could only identify a liver growth factor. Initially, no experiments to determine organ specificity were performed. When they were, it rapidly became evident that HGF stimulated growth in vitro of a wide variety of epithelial cell types, inciuding melanocytes,2Q renal tubular epithelial cells,30s31 keratinocytes,32.33 breast carcinoma cells, and hepatoblastoma cells.34 As with partial hepectomy, HGF levels increased following unilateral nephrectomy, and HGF is the most potent in vitro growth stimulator of kidney cells and keratinocytes as well. What was once viewed as a potent but organ-specific molecule has now emerged as a pleiotrophic epithelial cell growth factor. Scatter Factor While the characterization of HGF was proceeding apace, parallel work was occuring independently in an entirely different field. In 1985 Stocker and Perryman35 reported that conditioned medium from embryo fibroblasts was able to disburse tightly packed mammary epithelial cells. Because of its “scattering” effect this protein, which was able to induce disassociation and increase local cell motility of a variety of epithelial cells, including some cancer cells and endothelial cells, was termed “scatter factor.” The standard assay uses the Madin-Darby canine kidney (MDCK) cell line. When added to MDCK cell cultures, it changes their morphology, disrupts cell-cell junctions, and shows both chemotactic and chemokinetic activities. However, it is not mitogenic to MDCK cells. When the complete amino acid sequence of scatter factor was obtained, a search of computer data bases produced the astounding conclusion that scatter factor and HGF are identicaL3’
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The morphogenic effects of scatter factor were further highlighted by the recent demonstration of its ability to induce MDCK epithelial cells to form branching tubules when grown in collagen gels.37 This has stimulated interest in the possible role of HGF-scatter factor in embryogenesis. Because of the extensive literature that already existed on scatter factor, and this remarkably different property of the molecule as a “motogen” and “morphogen,” some investigators now refer to the factor as HGF-SF. Other Growth Effects Yet another growth factor was identified by Rubin et al.3* in their efforts to isolate and characterize growth factors produced by stromal cells that act as mitogens for epithelial cells. Their interest was spurred by the purification of keratinocyte growth factor, a member of the fibroblast growth factor family of heparin-binding growth factors specific for epithelial cells.3g They identified a factor produced by fibroblasts that showed mitogenic activity on melanocytes, epithelial cells, and endothelial cells. This activity was expressed in stromal fibroblasts from a variety of organs including adult skin, lung, gastrointestinal tract, and prostate, as well as embryonic lung. It was not mitogenic for fibroblasts and was felt to represent a possible paracrine mediator of cell proliferation. Sequence data showed that this fibroblastderived mitogen had an extremely high degree of homology with HGF.3* HGF Receptor During this same period, work in other laboratories showed the existence of a new oncogene, the MET oncogene. 40*41This gene was originally identified in HOS cells that had been transformed by treatment with the carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). These MNNG-HOS cells formed tumors in nude mice and had typical characteristics of morphologically transformed cells. The HOS cell line was derived from a human osteosarcoma but did not show a transformed morphology in cells, nor produce tumors in mice, until the MET gene was activated. Transfection of the MET gene into NIH 3T3 fibroblasts proved sufficient for transformation of the fibroblasts. The receptor has a structure similar to that of known members of the protein tyrosine kinase receptor family and is expressed by a wide variety of tissues and cell types including hepatocytes, epithelial cells, and solid tumors.42 A gastric carcinoma cell, GTL-16, has a highly amplified and overexpressed MET gene, and transformed NIH 3T3 fibroblasts appear to express a ligand that binds to the MET receptor, thus forming a possible autocrine loop for stimulation of their own growth.43 Given the presumed till then narrow speci-
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ficity of HGF, it was thus quite a shock when it was determined that the MET proto-oncogene product is the receptor for HGF.44 Studies had previously shown that HGF might produce its effects via a tyrosine kinase receptor, and subsequent work has clearly shown that this receptor is the MET oncogene product.43,44 Tumor Suppression This raises a whole new series of questions concerning the possible role of HGF in stimulating tumor growth. Experiments with transfected NIH 3T3 cells, which produce HGF but do not stimulate their own growth unless transfected with the MET receptor, suggest a possible role for autocrine and paracrine stimulation of tumor growth.43 Thus, possibly the greatest surprise in the HGF story thus far has been the recognition that a cytotoxic factor produced by IMR-90 human fibroblasts, which is toxic to most sarcoma cells and cytostatic to a number of other cell lines, was also identical to HGF.45 Subsequent work has shown that HGF inhibits the growth of a number of hepatoma cell lines, including the HEP G2 cell.46 Other hepatoma cells are unaffected or show mild stimulation. In an extensive study, Shiota et al. examined 35 tumor cell lines for HGF mRNA. Only 6 expressed HGF mRNA, and no HCC cell line expressed the message. The growth of 8 HCC cell lines was inhibited by exogenous HGF. When an albumin HGF expression vector was introduced into FAO HCC cells, their growth in vitro was inhibited and the tumors produced in nude mice by the FOA HGF cells were only 10% of the size of the resistant controls. In contrast, hepatocytes in transgenic mice that expressed HGF grew more rapidly than their normal controls. Thus, although HGF appears to be mitogenie for normal hepatocytes, it is clearly inhibitory for most hepatocellular carcinoma cells and many other malignant cells. HGF and Liver Regeneration In the short period of 10 years, HGF has gone from a molecule with a unique and specific role, that of stimulating liver growth, to one with an almost unlimited variety of actions. With such a multiplicity of effects, how could HGF possibly play a critical role in the initiation of liver regeneration, the search for which was the genesis of its original discovery? Liver regeneration is an extremely complex phenomenon involving the abrupt movement of a majority of liver parenchymal cells from a nonproliferating Go state to one of rapid proliferation. But the liver is made up of many other cells and structures, and the provision of new endothelial cells, bile duct cells,
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Kuppfer cells, a reticulin and collagen framework, etc. must be coordinated. Although HGF is the most potent hepatocyte mitogen, it has not been shown to stimulate the growth of nonparenchymal liver cells. Recently, however, Joplin et a1.48showed that HGF is a potent stimulator of bile canalicular cell proliferation However, following partial hepatectomy, parenchymal cells start to divide at least 24 hours before the proliferation of sinusoidal lining cells and bile duct cells. How is this selective, timed responsiveness achieved? The circumstantial evidence provided by the increased circulating levels of HGF during liver repair in response to injury suggest a cause-effect relationship, but it has been curious that the highest levels of HGF have always been found in patients who died of fulminant hepatic failure.23 This raises the possibility that the liver may merely serve to clear HGF from the circulation and that high levels are an indication of liver dysfunction rather than active efforts to regenerate. The paper in this issue of GASTROENTEROLOGY by Tomiya et al. 4g adds further ammunition to this argument. In their simple but elegent study, they examined serum HGF levels in surgical patients undergoing partial hepatectomy or major surgery not involving the liver. All patients showed increased levels of HGF following surgery, regardless of whether any liver was removed or not. Even in the hepatectomized patients, the levels showed no relationship to the volume of liver resected, and the levels returned to normal within 28 days of partial hepatectomy, even though the restored liver volume averaged only 25% and regeneration was clearly incomplete. Multiple regression analysis showed a direct relationship of HGF levels to the existence of liver cirrhosis preoperatively and the maximal levels of serum total bilirubin, alanine aminotransferase, and peripheral blood white cells postoperatively in the hepatectomized groups. In the nonhepatectomized surgical patients, correlations were found only with maximal peripheral white cell counts and serum C-reactive protein levels. These results are compatible with the hypothesis that elevated levels of HGF postoperatively are a systemic response and often reflect failure of a damaged or diseased liver to clear HGF, or release of HGF from inflammatory cells, rather than production of HGF in an attempt to stimulate regeneration. Michalopoulous et al. have suggested that this may in fact be the simple mechanism by which HGF produces its stimulation of liver regeneration.‘**” He has hypothesized that HGF is normally cleared by the liver. When the capacity of the liver to remove HGF is abruptly reduced by surgical resection or toxin damage, the rapid increase in HGF concentration
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may be sufficient to initiate liver regeneration. This also circumvents the problem raised by the increase in HGF concentration that occurs before the local increase in HGF message, as discussed above. Michalopoulous et al. further suggest that the concurrent increase in norepinephrine levels results from a similar decrease in clearance and possibly from increased production due to the stress of surgery or illness. Norepinephrine has been shown to augment the effects of HGF on hepatocytes in culture.1g*50 Further insight has come from the description of a presumed humoral factor that induces the production of HGF mRNA in distal organs. Yanagita et al. report that partial hepatectomy and unilateral nephrectomy both produced marked and rapid increases in HGF mRNA levels in the intact lung and spleen.51 More recently, Matsumoto et al. from the same laboratory5’ have purified a factor more than 200-fold from the serum of carbon tetrachloridetreated rats that increases dramatically within 3-6 hours of liver or kidney injury. This factor induces HGF mRNA expression in the lung of rats in vivo and HGF production by MRC 5 cells when it is added to the culture medium. This substance, which they called “injurin,” thus appears to be a humoral factor that is released by the damaged organ and causes production of HGF at distant sites. While this is not the only explanation for the above results, a scenerio can be proposed in which partial hepatectomy produces two immediate effects: (a) rapid accumulation of HGF (and possibly norepinephrine) due to failure of the reduced liver mass to clear circulating HGF; and (b) release of injurin, which stimulates further production in distant organs of HGF, which then returns via the circulation to help sustain the initial regenerative response. Clearly HGF is not the entire story. More than 20 substances have been shown to stimulate hepatocyte DNA synthesis. 53 The rapidly expanding literature on the roles of TGF-a, EGF, TGF+, acid fibroblast growth factor, and hepatic stimulator substance must be taken into account. The majority of HGF experiments have been carried out in vitro. Only one study has shown direct evidence of the in vivo effectiveness of HGF.54 The cell cycle moves through an orderly sequence of steps with many different control points, at each of which specific factors must be present for the cell to proceed to complete cell division, HGF is just one of the factors, albeit a critical one. The interplay of the mesenchyme and distant organs on liver cell growth is clearly an important area for continued research as well. There is no “master switch” or “magic bullet,” but the actions of HGF must certainly be accounted for in any attempt to understand the mechanisms of liver regeneration.
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Future Directions Mitogen, motogen, morphogen, metastagen (or suppressogen), . . . what role will HGF reveal next? At various times,HGF appears to act as an endocrine, a paracrine, and an autocrine factor. How does it carry out such a variety of different effects? In the past it has been felt that much of the control of second messenger activity was defined by the binding of a specific ligand to a specific receptor. To date only one receptor for HGF, the MET oncogene product, has been described. Clearly, much of the control must reside in steps beyond the initial binding and generation of message at the cell membrane. Already minor differences in HGF sequence and structure, and minor differences in the construction of the MET/HGF receptor, have been reported. How they produce different secondary and tertiary messages should be a .fruitful area of research into the next century. As the full picture of HGF is uncovered, a complex tapestry of effects has been revealed that should ultimately provide important insights into embryogenesis, morphogenesis, carcinogenesis, cellular growth control, cell motility, and the mechanisms by which the binding of a molecule at the cell surface specifies internal cellular actions. At the beginning of this century, it was often stated that “if one understood syphilis (or tuberculosis), one understood all of medicine.” More recently, acquired immunodeficiency syndrome has been substituted for tuberculosis and syphilis. In the future one might well be able to state “if you understand HGF, you understand biology.”
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DOUGLAS R. LABRECQUE, M.D.
Department of Internal
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Medicine University of Iowa College of Medicine and Veterans Administration Medical Center Iowa City, Iowa
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parenchymal hepatocytes in primary cultures. Proc Nat1 Acad Sci USA 1986;83:6489-6493. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama 0, Takahashi K, Miyazaki H, Hashimoto S, Daikuhara Y. Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J Clin Invest 1988;81:414-419. Zarnegar R, Michalopoulos G. Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res 1989;49:3314-3320. Miyazawa K, Tsubouchi H, Naka D, Takahashi K, Okigaki M, Arakaki N, Nakayama H, Hirono S, Sakiyama 0, Takahashi K, Gohda E, Daikuhara Y, Kitamura N. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem Biophys Res Commun 1989;163:967-973. Zarnegar R, Muga S, Enghild J, Michalopoulos G. NH,-terminal amino acid sequence of rabbit hepatopoietin A, a heparinbinding polypeptide growth factor for hepatocytes. Biochem Biophys Res Commun 1989;163:1370-1376. Nakamura R, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440-443. Michalopoulos GK, Zarnegar R. Hepatocyte growth factor (editorial). Hepatology 1992;15:149-155. Nakamura T. Structure and function of hepatocyte growth factor. Prog Growth Factor Res 1991;3:67-85. Tashiro K, Hagiya M, Nishizawa T, Seki T, Shimonishi M, Shimizu S, Nakamura T. Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues. Proc Nat1 Acad Sci USA 1990;87:3200-3204. Laguda B, Selden C, Jones M, Hodgson H, and Spurr NK. Assignment of the hepatocyte growth factor (HGF) to chromosome 7q22-qter. Ann Hum Genet 1991;55:213-216. Fukuyama R, Ichijoh Y, Minoshima S, Kitamura N, Shimizu N. Regional localization of the hepatocyte growth factor (HGF) gene to human chromosome 7 band q21.1. Genomics 1991;11:410-415. Zarnegar R, Petersen B, DeFrances MC, Michalopoulos G. Localization of hepatocyte growth factor (HGF) gene on human chromosome 7. Genomics 1992;12:147-150. Strain AJ, Ismail T, Tsubouchi H, Arakaki N, Hishida T, Kitamura N, Daikuhara Y, McMaster P. Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes. J Clin Invest 1991;87:1853-1857. Lindroos PM, Zarnegar R, Michalopoulos GK. Hepatic growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepato1ogy 1991;13:743-750. Asami 0, Ihara I, Shimidzu N, Shimizu S, Tomita Y, Ichihara A, Nakamura T. Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloridetreated rats. J Biochem 1991;109:8-13. Tsubouchi H, Niitani Y, Hirono S, Nakayama H, Gohda E, Arakaki N, Sakiyama 0, Takahashi K, Kimoto M, Kawakami S, Setoguchi M, Tachikawa T, Shin S, Arima T, Daikuhara Y. Levels of the human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay. Hepatology 1991;13:1-5. Shimizu I, Ichihara A, Nakamura T. Hepatocyte growth factor in ascites from patients with cirrhosis. J Biochem 1991; 109:14-18. Tsubouchi H, Hirono S, Gohda E, Nakayama H, Takahashi K, Sakiyama 0, Miyazaki H, Sugihara J, Tomita E, Muto Y, Daikuhara Y, Hashimoto S. Clinical significance of human hepa-
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growth factor in blood from patients with fulminant hepatic failure. Hepatology 1989;9:875-881. Schirmacher P, Geerts A, Pietrangelo A, Dienes HP, Rogler CE. Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology 1992;15:5-11. Ramadori G, Neubauer K, Odenthal M, Nakamura T, Knittel T, Schwljgler S, Meyer zum Bttschenfelde K-H. The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-p. Biochem Biophys Res Commun 1992;183:739-742. Wolf HK, Zarnegar R, Michalopoulos GK. Localization of hepatocyte growth factor in human and rat tissues: an immunohistochemical study. Hepatology 1991;14:488-494. Moolten FL, Bucher NLR. Regeneration of rat liver: transfer of humoral agents by cross circulation. Science 1967;158:272279. Grisham JW, Leong GF, Hole BV. Heterotropic partial autotransplantation of rat liver: technique and demonstration of structure and function of the graft. Cancer Res 1964;24:1474-
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and characterization of a newly identified growth factor specific for epithelial cells. Proc Nat1 Acad Sci USA 1989;86:802-806. Rhim JS, Kin C, Arnstein P, Heubner RH, Weisburger EK, Nelson-Rees WA. Transformation of human osteosarcoma cells by a chemical carcinogen. J Nat1 Cancer Inst 1975;55:12911295. Rhim JA, Park DK, Arnstein P, Huebner RJ, Weisburger EK. Transformation of human cells in culture by N-methyl-Nnitro-N-nitrosoguanidine. Nature 1975;256:751-753. Prat M, Narsimban RP, Crepaldi T, Nicotra PG, Natali PG, Comoglio PM. The receptor encoded by the human c-met oncogene is expressed in hepatocytes, in epithelial cells and in solid tumors. Int J Cancer 1991;49:323-328. Cooper CS. The met oncogene: from detection by transfection to transmembrane receptor for hepatocyte growth factor. Oncogene 1992;7:3-7. Bottaro DP, Rubin JS, Faletto DL, Chan AM-L, Kmiecik TE, Vande Woude GF, Aaronson SA. Identification of the hepatocyte growth factor receptor as the c-met proto-oncogene product. Science 1991;251:802-804. Higashio K, Shima N, Goto M, Itagaki Y, Nagao M, Yasuda H, Morinaga T. Identity of a tumor cytotoxic factor from human fibroblasts and hepatocyte growth factor. Biochem Biophys Res Commun 1990;170:397-404. Tajima H, Matsumoto K, Nakamura T. Hepatocyte growth factor has potent anti-proliferative activity in various tumor cell lines. FEBS Lett 1991;291:229-232. Shiota G, Rhoads DB, Wang TC, Nakamura T, Schmidt EV. Hepatocyte growth factor inhibits growth of hepatocellular carcinoma cells. Proc Nat1 Acad Sci USA 1992;89:373-377. Joplin R, Hishida T, Tsuboushi H, Daikuhara Y, Ayres R, Neubiliary epithelial herger JM, Strain AJ. Human intrahepatic cells proliferate in vitro in response to human hepatocyte growth factor. J Clin Invest 1992;90:1284-1289. Tomiya T, Tani M, Yamada S, Hayashi S, Umeda N, Fujiwara K. Serum hepatocyte growth factor levels in hepatectomized and nonhepatectomized surgical patients. Gastroenterology 1992;103:1621-1624. Cruise JL, Knechtle S, Bollinger RR, Kuhn C, Michalopoulos G. Alpha-l adrenergic effects and liver regeneration. Hepatology 1987;7:1189-1194. Yanagita K, Nagaike M, Ishibashi H, Niho Y, Matsumoto K, Nakamura T. Lung may have an endocrine function producing hepatocyte growth factor in response to injury of distal organs. Biochem Biophys Res Commun 1992;182:802-809, Matsumoto K, Tajima H, Hamanoue M, Kohno S, Kinoshita T, Nakamura T. Identification and characterization of “injurin,” an inducer of expression of the gene for hepatocyte growth factor. Proc Nat1 Acad Sci USA 1992;89:3800-3804, Bucher NLR, Strain AJ. Regulatory mechanisms in hepatic regeneration. In: Miller-Sadler GH, Wright R, Arthor MJP, eds. Wright’s liver and biliary disease. London: Saunders, 1992. Francavilla A, Starzl TE, Porter K, Scotti-Foglieni C. Michalopoulos GK, Carrieri G, Trejo J, Azzarone A, Barone M, Zeng QH. Screening for candidate hepatic growth factors by selective portal infusion after canine Eck’s fistula. Hepatology 1991:14:665-670.
Address requests for reprints to: Douglas R. LaBrecque, M.D., Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242. 0 1992 by the American
Gastroenterological
Association
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are told that these patients tended to have normalcaliber esophagi, suggesting that their stretch receptors in the esophageal wall had not been deactivated by chronic luminal distention. However, this cannot be the entire explanation, because other patients with nondilated esophagi still showed UES failure of relaxation after air instillation. Do the observations of Massey et al. explain the unusual achalasia in patients who develop a dilated esophagus with tracheal compression? Clearly, failure of UES relaxation would explain how the gas would be kept from leaving the esophagus, but how did the gas reach the esophagus in the first place? A study by Holloway et al.’ has shown that patients with achalasia in whom gas is placed in the stomach do not show transient lower esophageal sphincter relaxation and common cavity events as do control subjects in whom gas is instilled into the stomach.7 Therefore, the few unfortunate esophageal bloaters must either relax the lower esophageal sphincter in a manner that their achalasic colleagues do not or inject the air into the esophagus with the pharyngeal muscles during swallowing and then find themselves unable to vent the air. What questions remain? The authors mention that two of their patients had undergone lower esophageal myotomy. Did they have more trouble than the others because intragastric gas could reflux easily into the esophagus, or did they have less difficulty because a distal escape route for gas was now provided? Do patients with tumors infiltrating the lower esophageal sphincter zone producing a pseudoachalasia have a similar defect in UES function? We should be grateful to Massey et al. for emphasizing the clinical point that patients with achalasia are unable to belch. It certainly is an easy question to add to the diagnostic evaluation of swallowing difficulties. The implications of their study show the wonder of the control mechanisms of the esophagus. Who would think that such a simple-looking tube would manifest such complicated behavior? These investigators have extended the concept of achalasia (failure of relaxation) to the UES as well as to the
Unlike the lower esophageal lower sphincter. sphincter in achalasia, which does not relax for any stimulus, the fact that deglutition relaxes the UES but esophageal body distention does not relax the sphincter suggests that the defect in UES function in achalasia does not reside in the efferent limb of the neural control mechanism. Installation of air has certainly given food for thought! CHARLES E. POPE II, M.D.
VA Medical Center and University of Washington Seattle, Washington
References 1.
2.
3.
4. 5.
6.
7.
8.
Massey BT, Hogan WJ, Dodds WJ, Dantas RO. Alteration of the upper esophageal belch reflex in patients with achalasia. Gastroenterology 1992;103:1574-1579. Smith D, King NA, Waldron B, Cullen PT, Millar B, Fenwick M, Campbell FC. Study of belching ability in antireflux surgery patients and normal volunteers. Br J Surg 1991;78:32-35. McNally EF, Kelly JE, Ingelfinger FJ. Mechanism of belching: effects of gastric distension with air. Gastroenterology 1964;46:254-259. Dent J. A new technique for continuous sphincter pressure measurement. Gastroenterology 1976;71:263-267. Kahrilas PJ, Dent J, Dodds WJ, Hogan WJ, Arndorfer RC. A method of continuous monitoring of upper esophageal sphincter pressure. Dig Dis Sci 1987;32:121-128. Kahrilas PJ, Dodds WJ, Dent J, Wyman JB, Hogan WJ, Arndorfer RC. Upper esophageal sphincter function during belching. Gastroenterology 1986;91:133-140. Wyman JB, Dent J, Heddle R, Dodds WJ, Toouli J, Downton J. Control of belching by the lower esophageal sphincter. Gut 1990;31:639-646. Holloway RH, Wyman JB, Dent J. Failure of transient lower oesophageal sphincter relaxation in response to gastric distention in patients with achalasia: evidence for neural mediation of transient lower oesophageal sphincter relaxations. Gut 1989;30:762-767.
Address requests for reprints to: Charles E. Pope II, M.D., Gastroenterology Section (111 GI), Seattle VA Medical Center, 1660 South Columbian Way, Seattle, Washington 98108. This is a U.S. government work. There are no restrictions on its use.
Hepatocyte Growth Factor-How Thee? Let Me Count the Ways The existence of a platelet-derived growth factor for hepatocytes was first suggested by Strain et al. in 1982.l One year later, three laboratories simultaneously reported the presence of factors that stimulated DNA synthesis in cultured hepatocytes: Mi-
School of Medicine
Do I Know
chalopoulos et al.’ described hepatopoietin A in rabbit serum, Nakamura et a1.3 reported on hepatotrophin in regenerating rat serum, and Russell et al. expanded their report on hepatocyte growth factor in rat platelets.4 A similar activity was subsequently
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found by Gohda et al.’ in the plasma of patients with fulminant hepatic failure. Work in three different laboratories resulted in the purification of a molecule with similar characteristics from rat platelets,6 the plasma of a patient with fulminant hepatic failure,7 and rabbit serum.’ By 1989, a complete sequence from a human placental complementary DNA library,g a partial rabbit amino acid sequence,” and an entire human liver sequence, including a description of the molecule’s secondary structure,” had been published. The sequences were virtually identical. Because of its powerful mitogenic effect on hepatocytes in culture, the molecule was christened hepatocyte growth factor (HGF).12*13 Characterization
of HGF
HGF has an unusual structure for a growth factor. It is a large heterodimeric molecule made up of a heavy a chain of -69 kilodaltons and a light p chain of -34 kilodaltons. It is synthesized as a single prepro molecule of 728 amino acids and is cleaved to produce the mature heterodimer. HGF has a 38% sequence homology to plasminogen. The heavy chain has four kringle domains reminiscent of the five kringle domains in plasminogen and those in several other fibrinolytic and coagulation-related proteins. They are thought to play an important role in protein-protein interactions. The p chain contains the consensus sequence for serine proteases and has a 37% homology with the p chain of plasmin. However, two of the three amino acids in the catalytic site have been replaced in HGF, and it has no proteolytic activity. In contrast, neither plasminogen nor plasmin is mitogenic to hepatocytes.12*‘3 The gene for human HGF has recently been localized to chromosome 7.15-17 HGF is the single most potent in vitro mitogen for hepatocytes yet described, being loo-fold more active than transforming growth factor a (TGF-a) or epidermal growth factor (EGF).12*13*1* It is species indiscriminate. Increased levels of HGF in the rat following partial hepatectomy (15-Ii’-fold within 1-2 hours’? or carbon tetrachloride-induced liver dam1Q*20 and in patients with acute hepatitis or cirrhoage sis “J as well as its marked increase in patients with fuiminant hepatitis,23 strongly suggest that it plays a key role in liver regeneration. Its ability to stimulate cultured liver cells to proliferate in a completely defined medium containing no serum or growth factors, and its increase in vivo within 1 hour of partial hepatectomy long before the initial increase in DNA synthesis at 16-18 hours, suggests that it might be the long-sought “initiating factor” that triggers liver regeneration.“*13 Concerns about this logical interpretation of the data began to develop with the availability of molecu-
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lar probes and specific antibodies for HGF. The first surprise was the widespread distribution of HGF in vivo. HGF messenger RNA (mRNA) was found in human placenta’ and rat lung, kidney, brain, liver, and thymus.14 HGF protein was not found in liver parenchymal cells, but it was found (along with HGF mRNA) in the liver Ito ce11.24,25HGF protein was also found histochemically in the exocrine pancreas, throughout the gastrointestinal tract mucosa, and in all squamous epithelia and lining glandular epithelia as well as the ovum. It is not clear whether all of these cells produce HGF or whether its presence indicates passive uptake, because not all of them were found to contain HGF mRNA.26 If HGF is a liver-specific growth factor, why is it so universally distributed? HGF was identified during a specific search for the elusive “humoral factors” that had been implicated in liver regeneration since the early cross-circulation experiments of Moolten and Bucherz7 and the reverse-flow and autotransplantation studies of Grisham et aI.” By their very design, these studies could only identify a liver growth factor. Initially, no experiments to determine organ specificity were performed. When they were, it rapidly became evident that HGF stimulated growth in vitro of a wide variety of epithelial cell types, inciuding melanocytes,2Q renal tubular epithelial cells,30s31 keratinocytes,32.33 breast carcinoma cells, and hepatoblastoma cells.34 As with partial hepectomy, HGF levels increased following unilateral nephrectomy, and HGF is the most potent in vitro growth stimulator of kidney cells and keratinocytes as well. What was once viewed as a potent but organ-specific molecule has now emerged as a pleiotrophic epithelial cell growth factor. Scatter Factor While the characterization of HGF was proceeding apace, parallel work was occuring independently in an entirely different field. In 1985 Stocker and Perryman35 reported that conditioned medium from embryo fibroblasts was able to disburse tightly packed mammary epithelial cells. Because of its “scattering” effect this protein, which was able to induce disassociation and increase local cell motility of a variety of epithelial cells, including some cancer cells and endothelial cells, was termed “scatter factor.” The standard assay uses the Madin-Darby canine kidney (MDCK) cell line. When added to MDCK cell cultures, it changes their morphology, disrupts cell-cell junctions, and shows both chemotactic and chemokinetic activities. However, it is not mitogenic to MDCK cells. When the complete amino acid sequence of scatter factor was obtained, a search of computer data bases produced the astounding conclusion that scatter factor and HGF are identicaL3’
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The morphogenic effects of scatter factor were further highlighted by the recent demonstration of its ability to induce MDCK epithelial cells to form branching tubules when grown in collagen gels.37 This has stimulated interest in the possible role of HGF-scatter factor in embryogenesis. Because of the extensive literature that already existed on scatter factor, and this remarkably different property of the molecule as a “motogen” and “morphogen,” some investigators now refer to the factor as HGF-SF. Other Growth Effects Yet another growth factor was identified by Rubin et al.3* in their efforts to isolate and characterize growth factors produced by stromal cells that act as mitogens for epithelial cells. Their interest was spurred by the purification of keratinocyte growth factor, a member of the fibroblast growth factor family of heparin-binding growth factors specific for epithelial cells.3g They identified a factor produced by fibroblasts that showed mitogenic activity on melanocytes, epithelial cells, and endothelial cells. This activity was expressed in stromal fibroblasts from a variety of organs including adult skin, lung, gastrointestinal tract, and prostate, as well as embryonic lung. It was not mitogenic for fibroblasts and was felt to represent a possible paracrine mediator of cell proliferation. Sequence data showed that this fibroblastderived mitogen had an extremely high degree of homology with HGF.3* HGF Receptor During this same period, work in other laboratories showed the existence of a new oncogene, the MET oncogene. 40*41This gene was originally identified in HOS cells that had been transformed by treatment with the carcinogen N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). These MNNG-HOS cells formed tumors in nude mice and had typical characteristics of morphologically transformed cells. The HOS cell line was derived from a human osteosarcoma but did not show a transformed morphology in cells, nor produce tumors in mice, until the MET gene was activated. Transfection of the MET gene into NIH 3T3 fibroblasts proved sufficient for transformation of the fibroblasts. The receptor has a structure similar to that of known members of the protein tyrosine kinase receptor family and is expressed by a wide variety of tissues and cell types including hepatocytes, epithelial cells, and solid tumors.42 A gastric carcinoma cell, GTL-16, has a highly amplified and overexpressed MET gene, and transformed NIH 3T3 fibroblasts appear to express a ligand that binds to the MET receptor, thus forming a possible autocrine loop for stimulation of their own growth.43 Given the presumed till then narrow speci-
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ficity of HGF, it was thus quite a shock when it was determined that the MET proto-oncogene product is the receptor for HGF.44 Studies had previously shown that HGF might produce its effects via a tyrosine kinase receptor, and subsequent work has clearly shown that this receptor is the MET oncogene product.43,44 Tumor Suppression This raises a whole new series of questions concerning the possible role of HGF in stimulating tumor growth. Experiments with transfected NIH 3T3 cells, which produce HGF but do not stimulate their own growth unless transfected with the MET receptor, suggest a possible role for autocrine and paracrine stimulation of tumor growth.43 Thus, possibly the greatest surprise in the HGF story thus far has been the recognition that a cytotoxic factor produced by IMR-90 human fibroblasts, which is toxic to most sarcoma cells and cytostatic to a number of other cell lines, was also identical to HGF.45 Subsequent work has shown that HGF inhibits the growth of a number of hepatoma cell lines, including the HEP G2 cell.46 Other hepatoma cells are unaffected or show mild stimulation. In an extensive study, Shiota et al. examined 35 tumor cell lines for HGF mRNA. Only 6 expressed HGF mRNA, and no HCC cell line expressed the message. The growth of 8 HCC cell lines was inhibited by exogenous HGF. When an albumin HGF expression vector was introduced into FAO HCC cells, their growth in vitro was inhibited and the tumors produced in nude mice by the FOA HGF cells were only 10% of the size of the resistant controls. In contrast, hepatocytes in transgenic mice that expressed HGF grew more rapidly than their normal controls. Thus, although HGF appears to be mitogenie for normal hepatocytes, it is clearly inhibitory for most hepatocellular carcinoma cells and many other malignant cells. HGF and Liver Regeneration In the short period of 10 years, HGF has gone from a molecule with a unique and specific role, that of stimulating liver growth, to one with an almost unlimited variety of actions. With such a multiplicity of effects, how could HGF possibly play a critical role in the initiation of liver regeneration, the search for which was the genesis of its original discovery? Liver regeneration is an extremely complex phenomenon involving the abrupt movement of a majority of liver parenchymal cells from a nonproliferating Go state to one of rapid proliferation. But the liver is made up of many other cells and structures, and the provision of new endothelial cells, bile duct cells,
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1992
Kuppfer cells, a reticulin and collagen framework, etc. must be coordinated. Although HGF is the most potent hepatocyte mitogen, it has not been shown to stimulate the growth of nonparenchymal liver cells. Recently, however, Joplin et a1.48showed that HGF is a potent stimulator of bile canalicular cell proliferation However, following partial hepatectomy, parenchymal cells start to divide at least 24 hours before the proliferation of sinusoidal lining cells and bile duct cells. How is this selective, timed responsiveness achieved? The circumstantial evidence provided by the increased circulating levels of HGF during liver repair in response to injury suggest a cause-effect relationship, but it has been curious that the highest levels of HGF have always been found in patients who died of fulminant hepatic failure.23 This raises the possibility that the liver may merely serve to clear HGF from the circulation and that high levels are an indication of liver dysfunction rather than active efforts to regenerate. The paper in this issue of GASTROENTEROLOGY by Tomiya et al. 4g adds further ammunition to this argument. In their simple but elegent study, they examined serum HGF levels in surgical patients undergoing partial hepatectomy or major surgery not involving the liver. All patients showed increased levels of HGF following surgery, regardless of whether any liver was removed or not. Even in the hepatectomized patients, the levels showed no relationship to the volume of liver resected, and the levels returned to normal within 28 days of partial hepatectomy, even though the restored liver volume averaged only 25% and regeneration was clearly incomplete. Multiple regression analysis showed a direct relationship of HGF levels to the existence of liver cirrhosis preoperatively and the maximal levels of serum total bilirubin, alanine aminotransferase, and peripheral blood white cells postoperatively in the hepatectomized groups. In the nonhepatectomized surgical patients, correlations were found only with maximal peripheral white cell counts and serum C-reactive protein levels. These results are compatible with the hypothesis that elevated levels of HGF postoperatively are a systemic response and often reflect failure of a damaged or diseased liver to clear HGF, or release of HGF from inflammatory cells, rather than production of HGF in an attempt to stimulate regeneration. Michalopoulous et al. have suggested that this may in fact be the simple mechanism by which HGF produces its stimulation of liver regeneration.‘**” He has hypothesized that HGF is normally cleared by the liver. When the capacity of the liver to remove HGF is abruptly reduced by surgical resection or toxin damage, the rapid increase in HGF concentration
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may be sufficient to initiate liver regeneration. This also circumvents the problem raised by the increase in HGF concentration that occurs before the local increase in HGF message, as discussed above. Michalopoulous et al. further suggest that the concurrent increase in norepinephrine levels results from a similar decrease in clearance and possibly from increased production due to the stress of surgery or illness. Norepinephrine has been shown to augment the effects of HGF on hepatocytes in culture.1g*50 Further insight has come from the description of a presumed humoral factor that induces the production of HGF mRNA in distal organs. Yanagita et al. report that partial hepatectomy and unilateral nephrectomy both produced marked and rapid increases in HGF mRNA levels in the intact lung and spleen.51 More recently, Matsumoto et al. from the same laboratory5’ have purified a factor more than 200-fold from the serum of carbon tetrachloridetreated rats that increases dramatically within 3-6 hours of liver or kidney injury. This factor induces HGF mRNA expression in the lung of rats in vivo and HGF production by MRC 5 cells when it is added to the culture medium. This substance, which they called “injurin,” thus appears to be a humoral factor that is released by the damaged organ and causes production of HGF at distant sites. While this is not the only explanation for the above results, a scenerio can be proposed in which partial hepatectomy produces two immediate effects: (a) rapid accumulation of HGF (and possibly norepinephrine) due to failure of the reduced liver mass to clear circulating HGF; and (b) release of injurin, which stimulates further production in distant organs of HGF, which then returns via the circulation to help sustain the initial regenerative response. Clearly HGF is not the entire story. More than 20 substances have been shown to stimulate hepatocyte DNA synthesis. 53 The rapidly expanding literature on the roles of TGF-a, EGF, TGF+, acid fibroblast growth factor, and hepatic stimulator substance must be taken into account. The majority of HGF experiments have been carried out in vitro. Only one study has shown direct evidence of the in vivo effectiveness of HGF.54 The cell cycle moves through an orderly sequence of steps with many different control points, at each of which specific factors must be present for the cell to proceed to complete cell division, HGF is just one of the factors, albeit a critical one. The interplay of the mesenchyme and distant organs on liver cell growth is clearly an important area for continued research as well. There is no “master switch” or “magic bullet,” but the actions of HGF must certainly be accounted for in any attempt to understand the mechanisms of liver regeneration.
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GASTROENTEROLOGY
Future Directions Mitogen, motogen, morphogen, metastagen (or suppressogen), . . . what role will HGF reveal next? At various times,HGF appears to act as an endocrine, a paracrine, and an autocrine factor. How does it carry out such a variety of different effects? In the past it has been felt that much of the control of second messenger activity was defined by the binding of a specific ligand to a specific receptor. To date only one receptor for HGF, the MET oncogene product, has been described. Clearly, much of the control must reside in steps beyond the initial binding and generation of message at the cell membrane. Already minor differences in HGF sequence and structure, and minor differences in the construction of the MET/HGF receptor, have been reported. How they produce different secondary and tertiary messages should be a .fruitful area of research into the next century. As the full picture of HGF is uncovered, a complex tapestry of effects has been revealed that should ultimately provide important insights into embryogenesis, morphogenesis, carcinogenesis, cellular growth control, cell motility, and the mechanisms by which the binding of a molecule at the cell surface specifies internal cellular actions. At the beginning of this century, it was often stated that “if one understood syphilis (or tuberculosis), one understood all of medicine.” More recently, acquired immunodeficiency syndrome has been substituted for tuberculosis and syphilis. In the future one might well be able to state “if you understand HGF, you understand biology.”
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DOUGLAS R. LABRECQUE, M.D.
Department of Internal
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Medicine University of Iowa College of Medicine and Veterans Administration Medical Center Iowa City, Iowa
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parenchymal hepatocytes in primary cultures. Proc Nat1 Acad Sci USA 1986;83:6489-6493. Gohda E, Tsubouchi H, Nakayama H, Hirono S, Sakiyama 0, Takahashi K, Miyazaki H, Hashimoto S, Daikuhara Y. Purification and partial characterization of hepatocyte growth factor from plasma of a patient with fulminant hepatic failure. J Clin Invest 1988;81:414-419. Zarnegar R, Michalopoulos G. Purification and biological characterization of human hepatopoietin A, a polypeptide growth factor for hepatocytes. Cancer Res 1989;49:3314-3320. Miyazawa K, Tsubouchi H, Naka D, Takahashi K, Okigaki M, Arakaki N, Nakayama H, Hirono S, Sakiyama 0, Takahashi K, Gohda E, Daikuhara Y, Kitamura N. Molecular cloning and sequence analysis of cDNA for human hepatocyte growth factor. Biochem Biophys Res Commun 1989;163:967-973. Zarnegar R, Muga S, Enghild J, Michalopoulos G. NH,-terminal amino acid sequence of rabbit hepatopoietin A, a heparinbinding polypeptide growth factor for hepatocytes. Biochem Biophys Res Commun 1989;163:1370-1376. Nakamura R, Nishizawa T, Hagiya M, Seki T, Shimonishi M, Sugimura A, Tashiro K, Shimizu S. Molecular cloning and expression of human hepatocyte growth factor. Nature 1989;342:440-443. Michalopoulos GK, Zarnegar R. Hepatocyte growth factor (editorial). Hepatology 1992;15:149-155. Nakamura T. Structure and function of hepatocyte growth factor. Prog Growth Factor Res 1991;3:67-85. Tashiro K, Hagiya M, Nishizawa T, Seki T, Shimonishi M, Shimizu S, Nakamura T. Deduced primary structure of rat hepatocyte growth factor and expression of the mRNA in rat tissues. Proc Nat1 Acad Sci USA 1990;87:3200-3204. Laguda B, Selden C, Jones M, Hodgson H, and Spurr NK. Assignment of the hepatocyte growth factor (HGF) to chromosome 7q22-qter. Ann Hum Genet 1991;55:213-216. Fukuyama R, Ichijoh Y, Minoshima S, Kitamura N, Shimizu N. Regional localization of the hepatocyte growth factor (HGF) gene to human chromosome 7 band q21.1. Genomics 1991;11:410-415. Zarnegar R, Petersen B, DeFrances MC, Michalopoulos G. Localization of hepatocyte growth factor (HGF) gene on human chromosome 7. Genomics 1992;12:147-150. Strain AJ, Ismail T, Tsubouchi H, Arakaki N, Hishida T, Kitamura N, Daikuhara Y, McMaster P. Native and recombinant human hepatocyte growth factors are highly potent promoters of DNA synthesis in both human and rat hepatocytes. J Clin Invest 1991;87:1853-1857. Lindroos PM, Zarnegar R, Michalopoulos GK. Hepatic growth factor (hepatopoietin A) rapidly increases in plasma before DNA synthesis and liver regeneration stimulated by partial hepatectomy and carbon tetrachloride administration. Hepato1ogy 1991;13:743-750. Asami 0, Ihara I, Shimidzu N, Shimizu S, Tomita Y, Ichihara A, Nakamura T. Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloridetreated rats. J Biochem 1991;109:8-13. Tsubouchi H, Niitani Y, Hirono S, Nakayama H, Gohda E, Arakaki N, Sakiyama 0, Takahashi K, Kimoto M, Kawakami S, Setoguchi M, Tachikawa T, Shin S, Arima T, Daikuhara Y. Levels of the human hepatocyte growth factor in serum of patients with various liver diseases determined by an enzyme-linked immunosorbent assay. Hepatology 1991;13:1-5. Shimizu I, Ichihara A, Nakamura T. Hepatocyte growth factor in ascites from patients with cirrhosis. J Biochem 1991; 109:14-18. Tsubouchi H, Hirono S, Gohda E, Nakayama H, Takahashi K, Sakiyama 0, Miyazaki H, Sugihara J, Tomita E, Muto Y, Daikuhara Y, Hashimoto S. Clinical significance of human hepa-
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growth factor in blood from patients with fulminant hepatic failure. Hepatology 1989;9:875-881. Schirmacher P, Geerts A, Pietrangelo A, Dienes HP, Rogler CE. Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells. Hepatology 1992;15:5-11. Ramadori G, Neubauer K, Odenthal M, Nakamura T, Knittel T, Schwljgler S, Meyer zum Bttschenfelde K-H. The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-p. Biochem Biophys Res Commun 1992;183:739-742. Wolf HK, Zarnegar R, Michalopoulos GK. Localization of hepatocyte growth factor in human and rat tissues: an immunohistochemical study. Hepatology 1991;14:488-494. Moolten FL, Bucher NLR. Regeneration of rat liver: transfer of humoral agents by cross circulation. Science 1967;158:272279. Grisham JW, Leong GF, Hole BV. Heterotropic partial autotransplantation of rat liver: technique and demonstration of structure and function of the graft. Cancer Res 1964;24:1474-
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Address requests for reprints to: Douglas R. LaBrecque, M.D., Department of Internal Medicine, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242. 0 1992 by the American
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