Molecular mechanisms of liver development and differentiation.

Recent advances in identifying molecular signals that dictate liver development and differentiation have come from analysis of several experimental systems including the developing embryo, cell and tissue culture, knockout mice and transplantation of hepatic precursor cells. Fibroblast growth factors and several families of transcription factors including hepatocyte nuclear factors 1, 3 and 4 and CCAAT/enhancer-binding protein have been shown to be important components of the differentiation process that culminates in the fully functional liver.

Upregulation of interleukin 6 and granulocyte colony-stimulating factor receptors by transcription factor CCAAT enhancer binding protein alpha (C/EBP alpha) is critical for granulopoiesis.

Cytokines stimulate granulopoiesis through signaling via receptors whose expression is controlled by lineage-specific transcription factors. Previously, we demonstrated that granulocyte colony-stimulating factor (G-CSF) receptor mRNA was undetectable and granulocyte maturation blocked in CCAAT enhancer binding protein alpha (C/EBPalpha)-deficient mice. This phenotype is distinct from that of G-CSF receptor-/- mice, suggesting that other genes are likely to be adversely affected by loss of C/EBPalpha. Here we demonstrate loss of interleukin 6 (IL-6) receptor and IL-6-responsive colony-forming units (CFU-IL6) in C/EBPalpha-/- mice. The observed failure of granulopoiesis could be rescued by the addition of soluble IL-6 receptor and IL-6 or by retroviral transduction of G-CSF receptors, demonstrating that loss of both of these receptors contributes to the absolute block in granulocyte maturation observed in C/EBPalpha-deficient hematopoietic cells. The results of these and other studies suggest that additional C/EBPalpha target genes, possibly other cytokine receptors, are also important for the block in granulocyte differentiation observed in vivo in C/EBPalpha-deficient mice.

Monocyte-conditioned medium, interleukin-1, and tumor necrosis factor stimulate the acute phase response in human hepatoma cells in vitro.

Human hepatoma cells mimic the acute phase response after treatment with monocyte-conditioned medium. Levels of secreted fibrinogen, alpha-1 acid glycoprotein, C-reactive protein, haptoglobin, and the third component of complement were elevated compared with control levels after 48 h of incubation with conditioned supernatant medium from an enriched fraction of normal peripheral monocytes. Albumin levels declined and alpha-1 antitrypsin remained unchanged. Levels of specific mRNA were measured by hybridization to slot blots and Northern blots and changed in correspondence with protein alterations. Interleukin-1 and tumor necrosis factor stimulated the third component of complement, but did not elevate any other member of the acute phase group and were therefore only partially active in this system. The identification of an in vitro model of the human acute phase response will permit analysis of the molecular basis for coordinate regulation of this group of facultative genes.

Human serum albumin phenotype activation in mouse hepatoma--human leukocyte cell hybrids.

Murine hepatoma cells that secreted mouse serum albumin were fused with human leukocytes that did not produce albumin. The resulting hybrids secreted both mouse and human serum albumin, as demonstrated by immuno-electrophoretic techniques. The activation of the human genome suggests that mapping genes governing specialized functions in somatic cell hybrids may be accomplished by using adifferentiated human cells as a parental line.

Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage.

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.

Impaired energy homeostasis in C/EBP alpha knockout mice.

Mice homozygous for the targeted deletion of the c/ebp alpha gene, which expresses the CCAAT/enhancer-binding protein alpha (C/EBP alpha), did not store hepatic glycogen and died from hypoglycemia within 8 hours after birth. In these mutant mice, the amounts of glycogen synthase messenger RNA were 50 to 70 percent of normal and the transcriptional induction of the genes for two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, was delayed. The hepatocytes and adipocytes of the mutant mice failed to accumulate lipid and the expression of the gene for uncoupling protein, the defining marker of brown adipose tissue, was reduced. This study demonstrates that C/EBP alpha is critical for the establishment and maintenance of energy homeostasis in neonates.

C/EBP alpha: a critical regulator of genes governing integrative metabolic processes.

The role of C/EBPalpha in the developmental expression of a subset of genes governing essential metabolic processes has recently been elucidated using a mutant mouse model that lacks this transcription factor. The mutation results in a failure of the liver and white and brown adipose tissue to develop normal metabolic functions in the immediate perinatal period, including hepatic glycogen synthesis and gluconeogenesis and the synthesis and deposition of triglyceride in adipose tissue. The metabolic alterations are very similar to those of human infants born prior to the third trimester and suggest that many of the medical complications of prematurity are a result of the lack of activation of C/EBPalpha in development.

C/EBPalpha is critical for the neonatal acute-phase response to inflammation.

Members of the C/EBP (CCAAT/enhancer binding protein) family of transcription factors play important roles in mediating the acute-phase response (APR), an inflammatory process resulting from infection and/or tissue damage. Among the C/EBP family of proteins, C/EBPbeta and -delta were thought to be the primary mediators of the APR. The function of C/EBPalpha in the APR has not been fully characterized to date. Here, we investigate the role of C/EBPalpha in the APR by using neonatal mice that lack C/EBPalpha expression. Northern blot analysis of acute-phase protein gene expression in neonatal mice treated with purified bacterial lipopolysaccharide or recombinant interleukin 1beta as an inflammation stimulus showed a strong APR in wild-type mice, but a response in C/EBPalpha null animals was completely lacking. The C/EBPalpha knockout and wild-type mice demonstrated elevations in C/EBPbeta and -delta mRNA expression and DNA binding as well as increased DNA binding of NF-kappaB, all of which are known to be important in the APR. Null mice, however, failed to activate STAT3 binding in response to lipopolysaccharide. Our results provide the first evidence that C/EBPalpha is absolutely required for the APR in neonatal mice, is involved in STAT3 regulation, and cannot be compensated for by other C/EBP family members.

C/EBPalpha regulates generation of C/EBPbeta isoforms through activation of specific proteolytic cleavage.

C/EBPalpha and C/EBPbeta are intronless genes that can produce several N-terminally truncated isoforms through the process of alternative translation initiation at downstream AUG codons. C/EBPbeta has been reported to produce four isoforms: full-length 38-kDa C/EBPbeta, 35-kDa LAP (liver-enriched transcriptional activator protein), 21-kDa LIP (liver-enriched transcriptional inhibitory protein), and a 14-kDa isoform. In this report, we investigated the mechanisms by which C/EBPbeta isoforms are generated in the liver and in cultured cells. Using an in vitro translation system, we found that LIP can be generated by two mechanisms: alternative translation and a novel mechanism-specific proteolytic cleavage of full-length C/EBPbeta. Studies of mice in which the C/EBPalpha gene had been deleted (C/EBPalpha-/-) showed that the regulation of C/EBPbeta proteolysis is dependent on C/EBPalpha. The induction of C/EBPalpha in cultured cells leads to induced cleavage of C/EBPbeta to generate the LIP isoform. We characterized the cleavage activity in mouse liver extracts and found that the proteolytic cleavage activity is specific to prenatal and newborn livers, is sensitive to chymostatin, and is completely abolished in C/EBPalpha-/- animals. The lack of cleavage activity in the livers of C/EBPalpha-/- mice correlates with the decreased levels of LIP in the livers of these animals. Analysis of LIP production during liver regeneration showed that, in this system, the transient induction of LIP is dependent on the third AUG codon and most likely involves translational control. We propose that there are two mechanisms by which C/EBPbeta isoforms might be generated in the liver and in cultured cells: one that is determined by translation and a second that involves C/EBPalpha-dependent, specific proteolytic cleavage of full-length C/EBPbeta. The latter mechanism implicates C/EBPalpha in the regulation of posttranslational generation of the dominant negative C/EBPbeta isoform, LIP.

C/EBPalpha regulates formation of S-phase-specific E2F-p107 complexes in livers of newborn mice.

We previously showed that the rate of hepatocyte proliferation in livers from newborn C/EBPalpha knockout mice was increased. An examination of cell cycle-related proteins showed that the cyclin-dependent kinase (CDK) inhibitor p21 level was reduced in the knockout animals compared to that in wild-type littermates. Here we show additional cell cycle-associated proteins that are affected by C/EBPalpha. We have observed that C/EBPalpha controls the composition of E2F complexes through interaction with the retinoblastoma (Rb)-like protein, p107, during prenatal liver development. S-phase-specific E2F complexes containing E2F, DP, cdk2, cyclin A, and p107 are observed in the developing liver. In wild-type animals these complexes disappear by day 18 of gestation and are no longer present in the newborn animals. In the C/EBPalpha mutant, the S-phase-specific complexes do not diminish and persist to birth. The elevation of levels of the S-phase-specific E2F-p107 complexes in C/EBPalpha knockout mice correlates with the increased expression of several E2F-dependent genes such as those that encode cyclin A, proliferating cell nuclear antigen, and p107. The C/EBPalpha-mediated regulation of E2F binding is specific, since the deletion of another C/EBP family member, C/EBPbeta, does not change the pattern of E2F binding during prenatal liver development. The addition of bacterially expressed, purified His-C/EBPalpha to the E2F binding reaction resulted in the disruption of E2F complexes containing p107 in nuclear extracts from C/EBPalpha knockout mouse livers. Ectopic expression of C/EBPalpha in cultured cells also leads to a reduction of E2F complexes containing Rb family proteins. Coimmunoprecipitation analyses revealed an interaction of C/EBPalpha with p107 but none with cdk2, E2F1, or cyclin A. A region of C/EBPalpha that has sequence similarity to E2F is sufficient for the disruption of the E2F-p107 complexes. Despite its role as a DNA binding protein, C/EBPalpha brings about a change in E2F complex composition through a protein-protein interaction. The disruption of E2F-p107 complexes correlates with C/EBPalpha-mediated growth arrest of hepatocytes in newborn animals.

Glucocorticoid and developmental regulation of amylase mRNAs in mouse liver cells.

We characterized alpha-amylase expression in the hepatoma cell line Hepa 1-6 and in normal mouse liver. Both Amy-1 and Amy-2 were expressed in Hepa 1-6 and were regulated by glucocorticoids. Transcription in the hepatoma cells was initiated at the same start sites as in mouse tissues. Glucocorticoid treatment increased the abundance of Amy-1 and Amy-2 transcripts by 10 to 20-fold. This increase was detected within 4 h and was maximal by 24 h. The pattern of amylase expression in this hepatoma cell line accurately reflects amylase expression in the liver in vivo. During liver development, we observed a large increase in the abundance of Amy-1 transcripts just before birth, at a time when circulating glucocorticoids are also elevated. Adult mouse liver expressed Amy-1 and Amy-2 at levels comparable to those of fully induced hepatoma cells. Liver is thus a likely source of both amylase isozymes in mouse serum. These studies demonstrate that Amy-2 expression is not limited to the pancreas but also occurs at a low level in liver cells.

Multiple dispersed loci produce small cytoplasmic Alu RNA.

Alu repeats are short interspersed elements (SINEs) of dimeric structure whose transposition sometimes leads to heritable disorders in humans. Human cells contain a poly(A)- small cytoplasmic transcript of -120 nucleotides (nt) homologous to the left Alu monomer. Although its monomeric size indicates that small cytoplasmic Alu (scAlu) RNA is not an intermediary of human Alu transpositions, a less abundant poly(A)-containing Alu transcript of dimeric size and specificity expected of a transposition intermediary is also detectable in HeLa cells (A. G. Matera, U. Hellmann, M. F. Hintz, and C. W. Schmid, Mol. Cell. Biol. 10:5424-5432, 1990). Although its function is unknown, the accumulation of Alu RNA and its ability to interact with a conserved protein suggest a role in cell biology (D.-Y. Chang and R. J. Maraia, J. Biol. Chem. 268:6423-28, 1993). The relationship between the -120- and -300-nt Alu transcripts had not been determined. However, a B1 SINE produces scB1 RNA by posttranscriptional processing, suggesting a similar pathway for scAlu. An Alu SINE which recently transposed into the neurofibromatosis 1 locus was expressed in microinjected frog oocytes. This neurofibromatosis 1 Alu produced a primary transcript followed by the appearance of the scAlu species. 3' processing of a synthetic -300-nt Alu RNA by HeLa nuclear extract in vitro also produced scAlu RNA. Primer extension of scAlu RNA indicates synthesis by RNA polymerase III. HeLa-derived scAlu cDNAs were cloned so as to preserve their 5'-terminal sequences and were found to correspond to polymerase III transcripts of the left monomeric components of three previously identified Alu SINE subfamilies. Rodent x human somatic cell hybrids express Alu RNAs whose size, heterogeneous length, and chromosomal distribution indicate their derivation from SINEs. The coexpression of dimeric and monomeric Alu RNA in several hybrids suggests a precursor-product relationship.

A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.

We have cloned the promoter for the human third component of complement (C3) gene and have identified sequences involved in its regulation during the acute-phase response. A construct linking 199 bp of the C3 promoter to the firefly luciferase gene was found to be very responsive to interleukin-1 (IL-1) and modestly responsive to interleukin-6 (IL-6) by transfection analysis in the human hepatoma line Hep3B2. Simultaneous treatment with the two cytokines showed a strong synergy between the actions of the two molecules. A 58-bp fragment (-127 to -70 bp) was shown by 5' and 3' deletional mutagenesis to contain cis-acting elements that mediated both the IL-1 response and the IL-1-plus-IL-6 synergistic response of this promoter. When coupled to a heterologous promoter, this fragment enabled the synergistic induction by IL-1 plus IL-6. Sequences homologous to the palindrome ACATTGCACAATCT, which mediates the induction of the IL-6 gene by IL-1 (S. Akira, H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto, EMBO J. 9:1897-1906, 1990), and the core sequence of the IL-6-responsive element of the rat alpha 2-macroglobulin gene (CTGGGA; M. Hattori, L. J. Abraham, W. Northemann, and G. H. Fey, Proc. Natl. Acad. Sci. USA 87:2364-2368, 1990) are contained within this fragment in immediate juxtaposition and partially overlapping. Site-directed mutagenesis within this homology region drastically reduced the inducibility of the C3 promoter by either cytokine. DNase I footprinting analysis defined a binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP), which included the IL-1-responsive element-like sequence. No differences were seen between the footprints generated by using extracts from unstimulated and IL-1-stimulated Hep3B2 cells. However, gel retardation analyses revealed two IL-1-specific bands. The data suggest that the induction by IL-1 is mediated by a factor belonging to the family of C/EBP-related proteins.

A human Alu RNA-binding protein whose expression is associated with accumulation of small cytoplasmic Alu RNA.

Human Alu sequences are short interspersed DNA elements which have been greatly amplified by retrotransposition. Although initially derived from the 7SL RNA component of signal recognition particle (SRP), the Alu sequence has evolved into a dominant transposon while retaining a specific secondary structure found in 7SL RNA. We previously characterized a set of Alu sequences which are expressed as small cytoplasmic RNAs and isolated a protein that binds to these transcripts. Here we report that biochemical purification of this protein revealed it as the human homolog of the SRP 14 polypeptide which binds the Alu-homologous region of 7SL RNA. The human cDNA predicts an alanine-rich C-terminal tail translated from a trinucleotide repeat not found in the rodent homolog, which accounts for why the human protein-RNA complex migrates more slowly than its rodent counterpart in RNA mobility shift assays. The human Alu RNA-binding protein (RBP) is expressed after transfection of this cDNA into mouse cells. Expression of human RBP in rodent x human somatic cell hybrids is associated with substantial increase in endogenous small cytoplasmic Alu and scB1 transcripts but not other small RNAs. These studies provide evidence that this RBP associates with Alu transcripts in vivo and affects their metabolism and suggests a role for Alu transcripts in translation in an SRP-like manner. Analysis of hybrid lines indicated that the Alu RBP gene maps to human chromosome 15q22, which was confirmed by Southern blotting. The possibility that the primate-specific structure of this protein may have contributed to Alu evolution is considered.

Transactivation of pancreas-specific gene sequences in somatic cell hybrids.

Enhancer/promoter elements from two pancreas-specific genes, those encoding amylase and elastase, were ligated to the bacterial GPT gene. The resulting construct can be used to select for expression of gene products which activate these pancreas-specific promoters in hybrid cells. The selectable GPT construct was stably transferred into several cell lines either directly or by cotransfection with pSV2Neo. GPT was expressed when transferred to pancreatic cell lines but not when transferred to GPT-fibroblast (L) cells or hepatoma cells. When the transformed L cells and hepatoma cells were fused with pancreatic cell lines, GPT was activated in the hybrid cells. Endogenous pancreas-specific genes from the L-cell and hepatoma parents were also activated in the hybrids. In addition, a pancreas-specific nuclear protein, PTF1, was produced in pancreatic and hybrid cells, correlating with GPT expression. The transformed L cells and hepatoma cells thus contained a nonexpressed construct which could be activated in trans by factors present in pancreatic cells. The hepatoma hybrid also continued to produce albumin, demonstrating the coexpression of liver and pancreas-specific genes in the hybrid-cell population. Cell lines carrying the amylase/elastase/GPT construct may be useful as a selection system for cloning of pancreatic transcription activators.

Simultaneous expression of salivary and pancreatic amylase genes in cultured mouse hepatoma cells.

The tissue-specific expression of two types of mouse amylase genes does not overlap in vivo; the Amy-1 locus is transcribed in the parotid gland and the liver, while expression of Amy-2 is limited to the pancreas. We identified a mouse hepatoma cell line, Hepa 1-6, in which both amylase genes can be simultaneously expressed. Amy-1 is constitutively active in these cells and is inducible by dexamethasone at the level of mRNA. We demonstrated that the liver-specific promoter of Amy-1 is utilized by the dexamethasone-treated hepatoma cells, and that glucocorticoid consensus sequences are present upstream of this promoter. Amy-2 is not detectable constitutively, but can be activated if the cells are cultured in serum-free medium containing dexamethasone. Expression of Amy-2 in a nonpancreatic cell type has not previously been observed. We speculate that induction of Amy-1 and activation of Amy-2 may involve different regulatory mechanisms. Hepa 1-6 cells provide an experimental system for molecular analysis of these events.

Autoregulation of the human C/EBP alpha gene by stimulation of upstream stimulatory factor binding.

The human C/EBP alpha gene promoter shares significant sequence homology with that of the mouse but has a different mechanism of autoregulation. Activation of the murine promoter by direct binding of C/EBP alpha to a site within 200 bp of the transcriptional start was shown to elevate activity by approximately threefold (R. J. Christy, K. H. Kaestner, D. E. Geiman, and M. D. Lane, Proc. Natl. Acad. Sci. USA 88:2593-2597, 1991; K. Legraverend, P. Antonson, P. Flodby, and K. G. Xanthapoulos, Nucleic Acids Res. 21:1735-1742, 1993). Unlike its murine counterpart, the human C/EBP alpha gene promoter does not contain a cis element that binds the C/EBP alpha protein. Neither C/EBP alpha nor C/EBP beta (NF-Il-6) binds the human C/EBP alpha promoter within 437 bp. However, cotransfection studies show that C/EBP alpha stimulates transcription of a reporter gene driven by 437 bp of the C/EBP alpha promoter. Our studies show that the human C/EBP alpha protein stimulates USF to bind to a USF consensus element within C/EBP alpha promoter and activates it by two- to threefold. We propose that the human gene employs the ubiquitously expressed DNA-binding protein factor USF to carry out autoregulation. Autoregulation of the human C/EBP alpha promoter was abolished by deletion of the USF binding site, CACGTG. Expression of human C/EBP beta following transfection did not stimulate USF binding. These studies suggest a mechanism whereby tissue-specific autoregulation can be achieved via a trans-acting factor that is expressed in all cell types. Thus, direct binding of the C/EBP alpha protein to the promoter of the C/EBP alpha gene is not required for autoregulation.

CCAAT/enhancer binding protein alpha regulates p21 protein and hepatocyte proliferation in newborn mice.

CCAAT/enhancer binding protein alpha (C/EBP alpha) is expressed at high levels in quiescent hepatocytes and in differentiated adipocytes. In cultured cells, C/EBP alpha inhibits cell proliferation in part via stabilization of the p21 protein. The role of C/EBP alpha in regulating hepatocyte proliferation in vivo is presented herein. In C/EBP alpha knockout newborn mice, p21 protein levels are reduced in the liver, and the fraction of hepatocytes synthesizing DNA is increased. Greater than 30% of the hepatocytes in C/EBP alpha knockout animals continue to proliferate at day 17 of postnatal life when cell division in wild-type littermates is low (3%). p21 protein levels are relatively high in wild-type neonates but undetectable in C/EBP alpha knockout mice. The reduction of p21 protein in the highly proliferating livers that lack C/EBP alpha suggests that p21 is responsible for C/EBP alpha-mediated control of liver proliferation in newborn mice. During rat liver regeneration, the amounts of both C/EBP alpha and p21 proteins are decreased before DNA synthesis (6 to 12 h) and then return to presurgery levels at 48 h. Although C/EBP alpha controls p21 protein levels, p21 mRNA is not influenced by C/EBP alpha in liver. Using coimmunoprecipitation and a mammalian two-hybrid assay system, we have shown the interaction of C/EBP alpha and p21 proteins. Study of p21 stability in liver nuclear extracts showed that C/EBP alpha blocks proteolytic degradation of p21. Our data demonstrate that C/EBP alpha regulates hepatocyte proliferation in newborn mice and that in liver, the level of p21 protein is under posttranscriptional control, consistent with the hypothesis that protein-protein interaction with C/EBP alpha determines p21 levels.

Glucocorticoid receptor, C/EBP, HNF3, and protein kinase A coordinately activate the glucocorticoid response unit of the carbamoylphosphate synthetase I gene.

A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPalpha-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPbeta knockout mice and in HNF3alpha and -gamma double-knockout mice. (The role of HNFbeta could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway.

E2F/p107 and E2F/p130 complexes are regulated by C/EBPalpha in 3T3-L1 adipocytes.

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.