Growth factors in liver development, regeneration and carcinogenesis

in Growth Factor Research, Vol. 3. pp. 219-234. PrInted in Great Britain. All rights reserved.

Progrtw

GROWTH FACTORS REGENERATION

1991

095s2235/91 $0.00 + .50 ‘i’ 1992 Pergamon Pres, plc

IN LIVER DEVELOPMENT, AND CARCINOGENESIS Nelson Faust0

Department

of Pathology and Laboratory Medicine Brown University Box G, Providence, RI 02912. U.S.A.

Liver growth during regeneration is controlled by several growth factors which may be involved in the triggering, progression and termination of hepatocyte replication. It is likely that liver regeneration involves both circulatingfactors and those produced in hepatic tissue during the growth response. TGFu is an autocrine stimulator of hepatocyte proliferation which increases transiently in replicating hepatocytes both in vivo and in vitro. Constitutive TGFa overexpression in young transgenic mice causes liver hypertrophy and enhanced proliferation that progress to hepatic tumor development in the great majority of animals after 12 months of age. In contrast, HGF is present in normal blood in humans and animals and plasma concentrations increase after partial hepatectomy, liver injury and fulminant hepatic failure. In liver tissue, levels of HGF and its mRNA correlate better with the extent of injury than with the degree of proliferative activity. The factor is produced by nonparenchymal cells and presumably acts on hepatoeytes through paracrine or endocrine mechanisms. A transient increase of TGFPI in regenerating liver may promote the formation of extracellular matrix components and signal the end of hepatocyte proliferation. Prolonged overexpression of the factor in nonparenchymal cells causes liver fibrosis both in humans andexperimental animals. The liver contains TGFPI .2 and 3, all of which inhibit hepatoeyte DNA synthesis. Their mRNAs increase in the regenerating liver but with very d@ierent kinetics. Despite the enormous progress achieved in understanding the mechanisms that regulate liver regeneration, it is not known whether HGF, TGFr and TGFP interact with each other or with other factors or hormones during the growth process. Further, it remains to be established how the eflect of these factors ma) relate to the sequential changes in proto-oncogene expression that occur after partial hepatectomy.

Acknowledgemenfs-I author’s laboratory U.S.A.

thank Ms. Ann Baxter for her help in preparing the manuscript. was supported by grants CA 23226 and CA 35249 from the National 219

Work Cancer

from the Institute,

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INTRODUCTION The liver mass of mammals is in dynamic equilibrium with body size. When this equilibrium is disrupted by surgical removal of tissue or toxic injury, normally nonmitotic hepatocytes respond rapidly and proliferate to restore the appropriate organ size [ 1,2]. Clinical observations show that liver growth after transplantation in humans follows closely the expectations derived from the study of liver regeneration after partial hepatectomy in rodents. The similarity between the results obtained in clinical and experimental settings has led to the use of “split” transplants and the transplantation of hepatic tissue from living donors [3]. As predicted from animal studies, the organ of the partially hepatectomized donor as well as the liver transplanted into the new host grow until the liver size/body size ratio expected for each individual is reached. Because of its biological significance and applicability to clinical situations, the analysis of liver regeneration after partial hepatectomy in rats has gained special significance. In this article we will review work showing that some growth factors play an essential role in this process. However, before examining the effect of individual agents it is important to emphasize a few basic biological properties of liver regeneration. In the animal model, liver ‘regeneration’ is actually a process of compensatory hyperplasia but not one of true regeneration [2,4]. That is, the lobes of the liver removed by surgery (median and left lateral lobes, corresponding to approximately 67 percent of the liver weight) do not regrow. Instead, the remaining lobes increase in size and by lo-14 days after the operation in rats, the remnant liver which has 2 lobes (right lateral and caudate) reaches the mass of the original organ which had 4 lobes. Liver regeneration is often referred to as being a process ofwound healing. Indeed, in a certain sense, the liver ‘heals’ itself when undergoing compensatory growth. However, no liver wound is created by partial hepatectomy in rodents and none of the inflammatory and vascular phenomena usually associated with wound healing develop. In these animals, the lobes of the liver form separate units which are interconnected only through their vascular supply. By ligating the appropriate vessels, individual liver lobes can be removed intact without cutting through tissue or disrupting the circulation of the remaining units. Thus, the process of compensatory hyperplasia takes place in a ‘miniliver’ that is anatomically undamaged. Although partial hepatectomy is the most valuable model for the study of liver growth, clinically this situation is only encountered in transplantation cases. Liver regeneration in humans takes place most commonly in hepatic tissue that has been damaged by viruses or toxic agents. In these circumstances cell proliferation occurs in conjunction with necrosis, inflammation and fibrosis. Replenishment of liver mass in hepatic regeneration depends primarily on hepatocyte replication, but the regenerative response involves all of the other elements of liver tissue, that is, nonparenchymal cells (bile duct cells, Kupffer cells, lipocytes, endothelial cells and pit cells which together constitute about 35 percent of the total number of liver cells) the extracellular matrix, and vascular components. As more information is gained on the functions of nonparenchymal cells, it has become clear that they produce growth factors which have profound effects, both positive and negative, in hepatocyte replication [S-7]. Thus, while the mitogenic action of growth factors has been studied in primary cultures of hepatocytes, the interaction between the various cell types that produce

‘2 I

Growth Factors in the Liver

and respond to these factors as well as the inter-relationships between cells and the extracellular matrix can only be properly examined in vivo. Cell/matrix interactions and cross signaling between hepatocytes and nonparenchymal cells are essential components of the regulatory circuits that maintain the dynamic equilibrium between liver size and body size. GROWTH

FACTORS

IN LIVER

DEVELOPMENT

Major progress has been achieved in recent years in the study of growth factors in the early development of mammalian embryos. Unfortunately, much less information has been gathered on the role of growth factors in liver morphogenesis and development. IGF-II mRNA is particularly abundant in fetal rat liver and its levels decrease sharply during the first 2 postnatal weeks. IGF-I mRNA changes in the opposite direction, that is, it is very low during liver development but increases rapidly after birth [8,9]. Although there is no direct experimental proof, IGF-II has been considered as a major regulator of fetal liver growth by presumably acting as an enhancer of hepatocyte proliferation. Given that IGF-I mRNA levels in rats are low in fetal hepatic tissue but high in adult liver, it is surprising that adult liver contains few IGF-I receptors measurable by ligand binding assays which easily detect the fetal liver receptors. IGF-I receptors can however be demonstrated in adult liver by cross linking techniques [IO]. IGF-II is probably required for optimal fetal liver growth and IGF-I might be important in the neonatal period but more data are required to evaluate the precise role of IGFs and their receptors in liver development. Detailed studies on EGF/TGFcw and TGFPl receptors during liver development have been published by Gruppuso [1 I]. The number of EGF receptors increases sharply during the last 3 days of gestation and reach adult levels by the last day. As is the case for adult hepatocytes, fetal rat hepatocytes bind labeled EGF with high affinity. EGF and TGFacompete for the same site, although TGFa binds with 4-6-fold lower affinity. Analysis of the site of synthesis and timing of detection of EGF and TGF mRNAs in fetal and neonatal tissues indicate that TGFcris the physiological ligand for the EGF/TGFac receptor during liver development. Prepro-EGF mRNA is first detected in the kidney by two weeks post partum while only after weaning is EGF mRNA made in the salivary glands [ 121. In contrast, TGFa mRNA is present in many fetal tissues including the liver [6,13]. Hepatic TGFa mRNA levels increase in the first few days after birth and then decrease by lo-14 days. The peptide is detectable in rat liver at 20 days of gestation and decreases to the low adult levels by the second week after birth [14]. During the last few days of gestation and the first 2 postnatal weeks. liver TGFcY concentrations range from 24 to 70 pg/mg protein. By 6-8 weeks of age. hepatic TGFcr concentrations reach the low adult value of approximately 3 pg/mg protein [14]. Taken together, these data suggest that TGFa may function as an autocrine growth factor in fetal rodent liver. Although EGF is not produced by fetal tissues, a small amount of circulating maternal EGF might be transported to the fetus through the placenta and be metabolized in the liver. Similarly, the small amount of EGF present in hepatic tissue of adult rats probably originates from blood uptake [ 151. TGFPl binds to fetal rat liver cell membranes at higher levels than to preparations from adult animals [6,11,16]. The overall binding of TGF/?l to liver membranes does not change during the last days of gestation but affinity labeling of the M,SS,OOO component doubles between days 18 and 21 suggesting that this receptor may be

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physiologically important during this stage. Although TGF/31 inhibits DNA synthesis of fetal hepatocytes, the functions of TGFPl in fetal liver are not known. Both TGFPl mRNA and the peptide are present in the rat liver at 16 days of gestation and progressively decrease after that time [6]. GROWTH

FACTORS

IN LIVER

Stimulatory

REGENERATION

Factors

EGF

This peptide is a potent stimulator of DNA synthesis for hepatocytes in primary culture [18]. However, it is difficult to establish a physiological role for EGF in the regulation of liver regeneration in vivo because the peptide and its RNA are not synthesized by normal or regenerating livers. Furthermore, the peptide is not detectable by RIA in the portal blood of normal or partially hepatectomized rats [19]. Sialoadenectomy has no effect on liver regeneration while duodenectomy (salivary glands and Brunner glands in the duodenum are sites where EGF is produced in rodents) has a very slight effect on DNA synthesis after partial hepatectomy. Nevertheless, administration of EGF together with insulin or glucagon increases liver DNA synthesis in normal or partially hepatectomized rats [20]. These data indicate that EGF may not be a major participant of liver regeneration in vivo but that it has a strong effect on hepatocyte replication when injected into the animal or added to cells in culture. TGFa TGFa synthesis increases in liver hepatocytes during liver regeneration. TGFa mRNA changes are first detectable 48 h after partial hepatectomy and reach a maximum of about 8-lo-fold the normal level (sham-operated animals) at 20-24h [2 11. These changes slightly precede the major wave of DNA synthesis in the regenerating liver. The levels of TGFain the regenerating liver (measured by RIA with an antibody that recognizes the mature form of the peptide) approximately double between 12 and 18 h after partial hepatectomy (FitzGerald, Brown and Fausto, in preparation). TGFa is a potent stimulator of DNA synthesis for rat and human hepatocytes in primary culture and is generally 25-200 percent more active than EGF in its stimulatory effect [21,22]. In cultured hepatocytes the addition of TGFa causes the induction of a proliferative program [23] and a wave of DNA synthesis with a maximum at 48-72 h [21]. In the replicating cells, there is an increase of TGFa mRNA and release of the peptide in the culture medium. Neither the mRNA nor peptide levels change in nonstimulated cultures where they are barely detectable [21]. Thus both in vivo and in culture, TGFamRNA and the peptide increase in hepatocytes which have entered the cell cycle and are progressing toward DNA replication. EGF/TGFa

Receptors

(EGFr)

Earp and O’Keefe [24] have shown that the number of EGFr decreases during liver regeneration, an observation confirmed in many other laboratories [16]. Only the number but not the affinity of EGFr changes in the regenerating liver. TGFaand EGF compete for the same M,= 170,000 protein and although TGFa is a more potent

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stimulator of DNA replication, it binds with lower affinity than EGF [16]. As the EGF and TGFaeffects on cell proliferation do not directly correlate with the binding affinity of the ligands, it is possible that EGF and TGFa receptor complexes are processed in different ways or with different efficiency by the hepatocyte. The decrease in EGF receptor number during liver regeneration is not in dispute, but various hypotheses that attempt to explain the mechanisms responsible for the change have been offered. After partial hepatectomy or following the intraportal injection of EGF, the amount of EGFr mRNA increases during the first 2-4 h [25]. In this situation, it is likely that changes in receptor number are a consequence of increased turnover. The data on EGFr mRNA levels at later stages of liver regeneration are conflicting, with reports that they decrease [2&28] or increase [21,29] at 18-24 h after partial hepatectomy. Depending on the direction of the change observed in EGFr mRNA, the decrease in receptor number is explained as resulting from either lack of synthesis of receptor protein (decrease in EGFr mRNA) or, in an opposite way, as ligand induced down-regulation and turnover (increase in EGFr mRNA). Johansson et al. [25] made the interesting observation that in the regenerating liver the amount of EGF binding sites in prelysosomal endosomes is much less reduced than the membrane receptor sites. This suggests that endogenously produced TGFlrcould be bound to the receptor in intracellular compartments. The major difficulty in interpreting these data is that it is not known whether TGF6r synthesized in the regenerating liver remains in intracellular compartments, is anchored to the hepatocyte plasma membrane or is cleaved into the 50 amino acid peptide (or other forms) and released. Receptor dynamics and the relationship between the levels of receptor protein and its mRNA would differ significantly if TGFa effects were exerted by membrane anchored forms instead of the free, mature peptide. Hepatocyte growth factor

(HGF)

HGF is a heterodimer formed by 2 disulfide linked subunits of approximately 70,000 (z) and 34,000 (/3) M,. Mature HGF is derived from a 728 amino acid precursor and is encoded by an mRNA of approximately 6 kb [30-331. The Q subunit has 4 kringle structures, a configuration found in plasminogen, plasminogen activator, prothrombin and other components of the coagulation cascade [31]. The /J subunit shows homology with serine proteases but lacks proteolytic activity. HGF is identical to a broad-spectrum mitogen derived from human lung fibroblasts and also to the previously isolated scatter factor which greatly affects cell mobility [34,35]. HGF has been purified from many sources including rat, rabbit and human blood, platelets and fibroblasts [36]. Originally described as a hepatocyte-specific growth factor (the reason for its name) HGF is a mitogenic agent of broad specificity, capable of stimulating growth of many different cell types including keratinocytes, melanocytes, kidney tubule ceils and mammary gland cells [37]. The factor is present in many different tissues including liver, kidney, brain, placenta and skin [38]. In the liver, HGF is produced by nonparenchymal cells (Kupffer cells lipocytes and other sinusoidal lining cells) but not by hepatocytes and presumably acts on hepatocytes by a paracrine mechanism [39]. It is more potent that EGF and TGFcwas a stimulator of DNA synthesis for normal hepatocytes in primary culture [36,40]. Recent reports suggest that the factor is neither produced by, nor acts on, transformed hepatocytes. Blood HGF concentrations increase rapidly after toxic, ischemic and mechanical liver injury and after partial hepatectomy [41,42]. Although partial hepatectomy and

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CCI, induce regenerative responses of approximately the same magnitude, the increase in liver HGF after Ccl, injury is approximately 10 times larger than that observed after partial hepatectomy [43]. Similarly, after toxic injury there is an early, large increase in hepatic HGF mRNA while after partial hepatectomy the changes in liver HGF mRNA are smaller and occur only between 12-24 h after the operation [43]. Other investigators could not detect any change in liver HGF mRNA after partial hepatectomy [33]. Much of these data are still preliminary and the exact sources for the circulating factor in toxic injury or partial hepatectomy remain to be established. However. as after liver injury blood HGF increases at about the same time as serum aminotransferase activity, it is plausible to assume that blood HGF can be released from liver nonparenchymal cells as a consequence of, or as a response to, tissue injury. Similarly, the large increase in blood HGF found in patients with fulminant liver failure is likely to be caused by the release of HGF from the necrotic liver. Nevertheless, in the absence of more detailed data, it cannot be excluded that in liver injury blood HGF might be derived from extrahepatic tissues and that its accumulation is a consequence of decreased uptake. It has been suggested that release of HGF by extrahepatic tissues may account for the increase in blood HGF after partial hepatectomy and be a stimulus for the initiation of regeneration [43]. HGF receptor: c-met proto-oncogene

The met gene was originally described as an oncogene present in osteogenic sarcoma cells and found to originate from a rearrangement between two cellular genes, c-met and tpr [44]. Further characterization of the c-met proto-oncogene in nontransformed cells indicated that the gene encoded a receptor protein with tyrosine kinase activity. Receptor mRNA (c-met mRNA) has been detected in many different tissues and the constitutive overexpression of the gene can cause transformation of NIH 3T3 fibroblasts. Bottaro et al. [45] have recently demonstrated that HGF is the ligand for the c-met proto-oncogene receptor protein. HGF induces phosphorylation of a 145 kD protein with phosphotyrosine activity. This protein was identified as the c-met /J subunit and was found to be phosphorylated on tyrosine and serine residues within a minute after HGF addition to the culture medium. Higuchi and Nakamura [46] reported that the HGF receptor density in liver cell membranes is approximately 500-600 sites/cell with a dissociation constant of 24-32 PM. Both after partial hepatectomy and Ccl, injury the number of HGF binding sites decreases drastically within 3-12 h and remains low or undetectable for at least two days. Although the data suggest that the decrease in receptor number is a consequence of ligand induced down-regulation; no specific experiments have yet addressed this issue. It is essential to obtain more data on HGF-receptor interactions during liver regeneration or toxic injury because in these conditions HGF increases in the blood but a major mitotic response is found only in the liver. If HGF is responsible for triggering hepatocyte DNA synthesis, the specificity of this response requires explanation as HGF is a mitogen with broad specificity capable of inducing the proliferation of various cell types. Preliminary results show that there appears to be little or no increase in the levels of c-met mRNA after partial hepatectomy but these data do not exclude the possibility that other types of changes occur in the receptor.

-)‘F --_

Growth Factors in the Liver

Heparin-binding growth factor 1 (HBGF-1, aFGF) and IGFs

HBGF- 1 mRNA increases after partial hepatectomy before major changes in TGFcz gene expression are detectable [47]. The factor is produced by both parenchymal and nonparenchymal liver cells. It has been suggested that HBGF- 1 may constitute an early autocrine signal that initiates the prereplicative phase of liver regeneration. It has been postulated further that at later stages of the process a shift in HBGF-1 receptors causes the factor to inhibit hepatocyte proliferation. HBGF-1 has a relatively weak stimulatory effect on DNA synthesis of hepatocytes in culture and the effect is masked by EGF [47]. Studies of HBGF binding sites in liver membranes showed that a single hepatocyte may contain 6 to 12 homologous polypeptides which are members of a large family of receptor proteins [48]. Although the direct effect of HBGF-1 on hepatocyte DNA synthesis is relatively small, this factor might modulate the positive and negative action of other agents on hepatocyte replication. IGF-II and IGF-I mRNA expression do not change during liver regeneration [8]. but there is a rapid and transient increase in the expression of the mRNA for insulinlike growth factor-binding protein 1 (IGFBP-1) very shortly after partial hepatectomy [49]. This gene has been considered as a member of the immediate early gene family that is rapidly activated after a growth stimulus, but the function of IGFBP-1 in liver regeneration is unknown. Inhibitory Factors TGFP

effects on cell proliferation

TGFPl inhibits hepatocyte DNA replication in culture and blocks the stimulatory effects of EGF, TGFcrand HGF [reviewed in 6 and 501. That it also acts as an inhibitor in viva has been shown by experiments in which TGFPl was injected into partially hepatectomized animals in a free form or encapsulated in liposomes [5 1.521. In either case TGFpl reversibly inhibited the major wave of DNA synthesis in the regenerating liver. At later stages of regeneration there was little difference in the extent of DNA synthesis between TGFPl-injected and control animals. Two aspects of the in vivo inhibition of hepatocyte DNA synthesis in the regenerating liver are of interest. Firstly, it appears that TGFpl is an effective blocking agent even if given 18-22 h after partial hepatectomy at a time when most hepatocytes are in late G, or in the G,/S transition. This indicates that the effect of TGF/?l on hepatocyte DNA synthesis must be quite rapid and that its target is unlikely to be the expression of c-myc or other immediate early genes active at the Go/G, transition. This interpretation is consistent with results obtained with hepatocytes in primary culture showing that TGFPl blocks DNA synthesis without interfering with c-myc and p.53 expression [7]. Another interesting feature of TGFal inhibition of hepatocyte DNA synthesis in vivo is the apparently transient nature of the effect, that is, even when TGFPl is presumably continuously present in the tissue, the inhibition is most effective at the time of maximum DNA synthesis. This could result from the inability of hepatocytes to respond to TGF/?l at later stages of regeneration, because of changes in receptor number, decrease in receptor affinity or alterations in postreceptor mechanisms [I 11. However, as the TGF/3 receptor affinity increases rather than decreases at later stages of liver regeneration [ 1 l] and no changes in postreceptor pathways have been demonstrated, it is possible that the main target for TGFQl is an event that occurs only at the

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prereplicative stage of the first wave of DNA replication. Blockage of this event by TGF/?l would then lead to DNA synthesis inhibition. Experiments with keratinocytes and hepatocytes in culture suggest that TGFpl may cause the activation of Type 1 phosphatases [53]. An effect on this enzyme leading to dephosphorylation of key molecules required in the cell cycle could account for the rapid effect of TGF/?l on DNA synthesis. However the physiological substrates for the dephosphorylation effect have not been identified. Given that TGF/Jl acts as an inhibitor of hepatocyte DNA synthesis in vivo and in vitro, it is puzzling that TGFpl mRNA increases during liver regeneration [5]. The most logical role for TGFPl in liver regeneration would be for it to function as a stop signal to prevent unrestrained cell proliferation. If indeed this is the physiological role of TGFPl in this process, explanations must be found for the observation that the start in the increase in TGFPl mRNA precedes the peak of DNA synthesis. It is possible that TGFPl, while present in the regenerating liver throughout regeneration, acts only after the first wave of cell replication. This interpretation might be at variance with the data discussed above on the inhibition of hepatocyte DNA synthesis caused by injections of TGFPl into partially hepatectomized animals [5 1,521. The explanation for these apparent discrepancies might rest on knowledge of the relative amounts of latent and active TGFPl present in the tissue at various stages of regeneration. Although rat liver contains approximately 10 times more TGFPl than TGFP2, no measurements of the amounts of active and latent TGFPl in normal and regenerating liver are available [7]. It is conceivable that activation of latent complexes produced in the liver might take place only after the major wave of hepatocyte and nonparenchymal cell replication. In culture, hepatocytes from normal liver have little, if any capacity to activate latent TGF/?l complexes but, hepatocytes obtained from regenerating liver are able to release active TGFPl from the complex [7]. It is however difficult to extrapolate the results obtained with this system to the process of TGF/31 activation in vivo during liver regeneration. Liver tissue contains TGF/?l, 82 and p3, all of which are synthesized by nonparenchymal cells, but not by hepatocytes [7]. The 3 isoforms inhibit DNA synthesis in cultured hepatocytes with approximately the same potency. During liver regeneration, mRNAs for the 3 isoforms increase but with very different kinetics. TGFPl mRNA shows a sustained change starting at about 4 h and reaching a peak at 24-72 h after partial hepatectomy [5,54,55]. In contrast, TGF/32 and p3 mRNAs increase at 4 h but return to normal levels by 8-12 h. The relationship between the early changes in TGFP2 and TGFP3 mRNAs and other events that occur at the same time during the prereplicative phase of liver regeneration is unknown at this time. TGFj31 and liver fibrosis

TGF/?l expression is associated with fibrogenesis in the liver tissue [5658]. Although there is some dispute about the site of synthesis of matrix components in the liver, the lipocyte (perisinusoidal cell, stellate cell or Ito cell in its various nomenclatures) is a major site of collagen Type I and Type III production on the fibrotic liver. TGF@ induces collagen mRNA in cultured liver cells and both in rat and human liver, TGFPl has been localized in lipocytes by immunohistochemical staining and in situ hybridization [59-611. The expression of TGF/31 mRNA correlates well with the amounts of collagen Type I mRNA in human liver biopsies [62,63]. The expression of both TGF/31 and collagen

Growth Factors in the Liver

,727

Type I mRNA increase in patients with chronic liver disease. In patients with high fibrogenic activity (determined by serum levels of procollagen Type III aminoterminal peptide) TGFPl mRNA levels were 2-14 times higher than in normal controls or in biopsies from patients with liver disease with low fibrogenic activity [62]. In a group of patients with chronic hepatitis C virus infection, treatment with interferon acaused a decrease in TGFPI and collagen Type I mRNA levels measured in liver biopsies. These results suggest that TGFPl may have an important role in the pathogenesis of fibrosis in patients with chronic liver disease. In this situation, TGFPI synthesis may be induced directly by hepatitis B or C viruses, by products of necrotic cells and tissue disruption or by other cytokines produced by nonparenchymal liver cells or cells of the inflammatory infiltrate [62]. Whatever the initiating agent, the increase in TGFpl in lipocytes probably induces the expression of Type I collagen mRNA in these cells by transcriptional and post-transcriptional mechanisms. REGULATION

OF LIVER

GROWTH

IN TGFa TRANSGENIC

MICE

The expression of TGFa in the regenerating liver is a transient event that is associated with the major wave of DNA synthesis. It is important to determine what effects the constitutive overexpression of the gene in viva might have on liver growth. Transgenic mice carrying TGFa constructs are ideally suited for these studies. Lines that overexpress human TGFa under the control of various promoter elements have been established and those using the mouse metallothionein promoter proved to be particularly useful for analysis of the role of TGFa in liver growth regulation. Both Sandgren et al. [64] and Jhappan et al. [65] found that TGFcx overexpression causes an increase in liver size in young animals (2-6 weeks old). Older transgenic and nontransgenic mice had similar liver weights suggesting that the TGFor effect is most important during the postnatal growth period. The major difference in the findings reported by these two groups of investigators is that the incidence of tumor in the CD 1 transgenic mice used by Jhappan et al. [65] was very high (about 80%) while it was low in the strain (C57B1/6 x 5JL, F2) used by Sandgren et al. [64]. In the CD1 transgenic mice tumors appeared after 12 months of age but studies with very old animals of the C57BL/6 x 5JL strain have not yet been reported. The difference in tumor incidence between these strains is of interest because normal CD 1 mice have a 5-8% spontaneous incidence of hepatocellular tumors (all benign tumors appearing after one year) while the incidence of spontaneous tumors is negligible in the strain used by Sandgren et al. [641. The increase in liver weight in the young transgenic animals is caused by liver cell hypertrophy, changes in ploidy and hyperplasia. The livers of these animals have very high mitotic activity and higher DNA content/mg tissue than nontransgenic mice. The most obvious morphologic change in the liver of the transgenic animals is centrolobular hypertrophy, which is similar to that observed after phenobarbital administration. Immunohistochemical staining with an antibody that detects the TGFlr precursor molecule showed that cells overexpressing TGFol in the liver of transgenic mice have a patchy distribution with heavily stained hepatocytes forming nests located in centrolobular or periportal areas. Northern blot analysis, immunohistochemistry and in situ hybridization techniques demonstrated that the tumors formed in these animals generally have a much higher level of TGFcr expression than

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the surrounding tissue [65]. On the other hand, there were areas of very high TGFa expression by apparently normal hepatocytes suggesting that increased expression of TGFa! by itself may not be sufficient for transformation (Lee, Merlin0 and Fausto, submitted). Hepatocytes from TGFa transgenic mice can be maintained in primary culture in serum free media without any growth factors and undergo l-3 rounds of synchronous replication. These cells secrete active TGFa in the culture medium and show an increase in the mRNA for the endogenous murine TGFdlas they replicate. Addition of either EGF or HGF to the medium does not enhance the level of DNA synthesis. Thus, hepatocytes from TGFcx transgenic animals have the capacity to replicate autonomously in culture (within certain limits) most likely because of TGFaautocrine activity. Several cell lines have now been isolated from the liver of TGFa transgenic animals. Preliminary results indicate that some of these lines retain differentiated hepatocyte traits and morphology for at least 2-3 months (Wu, Merlin0 and Fausto, in preparation). Could the development of liver tumors in TGFa transgenic mice be the result of transgene overexpression alone? This is an unlikely possibility because the tumors formed in these animals are of a focal nature, take at least 12 months to develop and generally have higher levels of TGFa expression than the surrounding tissue. More likely, liver neoplasia in TGFcr transgenic mice requires the interaction between TGFa overexpression and other events. It is then plausible to suggest that the transgene functions as a promoter rather than an initiator of carcinogenesis either by inducing the proliferation of already altered cells or by increasing the risk for the development of genetic alterations in normal cells. So far, however, neither ras nor ~53 mutations and rearrangements have been found to occur at a higher frequency in the tumors. A potentially interesting clue to the pathogenesis of the tumors is that they develop in high frequency in CD1 males but rarely in females. Whether this difference is due to the smaller number of EGFr sites found in the liver of female mice or is related to specific hormonal changes remains to be determined. GROWTH

FACTOR

EXPRESSION DURING CARCINOGENESIS HEPATOCELLULAR CARCINOMAS

AND IN

TGFct

Urinary TGFa excretion has been reported to be much higher in patients with hepatocellular carcinoma than in normal controls, although there is large individual variation [66]. Yeh et al. found a significant difference in TGFacexcretion (expressed as pgTGFcl/g creatinine) between patients with primary liver cancer and controls. The same authors reported that urinary EGF levels were not changed in these patients and showed that EGF/TGFa: ratios in the urine of patients with hepatoceihtlar carcinomas were significantly decreased. On the basis of these results they suggested that the measurement of urinary TGFcwmay be a useful tumor marker, particularly for patients with hepatocellular carcinomas who have low serum alphafetoprotein levels [66]. Studies with liver epithelial cells which can function as precursors to hepatocellular carcinomas indicate that transformation of these cells is generally associated with increased TGFa gene expression [67]. As shown in various laboratories, immortalized nontumorigenic liver epithelial cells do not generally produce TGFol. However, their

Growth

Factors in the Liver

32-J

transformation by chemicals, oncogene transfection or manipulation of culture conditions causes an increase in TGFamRNA and the secretion of active peptide in the culture medium (Laird and Fausto, in preparation). In the WB liver epithelial cell line extensively analyzed by Grisham, Tsao and their colleagues, TGFa expression and tumorigenicity cosegregate in many different clonal lines [68]. The correlation between overexpression of TGFaand tumorigenic capacity is particularly strong in clones that overexpress c-myc in addition to TGFa. Although the expression of c-myc and TGFcw are likely to be independently controlled, they act synergistically in promoting tumorigenesis. Perhaps, high levels of MYC sensitize the cells to the mitogenic effect of TGFcr. These studies are further confirmation that TGFa functions through an autocrine loop to regulate liver growth. Transient expression of the factor is associated with a wave of DNA synthesis in the regenerating liver, but its constitutive expression leads to transformation of liver epithelial cells in culture and to the formation of hepatocellular carcinomas in TGFa transgenic mice. TGFd

Elevated amounts of TGFPl mRNA and its peptide have been found in human hepatocellular carcinomas by Ito et al. [69]. The peptide was localized in transformed hepatocytes, but it is not known whether it was synthesized in parenchymal or nonparenchymal cells. An analysis of the expression of TGFpl mRNA by in situ hybridization during rat hepatocarcinogenesis showed that TGFPl is synthesized in nonparenchymal cells and primarily in desmin-positive perisinusoidal cells [70]. Thus, both in liver regeneration and in hepatocarcinogenesis TGFPl acts by a paracrine mechanism. However, the significance of the overexpression of TGFPl by perisinusoidal cells during hepatocarcinogenesis is unclear. TGFPl production in the liver during carcinogenesis might be seen as a protective mechanism that ultimately fails because liver cells lose their sensitivity to TGFPl [59] at the early stages of the transformation process (at least in culture but in viva data are lacking). The net result is that TGF/31 may act as a selective agent that permits proliferation of altered cells but inhibits normal cell replication. It is also conceivable that TGFpl released during hepatocarcinogenesis acts mainly on cell matrix formation without having a direct effect on cell proliferation [71]. IGF-II

Expression of IGF-II mRNA increases in woodchuck, rat and human hepatocellular carcinomas [72,73]. In woodchuck infected with woodchuck hepatitis virus, the development of hepatocellular carcinomas involves lesions similar to those described in rat hepatocarcinogenesis. Yang and Rogler have found by in situ hybridization that IGF-II is expressed in some cell clusters in neoplastic nodules and that most carcinomas show high IGF-II mRNA expression although with a variable pattern [72]. The major IGF-II mRNA expressed in nodules and tumors is a 3.4 kb transcript which is one of the two IGF-II mRNA forms detected in fetal woodchuck liver. Thus, in these animals, IGF-II has the pattern of expression expected from an oncofetal marker. It remains to be established whether IGF-II re-expression in tumors contributes to the development of the tumors or if it is a consequence of the proliferation of immature liver cells forming the tumor.

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SUMMARY

AND FUTURE PERSPECTIVES

Major progress has been achieved in the study of the role of growth factors in liver growth regulation both in vivo and in liver cells in culture. Two factors, TGFa and HGF appear to play key roles as positive mediators of liver regeneration while TGFPl may function as an inhibitor. As shown in hepatocyte cultures and in a transgenic mouse model, TGFais a strong liver mitogen. Its transient production and expression in hepatocytes is associated with proliferation of these cells both in vivo and in culture. Constitutive overexpression of the factor causes liver hypertrophy and, at least in the CD1 mouse strain, leads to tumor formation in a large majority of animals after 12 months of age. HGF is produced by nonparenchymal cells and is a very potent mitogen for hepatocytes in culture. In contrast to TGFq HGF is present in the blood and its concentration increases after liver injury and partial hepatectomy. In these conditions HGF mRNA and the peptide also increase in liver tissue but the changes are much larger following injury than after partial hepatectomy. It is of major interest to determine how HGF and TGFormay interact in inducing liver growth and whether the mechanisms that regulate liver regeneration after partial hepatectomy differ from those which govern liver growth in response to cell death. Based on the timing of the changes of HGF and TGFaafter partial hepatectomy, it is plausible to suggest that HGF might work as a triggering stimulus while TGFcz acts on hepatocytes which have already entered the cell cycle. TGFa appears to play an important role in the development of hepatocellular carcinomas while little is known about HGF effects in this process. Preliminary data suggest that HGF may in fact inhibit the proliferation of liver tumor cells. It is unlikely that TGFol by itself can function as a transforming agent. Its overexpression is part of a constellation of changes occurring during hepatocarcinogenesis that may involve c-myc overexpression and at least in some cases, ~53 mutations. It is not known whether high, constitutive expression of TGFar increases the risk for the development of such alterations or if it promotes the proliferation of “initiated” cells that pre-exist in the tissue. The inter-relationships between growth factor overexpression and protooncogene alterations in the development of hepatocellular carcinomas can now be studied in culture using both liver epithelial cell lines with stem-cell capacities and hepatocytes isolated from TGFa transgenic mice. TGFpl may function as a stop signal for liver regeneration through its inhibitory effect or hepatocyte proliferation. It also induces fibrogenesis, an effect which is of particular importance in the development of chronic liver disease in humans. An interesting speculation regarding the multifunctional roles of TGFBl in liver is that moderate, transient increases in active TGFPl in hepatic tissue after partial hepatectomy promote the formation of extracellular matrix components which signal the end of hepatocyte proliferation. In contrast, chronic TGFpl overexpression localized in lipocytes leads to liver fibrosis. In addition to TGFq HGF and TGFPl, several other agents may directly or indirectly influence liver regeneration. They include growth factors such as HBGF- 1, binding proteins such as IGFBP-1 and various hormones and nutritional factors [74]. In addition new growth factors that stimulate or inhibit hepatocyte proliferation have been described and are being purified [75- 781. In a recent publication Francavilla et al. [79] reported that among many factors tested, infusion of insulin, TGFcr (but not

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EGF), HGF, IGF-II and HSS (‘hepatic stimulatory substance’) in vivo had mitogenic activity in livers made atrophic by portal blood diversion. Liver regeneration is a very complex process which is likely to have multiple levels of regulation. Individual growth factors that have major roles in the regenerative response have been identified and new systems have been devised to study the effect of these factors on liver regeneration and carcinogenesis. The interaction between these factors, as well as their relationships with proto-oncogene expression during liver growth are exciting topics for future research.

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