Purification and subunit structure of hepatocyte growth factor from rat platelets

A hepatocyte growth factor (HGF) that stimulates DNA synthesis of adult rat hepatocytes in primary culture was purified as a homogeneous material from platelets of 1000 rats by a four-step procedure: stimulation of its release from platelets by thrombin, cation-exchanger fast protein liquid chromatography (FPLC) on a Mono S column, heparin-Sepharose CL-6B chromatography, and reverse-phase HPLC on a C4 column. The purified HGF stimulated DNA synthesis of adult rat hepatocytes in primary culture at 1 ng/ml and was maximally effective at 5 ng/ml, being about twice as potent as EGF at this concentration. HGF did not stimulate DNA synthesis of Swiss 3T3 cells. It was found to be a heat- and acid-labile protein that was inactivated by reduction with dithiothreitol. The purified HGF had a molecular mass of 82 kDa, as estimated by SDS-PAGE, and was found to be a heterodimer which dissociated into a large subunit of 69 kDa and a small one of 34 kDa by SDS-PAGE under reducing conditions. These biological and chemical properties showed that HGF was not identical with any known growth factors, including platelet-derived growth factor (PDGF).     

Inhibitory effect of transforming growth factor-beta on DNA synthesis of adult rat hepatocytes in primary culture

A transforming growth factor-beta (TGF-beta) found in platelets strongly inhibited DNA synthesis of adult rat hepatocytes in primary culture stimulated by insulin plus EGF or by hepatocyte growth factor (HGF) from rat platelets, but not the syntheses of secretory and intracellular proteins by the cells. TGF-beta had no cytotoxic effect, as judged by phase-contrast microscopic examination of the cell morphology. The inhibition of DNA synthesis by TGF-beta was correlated with marked decrease in the labeling index. TGF-beta did not inhibit growth of hepatoma cell line. These findings indicate that TGF-beta is a strong growth inhibitor of adult rat hepatocytes and may block their shift from the G1 phase to the S phase. The physiological role of TGF-beta in inhibiting growth of adult hepatocytes during liver regeneration is discussed.     

Biological properties of a hepatocyte growth factor from rat platelets

In an accompanying communication we demonstrated that about half of the potency of rat serum to stimulate DNA synthesis in cultured adult rat hepatocytes resides in a polypeptidelike substance from the platelets. A lysate of rat platelets was able to restore the potency of platelet-poor rat serum, whereas a lysate of human platelets inhibited thymidine incorporation by the hepatocytes. Moreover, addition to these cultures of either highly purified human platelet-derived growth factor (PDGF) or human platelet factor 4 (PF-4) failed to influence DNA synthesis either alone or in the presence of rat or human platelet-poor serum, which is required for expression of PDGF activity. Unlike the human platelet factors, rat platelet lysate (RPL) was moderately active by itself and was augmented equally well by platelet-poor serum from either source. At concentrations below 5%, platelet-poor serum from hypophysectomized rats was as potent as that from normal rats in augmenting RPL activity. This suggests that, unlike PDGF, which is not activated by hypophysectomized rat serum, the hepatotrophic component of RPL does not require the presence of exogenous somatomedins for activity, but interacts instead with other plasma constituents or with somatomedins produced by the hepatocytes in vitro. Rat platelets do, however, appear to contain PDGF or its rat equivalent in addition to the hepatocyte growth factor, since if they are heated to 100 degrees C for 10 min, their ability to stimulate nuclear labeling in confluent BALB/c 3T3 cells is not impaired, while their ability to stimulate DNA synthesis in rat hepatocytes is destroyed. These studies indicate that the hepatocyte growth factor from rat platelets differs from PDGF in its biological as well as physical characteristics, but that rat platelets also contain PDGF or an equivalent substance.     

Partial purification and characterization of masking protein for beta-type transforming growth factor from rat platelets

beta-Transforming growth factor (TGF-beta) is stored in platelets and secreted as a high molecular weight latent form associated with a carrier protein of about 440 KD. This carrier protein could be separated from TGF-beta in 1 N acetic acid and could again mask the activity of TGF-beta under neutral conditions. Therefore, it was named the masking protein of TGF-beta. The masking protein was separated from TGF-beta by gel filtration on a Sephacryl S-300 column or by anion-exchanger FPLC on a Mono Q column in the presence of 6 M urea. Partially purified masking protein from rat platelets neutralized the activity of TGF-beta dose-dependently and was effective at 0.3 microgram/ml. This masking protein could also mask the activity of human TGF-beta, suggesting that it was not species specific. The masking protein was a heat- and acid-stable protein, but was inactivated by treatment with dithiothreitol. The Physiological role of the masking protein in the mechanisms of wound healing and liver regeneration is discussed.     

Hepatocyte growth factor in ascites from patients with cirrhosis.

Hepatocyte growth factor (HGF) stimulating DNA synthesis of adult rat hepatocytes in primary culture was found in the ascites and plasma from patients with liver cirrhosis, but not in those from patients without cirrhosis. HGF was purified about 400-fold in 10% yield from cirrhotic ascites by ultrafiltration, cation-exchange chromatography on a S-Sepharose column, and affinity chromatography on a heparin-Sepharose CL-6B column. The partially purified factor was a heat- and acid-labile cationic protein with a molecular weight of 100,000-150,000. Its effect was half-maximal at 3.8 micrograms/ml, and was additive with those of insulin and epidermal growth factor. HGF in ascites from patients with cirrhosis had the same properties as HGF purified and characterized from rat platelets. These findings suggest that HGF is secreted into the ascites from the plasma or liver of patients with cirrhosis and may increase in the plasma with the development of hepatic impairment and act in repair of the damaged liver of patients with chronic liver disease.     

Rat platelets contain growth factor(s) distinct from PDGF which stimulate DNA synthesis in primary adult rat hepatocyte cultures

Adult rat hepatocytes in primary cultures are stimulated to synthesize DNA in response to rat serum, whereas rat plasma is considerably less active. Biological activity is present in rat platelets and is secreted during aggregation in response to thrombin. The material secreted by rat platelets is heat labile and is sensitive to digestion with trypsin, suggesting that it is a protein. When assayed on 3T3 cells this material also stimulates DNA synthesis; however, the trypsin-sensitive activity is heat stable (100 degrees C, 10 min). These results indicate that rat platelets contain hepatotrophic activities which by virtue of their heat sensitivity are distinct from heat-stable platelet-derived growth factor (PDGF)-like mitogenic activities required by 3T3 cells for growth. It is possible that hepatotrophic platelet factors might be involved in mediating liver regeneration in the rat after partial hepatectomy.     

Partial purification and characterisation of an inhibitor of hepatocyte proliferation derived from nonparenchymal cells after partial hepatectomy.

We have investigated the influences that nonparenchymal cells from regenerating rat liver exert on hepatocyte proliferation. When primary adult rat hepatocytes isolated from resting liver were co-cultured with nonparenchymal cells (NPCs) from resting liver of a different syngeneic animal, the proliferative response of hepatocytes to epidermal growth factor (EGF) was unaffected by the presence of NPCs. In the presence of NPCs taken from livers that had undergone partial hepatectomy 24 hours before (regen-NPCs), the response of hepatocytes from resting liver to EGF, TGF-alpha, and hepatocyte growth factor (HGF) was markedly inhibited. Inhibitory activity was not dependent on cell-to-cell contact, and conditioned-medium from regen-NPCs, but not normal NPCs, inhibited EGF-induced hepatocyte DNA synthesis by approximately 50%. After concentration by gel chromatography and lyophilisation, inhibition was 98%. The inhibitory activity migrated on SDS-PAGE gel electrophoresis with an apparent molecular weight of 14 to 17 kDa and was trypsin-sensitive but relatively heat-stable. The effects of blocking antibodies established that it was not TGF-beta 1, IL1-beta, or IL6. Investigations of regen-NPCs taken at different time points demonstrated that inhibitory activity was released into conditioned medium of cells harvested at 24 and 48 hours after partial hepatectomy, but not 10 or 72 hours. This powerful inhibitor of hepatocyte response to proliferogens is released by cultures of NPCs with a time course suggesting that it may be involved in terminating the surge of hepatocyte replication induced by partial hepatectomy.     

Autocrine mechanism of growth of neonatal rat hepatocytes in primary culture

Hepatocytes from neonatal rats of 0 to 3 days old grew actively in primary culture without added serum or growth factors. In these culture conditions, growth of hepatocytes decreased progressively with increase in age of the rats from which they were isolated, and hepatocytes from rats of 2 weeks old showed scarcely any growth. Actively growing hepatocytes were found to secrete a growth factor that promoted their growth and that of Swiss 3T3 cells, but not that of adult hepatocytes. This growth factor in conditioned medium of growing hepatocytes was heat- and acid-stable, but sensitive to trypsin, and had a molecular weight of over 10,000. It did not inhibit the binding of [125I]epidermal growth factor to its receptor, and its growth promoting activity was not inhibited by monoclonal antibody against insulin-like growth factor II. Therefore, it seems to be a new growth factor. These results, together with previous findings (Nakamura, T., Nagao, M., & Ichihara, A. (1987) Exp. Cell Res. 169, 1-14) demonstrated a reciprocal relation between growth and maturation of neonatal hepatocytes during development, like that of adult cells, but indicated that unlike growth of the latter, growth of neonatal cells is induced by an autocrine mechanism.     

Partial characterization of a hepatocyte growth factor from rat platelets

Rat serum has been shown to stimulate DNA synthesis in primary cultures of adult rat hepatocytes 2-3 times more potently than serum from several other mammalian sources, including humans. Parallel to its stimulation of thymidine incorporation into DNA, rat serum increased the total DNA content of the hepatocyte cultures over time, and also increased the frequency of nuclear labeling and mitosis. Moreover, normal rat serum, derived from whole blood (NRS), stimulated DNA synthesis in hepatocytes twice as effectively as platelet-poor rat serum, derived from plasma (ppNRS). Addition of a rat platelet lysate (RPL) to ppNRS restored the activity to equal that of NRS. The avid binding of the active principle to CM Sephadex and its sensitivity to trypsin digestion suggest that it is a cationic polypeptide with an apparent molecular weight of about 65,000, as determined by gel filtration. It was inactivated by reduction of disulfide bonds, or by exposure to pH below 5.5, to NaCl concentration below 0.05 M, to 65 degrees C for 30 min, or to 100 degrees C for 10 min. Although it resembles the human platelet-derived mitogen platelet-derived growth factor (PDGF) in several of its properties, it differs in others. Hence the hepatocyte growth factor from rat platelets, which accounts for 50% of the DNA synthesis-stimulatory activity of rat serum, appears to be a distinct entity.     

Transforming growth factor-beta-like activity is secreted by rabbit renal cortical tubular cells in primary culture.

The activity of an autocrine growth factor in a medium conditioned by cultured rabbit renal cortical tubular cells was investigated. Little stimulatory growth activity for tubular cells was observed in the conditioned medium, and inhibitory activity was seen only in acidified conditioned medium. This factor stimulated the colony formation of NRK 49F cells in soft agar only with epidermal growth factor and inhibited the DNA synthesis of primary cultured rat hepatocytes, and its molecular weight was about 25 kDa. The factor was neutralized by the specific antibody to transforming growth factor (TGF)-beta 1. These results indicate that renal tubular epithelial cells can produce latent TGF-beta in primary culture.     

DNA synthesis in rat hepatocytes: Inhibition by a platelet factor and stimulation by an endogenous factor

Primary monolyer cultures of adult rat hepatocytes can be induced to undergo DNA synthesis in serum-free medium in the presence of insulin, glucagon, and epidermal growth factor (three factors). We have found that hepatocyte DNA synthesis is affected not only by an endogenous stimulant produced by the hepatocytes and released into the culture medium. Serum has a strong inhibitory effect on hepatocyte DNA synthesis. Partially purified human platelet extract (“platelet inhibitor”) inhibits the three-factor-induced DNA synthesis in a concenration-dependent manner. Pure βTGF at 0.5 ng/ml as well as HPLC-purified PDGF at 10 ng/ml completely inhibit the three-factor-induced DNA synthesis. Determination of the time required for the presence of the three factors and the platelet inhibitor to exert their effects indicated that the inhibition of DNA synthesis is caused not by competition of the platelet inhibitor with any of the three factors but through an independent pathway. Hepatocyte DNA synthesis is density-dependent and is greater if medium is not changed during the course of an experiment than if medium is changed daily. Hepatocyte-conditioned medium is also affective in stimulating DNA synthesis beyond the level induced by the three factors. These results suggest that an endogenous stimulant for hepatocyte DNA synthesis is produced by the hepatocytes themselves. Our studies demonstrate that hepatocyte DNA synthesis is subject to both stimulatory and inhibitory controls. Unlike the three factors, the endogenous stimulant can overcome the inhibition by the platelet inhibitor, suggesting the importance of these factors in the physiological control of hepatocyte DNA synthesis.     

Altered growth factor requirements and cell cycle control in rat hepatoma cells versus adult rat hepatocytes in culture

Adult rat hepatocytes multiply in primary cultures when incubated in arginine-free MX-83 medium supplemented with dialyzed fetal calf serum, insulin, glucagon, hydrocortisone, epidermal growth factor, and transferrin. In the absence of mitogens, the fraction of the cells engaged in DNA synthesis dropped sharply. However, cells initiated DNA synthesis in response to the mitogenic mixture indicating that hepatocyte proliferation is controlled by G1----S transition rates. In contrast, rat hepatoma line DTH-3, derived from Morris 7777 "minimal deviation" hepatoma, required only insulin for proliferation in chemically defined MX-83 medium. The lengths of their cell cycle phases varied with the growth rate. The phases of the growth cycle were proportionately shortened (expanded) when the growth rate was increased (decreased). It is concluded that DTH-3 hepatoma cells, which display a decreased growth factor requirement as compared with adult rat hepatocytes differ from normal hepatocytes by fundamental alterations in the mechanisms controlling the progression of the cell cycle.     

Characteristics of a thrombin inhibitor secreted by activated platelets

A 77-kDa complex of thrombin and a protein secreted by activated platelets had little if any thrombin amidolytic activity, indicating that the secreted protein is an inhibitor. The molecular weight of the inhibitor before reaction with thrombin was approximately 50,000. The apparent second-order rate constant for complex formation was estimated to be 1.3 x 10(6) M-1 s-1 (mean of four measurements); it was not affected by heparin or heparinase. These properties distinguish this inhibitor from other protease inhibitors secreted by platelets. The inhibitor reacted with trypsin and possibly with urokinase but not with factor Xa.     

Purification and characterization of hepatocyte growth factor from injured liver of carbon tetrachloride-treated rats.

A hepatocyte growth factor (HGF)-like substance that strongly stimulated DNA synthesis of adult rat hepatocytes in primary culture was found to increase markedly in liver of rats treated with carbon tetrachloride (CCl4). This increase of HGF-like activity was time- and dose-dependent, and 36 h after a dose of CCl4 of 0.2 ml per 100 g body weight the activity was about 20-times the normal level. The extent of induction of HGF-like factor correlated well with the extent of liver damage. The HGF-like factor was purified to homogeneity from the liver of CCl4-treated rats by a four-step procedure. The purified HGF-like factor had a molecular weight of 82-85 kDa, as estimated by SDS-PAGE, and was a heterodimer composed of a large subunit of about 69 kDa and a small subunit of 34 kDa linked by disulfide bridges. This factor had similar biological and chemical properties to HGF purified from rat platelets. Moreover, the N-terminal amino acid sequence of its 34-kDa subunit was identical to that of the small subunit of rat HGF. These findings indicate that the HGF-like factor in damaged liver of CCl4-treated rats is HGF and that liver itself can produce HGF when injured.     

The prevalence and purification of hepatoproliferin: a liver regeneration factor from rat hepatocytes

Hepatoproliferin (HPF), a liver regeneration factor that was able to augment the growth of hepatocytes in the presence of EGF, was produced by young rat livers and hepatectomized adult livers (70%), but not by adult intact livers. Therefore only growing and regenerating livers produce HPF. This growth factor was purified into two homogeneous bioactive species having different single SDS-PAGE bands at 18.5 and 14 kDa, and different single pI-bands at pH 4.3 and 8.7, respectively. HPF was synthesized de novo by hepatocytes in the liver as shown by the in vivo incorporation of radiolabeled 35S-sulfate and 14C/3H-glucosamine. This radioactive HPF was secreted ex vivo by hepatocytes, probably to act as an autocrinal hepatomitogen since 90% was found in the growth medium. HPF was neither a classical peptido-mitogen nor a heparin binding growth factor, but a liver-originated non-proteinaceous factor, which probably contains sulfonated saccharides such as glucosamine sulfate. HPF was neither a polyglycan nor a glycopeptide nor a peptidoglycan.     

Stimulation of hepatocyte DNA synthesis by neurotensin

Epidermal growth factor and transforming growth factor alpha stimulated DNA synthesis in primary cultures of adult rat hepatocytes. Neurotensin amplified epidermal growth factor-stimulated or transforming growth factor alpha-stimulated DNA synthesis by three- to eightfold. Neurotensin by itself did not stimulate DNA synthesis. Amplification of DNA synthesis by neurotensin was observed as low as 10^?10 M, and it was increased in a dose-dependent manner with maximal effects at 10^–8 M. These results were obtained when hepatocytes were cultured in Williams' medium E, but not in Leibovitz L-15 medium, suggesting that a minor component(s) in the medium is required for hepatocytes to fully respond to neurotensin. Neurotensin effect on DNA synthesis was observed not only in normal rat hepatocytes but also in partially hepatectomized rat hepatocytes, although its effect was stronger in normal hepatocytes. Amplified DNA synthesis was inhibited by transforming growth factor β. Secondary mitogens (co-mitogens) such as insulin, vasopressin, or angiotensin II interacted additively with low concentrations of epidermal growth factor as well as with neurotensin. Neurotensin-related peptides such as kinetensin or neuromedin-N, which was released from blood plasma by pepsin digestion, did not have this amplifying effect on DNA synthesis at any concentrations tested. Neurotensin mRNA was found in several organs including brain and intestine, but not liver. These results suggest that neurotensin can be regarded as a new secondary mitogen and that it may be involved in cell proliferation, including regenerating liver as a gastrointestinal hormone and/or a neurotransmitter. © 1994 Wiley-Liss, Inc.     

Partial purification and characterization of hepatocyte growth factor from serum of hepatectomized rats

When rat serum was subjected to gel filtration on a Sephadex G-200 column, a factor, "hepatotropin", that promoted hepatocyte growth in primary culture was separated. Its Mr was about 150 KD and it was an anionic protein that was unstable on acid- and heat-treatments. Hepatotropin was purified 20-fold further by affinity chromatography on heparin-Sepharose CL-6B. The purified hepatotropin was effective at 20 micrograms/ml and maximally effective at 120 micrograms/ml, and its effect was additive with that of insulin plus epidermal growth factor. In rats after partial hepatectomy, the hepatotropin activity in the serum increased time-dependently reaching a maximum of about 5-fold the initial level 24 h after the operation. Various known growth factors, such as fibroblast growth factor, platelet derived growth factor, somatomedin, thrombin and transferrin, did not stimulate DNA synthesis in cultured hepatocytes. These results suggest that hepatotropin is a new growth factor.     

Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells

In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nerve growth factor activates mast cells through the collaborative interaction with lysophosphatidylserine expressed on the membrane surface of activated platelets

Effect of nerve growth factor (NGF) on platelet-associated mast cell activation was investigated. Although neither NGF alone nor platelets alone induced significant 5-hydroxytriptamine (5-HT) release from rat peritoneal mast cells, marked 5-HT release was detected when costimulated with NGF and calcium ionophore-activated platelets. This response reached maximal levels as early as 5 min after the initiation of the coincubation and was completely blocked by anti-NGF Ab or by an inhibitor for a tyrosine kinase of the trkA NGF receptor. Paraformaldehyde-fixed platelets activated with either calcium ionophore or thrombin exhibited the collaborative ability, suggesting the possible involvement of some membrane molecules expressed on activated platelets in mast cell activation. Because activation of platelets induced expression of phosphatidylserine (PS) and/or lysoPS on membrane surface, and since lysoPS, unlike PS, initiated the NGF-induced 5-HT release, lysoPS expressed on activated platelets may be involved in the mast cell activation. Moreover, intradermal injection of NGF and activated platelets into the rat skin increased local vascular permeability. These findings suggested that NGF collaboratively worked with membrane lysoPS of activated platelets to induce mast cell activation. Thus, NGF released in response to inflammatory stimuli may contribute to mast cell activation in collaboration with locally activated platelets in the process of inflammations and tissue repair.     

Release of a product of growth arrest-specific gene 6 from rat platelets

A product of growth arrest-specific gene 6 (Gas6) is known to be synthesized by growth-arrested cells. In this study, we found that several rat tissues including platelets contain Gas6 and activation of the platelets with thrombin provoked the release of Gas6. ADP and collagen, which as well as thrombin stimulated release of ATP from platelets, also enhanced the release of Gas6, suggesting that the mechanism of its release was similar to that of ATP release. This study provides the first evidence of growth arrest-independent secretion of Gas6 and suggests the involvement of Gas6 in vascular diseases as well as hemostasis.