Biochimica et Biophysica Acta, 1132 (1992) 119-126
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
Review
BBAEXP 92417
Transcription factors and liver-specific genes Vincenzo De Simone
a
and Riccardo Cortese b
a 'CEINGE Centro di Ingegneria Genetica' and Dipartimento di Biochimica e Biotecnologie Mediche, Universith degli Studi 'Federico II' Napoli (Italy) and b IRBM - lstituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia (Italy)
(Received 16 June 1992)
Key words: Transcriptional regulation; Liver-specificity;DNA binding protein
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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If. Liver-specific transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The C/EBP gene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. HNF1/LFB1 and vHNF1/LFB3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The HNF-3 gene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. HNF-4/LFA1 and ARP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119 120 121 122 123
III. More complex networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
IV. Hepatocyte differentiation and the role of tissue-architecture . . . . . . . . . . . . . . . . . . . . . . . . . . .
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V. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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I. Introduction T h e liver is where a large n u m b e r of metabolic regulatory processes take place. Here, a set of enzymes are constitutively expressed or induced whose function is to metabolize the foreign substances absorbed by the intestinal surface. T h e liver is also the major site of plasmaprotein synthesis, which can be subdivided in several ' g r o u p s ' according to their function and intercorrelation, i.e., the coagulation cascade, the complem e n t system, the transport of lipids, etc. O t h e r specialized functions of the liver are mediated by specific groups of proteins involved in the regulation of carbohydrate and lipid metabolism and the biosynthesis of
Correspondence to: R. Cortese, IRBM - Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina, Kn 30.6, 00044 Pomezia, Roma, Italy.
urea. All these functions rely on a highly specialized system of intracellular transport and molecular sorting t h r o u g h two separate m e m b r a n e compartments: the apical and basolateral regions. M a n y of the genes encoding these functions are only expressed in liver, whilst others are m o r e widely expressed. It is clear, however, that all of t h e m are coordinately activated and sustained by c o m m o n mechanisms, triggered by hepatocyte differentiation.
II. Liver-specific transcription factors T h e bulk of our present knowledge about the transcriptional regulation of liver-specific genes comes from the study of p r o m o t e r and e n h a n c e r elements of genes specifically expressed in terminally-differentiated hepatocytes. T h e c i s - a c t i n g elements which control the transcription of the albumin (ALB), a l - a n t i t r y p s i n ( a l A T ) , a - f e t o p r o t e i n ( A F P ) and transthyretin ( T T R ) are shown
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ALB
~
TTR
Fig. 1. A diagram of the interactions between c/s-acting elements and trans-acting factors in the promoters of albumin (ALB), al-antitrypsin (al-AT) and transtbyretin (TTR) [4,37,39].
in Fig. 1. Other genes are still under investigation, but the general picture seems quite coherent: several short target sequences for DNA binding proteins are scattered mainly in the 5' untranscribed region, each of them contributing to a certain extent to the overall transcriptional activity of the gene. As we will discuss later, none of these DNA binding proteins are sufficient to confer tissue-specificity, nor are they restricted only to hepatocytes. Nevertheless, the unique combination of their binding sites dictates when and where the gene is transcribed. The identification of the target sequences has allowed the cognate DNA binding proteins to be purified and, ultimately, the corresponding genes to be isolated. As a result, four gene families which play a major rote in liverspecificity have been identified so far. All these proteins, with a few specific exceptions, are pleiotropic transcriptional activators and will be the main subject of this review.
H-A. The C / EBP gene family C / E B P is a heat-stable DNA-binding protein originally identified in rat liver nuclei, which plays an important role in the transcriptional regulation of several liver-specific genes. C / E B P dependent regulatory elements have been identified in the promoter and the enhancer of the albumin [1-3], transthyretin (TTR) and cd-antitrypsin (al-AT) [4,5], phosphoenolpyruvate carboxykinase (PEPCK) [6,7], ornithine transcarbamylase (OTC) [8], apoA-I [9], apolipoprotein B-100 (apoB-100) [10], serum amyloid A (SAA) [11] and many other genes. The canonical C / E B P target sequence is the perfect palyndrome ATTGCGCAAT, which recalls the CCAAT homology motif and is therefore responsible for the original classification of C / E B P as a
CCAAT-binding protein. However, C / E B P can also bind more degenerated sequences; the comparison of the C / E B P binding sites of several cis-acting elements results in the T T / c N N G C / T A A T / ( ~ consensus sequence. C / E B P is the prototype of the B-ZIP proteins, binding to DNA as a dimeric protein via a 'leucine zipper' dimerization interface and a 'basic' DNA contact surface [12-14]. The recombinant C / E B P protein behaves as a transcriptional activator both in transient transfection and in vitro transcription assays [15]. Two regions of the C / E B P N-terminal portion are required for transcriptional activation; these regions retain function when fused to the DNA-binding domain of a different protein [16,17]. C / E B P gene expression is not restricted to the liver. High levels of C / E B P mRNA have been observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These include liver, fat, intestine, lung, adrenal gland and placenta. In liver and fat, C / E B P expression is restricted to fully differentiated cells. Many eukaryotic virus promoters [6] and enhancers [18] contain C / E B P binding sites which are essential for the expression of viral genes [19]. Moreover, C / E B P is a pleiotropic activator of a group of adipose-specific genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein, and stearoyl-CoA desaturase 1 (SCD1) [20-22]. No information is available about the mechanisms which regulate C / E B P expression. The findings that C / E B P mRNA, but no corresponding protein, can be detected in some tissues like kidney and brain, suggests posttranscriptional regulation. However, during hepatocyte and adipocyte differentiation, C / E B P expression is mainly regulated at transcriptional level, and there is evidence to suggest that C / E B P may regulate the transcription of its own gene [23]. As in the case of all the transcriptional factors which regulate liver-specific genes, ectopic expression of C / E B P in non liver cells and overexpression in hepatoma cells does not affect either their differentiation pattern, or the expression of the endogenous target genes. The only effect of deregulated C / E B P expression in differentiation has been observed in pre-adipocytes, where premature expression of C / E B P causes a direct cessation of mitotic growth and, in conjunction with three adipogenic hormones, promotes terminal cell differentiation [24]. On the basis of these observations, it has been speculated that C / E B P may play a role in establishing and maintaining the differentiated, nonproliferative state, by regulating the balance between cell growth and differentiation [15]. C/EBP-Iike proteins, defined as trans-acting factors which bind to the same recognition sequence as C/EBP, have been described. One member of this
121 family is DBP, a transcriptional activator that binds to the D site of the albumin promoter. Among several rat tissues tested, significant levels of DBP protein are only observed in liver; yet, with the exception of testis, DBP mRNA was found in all of the examined tissues [25]. Both DBP and the related chicken protein VBP belong to the bZIP family. The original report of DBP lacking the leu zipper domain was probably due to a mistake in the first published sequence [26]. A C/EBP-Iike protein mediates the interleukin-6 (IL-6) dependent change in the rate of transcription of several liver genes, the Acute-_Phase (AP) genes, during the acute phase of infections. IL-6 has been shown to induce transcription of several AP genes through an IL-6 responsive element (IL-6RE) present in the promoter of the responsive genes [27]. This protein, called IL-6 DBP or LAP in rat [28,29], NF-IL6 in humans [30] and A G P / E B P in mouse [31], recognizes the IL-6RE sequence and trans-activates the relative genes in a IL-6 dependent fashion. IL-6DBP and NF-IL6 activities are induced by IL-6 treatment of human hepatoma cell lines through a posttranslational mechanism [28,30]. It has been recently reported that cAMP treatment of PC12 neuroblastoma cells induces IL-6DBP/NF-IL6 translocation from cytoplasm to the nucleus [32]. However, IL-6DBP/NF-IL6 is constitutively nuclear in hepatoma cell lines [33]. Other trans-acting proteins have been described which belong to the C / E B P family. The transcription factor E2BP binds to a sequence in the U3 region of Rous Sarcoma virus LTR which is identical to that recognized by C / E B P [34]. The EBV transactivator protein BZLF1 can bind many C/EBP-Iike target sequences in the EBV genome, and can also recognize the binding site for C / E B P [35]. Ig/EBP-1 is a protein which recognizes the same DNA binding sites as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E) in both the IgH enhancer and the VH1 promoter. Ig/EBP-1 and C / E B P are highly homologous throughout the DNA-binding domain and leucine zipper regions, and they can form heterodimers. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells [36].
II-B. HNFla /LFB1 and HNFlfl /LFB3 The g / a G T F A A T N A T I ' A A C c / a cis-acting regulatory element was originally identified in the promoter of the al-antitrypsin [37], a and /3-fibrinogen [38] and albumin genes [39]. Subsequently, it has been found in the promoters and enhancers of several other liverspecific genes, like a-fetoprotein (AFP) [40]; transthyretin (T-FR) [4]; hepatitis B virus (HBV) [41,42];
aldolase B [43]; L-type pyruvate kinase (L-PK) [44]; A2 vitellogenin gene of Xenopus laevis [45]; human C-reactive protein (CRP) [46] and many others. The cognate DNA binding protein has been called variously HNF-1 [47], LFB1 [48], APF [49] or, more recently, H N F - l a [50]. Another DNA binding activity recognizing the same target sequence was identified in the dedifferentiated H5 and C2 rat hepatoma cell lines or in hepatoma/fibroblasts cell hybrids, and called vHNF1 [51], vAPF [49], LFB3 [52] or HNF-lfl [50]. In this review, we will refer to these two proteins as L F B 1 / H N F - l a and LFB3/HNF-1/3, respectively. cDNA coding for L F B 1 / H N F - l a [53,54,55] and LFB3 /HNF-1/3 [50,53,56] have been isolated, showing that they are highly homologous in three regions. The first 34 amino acids of the N-terminal region correspond to a dimerization domain. The other two homologous regions have been referred to as the pseudo-pou domain (6-pou) and the extra-large homeodomain (XLHD), respectively, and they are the two components of a bipartite DNA binding domain [57]. Deletion analysis of the L F B 1 / H N F - l a DNA binding domain revealed that DNA binding as such is mediated by the 80 amino acids long XL-HD. This polypeptide is capable of recognizing a collection of DNA sequences having a common AT rich consensus sequence and binds DNA as a monomer, with a k d in the nanomolar range. A peptide containing the XL-HD plus the additional 90 amino acids of the $-pou domain no longer binds to any AT rich DNA sequences, but instead recognizes only the 12 bases palindromic HNF-1/LFB1 recognition site. The presence of the th-pou domain drastically increases the 'specificity' of the DNA protein interaction without significantly changing the 'affinity' (Tomei and De Francesco, unpublished results). L F B 1 / H N F - l a dimerization is required to bind the DNA. The dimerization domain at the N-termini of the protein forms a kinked a-helix, with several hydrophobic residues (mainly leucines) aligned on the same side. Dimerization occurs by interaction of the hydrophobic surfaces of the two monomers [58,59]. Adding these 34 amino acids to a peptide containing both ~b-pou and XL-HD significantly increases affinity for the target sequence and allows the dimers to form also in the absence of DNA (Tomei and De Francesco, unpublished). The same region mediates the formation of heterodimers between L F B 1 / H N F - l a and LFB3/HNF-1/3. A protein factor that selectively stabilizes the L F B 1 / H N F - l a dimers has been recently described [60]. This factor, called DCoH, has no direct DNA binding or transcriptional activity, but it strongly enhances the transcriptional activity of LFB1/HNF-1 a . Both L F B 1 / H N F - l a and LFB3/HNF-1/3 recombinant proteins behave as transcriptional activators in vivo and in vitro [57,52]. Two domains of LFB1/HNF-
122 1c~ which are required for the transcriptional activity in vitro have been identified, one at the C-terminus and the second adjacent to the XL-homeodomain [57]. Interestingly, the LFB3/HNF-1/3 protein is shorter than LFB1/HNF-1 a, missing the region which in the latter corresponds to the C-terminal activation domain. The LFB3/HNF-1/3 activation domain is located in a region which shares no homology with L F B 1 / H N F - l a ; a deletion mutant lacking the last 87 amino acids of the C-terminal part of the protein fails to trans-activate the target promoter in co-transfection assays (De Simone, unpublished data). As for C/EBP, L F B 1 / H N F - l a and LFB3/HNF-1/3 expression is not restricted to liver cells. In the adult rat and mouse, they are expressed in several tissues like kidney, intestine, stomach and pancreas. LFB3/HNF-1/3 is also expressed in lungs. These tissues are not embryologically related; their only common feature is to have a specialized epithelium. In situ hybridization experiments show clearly that both genes are indeed expressed in the polarized epithelia of these organs. It has been suggested that these factors play a role in implementing an 'epithelial' genetic program, similar to that observed for crumbs in Drosophila melanogaster [52]. LFB3/HNF-1/3 is expressed earlier than LFB1/ HNF-I~ during mouse, rat and Xenopus development (Refs. 52, 61 and De Simone, unpublished observations). This sequential expression of the two genes has been monitored during organogenesis of rat kidney, in which mesoderm-derived cells are induced to differentiate into a polarized epithelium by a morphogenetic signal released by the growing ureter bud. LFB3/HNF-1/3 transcripts can be detected in mesoderm-derived cells as soon as they are induced to differentiate into a polarized epithelium, while LFB1/ HNF-la transcripts appear only at a later stage, when the three different segments of the nephron become apparent [61]. Differential expression of LFB1/HNF-lc~ and LFB3/HNF-1/3 has been observed in a set of rat hepatoma cell lines. These cells consist of two lines (H4II and C2rev7), which retain a fully differentiated phenotype, and two 'dedifferentiated' subclonal derivatives (H5 and C2) which have lost the capacity to express several liver-specific genes [62]. L F B 1 / H N F - l a is only expressed in the fully differentiated H4II and C2rev7, and not in the dedifferentiated H5 and C2 cell lines. It is also absent in cell hybrids between hepatoma and fibroblast cell lines, which have lost the ability to express some liver-specific genes [63]. On the other hand, LFB3/HNF-1/3 is expressed in all of them, irrespective of whether the target genes are active or not. LFB3/HNF-1/3 and L F B 1 / H N F - l a are also differentially regulated in F9 murine teratocarcinoma cells. F9 can be induced to differentiate into endoder-
mal-like cultures by retinoic acid (RA) treatment. After 24 h of RA treatment, LFB3/HNF-1/3 is strongly induced, while HNF1/LFB1 levels do not change until 5 days after RA administration [52,64]. The sequential expression of LFB3/HNF-1/3 and L F B 1 / H N F - l a suggests that they play a different role in hepatocyte (or epithelia) differentiation; perhaps the first is involved in the onset and the second in the maintenance of the expression of tissue-specific genes: However, no direct evidence is available yet to support this hypothesis.
II-C. The HNF-3 gene family Hepatocyte nuclear factor 3 (HNF-3) was described as a DNA binding activity interacting with the mouse transthyretin (TTR) gene promoter, the a 1-AT proximal enhancer region and the far upstream region of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene [4,65]. It was subsequently shown that this activity consists of three major DNA binding proteins, called HNF-3a, HNF-3/3 and HNF-3y [66]. The DNA binding domain and the activation domain of HNF-3a protein have been mapped respectively to the 157/267 and 272/326 amino acids regions. However, neither of these regions show any recognizable feature that allows their classification within one of the already known classes of DNA binding or transcriptional activation elements. All the three HNF-3 proteins behave as transcriptional activators in cotransfection experiments. The DNA binding domains of HNF-3a,/3 and -/are highly conserved in rat (93 out of 110 identical amino adds). Moreover, they are strikingly homologous to a similar region in the Drosophila homeotic gene fork head (88 of the 93 residues that are conserved among the three rat genes) [64]. Nevertheless, some of the differences between these DNA binding domains must be important: experiments comparing the affinity of the three HNF-3 proteins for two different oligos (S and W, corresponding respectively to a 'weak' and a 'strong' site for HNF-3 in the TTR promoter) resulted in HNF-3/3 having an inverted affinity compared to HNF-3a and HNF-3T [67]. The HNF-3 family genes are expressed in cells that derive from the lining of the primitive gut. Some of the embryonic Drosophila cells in which fork head is expressed also give rise to gut and salivary glands. It has been proposed that this gene family may contribute to differentiation of organs and structures derived from the endodermal tissues of the primitive intestine [68]. However, there are differences in the expression pattern of the three genes: while all of them are expressed in liver and intestine, only a and /3 are expressed in lungs and only y in testis [67].
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II-D. HNF-4 / LFA1 and ARP-1 HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds an essential region of the transthyretin and apolipoprotein CIII promoters [4]. HNF-4 is a member of the thyroid-steroid hormone receptor superfamily; the 50/133 amino acids region corresponds to the DNA binding domain, which belongs to the zinc finger group and is 40 to 65% homologous to the other members of the family. The 133/373 amino acids region contains the ligand binding domain, which is 20 to 35% homologous to the corresponding region of other receptor-like proteins. HNF-4 protein binds to DNA as a dimer, and it activates transcription in cotransfection assays. HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues [4]. It has been suggested that HNF-4 could be identical to the previously described liver factor Al (LF-A1), a DNA-binding activity implicated in the transcriptional regulation of the al-antitrypsin, apolipoprotein A1 [46] and pyruvate kinase genes [69]. The apoAI regulatory protein-1 (ARP-1), whose gene has been recently cloned, binds to the apolipoprotein Al gene promoter at the same sequence which is also recognized by HNF-4/LF-A1 [70]. ARP-1 also belongs to the steroid hormone receptor superfamily. HNF-4 and ARP-1 genes are clearly different, yet they are much more alike than the other members of the family. ARP-1 binds to DNA as a dimer, and its dimerization domain is localized to the COOH-terminal region. However, in cotransfection experiments, ARP-I downregulates both the apoAI and the apoCIII gene expression. A 3-4-fold excess of ARP-1 prevents the activating effect of HNF-4, probably due to a competition for the same target sequence, since both ARP-1 and HNF-4 bind to the apoAI recognition site with the same affinity. Although both bind DNA as homodimers, no heterodimer formation occurs between the two proteins (Darnell, personal communication).
IlL More complex regulatory networks The notion of a positive regulation of liver-specific genes by a combination of transcriptional activators contrasts with the results of somatic cell genetic experiments, which were originally explained through negative regulation. In fibroblast x hepatoma cell hybrids several liver-specific functions are extinguished, and epistatic genes which negatively regulate sets of liverspecific functions have been described [71]. These two lines of evidence have been reconciled by the recent cloning of the TSE1 (tissue-specific extinguisher) gene, a locus responsible for the extinction of tyrosine
RETINOIC ACID
STEROIDS (?)
IL-6
HNF-4
HNF-3
/1
LFB1 " ~ LAP HNF-1~J IL6-DBP / t heter°dimersJ t formation LFB3 / C/EBP HNF- 1f ~ I
LIVER-SPECIFIC GENES Fig. 2. A scheme of the transcriptional regulatory network. Thin arrows = transcriptional activation; double-headed arrows = heterodimer formation; loops= self-regulation; stippled arrows= modulationof activityby extracellularsignals.
aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK) genes. TSE1 encodes for the R I a regulatory subunit of protein kinase A (PKA). R I a increased expression in hybrids causes the dephosphorylation of the CREB transcriptional activator, resulting in repression of the target genes [72]. However, it appears clear that the extinction of liver-specific genes in somatic cell hybrids is a complex phenomenon, involving multiple mechanisms [73]. Some evidence about cross-talk between the different factors are now emerging. Two groups have independently reported that recombinant HNF-4 can trans-activate the L F B 1 / H N F - l a promoter [74,75]. HNF-4, which belongs to the thyroid/steroid hormone receptor superfamily, is a valid candidate for mediating the response of hepatocytes to as yet unidentified external stimuli which could trigger or modulate differentiation. Moreover, transcriptional self-regulation has been postulated for C/EBP, HNF-3# and L F B 1 / H N F - l a (Refs. 23,76 and Piaggio, unpublished results). A hypothetical model in which transcription factors are selfregulated and organized into a hierarchy is shown schematically in Fig. 2.
124 IV. Hepatocyte differentiation and the role of tissue-architecture Although several details have still to be clarified, the general mechanisms which govern the transcriptional regulation of liver-specific gene expression have been understood. What still remains completely obscure is the cascade of events that initiates the differentiation of hepatocytes. As we have already mentioned, none of these transcriptional factors are capable of starting the hepatocyte differentiation program. However, although unlikely, the possibility that expressing all of them in a non-liver cell could trigger differentiation still formally exists. The liver originates from an endodermal diverticulum of the foregut, when epithelial 'cords' deriving from this endodermal rudiment invade the surrounding mesenchymal mass and eventually differentiate into hepatocytes [77]. These morphogenetic events are induced by the mesenchymal portion in a two-step process: an initial message from the mesoderm results in the 'determination' or commitment of the epithelial primordium to the liver fate, without any apparent morphological modification. Later, a second message from mesoderm triggers the 'induction' of liver organogenesis [78]. The recent identification in mesenchymal cells of a membrane protein, termed epimorphin, which seems to play a role in the induction of epithelial cell differentiation opens the possibility to identify and to study the molecules which are responsible for this exchange of messages between mesoderm and endoderm [79]. In situ hybridization studies have recently shown that albumin m R N A can be first detected in the liver bud of 9.5-day-old mouse embryos [80]. This stage precedes liver formation, and coincides with endoderm-mesoderm interactions, indicating that liverspecific terminal genes are activated as soon as the precursor cells become committed to the hepatocyte fate. However, the same authors describe a 15-20 fold increase in steady-state albumin m R N A levels following the definitive formation of the liver, suggesting that this increase could be due to the establishment of the right cell-cell contact. The authors propose a model in which at least one subset of adult transcriptional factors is switched on at the e n d o d e r m / m e s o d e r m induction stage, and cell assembly into an organ serves to amplify a transcriptional state established prior to organ formation. One implication of this model is that liver-specific gene expression begins when cells enter a rapidly dividing stage, unlike what happens in other systems such as muscular differentiation. The dependence of liver gene expression on cell-cell and cell-extracellular matrix (ECM) contacts has been described by several groups in the past [81-84]. Some recent reports demonstrate how interactions between
the hepatocyte and the extracellular matrix coordinately modulate cell architecture and liver-specific gene expression, by affecting the transcriptional efficiency of the albumin enhancer element [85] or the amount of some transcriptional factors like HNF-3a [80]. V. Conclusions and perspectives As more details about the transcriptional regulation of liver-specific genes are known, new interesting features are emerging. First, each cis-acting signal can usually be recognized by more than one class of transacting factors which co-exist in the same cell and which can bind in a mutually-exclusive manner. Moreover, in many cases the different members of these families can form heterodimeric complexes, adding new potential to the system. Second, the recognition sequence is often very degenerate, yet the affinity remains very high, as if several alternative ways of binding the same protein have evolved independently in different genes (or at different positions in the same gene). Whether these differences play any role in the fine regulation of these genes remains to be seen. Finally, the transcriptional effects of different cis-acting elements in the same promoter are usually additive and independent, although some cases of cooperative effects have been described. Despite the mass of infomation which has been acquired about the molecules that regulate the transcription of liver-specific genes, it is a matter of fact that our knowledge of hepatocyte differentiation and liver organogenesis is still very preliminary. This may reflect the complexity of the process, and the inadequacy of simple cell culture systems (mainly hepatoma cell lines) in approaching differentiation problems. To investigate the nature of the signals that mesoderm and pre-hepatic endoderm exchange, which trigger hepatocyte differentiation, may require going back to organ culture techniques and trying to identify and isolate a 'morphogen' and possibly its targets in pre-hepatocytes. An indication about how misleading experiments with cell cultures can be comes from the biological effect of the hepatic growth factor (HGF, a long known molecule), on kidney-derived epithelial ceils. The addition of H G F to epithelial ceils in culture (including hepatocytes) results in cell growth stimulation. Montesano et al. have recently shown that when MDCK canine renal cells are grown on a collagen matrix which allows cells to spread tridimensionally, of adding H G F results in the induction of tubulogenesis, and the mitogenic activity is only a consequence of the main morphogenic effect [86]. Similar phenomena in liver cell culture might have been overlooked simply because of the lack of such 'more physiological' assay conditions. This is a direction that clearly deserves to be pursued.
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Acknowledgements This work was partially supported by grants related to the contract between CEINGE and the Agenzia per 1o Sviluppo del Mezzogiorno (Rome), No. PS35126/IND-AVV and by the EEC-BRIDGE grant No. BIOT-CT91-0260 (TSTS) to V.D.S.
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126 61 Lazzaro, D., De Simone, V., De Magistris, L., Lehtonen, E. and Cortese, R. (1991) Development 114, 469-479. 62 Deschatrette, J., Fougere-Deschatrette, C., Corcos, L. and Schimke, R.T. (1985) Proc. Natl. Acad. Sci. USA 82, 765-769. 63 Cereghini, S., Yaniv, M. and Cortese, R. (1990) EMBO J. 9, 2257-2263. 64 Kuo, C.J., Mendel, D.B., Hansen, L.P. and Crabtree, G.R. (1991) EMBO J. 10, 2231-2236. 65 lp, Y.T., Poon, D., Stone, D., Granner, D.K. and Chalkley, R. (1990) Mol. Cell Biol. 10, 3770-3781. 66 Lai, E., Prezioso, V.R., Smith, E., Litvin, O., Costa, R.H. and Darnell, J.E.Jr. (1990) Genes Dev. 4, 1427-1436. 67 Lai, E., Prezioso, V.R., Tao, W.F., Chen, W.S., Darnell, J.E.Jr. (1991) Genes Dev. 5, 416-27. 68 Weigel, D., Jiirgens, G., Kuttner, F., Seifert, E. and J~ickle, H. (1989) Cell 57, 645-658. 69 Vaulont, S., Puzenat, N., Kahn, A. and Raymondjean, M. (1989) Mol. Cell Biol. 9, 4409-4415. 70 Ladias, J.A. and Karathanasis, S.K. (1991) Science 251. 561-565. 7l Chin, A.C. and Fournier, R.E.K. (1987) Proc. Natl. Acad, Sci. USA 84, 1614-161618. 72 Boshart, M., Welch, F., Nichols, M. and Schiitz, G. (1991) Cell 66, 849-859. 73 Ruppert, S., Boshart, M., Bosch, F.X., Schmid, W., Fournier, R.E. and Schlitz, G. (1990) Cell 61,895-904.
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© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
Review
BBAEXP 92417
Transcription factors and liver-specific genes Vincenzo De Simone
a
and Riccardo Cortese b
a 'CEINGE Centro di Ingegneria Genetica' and Dipartimento di Biochimica e Biotecnologie Mediche, Universith degli Studi 'Federico II' Napoli (Italy) and b IRBM - lstituto di Ricerche di Biologia Molecolare P. Angeletti, Pomezia (Italy)
(Received 16 June 1992)
Key words: Transcriptional regulation; Liver-specificity;DNA binding protein
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119
If. Liver-specific transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The C/EBP gene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. HNF1/LFB1 and vHNF1/LFB3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The HNF-3 gene family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. HNF-4/LFA1 and ARP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
119 120 121 122 123
III. More complex networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
123
IV. Hepatocyte differentiation and the role of tissue-architecture . . . . . . . . . . . . . . . . . . . . . . . . . . .
124
V. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
124
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
125
I. Introduction T h e liver is where a large n u m b e r of metabolic regulatory processes take place. Here, a set of enzymes are constitutively expressed or induced whose function is to metabolize the foreign substances absorbed by the intestinal surface. T h e liver is also the major site of plasmaprotein synthesis, which can be subdivided in several ' g r o u p s ' according to their function and intercorrelation, i.e., the coagulation cascade, the complem e n t system, the transport of lipids, etc. O t h e r specialized functions of the liver are mediated by specific groups of proteins involved in the regulation of carbohydrate and lipid metabolism and the biosynthesis of
Correspondence to: R. Cortese, IRBM - Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina, Kn 30.6, 00044 Pomezia, Roma, Italy.
urea. All these functions rely on a highly specialized system of intracellular transport and molecular sorting t h r o u g h two separate m e m b r a n e compartments: the apical and basolateral regions. M a n y of the genes encoding these functions are only expressed in liver, whilst others are m o r e widely expressed. It is clear, however, that all of t h e m are coordinately activated and sustained by c o m m o n mechanisms, triggered by hepatocyte differentiation.
II. Liver-specific transcription factors T h e bulk of our present knowledge about the transcriptional regulation of liver-specific genes comes from the study of p r o m o t e r and e n h a n c e r elements of genes specifically expressed in terminally-differentiated hepatocytes. T h e c i s - a c t i n g elements which control the transcription of the albumin (ALB), a l - a n t i t r y p s i n ( a l A T ) , a - f e t o p r o t e i n ( A F P ) and transthyretin ( T T R ) are shown
120
ALB
~
TTR
Fig. 1. A diagram of the interactions between c/s-acting elements and trans-acting factors in the promoters of albumin (ALB), al-antitrypsin (al-AT) and transtbyretin (TTR) [4,37,39].
in Fig. 1. Other genes are still under investigation, but the general picture seems quite coherent: several short target sequences for DNA binding proteins are scattered mainly in the 5' untranscribed region, each of them contributing to a certain extent to the overall transcriptional activity of the gene. As we will discuss later, none of these DNA binding proteins are sufficient to confer tissue-specificity, nor are they restricted only to hepatocytes. Nevertheless, the unique combination of their binding sites dictates when and where the gene is transcribed. The identification of the target sequences has allowed the cognate DNA binding proteins to be purified and, ultimately, the corresponding genes to be isolated. As a result, four gene families which play a major rote in liverspecificity have been identified so far. All these proteins, with a few specific exceptions, are pleiotropic transcriptional activators and will be the main subject of this review.
H-A. The C / EBP gene family C / E B P is a heat-stable DNA-binding protein originally identified in rat liver nuclei, which plays an important role in the transcriptional regulation of several liver-specific genes. C / E B P dependent regulatory elements have been identified in the promoter and the enhancer of the albumin [1-3], transthyretin (TTR) and cd-antitrypsin (al-AT) [4,5], phosphoenolpyruvate carboxykinase (PEPCK) [6,7], ornithine transcarbamylase (OTC) [8], apoA-I [9], apolipoprotein B-100 (apoB-100) [10], serum amyloid A (SAA) [11] and many other genes. The canonical C / E B P target sequence is the perfect palyndrome ATTGCGCAAT, which recalls the CCAAT homology motif and is therefore responsible for the original classification of C / E B P as a
CCAAT-binding protein. However, C / E B P can also bind more degenerated sequences; the comparison of the C / E B P binding sites of several cis-acting elements results in the T T / c N N G C / T A A T / ( ~ consensus sequence. C / E B P is the prototype of the B-ZIP proteins, binding to DNA as a dimeric protein via a 'leucine zipper' dimerization interface and a 'basic' DNA contact surface [12-14]. The recombinant C / E B P protein behaves as a transcriptional activator both in transient transfection and in vitro transcription assays [15]. Two regions of the C / E B P N-terminal portion are required for transcriptional activation; these regions retain function when fused to the DNA-binding domain of a different protein [16,17]. C / E B P gene expression is not restricted to the liver. High levels of C / E B P mRNA have been observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These include liver, fat, intestine, lung, adrenal gland and placenta. In liver and fat, C / E B P expression is restricted to fully differentiated cells. Many eukaryotic virus promoters [6] and enhancers [18] contain C / E B P binding sites which are essential for the expression of viral genes [19]. Moreover, C / E B P is a pleiotropic activator of a group of adipose-specific genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein, and stearoyl-CoA desaturase 1 (SCD1) [20-22]. No information is available about the mechanisms which regulate C / E B P expression. The findings that C / E B P mRNA, but no corresponding protein, can be detected in some tissues like kidney and brain, suggests posttranscriptional regulation. However, during hepatocyte and adipocyte differentiation, C / E B P expression is mainly regulated at transcriptional level, and there is evidence to suggest that C / E B P may regulate the transcription of its own gene [23]. As in the case of all the transcriptional factors which regulate liver-specific genes, ectopic expression of C / E B P in non liver cells and overexpression in hepatoma cells does not affect either their differentiation pattern, or the expression of the endogenous target genes. The only effect of deregulated C / E B P expression in differentiation has been observed in pre-adipocytes, where premature expression of C / E B P causes a direct cessation of mitotic growth and, in conjunction with three adipogenic hormones, promotes terminal cell differentiation [24]. On the basis of these observations, it has been speculated that C / E B P may play a role in establishing and maintaining the differentiated, nonproliferative state, by regulating the balance between cell growth and differentiation [15]. C/EBP-Iike proteins, defined as trans-acting factors which bind to the same recognition sequence as C/EBP, have been described. One member of this
121 family is DBP, a transcriptional activator that binds to the D site of the albumin promoter. Among several rat tissues tested, significant levels of DBP protein are only observed in liver; yet, with the exception of testis, DBP mRNA was found in all of the examined tissues [25]. Both DBP and the related chicken protein VBP belong to the bZIP family. The original report of DBP lacking the leu zipper domain was probably due to a mistake in the first published sequence [26]. A C/EBP-Iike protein mediates the interleukin-6 (IL-6) dependent change in the rate of transcription of several liver genes, the Acute-_Phase (AP) genes, during the acute phase of infections. IL-6 has been shown to induce transcription of several AP genes through an IL-6 responsive element (IL-6RE) present in the promoter of the responsive genes [27]. This protein, called IL-6 DBP or LAP in rat [28,29], NF-IL6 in humans [30] and A G P / E B P in mouse [31], recognizes the IL-6RE sequence and trans-activates the relative genes in a IL-6 dependent fashion. IL-6DBP and NF-IL6 activities are induced by IL-6 treatment of human hepatoma cell lines through a posttranslational mechanism [28,30]. It has been recently reported that cAMP treatment of PC12 neuroblastoma cells induces IL-6DBP/NF-IL6 translocation from cytoplasm to the nucleus [32]. However, IL-6DBP/NF-IL6 is constitutively nuclear in hepatoma cell lines [33]. Other trans-acting proteins have been described which belong to the C / E B P family. The transcription factor E2BP binds to a sequence in the U3 region of Rous Sarcoma virus LTR which is identical to that recognized by C / E B P [34]. The EBV transactivator protein BZLF1 can bind many C/EBP-Iike target sequences in the EBV genome, and can also recognize the binding site for C / E B P [35]. Ig/EBP-1 is a protein which recognizes the same DNA binding sites as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E) in both the IgH enhancer and the VH1 promoter. Ig/EBP-1 and C / E B P are highly homologous throughout the DNA-binding domain and leucine zipper regions, and they can form heterodimers. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells [36].
II-B. HNFla /LFB1 and HNFlfl /LFB3 The g / a G T F A A T N A T I ' A A C c / a cis-acting regulatory element was originally identified in the promoter of the al-antitrypsin [37], a and /3-fibrinogen [38] and albumin genes [39]. Subsequently, it has been found in the promoters and enhancers of several other liverspecific genes, like a-fetoprotein (AFP) [40]; transthyretin (T-FR) [4]; hepatitis B virus (HBV) [41,42];
aldolase B [43]; L-type pyruvate kinase (L-PK) [44]; A2 vitellogenin gene of Xenopus laevis [45]; human C-reactive protein (CRP) [46] and many others. The cognate DNA binding protein has been called variously HNF-1 [47], LFB1 [48], APF [49] or, more recently, H N F - l a [50]. Another DNA binding activity recognizing the same target sequence was identified in the dedifferentiated H5 and C2 rat hepatoma cell lines or in hepatoma/fibroblasts cell hybrids, and called vHNF1 [51], vAPF [49], LFB3 [52] or HNF-lfl [50]. In this review, we will refer to these two proteins as L F B 1 / H N F - l a and LFB3/HNF-1/3, respectively. cDNA coding for L F B 1 / H N F - l a [53,54,55] and LFB3 /HNF-1/3 [50,53,56] have been isolated, showing that they are highly homologous in three regions. The first 34 amino acids of the N-terminal region correspond to a dimerization domain. The other two homologous regions have been referred to as the pseudo-pou domain (6-pou) and the extra-large homeodomain (XLHD), respectively, and they are the two components of a bipartite DNA binding domain [57]. Deletion analysis of the L F B 1 / H N F - l a DNA binding domain revealed that DNA binding as such is mediated by the 80 amino acids long XL-HD. This polypeptide is capable of recognizing a collection of DNA sequences having a common AT rich consensus sequence and binds DNA as a monomer, with a k d in the nanomolar range. A peptide containing the XL-HD plus the additional 90 amino acids of the $-pou domain no longer binds to any AT rich DNA sequences, but instead recognizes only the 12 bases palindromic HNF-1/LFB1 recognition site. The presence of the th-pou domain drastically increases the 'specificity' of the DNA protein interaction without significantly changing the 'affinity' (Tomei and De Francesco, unpublished results). L F B 1 / H N F - l a dimerization is required to bind the DNA. The dimerization domain at the N-termini of the protein forms a kinked a-helix, with several hydrophobic residues (mainly leucines) aligned on the same side. Dimerization occurs by interaction of the hydrophobic surfaces of the two monomers [58,59]. Adding these 34 amino acids to a peptide containing both ~b-pou and XL-HD significantly increases affinity for the target sequence and allows the dimers to form also in the absence of DNA (Tomei and De Francesco, unpublished). The same region mediates the formation of heterodimers between L F B 1 / H N F - l a and LFB3/HNF-1/3. A protein factor that selectively stabilizes the L F B 1 / H N F - l a dimers has been recently described [60]. This factor, called DCoH, has no direct DNA binding or transcriptional activity, but it strongly enhances the transcriptional activity of LFB1/HNF-1 a . Both L F B 1 / H N F - l a and LFB3/HNF-1/3 recombinant proteins behave as transcriptional activators in vivo and in vitro [57,52]. Two domains of LFB1/HNF-
122 1c~ which are required for the transcriptional activity in vitro have been identified, one at the C-terminus and the second adjacent to the XL-homeodomain [57]. Interestingly, the LFB3/HNF-1/3 protein is shorter than LFB1/HNF-1 a, missing the region which in the latter corresponds to the C-terminal activation domain. The LFB3/HNF-1/3 activation domain is located in a region which shares no homology with L F B 1 / H N F - l a ; a deletion mutant lacking the last 87 amino acids of the C-terminal part of the protein fails to trans-activate the target promoter in co-transfection assays (De Simone, unpublished data). As for C/EBP, L F B 1 / H N F - l a and LFB3/HNF-1/3 expression is not restricted to liver cells. In the adult rat and mouse, they are expressed in several tissues like kidney, intestine, stomach and pancreas. LFB3/HNF-1/3 is also expressed in lungs. These tissues are not embryologically related; their only common feature is to have a specialized epithelium. In situ hybridization experiments show clearly that both genes are indeed expressed in the polarized epithelia of these organs. It has been suggested that these factors play a role in implementing an 'epithelial' genetic program, similar to that observed for crumbs in Drosophila melanogaster [52]. LFB3/HNF-1/3 is expressed earlier than LFB1/ HNF-I~ during mouse, rat and Xenopus development (Refs. 52, 61 and De Simone, unpublished observations). This sequential expression of the two genes has been monitored during organogenesis of rat kidney, in which mesoderm-derived cells are induced to differentiate into a polarized epithelium by a morphogenetic signal released by the growing ureter bud. LFB3/HNF-1/3 transcripts can be detected in mesoderm-derived cells as soon as they are induced to differentiate into a polarized epithelium, while LFB1/ HNF-la transcripts appear only at a later stage, when the three different segments of the nephron become apparent [61]. Differential expression of LFB1/HNF-lc~ and LFB3/HNF-1/3 has been observed in a set of rat hepatoma cell lines. These cells consist of two lines (H4II and C2rev7), which retain a fully differentiated phenotype, and two 'dedifferentiated' subclonal derivatives (H5 and C2) which have lost the capacity to express several liver-specific genes [62]. L F B 1 / H N F - l a is only expressed in the fully differentiated H4II and C2rev7, and not in the dedifferentiated H5 and C2 cell lines. It is also absent in cell hybrids between hepatoma and fibroblast cell lines, which have lost the ability to express some liver-specific genes [63]. On the other hand, LFB3/HNF-1/3 is expressed in all of them, irrespective of whether the target genes are active or not. LFB3/HNF-1/3 and L F B 1 / H N F - l a are also differentially regulated in F9 murine teratocarcinoma cells. F9 can be induced to differentiate into endoder-
mal-like cultures by retinoic acid (RA) treatment. After 24 h of RA treatment, LFB3/HNF-1/3 is strongly induced, while HNF1/LFB1 levels do not change until 5 days after RA administration [52,64]. The sequential expression of LFB3/HNF-1/3 and L F B 1 / H N F - l a suggests that they play a different role in hepatocyte (or epithelia) differentiation; perhaps the first is involved in the onset and the second in the maintenance of the expression of tissue-specific genes: However, no direct evidence is available yet to support this hypothesis.
II-C. The HNF-3 gene family Hepatocyte nuclear factor 3 (HNF-3) was described as a DNA binding activity interacting with the mouse transthyretin (TTR) gene promoter, the a 1-AT proximal enhancer region and the far upstream region of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene [4,65]. It was subsequently shown that this activity consists of three major DNA binding proteins, called HNF-3a, HNF-3/3 and HNF-3y [66]. The DNA binding domain and the activation domain of HNF-3a protein have been mapped respectively to the 157/267 and 272/326 amino acids regions. However, neither of these regions show any recognizable feature that allows their classification within one of the already known classes of DNA binding or transcriptional activation elements. All the three HNF-3 proteins behave as transcriptional activators in cotransfection experiments. The DNA binding domains of HNF-3a,/3 and -/are highly conserved in rat (93 out of 110 identical amino adds). Moreover, they are strikingly homologous to a similar region in the Drosophila homeotic gene fork head (88 of the 93 residues that are conserved among the three rat genes) [64]. Nevertheless, some of the differences between these DNA binding domains must be important: experiments comparing the affinity of the three HNF-3 proteins for two different oligos (S and W, corresponding respectively to a 'weak' and a 'strong' site for HNF-3 in the TTR promoter) resulted in HNF-3/3 having an inverted affinity compared to HNF-3a and HNF-3T [67]. The HNF-3 family genes are expressed in cells that derive from the lining of the primitive gut. Some of the embryonic Drosophila cells in which fork head is expressed also give rise to gut and salivary glands. It has been proposed that this gene family may contribute to differentiation of organs and structures derived from the endodermal tissues of the primitive intestine [68]. However, there are differences in the expression pattern of the three genes: while all of them are expressed in liver and intestine, only a and /3 are expressed in lungs and only y in testis [67].
123
II-D. HNF-4 / LFA1 and ARP-1 HNF-4 (hepatocyte nuclear factor 4) is a protein enriched in liver extracts that binds an essential region of the transthyretin and apolipoprotein CIII promoters [4]. HNF-4 is a member of the thyroid-steroid hormone receptor superfamily; the 50/133 amino acids region corresponds to the DNA binding domain, which belongs to the zinc finger group and is 40 to 65% homologous to the other members of the family. The 133/373 amino acids region contains the ligand binding domain, which is 20 to 35% homologous to the corresponding region of other receptor-like proteins. HNF-4 protein binds to DNA as a dimer, and it activates transcription in cotransfection assays. HNF-4 mRNA is present in kidney and intestine, as well as liver, but is absent in other tissues [4]. It has been suggested that HNF-4 could be identical to the previously described liver factor Al (LF-A1), a DNA-binding activity implicated in the transcriptional regulation of the al-antitrypsin, apolipoprotein A1 [46] and pyruvate kinase genes [69]. The apoAI regulatory protein-1 (ARP-1), whose gene has been recently cloned, binds to the apolipoprotein Al gene promoter at the same sequence which is also recognized by HNF-4/LF-A1 [70]. ARP-1 also belongs to the steroid hormone receptor superfamily. HNF-4 and ARP-1 genes are clearly different, yet they are much more alike than the other members of the family. ARP-1 binds to DNA as a dimer, and its dimerization domain is localized to the COOH-terminal region. However, in cotransfection experiments, ARP-I downregulates both the apoAI and the apoCIII gene expression. A 3-4-fold excess of ARP-1 prevents the activating effect of HNF-4, probably due to a competition for the same target sequence, since both ARP-1 and HNF-4 bind to the apoAI recognition site with the same affinity. Although both bind DNA as homodimers, no heterodimer formation occurs between the two proteins (Darnell, personal communication).
IlL More complex regulatory networks The notion of a positive regulation of liver-specific genes by a combination of transcriptional activators contrasts with the results of somatic cell genetic experiments, which were originally explained through negative regulation. In fibroblast x hepatoma cell hybrids several liver-specific functions are extinguished, and epistatic genes which negatively regulate sets of liverspecific functions have been described [71]. These two lines of evidence have been reconciled by the recent cloning of the TSE1 (tissue-specific extinguisher) gene, a locus responsible for the extinction of tyrosine
RETINOIC ACID
STEROIDS (?)
IL-6
HNF-4
HNF-3
/1
LFB1 " ~ LAP HNF-1~J IL6-DBP / t heter°dimersJ t formation LFB3 / C/EBP HNF- 1f ~ I
LIVER-SPECIFIC GENES Fig. 2. A scheme of the transcriptional regulatory network. Thin arrows = transcriptional activation; double-headed arrows = heterodimer formation; loops= self-regulation; stippled arrows= modulationof activityby extracellularsignals.
aminotransferase (TAT) and phosphoenolpyruvate carboxykinase (PEPCK) genes. TSE1 encodes for the R I a regulatory subunit of protein kinase A (PKA). R I a increased expression in hybrids causes the dephosphorylation of the CREB transcriptional activator, resulting in repression of the target genes [72]. However, it appears clear that the extinction of liver-specific genes in somatic cell hybrids is a complex phenomenon, involving multiple mechanisms [73]. Some evidence about cross-talk between the different factors are now emerging. Two groups have independently reported that recombinant HNF-4 can trans-activate the L F B 1 / H N F - l a promoter [74,75]. HNF-4, which belongs to the thyroid/steroid hormone receptor superfamily, is a valid candidate for mediating the response of hepatocytes to as yet unidentified external stimuli which could trigger or modulate differentiation. Moreover, transcriptional self-regulation has been postulated for C/EBP, HNF-3# and L F B 1 / H N F - l a (Refs. 23,76 and Piaggio, unpublished results). A hypothetical model in which transcription factors are selfregulated and organized into a hierarchy is shown schematically in Fig. 2.
124 IV. Hepatocyte differentiation and the role of tissue-architecture Although several details have still to be clarified, the general mechanisms which govern the transcriptional regulation of liver-specific gene expression have been understood. What still remains completely obscure is the cascade of events that initiates the differentiation of hepatocytes. As we have already mentioned, none of these transcriptional factors are capable of starting the hepatocyte differentiation program. However, although unlikely, the possibility that expressing all of them in a non-liver cell could trigger differentiation still formally exists. The liver originates from an endodermal diverticulum of the foregut, when epithelial 'cords' deriving from this endodermal rudiment invade the surrounding mesenchymal mass and eventually differentiate into hepatocytes [77]. These morphogenetic events are induced by the mesenchymal portion in a two-step process: an initial message from the mesoderm results in the 'determination' or commitment of the epithelial primordium to the liver fate, without any apparent morphological modification. Later, a second message from mesoderm triggers the 'induction' of liver organogenesis [78]. The recent identification in mesenchymal cells of a membrane protein, termed epimorphin, which seems to play a role in the induction of epithelial cell differentiation opens the possibility to identify and to study the molecules which are responsible for this exchange of messages between mesoderm and endoderm [79]. In situ hybridization studies have recently shown that albumin m R N A can be first detected in the liver bud of 9.5-day-old mouse embryos [80]. This stage precedes liver formation, and coincides with endoderm-mesoderm interactions, indicating that liverspecific terminal genes are activated as soon as the precursor cells become committed to the hepatocyte fate. However, the same authors describe a 15-20 fold increase in steady-state albumin m R N A levels following the definitive formation of the liver, suggesting that this increase could be due to the establishment of the right cell-cell contact. The authors propose a model in which at least one subset of adult transcriptional factors is switched on at the e n d o d e r m / m e s o d e r m induction stage, and cell assembly into an organ serves to amplify a transcriptional state established prior to organ formation. One implication of this model is that liver-specific gene expression begins when cells enter a rapidly dividing stage, unlike what happens in other systems such as muscular differentiation. The dependence of liver gene expression on cell-cell and cell-extracellular matrix (ECM) contacts has been described by several groups in the past [81-84]. Some recent reports demonstrate how interactions between
the hepatocyte and the extracellular matrix coordinately modulate cell architecture and liver-specific gene expression, by affecting the transcriptional efficiency of the albumin enhancer element [85] or the amount of some transcriptional factors like HNF-3a [80]. V. Conclusions and perspectives As more details about the transcriptional regulation of liver-specific genes are known, new interesting features are emerging. First, each cis-acting signal can usually be recognized by more than one class of transacting factors which co-exist in the same cell and which can bind in a mutually-exclusive manner. Moreover, in many cases the different members of these families can form heterodimeric complexes, adding new potential to the system. Second, the recognition sequence is often very degenerate, yet the affinity remains very high, as if several alternative ways of binding the same protein have evolved independently in different genes (or at different positions in the same gene). Whether these differences play any role in the fine regulation of these genes remains to be seen. Finally, the transcriptional effects of different cis-acting elements in the same promoter are usually additive and independent, although some cases of cooperative effects have been described. Despite the mass of infomation which has been acquired about the molecules that regulate the transcription of liver-specific genes, it is a matter of fact that our knowledge of hepatocyte differentiation and liver organogenesis is still very preliminary. This may reflect the complexity of the process, and the inadequacy of simple cell culture systems (mainly hepatoma cell lines) in approaching differentiation problems. To investigate the nature of the signals that mesoderm and pre-hepatic endoderm exchange, which trigger hepatocyte differentiation, may require going back to organ culture techniques and trying to identify and isolate a 'morphogen' and possibly its targets in pre-hepatocytes. An indication about how misleading experiments with cell cultures can be comes from the biological effect of the hepatic growth factor (HGF, a long known molecule), on kidney-derived epithelial ceils. The addition of H G F to epithelial ceils in culture (including hepatocytes) results in cell growth stimulation. Montesano et al. have recently shown that when MDCK canine renal cells are grown on a collagen matrix which allows cells to spread tridimensionally, of adding H G F results in the induction of tubulogenesis, and the mitogenic activity is only a consequence of the main morphogenic effect [86]. Similar phenomena in liver cell culture might have been overlooked simply because of the lack of such 'more physiological' assay conditions. This is a direction that clearly deserves to be pursued.
125
Acknowledgements This work was partially supported by grants related to the contract between CEINGE and the Agenzia per 1o Sviluppo del Mezzogiorno (Rome), No. PS35126/IND-AVV and by the EEC-BRIDGE grant No. BIOT-CT91-0260 (TSTS) to V.D.S.
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