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RESEARCH PAPER
growth inhibitory activity
1 Huffington Center on Aging and Department of Pathology, and 2 Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA
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Abstract |
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maintains quiescence of hepatocytes through two pathways: inhibition of cdks and repression of E2F. Nevertheless, liver tumors and cultured hepatoma cell lines proliferate in the presence of C/EBP. In this paper, we present evidence that the activation of the PI3K/Akt pathway in liver tumor cells blocks the growth inhibitory activity of C/EBP through the PP2A-mediated dephosphorylation of C/EBP on Ser 193, leading to a failure of C/EBP to interact with and inhibit cdks and E2F. Mutation of Ser 193 to Ala also abolishes the ability of C/EBP to cause growth arrest because of a lack of interactions with cdk2 and E2F–Rb complexes. These data provide a molecular basis for the development of liver tumors in which the activation of PI3K/Akt pathway neutralizes C/EBP growth inhibitory activity.
[Keywords: Hepatoma; C/EBP; cell proliferation; Akt; insulin]
Received December 31, 2003; revised version accepted March 15, 2004.
; Sage et al. 2003). In the liver and in myeloid cells, CCAAT/enhancer binding protein alpha (C/EBP) is a key inhibitor of cell proliferation. A number of studies have demonstrated that liver quiescence is mediated by growth inhibitory activity of C/EBP. C/EBP knockout livers have an increased rate of proliferation and express high levels of cell cycle proteins (Timchenko et al. 1999). Examination of cultured hepatocytes derived from C/EBP knockout mice also showed that the lack of C/EBP leads to increased proliferation (Soriano et al. 1998). A detailed analysis of the mechanisms by which C/EBP inhibits liver growth revealed that protein–protein interactions are the major pathway of C/EBP-mediated growth arrest in liver (Timchenko et al. 1999; Wang et al. 2001; Iakova et al. 2003). C/EBP inhibits the proliferation of myeloid cells through mechanisms that differ from those operating in liver and involve the transcriptional activity of C/EBP (Porse et al. 2001; Keeshan et al. 2003; Wang et al. 2003). Although mutations within the DNA binding region of C/EBP cause the development of leukemia, no liver tumors were associated with mutations within the DNA binding region of C/EBP (Pabst et al. 2001). The lack of proliferation abnormalities in the liver of patients with mutations/deletions within the DNA binding region of C/EBP is consistent with findings showing that, in liver, C/EBP inhibits proliferation through protein–protein interactions and that this inhibition does not require the transcriptional activity of C/EBP (Wang et al. 2001; Iakova et al. 2003).
C/EBP interacts with a number of cell cycle proteins and potentially might inhibit cell proliferation via different pathways, including inhibition of cdk2 and cdk4, upregulation of p21, and repression of E2F transcription (Timchenko et al. 1996; Johansen et al. 2001; Porse et al. 2001; Wang et al. 2001; Iakova et al. 2003). Given these multiple pathways, one can suggest that the growth inhibitory activity of C/EBP is tightly regulated in tissues during prenatal development and in adults. Despite the fact that significant progress in the identification of downstream targets of C/EBP has been made, very little is known about molecular mechanisms that regulate the ability of C/EBP to cause growth arrest. A growing number of observations suggest that posttranslational modifications of C/EBP or interactions of C/EBP with other proteins influence its growth inhibitory activity. Several publications suggested that biological activities of C/EBP might be controlled on the level of posttranslational modifications (Hemati et al. 1997; Ross et al. 1999). These studies showed that insulin signaling leads to a dephosphorylation C/EBP via the PI3K/Akt pathway, suggesting that C/EBP is a downstream target of this pathway. A recent paper with Akt1/Akt2 double knockout animals showed that the ablation of these kinases reduces expression and activities of C/EBP, which correlates with impaired adipogenesis in these animals (Peng et al. 2003).
Here we present evidence that the growth inhibitory activity of C/EBP is blocked in liver tumors and in cultured hepatoma cells by activation of the PI3K/Akt pathway. We found that the activation of PI3K/Akt pathway leads to the nuclear accumulation of PP2A, which dephosphorylates mouse C/EBP on Ser 193, leading to a failure of C/EBP to cause growth arrest. Phosphorylation of C/EBP on Ser 193 is required for the interactions of C/EBP with cdks and with E2F–Rb–Brm complexes and for the inhibition of these targets. Both mutation of Ser 193 and dephosphorylation of Ser 193 block the growth inhibitory activity of C/EBP. Our data provide a molecular basis for the development of liver tumors: neutralization of C/EBP growth inhibitory activity by the PI3K/Akt pathway.
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Results |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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growth arrest
C/EBP inhibits liver growth through multiple pathways, suggesting that liver should not proliferate in the presence of C/EBP. However, under several biological situations, liver proliferates and expresses high levels of C/EBP. As can be seen in Figure 1A, protein levels of C/EBP remain high in liver proliferating after partial hepatectomy (PH). Surprisingly, we found that liver tumors also proliferate in the presence of C/EBP. Western blotting of two tumor samples with antibodies to C/EBP showed no reduction of C/EBP in tumor sections of the livers (Fig. 1B). A parallel examination of cell cycle proteins in the tumor samples revealed that livers proliferate, because they expressed high levels of cell cycle proteins such as cyclin D1 and S-phase-specific protein PCNA (Fig. 1B). In addition to these observations, our group and others previously showed that hepatoma cell lines derived from liver tumors Hep3B2 and HepG2 express endogenous C/EBP and may be arrested by C/EBP only when C/EBP is expressed to very high levels from strong RSV or CMV promoters (Timchenko et al. 1996; Wang et al. 2001). However, biologically relevant levels of C/EBP do not cause inhibition of proliferation in these cells (see following). How can liver tumors and hepatoma cell lines proliferate in the presence of C/EBP? We hypothesized that, during malignant transformation, cells in liver tumors may have developed a mechanism to neutralize the inhibitory activity of C/EBP. To determine the molecular basis of the resistance of hepatoma cells to C/EBP growth arrest, we generated stable Hep3B2 clones expressing different levels of C/EBP. Figure 1C shows that the levels of IPTG-induced expression of C/EBP in these clones are significantly higher than endogenous levels of C/EBP. However, the examination of growth in these clones showed that these lines still proliferate in the presence of exogenous C/EBP (Fig. 1C). A comparison of these Hep3B2 clones and stable clones previously generated in human fibrosarcoma cells HT1080 and in SAOS2 cells showed that similar levels of C/EBP are sufficient to cause growth arrest in the latter cell lines (Timchenko et al. 1996; Wang et al. 2001). The failure of C/EBP to inhibit Hep3B2 cells under physiological conditions confirmed the hypothesis that hepatoma cells developed a mechanism to block growth inhibitory activity of C/EBP.
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in cultured hepatoma cells
If our hypothesis is correct, one would expect that other hepatoma cell lines also escape negative control of proliferation by the block of C/EBP growth inhibitory activity. Therefore, we used three hepatoma cell lines, Hep3B2, HepG2, and SK-Hep1, to examine this hypothesis. Figure 2A (upper) shows that all three hepatoma cell lines express C/EBP. Parallel examinations of growth inhibitory activity of C/EBP in 3T3-L1 cells indicated that insulin blocks growth inhibitory activity of C/EBP in these cells (data not shown; see Fig. 6A, below). Because insulin affects many biological processes through activation of the PI3K/Akt pathway (Lawlor and Alessi 2001; Shamji et al. 2003), we examined whether PI3K/Akt is active in hepatoma cells. Western blotting with antibodies to ph-Akt showed that the active form of Akt is abundant in hepatoma cells, whereas in 3T3-L1 cells ph-Akt is not detectable, but can be activated by insulin (Fig. 2A,B). The activation of Akt in hepatoma cells is mediated by PI3K, because the treatment of these cells with the PI3K inhibitor wortmannin (WM) leads to the reduction of the active Akt (Fig. 2B). We next examined whether the inhibition of PI3K/Akt pathway by specific inhibitors might restore growth inhibitory activity of C/EBP. Colony formation assay (Fig. 2C) and cell counting (Fig. 2D) showed that hepatoma cell lines are arrested by treatment with WM. Because WM is a specific inhibitor of PI3K and because WM restores growth inhibitory activity of C/EBP (see Fig. 6C, below), this result suggests that hepatoma cells block growth inhibitory activity of C/EBP via the PI3K/Akt pathway. To confirm the role of Akt in the PI3K-mediated blocking C/EBP, we applied an additional approach: inhibition of Akt by siRNA technique. It has been recently demonstrated that the inhibition of both Akt1 and Akt2 by siRNA is required for efficient blockage of downstream targets of Akts (Jiang et al. 2003). Therefore, we expressed siAkt1 and siAkt2 RNA oligomers in 3T3-L1 cells, and then transfected these cells with C/EBP. 3T3-L1 cells were chosen for these experiments because the PI3K/Akt pathway is not active in these cells, but might be activated by insulin (Ross et al. 1999; see Fig. 2A). Growth inhibitory activity of C/EBP was measured in untreated cells and in cells treated with insulin. As can be seen, the inhibition of Akts by si RNAs abolishes the ability of insulin to block C/EBP growth arrest (Fig. 2E).
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, we inhibited C/EBP Hep3B2 cells by siRNA, as shown in Figure 2F. siRNA-mediated inhibition of C/EBP abolishes WM-dependent growth arrest in Hep3B2 cells (Fig. 2G). Similar results were obtained for HepG2 and SK-Hep1 cells (data not shown). Thus, these data demonstrate that hepatoma cells block the growth inhibitory activity of C/EBP by activating the PI3K/Akt pathway.
A mutation of Ser 193 to Ala blocks the interaction of C/EBP with cdks and abolishes C/EBP-mediated growth arrest
Given that we have identified a potential pathway by which hepatoma cells neutralize C/EBP, we further determined a precise molecular mechanism by which insulin/PI3K/Akt blocks growth inhibitory activity of C/EBP. We previously mapped a short region of mouse/rat C/EBP that interacts with cdk2 and cdk4 and alone is sufficient to cause growth arrest (Wang et al. 2001). Figure 3A shows that amino acid sequences of growth inhibitory regions of human and mouse C/EBP have a very high level of homology including identical proline-rich sequences and a Ser residue surrounded by the prolines. Given this high level of homology and similar growth inhibitory activities of the mouse and human C/EBP, we focused our mutational studies on mouse C/EBP. We generated mouse C/EBP constructs with mutations that substitute prolines (mut182 and mut184) and a mutation that replaces Ser 193 with Ala (Fig. 3A). The mutant C/EBP molecules were cloned into the pcDNA vector for expression in mammalian cells and fused to GST to investigate protein–protein interactions. First, we determined whether the C/EBP mutants interact with C/EBP DNA consensuses by using a gelshift assay. Figure 3B shows that all constructs are expressed in mammalian cells at approximately equal levels and bind to C/EBP consensus. Then, we examined the interactions of these mutants with cdk2 and cdk4 in cultured cells. 3T3-L1 cells were transfected with C/EBP constructs, C/EBP was precipitated with specific antibodies, and cdk2 and cdk4 were examined in C/EBP IPs. Figure 3C shows a typical picture of these experiments. The mutations of prolines do not affect the interactions of C/EBP with cdk2 and cdk4; however, the substitution of Ser 193 with Ala abolishes the interactions of C/EBP with cdks. To verify these observations, we applied a GST pull-down approach by using GST–C/EBP mutants and nuclear extracts from 3T3-L1 adipocytes. Consistent with Co-IP results, the GST pull-down assay showed that the C/EBP-S193A mutant fails to interact with cdk2 and cdk4, whereas C/EBP mut182 and mut184 interact with these kinases. On the basis of these observations, Ser 193 is a key residue that is required for the interactions with cdks.
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mutants by using cotransfections with a 
-gal plasmid (ratio 10:1) in 3T3-L1 cells. Growth-arrested (single cells) and dividing cells (two, three, and more cells per colony) were counted at day 1 and day 3 after transfections. Results of these studies are shown in Figure 3D. At day 3 after transfection, 85%–90% of cells expressing wild-type C/EBP, C/EBP-182, and C/EBP-184 mutants are arrested and stay as single colonies. However, cells expressing C/EBP-S193A mutant divide and form colonies containing multiple cells. In the course of these studies, we observed that cells expressing the C/EBP-S193A mutant proliferate faster than cells transfected with an empty vector. As can be seen in Figure 3D, >50% of cells transfected with C/EBP-S193A form two and three cell colonies at day 1. At day 3, 
35% of the colonies contain multiple cells, whereas in control cells only 7%–10% of the colonies contain four-cell clusters. A typical picture of these multicell colonies is shown in Figure 3D (bottom). The increased number of dividing cells in C/EBP-S193A transfections suggested that this mutant promotes cell proliferation. To better investigate this possibility, we used constructs in which wild-type C/EBP and C/EBP mutants were cloned into pAdTrack-CMV plasmid that also expresses a green fluorescent protein (GFP) from a separate mRNA. Using this approach, one can monitor growth arrest without fixation of cells and test the levels of C/EBP within each experimental plate at the end of experiments. A summary of these studies is shown in Figure 3E (bar graphs). Consistent with previous experiments, this assay confirmed that C/EBP-S193A mutant promotes cell proliferation. A typical picture of cell images is shown in Figure 3E. Western blotting showed that wild-type C/EBP and the C/EBP-S193A are expressed at approximately equal levels. Western blotting for GFP was used as a control for loading. These data and further studies of BrdU uptake and FACS analysis of DNA content (Fig. 4) showed that the C/EBP-S193A mutant is not capable of inhibiting cell proliferation.
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-S193A mutant accelerates cell proliferation
Because the data for the colony growth assay (Fig. 3D,E) suggested that the C/EBP-S193A mutant enhances cell proliferation, we performed a detailed analysis of biological activities of this mutant. First, DNA synthesis (BrdU uptake) was measured in cells transfected with empty vector, wild-type C/EBP, and C/EBP-S193A mutant. C/EBP-S193A transfected plates contain 40% BrdU-positive cells, whereas cells transfected with an empty vector contain 15%–20% BrdU-positive cells (Fig. 4A). This result confirms data of colony growth arrest, showing that the mutant accelerates proliferation. To obtain more evidence for this conclusion, we examined the cell cycle distribution of transfected cells by using FACS analysis. A typical picture of these studies is shown in Figure 4B. In agreement with colony growth assays and BrdU uptake, the majority of cells transfected with wild-type C/EBP are arrested in G1. In contrast, the C/EBP-S193A mutant drives a significant portion of cells (38%–40%) into S phase. The summary of three experiments showed that the number of C/EBP-S193A transfected cells in the S phase is twofold higher than the number of cells in S phase on the plates transfected with the empty vector. Taken together, these data revealed that the C/EBP-S193A mutant accelerates cell proliferation by driving a higher percentage of cells into S phase. Thus, the mutation of Ser 193 to Ala not only abolishes growth inhibitory activity of C/EBP but also accelerates cell proliferation.
Given the acceleration of proliferation by C/EBP-S193A mutant, we examined mechanisms by which this mutant causes increased proliferation. Because the C/EBP-S193A mutant binds to DNA, it might accelerate proliferation by transcriptional activity independent of binding and inhibiting cdks. To test this possibility, we incorporated an additional point mutation, R290A, into C/EBP-S193A. This mutation abolishes the binding of C/EBP to DNA (Miller et al. 2003). As can be seen in Figure 4C, this additional mutation blocks the binding of the C/EBP-S193A to DNA. Colony growth assays indicated that the double-mutant (DM) C/EBP retains the ability to accelerate cell proliferation (Fig. 4D). Examination of the protein levels of C/EBP showed that wild-type and the mutant C/EBP are expressed at approximately equal levels (Fig. 4D, bottom image). To further examine the possible role of transcriptional activity of the C/EBP-S193A mutant in the promotion of proliferation, we examined the activation of C/EBP-dependent promoters by wild-type, C/EBP-S193A, and DM. These proteins were cotransfected with a reporter C3-luciferase construct (C3 promoter linked to luciferase; Timchenko et al. 1996) into 3T3-L1 cells. The activation of C3 promoter by C/EBP mutants was examined in untreated cells and in cells treated with insulin (in which C/EBP inhibitory activity is blocked, see below). C3 promoter contains a C/EBP binding site and has been shown to be activated by C/EBP (Timchenko et al. 1996). As can be seen in Figure 4E, wild-type C/EBP and C/EBP-S193A activate C3-luc promoter, whereas the DM construct fails to increase activity of the C3 promoter. We also observed that insulin slightly induces transcriptional activity of C/EBP; however, this induction is not dependent on Ser 193. Because the DM does not activate transcription, but still accelerates cell growth, these data clearly demonstrate that the transcriptional activity of the C/EBP-S193A mutant is not required for the acceleration of growth. Our investigations of biological activities of the C/EBP-S193A mutant suggest that this mutant accelerates proliferation through sequestering of Rb (data not shown).
Dephosphorylation of Ser 193 inhibits the interaction of C/EBP with cdk2 and Brm
The failure of C/EBP-S193A mutant to inhibit cell proliferation suggested that Ser 193 plays a key role in the regulation of C/EBP growth inhibitory activity. Therefore, we examined whether dephosphorylation of C/EBP might affect its interactions with cdks and Brm and its ability to cause growth arrest. Because C/EBP inhibits liver proliferation through interactions with cdk2 and cdk4 (Wang et al. 2001, 2002), we tested the phosphorylation status of C/EBP in young mouse livers, where C/EBP forms complexes with cdk2 (Wang et al. 2001, 2002). After immunoprecipitation from mouse livers, C/EBP was separated by 2D gel electrophoresis and examined by Western blotting. Figure 5A shows that C/EBP in nuclear extracts from mouse livers migrates as five isoforms, three of which are eliminated after treatment with CIP. The shift of these three isoforms after CIP treatment demonstrates that C/EBP is phosphorylated in livers. Then, we examined the effects of dephosphorylation on the ability of C/EBP to form C/EBP–cdk2 complexes. Liver nuclear extracts were treated with CIP, and C/EBP–cdk complexes were examined by size exclusion chromatography as described in our previous papers (Wang et al. 2001; Iakova et al. 2003). Results of these experiments are presented in Figure 5B. Immunoprecipitation of C/EBP from gel-filtration fractions of untreated nuclear extracts and Western blotting with Abs to cdk2 showed that C/EBP forms complexes with cdk2 (Fig. 5B). After the incubation of nuclear extracts with CIP, C/EBP is dephosphorylated (Fig. 5A) and is shifted to low-molecular-weight fractions of gel filtration. Co-IP studies revealed that the CIP treatment destroys C/EBP–cdk2 complexes because cdk2 is no longer detectable in C/EBP IPs (Fig. 5B, bottom).
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and blocks the ability of C/EBP to interact with cdks and Brm
Given the observations that the C/EBP-S193A mutant does not inhibit cell proliferation (Figs. 3, 4) and that the dephosphorylation destroys cdk2–C/EBP interactions, we suggested that hepatoma cells might block C/EBP growth inhibitory activity by dephosphorylation C/EBP at Ser 193. To test this hypothesis, we used 3T3-L1 cells in which the PI3K/Akt pathway is not detectable, but might be activated by insulin (Ross et al. 1999). We have initially verified that the activation of PI3K/Akt dephosphorylates C/EBP in these cells. Wild-type C/EBP and C/EBP-S193A were immunoprecipitated from insulin-treated and untreated cells and examined by 2D gel electrophoresis. Results of these studies are shown in Figure 5C. Two acidic isoforms of wild-type C/EBP (a and b) are not detectable in cells treated with insulin. Parallel examination of the C/EBP-S193A mutant revealed that these acidic isoforms are the result of phosphorylation of wild-type C/EBP on Ser 193, because they are not detectable within the C/EBP-S193A mutant. Thus, these data demonstrate that treatment of 3T3-L1 cells with insulin causes dephosphorylation of C/EBP on Ser 193.
We next examined whether the insulin-mediated dephosphorylation of Ser 193 affects the interactions of C/EBP with cdks and Brm. After transfections with wild-type C/EBP and insulin treatment, C/EBP was precipitated with specific antibodies. Western blotting of C/EBP IPs revealed that, in insulin-treated cells, dephosphorylated C/EBP does not interact with cdks and Brm (Fig. 5D). Because insulin affects several biochemical pathways, the lack of the interactions with cdks might also be due to alterations with cdks. To examine this possibility, we determined whether insulin affects interactions of cdk2 with p21. Co-IP studies showed that p21 interacts equally well with cdk2 in untreated cells and in cells treated with insulin (Fig. 5D, right). These results show that C/EBP does not interact with cdks and Brm because of dephosphorylation on Ser 193.
Activation of PI3K/Akt pathway blocks C/EBP growth inhibitory activity in 3T3-L1 cells
We next determined whether activation of the PI3K/Akt pathway abolishes C/EBP growth arrest in 3T3-L1 cells in which the PI3K/Akt pathway is not active (see Fig. 2). Western blotting with antibodies specific to Akt–Ser 473-ph showed a dramatic induction of the active, phosphorylated Akt by insulin, whereas there was no change in the total protein levels of Akt (Fig. 6A). Incorporation of a specific inhibitor of PI3K, WM, blocks activation of Akt (Fig. 6A). To examine whether Akt activation leads to a dephosphorylation of C/EBP and subsequent release of growth arrest, we transfected 3T3-L1 cells with wild-type C/EBP, treated with insulin (100 nM) or with insulin + WM. Figure 6A (right image) shows that insulin signaling leads to a dephosphorylation of C/EBP and that WM blocks insulin-mediated C/EBP dephosphorylation. Examination of the growth inhibitory activity of C/EBP showed that WM-mediated inhibition of Akt also restores the ability of C/EBP to cause growth arrest (Fig. 6A, bottom). Thus, these data demonstrate that insulin releases C/EBP growth arrest in 3T3-L1 cells through activation of PI3K/Akt, which results in dephosphorylation of C/EBP on Ser 193.
It has been shown that insulin signaling causes a dephosphorylation of C/EBP on several residues (Ross et al. 1999), suggesting that, in addition to Ser 193, dephosphorylation of other residues might contribute to the lack of growth arrest. To examine whether Ser 193 is the critical target of this pathway, we cloned a short growth inhibitory region (SGIR, previously shown to be sufficient for growth arrest) of C/EBP into pcDNA6, fusing it to his/myc tag (Fig. 6B), and performed functional analysis of growth inhibitory activity of this region in insulin-treated cells. Figure 6B shows that insulin does not affect the protein levels of the SGIR. Examination of phosphorylation of the SGIR by 2D gel electrophoresis revealed that insulin signaling dephosphorylates the SGIR, and WM causes the accumulation of ph-SGIR. We next examined the growth inhibitory activity of the SGIR in cells treated with insulin or with insulin + WM. Figure 6B (bottom) represents a summary of these studies. Expression of SGIR in 3T3-L1 cells inhibits proliferation, and insulin-mediated de-phosphorylation of the SGIR leads to a block of growth inhibitory activity of the SGIR. WM reverses the growth inhibitory activity of the SGIR of C/EBP. Because the SGIR contains only one residue (Ser 193) that can be phosphorylated, these data demonstrate that insulin/WM regulates the growth inhibitory activity of C/EBP through regulation of Ser 193 phosphorylation.
Inhibition of PI3K/Akt in stable Hep3B2 clones leads to the restoration of the growth inhibitory activity of C/EBP
We next examined whether the block of PI3K/Akt might restore growth inhibitory activity of C/EBP in stable clones, Hep3B2-A2, and Hep3B2-b10 (see Fig. 1). Examination of C/EBP in control and in WM-treated Hep3B2 cells by 2D gel-Western shows that, in control samples, C/EBP is not phosphorylated on Ser 193 and migrates as three isoforms (Fig. 6C). However, WM dramatically increases phosphorylation of C/EBP and leads to the accumulation of growth inhibitory isoforms of C/EBP in stable clones and in original Hep3B2 cells. Colony growth assay demonstrated that the accumulation of the growth inhibitory isoform of C/EBP in WM-treated Hep3B2-b10 and Hep3B2-A2 cells leads to growth arrest (Fig. 6C; data for Hep3B2-A2 clone are not shown). To examine whether WM causes growth arrest in stable clones through activation of C/EBP we inhibited C/EBP expression by siRNA as shown in Figure 2. Cells were transfected with pAdTrack-siRNA, and C/EBP was induced by IPTG. In control samples, cells were transfected with an empty pAdTrack-CMV vector that expresses only GFP. As can be seen in Figure 6D, WM inhibits Hep3B2-b10 cells through the activation of C/EBP, because WM fails to induce growth arrest in cells that do not express C/EBP.
PP2A is responsible for insulin/Akt-mediated block of C/EBP growth inhibitory activity in 3T3-L1 cells
We next determined a phosphatase that dephosphorylates C/EBP. Because insulin activates PP1 and PP2A (Hemati et al. 1997), we first examined whether these phosphatases might be involved in the insulin-dependent dephosphorylation of C/EBP. We initially examined whether a specific inhibitor of PP2A and PP1, okadaic acid, might abolish insulin-mediated effects. Figure 7A shows that insulin is not able to block C/EBP growth arrest in 3T3-L1 cells treated with okadaic acid. In control cells, the majority of PP2A is located in the cytoplasm (Fig. 7B). Western blotting shows that insulin causes accumulation of PP2A in the nuclei of 3T3-L1 cells, whereas PP1 is not affected in insulin-treated cells (Fig. 7B). Coimmunoprecipitation studies revealed that the accumulation of PP2A in nuclei leads to the interaction of PP2A with C/EBP and to dephosphorylation of C/EBP. The inhibition of PP2A by okadaic acid blocks insulin-mediated dephosphorylation of C/EBP (Fig. 7B,C). Thus, these studies suggested that, in 3T3-L1 cells, the insulin/Akt pathway inhibits C/EBP activities through PP2A.
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is blocked in regenerating livers by Akt–PP2A pathway
We next examined whether the PI3K/Akt/PP2A pathway might be involved in the neutralization of the growth inhibitory activity of C/EBP in liver when the liver proliferates after surgery. A removal of a portion of the liver by surgery (PH) leads to the initiation of liver proliferation (Iakova et al. 2003). Although expression of C/EBP is reduced after PH, protein levels of C/EBP remain relatively high during liver proliferation (Timchenko et al. 1999; Figs. 1A, 7D), suggesting that additional mechanisms are activated to neutralize the growth inhibitory activity of the remaining C/EBP. We examined whether the Akt-mediated dephosphorylation of C/EBP might be such a mechanism. Western blotting with ph-Akt-specific antibodies showed that Akt is activated in mouse livers at 4 and 8 h after PH (Fig. 7D). Consistent with data for cultured cells, the activation of Akt also leads to accumulation of PP2A in nuclei after PH. Precipitation of C/EBP from nuclear extracts and analysis of C/EBP IPs with antibodies to PP2A revealed that increasing levels of PP2A bind to C/EBP (Fig. 7E). This suggested that PP2A might dephosphorylate C/EBP on Ser 193 to allow liver proliferation. Therefore, we next examined C/EBP status after PH. Examination of C/EBP isoforms by 2D gel electrophoresis showed that growth inhibitory isoforms of C/EBP are abundant in quiescent livers; however, these isoforms are not observed at 4 h after PH (Fig. 7F). To examine whether PP2A is responsible for the dephosphorylation of C/EBP in liver after PH and in insulin-treated 3T3-L1 cells, we immunoprecipitated PP2A from corresponding nuclear extracts and incubated it with C/EBP overexpressed in untreated 3T3-L1 cells, where it is properly phosphorylated. Examination of C/EBP isoforms after this incubation shows that PP2A isolated from insulin-treated 3T3-L1 cells and PP2A isolated from livers 4 h after PH dephosphorylates C/EBP, leading to the disappearance of the growth inhibitory isoforms of C/EBP (Fig. 7G). Taken together, these data show that the accumulation of PP2A in nuclei causes dephosphorylation of C/EBP, leading to the neutralization of the growth inhibitory activity of C/EBP.
Human liver tumors block growth inhibitory activity of C/EBP through the activation of Akt–PP2A
Having established the pathway by which hepatoma cells and regenerating liver neutralize growth inhibitory activity of C/EBP, we asked whether the Akt/PP2A-mediated block of C/EBP is relevant for liver tumors in vivo. Therefore, we examined the PI3K/Akt/PP2A pathway and phosphorylation status of C/EBP in two human liver tumor samples that proliferate and express high levels of C/EBP (see Fig. 1B). We initially tested whether the tumor samples have mutations/deletions within the growth inhibitory region of C/EBP by amplifying a fragment of C/EBP from amino acid 110 to amino acid 200 (which covers the growth inhibitory region of C/EBP) from each tumor sample. No mutation/deletion was observed in the growth inhibitory region of C/EBP amplified from the tumor cells (data not shown). Therefore, we next examined the PI3K/Akt/PP2A pathway in control and tumor samples. Western blotting with antibodies to the active, phosphorylated form of Akt showed that Akt is activated in both liver tumor samples (Fig. 8A). As can be seen in Figure 1B, the activation of Akt in the tumors correlates with the induction of the S-phase-specific protein PCNA and cyclin D1, whereas protein levels of cdk2, Brm, and cdk4 are not altered (Fig. 1B; cdk4 data are not shown). Because activation of Akt in liver after PH and in cultured cells neutralizes C/EBP through dephosphorylation on Ser 193, we next analyzed C/EBP in liver tumors by 2D gel-Western assay. In control human samples, growth inhibitory isoforms of C/EBP (a and b) are abundant; however, liver tumors do not contain detectable amounts of these isoforms (Fig. 8A). Because PP2A dephosphorylates C/EBP in cultured cells and in regenerating livers, we determined protein levels of PP2A in nuclear extracts of human liver tumors and the interactions of PP2A with C/EBP. Figure 8B shows that, in control samples, the majority of PP2A is observed in the cytoplasm, whereas in liver tumor samples PP2A is accumulated in the nuclei and interacts with C/EBP (Fig. 8B, C/EBP IP). GST pull-down assay with nuclear extracts from the livers confirmed that wild-type C/EBP is interacting with PP2A. Interestingly, the mutant C/EBP-S193A also interacts with PP2A in GST pull-down assay, suggesting that the presence of a phosphate on Ser 193 is not required for efficient interactions. To further examine C/EBP–PP2A interactions in liver tumors, we fractionated nuclear extracts from liver tumor by size exclusion chromatography and examined them by Western blotting and Co-IP assays. These studies identified a complex C/EBP–PP2A that is located in the region of 300 kD (Fig. 8C). Thus, these data demonstrate that PP2A interacts with C/EBP in tumor cells. We next precipitated PP2A from liver tumors and incubated it with phosphorylated C/EBP. Examination of C/EBP by 2D gel electrophoresis after this incubation shows that the interaction of PP2A with C/EBP leads to dephosphorylation of C/EBP on Ser 193 (Fig. 7G).
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with cdks and Brm in control and tumor samples by coimmunoprecipitation assay. Figure 8D shows a typical result of these studies. In control samples, C/EBP forms complexes with cdks and with Brm; however, no interactions with these proteins were detected in protein extracts isolated from liver tumors. The failure of C/EBP to interact with cdks and Brm in liver tumor cells correlates with the lack of a growth inhibitory form of C/EBP, C/EBP-S193-ph, and with the proliferative status of the livers. Thus, these investigations suggest that liver tumors in humans proliferate because growth inhibitory activity of C/EBP is neutralized by dephosphorylation on Ser 193. |
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on Ser 193, leading to proliferation of cells. Akt was originally identified as an oncogene that can transform rodent cells (Bellacosa et al. 1991). Examination of mouse transgenic models overexpressing Akt in heart showed that Akt causes an increased proliferation and increased heart size (Shioi et al. 2002). Interestingly, Akt promotes proliferation of cells under conditions in which cells should normally be growth arrested (Lawlor and Alessi 2001; Shamji et al. 2003). Our findings suggest a pathway by which Akt displays its growth promotion function in liver: through inactivation of C/EBP. In agreement with this hypothesis, a number of previous investigations indicated that the inhibition of Akt in many hepatoma cell lines leads to growth arrest (Lin and Chou 1998; Zou et al. 2002; Shi et al. 2003). Examination of liver tumors in humans revealed that C/EBP growth inhibitory activity is also blocked in tumors by activation of Akt. Our hypothetical model for the mechanism by which tumor cells block C/EBP activity is shown in Figure 8E. We suggest that the activation of the PI3K/Akt pathway in liver tumors causes a translocation of PP2A into nuclei, where PP2A binds to and dephosphorylates C/EBP. As a result, liver loses a negative control of proliferation and may proliferate or develop tumors. Although our experimental data favor the hypothesis that the PI3K/Akt pathway activates PP2A to dephosphorylate C/EBP, it is also possible that inhibition of an additional kinase might contribute to the dephosphorylation of C/EBP.
C/EBP family proteins regulate transcription of genes in specific tissues. Recent studies demonstrated that two members of the C/EBP family, C/EBP and C/EBP, control cell proliferation and cell survival through protein–protein interactions (Buck et al. 2001; McKnight 2001; Wang et al. 2001; Timchenko 2003). These new activities of C/EBP proteins are mainly regulated at the level of posttranslational modifications. In this paper, we determined mechanisms that block the growth inhibitory activity of C/EBP in hepatoma cells. We have previously shown that a short region of C/EBP interacts with cdks and alone is sufficient to inhibit cell proliferation (Wang et al. 2001). Within this region, we found a residue (Ser 193 for mouse/rat protein and Ser 190 for human protein) that is crucial for the regulation of growth inhibitory activity of C/EBP and for the interactions with cdk2 and with Rb–E2F complexes. A mutation of Ser 193 to Ala makes C/EBP unable to cause growth arrest. Surprisingly, this mutant molecule displays an opposite activity, which is the acceleration of proliferation. Our unpublished observations suggest that the C/EBP-S193A accelerates cell proliferation via sequestering of Rb. Further work is required to understand whether this acceleration is relevant to in vivo conditions.
C/EBP interacts with several cell cycle proteins and might cause growth arrest through multiple pathways (Timchenko et al. 1996; Johansen et al. 2001; Porse et al. 2001; Wang et al. 2001; Iakova et al. 2003). In addition to growth arrest, C/EBP is also a critical regulator of expression of adipose- and liver-specific genes. How might all of these activities (pathways) be regulated in vivo? Our previous papers showed that one possible mechanism is a switch of protein partners that interact with C/EBP (Iakova et al. 2003; Timchenko 2003). In this paper, we show that substitution of Ser 193 with Ala abolishes C/EBP-mediated growth arrest. On the other hand, the S193A mutation does not affect the ability of C/EBP to interact with DNA and to activate transcription of target genes (Fig. 4). This finding suggests a pathway by which cells might distinctly regulate growth inhibitory and transcriptional activities of C/EBP. Insulin-mediated dephosphorylation of Ser 193 specifically blocks the growth inhibitory function of C/EBP, but does not affect the binding to DNA and presumably would not affect the activation of corresponding promoters.
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Antibodies to C/EBP (14AA and N19), cdk4 (C-22), cdk2 (M2), Brm, and Rb (C-15) were purchased from Santa Cruz Biotechnology. Antibodies to PP2A and PP1 are from Signal Transduction Laboratories. Expression vectors for wild-type mouse CEBP and mutations were generated by PCR-based amplification of the coding region of C/EBP from genomic clone pBS-Bam#6 (Wang et al. 1995). The full-length C/EBP cDNA was subcloned into the pcDNA3.1(+) expression vector. Based on the pcDNA3.1(+)-mCEBP construct, mutations (shown in Fig. 3A) were created using point mutational PCR. For growth arrest assays, wild-type CEBP and mutants were cloned into a pAdTrack-CMV vector that expresses GFP from a separate CMV promoter. Generation of pcDNA6/myc-His(B)-SGIR: An SGIR of C/EBP (see Fig. 6) was generated by fusing a synthetic DNA oligomer (amino acids 180–211) to the AUG codon on the 5' end, and cloned it into KpnI and Not I sites of pcDNA6/myc-His expression vector. Generation of mCEBP recombinant adenoviruses: pAdTrack–C/EBP constructs were cotransformed into BJ 5183 cells. Recombinant virus DNAs were selected from the kanamycin plates. The recombinant adenoviruses were packaged and produced in HEK 293 cells. Purified high-titer viral supplies were used in culture cell infections as described (He et al. 1998). Constructs with small interfering RNAs against C/EBP were generated as follows: Two siRNA primers (specific for mouse and human CEBP) were synthesized. Each primer contained 19 nt of CEBP sequences (828–846 in mouse and 825–843 in human). The annealed fragment was cloned into BglII/HindIII sites of pSUPER vector. For growth arrest assay, the siRNA fragment was subcloned into pAdTrack-CMV expression vector. All constructs were verified by sequencing.
Human liver tumor samples and liver regeneration
Human liver samples were obtained as part of the IRB approved protocol, where tumor and normal sections were collected from resected samples. Liver regeneration and examination of C/EBP were performed as described in our previous paper (Iakova et al. 2003).
Transient transfection assay
Analysis of C/EBP growth arrest was performed in Hep3B2, HepG2, SK-Hep1, HT1080, and 3T3-L1 cells by using C/EBP mutants described earlier. Transient transfection assay was performed with two approaches: cotransfections of pcDNA–C/EBP with -gal and single transfection of pAdTrack–C/EBP, which expresses both GFP and C/EBP from distinct mRNAs. C/EBP vectors were cotransfected into cells with CMV--gal at a ratio of 10:1. Cells were stained for -gal activity at days 1 and 3 following transfection. Cell growth was calculated by counting the number of blue-stained cells in each colony. In experiments with pAdTrack–C/EBP, the inhibition was calculated by counting the number of green cells in each colony.
BrdU up
