• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Exp Cell Res. Author manuscript; available in PMC Dec 1, 2011.
Published in final edited form as:
PMCID: PMC3228643
NIHMSID: NIHMS45994

Regulation of apoptotic and growth inhibitory activities of C/EBPα in different cell lines

Abstract

C/EBPα is expressed in many tissues and inhibits cell growth. In this paper, we have examined mechanisms which regulate activities of C/EBPα in cell lines derived from different tissues. We found that C/EBPα possesses strong pro-apoptotic activity in NIH3T3 cells, while this activity is not detected in 3T3-L1, Hep3B2 and HEK293 cells. Micro array data show that C/EBPα activates many genes of apoptosis signaling in NIH 3T3 cells. One of these genes, ARL6IP5, is a direct target of C/EBPα and is a key mediator of the apoptosis. Using C/EBPα mutants which do not cause cell death; we have found that C/EBPα does not arrest proliferation of NIH3T3 cells. The lack of growth arrest in NIH3T3 cells correlates with the inhibition of p16INK4 and with low levels of cyclin D3. The limited growth inhibitory activity of C/EBPα is also observed in Hep3B2 cells which express low levels of cyclin D3. Elevation of cyclin D3 restores growth inhibitory activity of C/EBPα in NIH3T3 and in Hep3B2 cells. These data show that apoptotic and growth inhibitory activities of C/EBPα are differentially regulated in different cells and that cooperation of cyclin D3 and C/EBPα is required for the inhibition of proliferation.

INTRODUCTION

A member of C/EBP family, C/EBPα, is expressed in many tissues and is involved in the regulation of differentiation and in growth arrest [13]. C/EBPα regulates gene expression via direct interactions with the promoters, while C/EBPα-mediated growth arrest requires direct interactions of C/EBPα with cell cycle proteins. The growth inhibitory activity of C/EBPα in the liver, adipose tissue and in myeloid cells is displayed through different mechanisms [3]. Molecular mechanisms regulating growth inhibitory activity of C/EBPα in the liver present an example of a complicated network of several pathways operating up-stream and downstream of C/EBPα. Expression of C/EBPα is first detected in livers at days 13–14 of gestation [4, 5, 6]. At that time, C/EBPα plays a critical role in transcriptional activation of the genes required for assembly of the liver architecture. Since liver needs to grow before birth, the growth inhibitory activity of C/EBPα is significantly reduced in prenatal livers and is displayed in restricted cell types of the liver. Sato and co-workers have shown that, in prenatal livers, a transient sumoylation of C/EBPα on 159-lysine blocks the interaction of C/EBPα with Brg1/Brm and blocks its inhibitory effect on liver proliferation [6]. After the birth, C/EBPα is not sumoylated and inhibits liver proliferation [6, 7, 8]. In addition, recent publications show that the biological functions of C/EBPα during prenatal development of the liver depend on the cell type in which C/EBPα is expressed. At day 17 of gestation, the deletion of C/EBPα causes a 3.5-fold increase of proliferation in hepatocytes; while billiary epithelial cells do not show significant change in the rate of proliferation [9]. In young adult animals, C/EBPα is associated with cdk2 and p21 and inhibits liver proliferation by inhibiting cdk2 [2, 7, 10]. With age, C/EBPα changes the pathway of growth arrest and inhibits E2F-dependent promoters as the component of a high MW C/EBPα-Brm complex [11]. In the liver, cyclin D3 is a key positive regulator of C/EBPα inhibitory activity and the expression of cyclin D3 is increased with age leading to enhancement of growth inhibitory activity of C/EBPα [12]. Cyclin D3 belongs to a family of D-type cyclins which consists of three members: cyclins D1, D2 and D3. On the contrary to cyclins D1 and D2, cyclin D3 is expressed in a number of quiescent tissues and displays functions that support differentiation status of the tissues [1315]. Protein levels of cyclin D3 are increased during differentiation of myocytes and are involved in the regulation of the MyoD-mediated program of gene expression [16]. Cyclin D3 is also increased during differentiation of 3T3-L1 cells [17] and contributes to differentiation of adipocytes by activation of PPARγ [18]. High levels of Cyclin D3 are observed in the quiescent liver [19]. These studies showed that cyclin D3 supports growth arrest and differentiation of several tissues through activation of certain signal transduction pathways.

The initial goal of the work presented in this paper was to determine the mechanisms which regulate growth inhibitory activities of C/EBPα in different cell lines. In the course of these studies, we have found that C/EBPα also possesses pro-apoptotic activity which is very strong in NIH3T3 cells and which prevents investigations of C/EBPα growth arrest in these cells. Searching for the molecular mechanisms by which C/EBPα causes death of NIH3T3 cells, we have performed micro array studies and found that C/EBPα increases expression of several apoptotic proteins in NIH3T3 cells, while in 3T3-L1 cells these proteins are not activated. Among activated proteins, ARL6IP5 was increased up to 10–15 folds. We have found that the ARL6IP5 is a key regulator of apoptotic activities of C/EBPα in NIH 3T3 cells and that C/EBPα activates this protein through direct binding to its promoter. We have generated C/EBPα mutant which is not able to cause cell death and used this mutant for examination of the inhibitory activities of C/EBPα in NIH3T3 cells. Our further data demonstrated a critical role of the cooperation of cyclin D3 and C/EBPα in the inhibition of cell proliferation.

Materials and Methods

Growth arrest studies in cultured cells

In these studies, we have used NIH3T3, 3T3-L1, Hep3B2 and HEK293 cells. Examination of growth arrest in these cells was performed as described in our previous papers [11, 20]. Briefly, WT C/EBPα and C/EBPα mutants (shown in Fig 1A) and mouse cyclin D3 were cloned into pAdTrack plasmid under CMV promoter. This plasmid also expresses green fluorescent protein (GFP) which allows a visualization of transfected cells. The pAdTrack-cyclin D3 or pAdTrack-C/EBPα plasmids were transfected into cells and the levels of corresponding protein were examined in transfected cells by Western blotting and calculated as ratios to β-actin. Number of single green colonies (growth arrest) and number of colonies containing two, three and more cells per colony was counted. The efficiency of transfections did not differ significantly between Hep3B2, NIH3T3 and 3T3-L1 cell lines and was in the range of 15–25%. For HEK293 cells, we have higher efficiency which reached up to 40–60%. Since C/EBPα is expressed from CMV promoter, the levels of C/EBPα as ratios to β-actin. were approximately identical in HEK293, Hep3B2 and 3T3-L1 cells In NIH3T3 cells; however, C/EBPα/β-actin ratio was lower due to apoptotis-mediated selection of un-transfected cells. Since the WT C/EBPα induces apoptosis in cultured cells at days 4–5 after transfections (see Fig 1), the C/EBPα growth arrest was examined at days 1, 2 and 3 after transfections. Data in the manuscript present summaries with at least three independent experiments. 200–300 colonies were calculated in each experiment.

Figure 1
Expression of C/EBPα in NIH3T3 cells causes a massive death of the cells

Examination of cell death and apoptosis

Survival studies

pAdTrack -C/EBPα plasmids and an empty vector were transfected in cultured cells. Both empty vector and pAdTrack-C/EBPα plasmids express GFP protein; and the transfected cells are visualized by green fluorescence. We have also examined expression of GFP protein from the control vector and C/EBPα plasmid by Western blotting (Fig 1C). At day one, the majority of cells in all transfections were single. At later time points, proliferating cells formed colonies containing 2, 3 and more cells, while cells inhibited by C/EBPα remain as single cell colony. Therefore, we have calculated number of green colonies (transfected cells) at days 1, 2, 3, 4 and 5 after transfections independently on number of cells in the colonies. The percent of survived colonies was calculated as ratio to a number of colonies observed at day one after transfections. Apoptosis was examined by measuring DNA laddering.

Inhibition of ARL6IP5 and C/EBPα by siRNAs

To inhibit expression of ARL6IP5 or C/EBPα, siRNAs to mRNAs of these proteins were transfected into cells and mRNA and protein levels of ARL6IP5 and C/EBPα were examined by RT-PCR and by Western blotting. Nucleotide sequence of siRNA to ARL6IP5 is as follows. 5′-GGGACATTTCCAAATGGAACAACCG-3′. The sequence of siRNA to C/EBPα and conditions for the inhibition are described in our previous papers [1, 20].

C/EBPα constructs and antibodies

Generation of C/EBPα mutant constructs was described in our previous papers [1, 20]. Antibodies to cyclin D3, C/EBPα (14AA and N19), cdk4 (C-22), cdk2 (M2), Brm and Rb (C-15), p21 (H164), p16 and HDAC1 were purchased from Santa Cruz Biotechnology. Antibodies to ARL6IP5 (JWA) and to β-actin are from Abcam and Sigma. The generation and characterization of S193-ph-C/EBPα antibodies was described in our previous paper [12].

Examination of C/EBPα-mediated regulation of ARL6IP5 promoter, mRNA and protein

Gelshift assay was performed with DNA probes which cover C/EBPα consensuses within the ARL6IP5 promoter (see Fig 4A). The conditions for EMSA were described in our previous papers [1, 11]. To examine activation of the ARL6IP5 promoter by C/EBPα, DNA fragment containing 216 nucleotide of the promoter was linked to luciferase. The ARL6IP5-luc construct was co-transfected with WT C/EBPα or with C/EBPα-R290A mutant into cells. The luciferase activity was determined in 16 hours after transfections. To examine effects of C/EBPα on the expression of ARL6IP5 in NIH3T3 cells, C/EBPα was delivered into cells by adenovirus based vectors, mRNA and proteins were isolated and examined by semi quantitative RT-PCR and by Western blotting correspondingly. Note that examination of the ARL6IP5 promoter, levels of mRNA and protein in NIH3T3 cells were performed within 16 hours after transfections since cells die in two-three days after transfections.

Figure 4
C/EBPα increases expression of a pro-apoptotic protein ARL6IP5 through the activation of ARL6IP5 promoter

Protein isolation, Western blotting and Co-Immunoprecipitation

Nuclear extracts were isolated as described in previous papers [1, 7]. Proteins (50–100ug) were loaded on gradient (4–20% or 8–16%) poly acrylamide gel, transferred on the membrane and probed with antibodies to C/EBPα, cdk2, cyclin D3, E2F4 or Brm. To verify protein loading, each filter was re-probed with β-actin and then stained with coomassie. C/EBPα was immunoprecipitated from nuclear extracts with polyclonal antibodies (14AA, Santa Cruz), and the presence of Brm, E2F4, cdk2 in C/EBPα IPs was examined by Western blotting with monoclonal antibodies to mentioned proteins. Different conditions were used for detection of endogenous C/EBPα in Hep3B2 cells and in cells transfected with C/EBPα. For detection of endogenous C/EBPα, the filter was incubated with antibodies for C/EBPα for overnight and then for 2 hours with secondary antibodies. For examination of C/EBPα after transfection in cultured cells, the membranes were incubated for 30 min with primary antibodies and then for 1 hour with secondary antibodies. Under these conditions, the signals of endogenous C/EBPα in Hep3B2 cells are weak or not detectable (see Fig 1C).

Kinase assay

To determine activities of kinases associated with cyclin D3, we have precipitated cyclin D3 from nuclear extracts of 3T3-L1 and NIH3T3 cells and added to a kinase reaction with GST-Rb substrate. Mock agarose was used as a negative control. Baculovirus expressed purified cyclin D1-cdk4 complex was used as a positive control. The mixtures were separated by gradient PAGE and transferred on the membrane. The membrane was stained with coomassie blue to verify loading of GST-Rb and exposed with the X ray film.

Examination of gene expression by micro array

NIH 3T3 and 3T3-L1 cells were transfected with WT C/EBPα or with C/EBPα-R290A mutant by adenovirus delivery. The efficiency of transfections was 85–90%. Total RNA was isolated and used for micro array assay. Two hundred nanograms of total RNA were amplified and purified using Illumina TotalPrep RNA Amplification Kit (Ambion, Cat# IL1791) following kit instructions. Briefly, first strand cDNA was synthesized by incubating RNA with T7 oligo(dT) primer and reverse transcriptase mix at 42 °C for 2 hours. RNAse H and DNA polymerase master mix were immediately added into the reaction mix following reverse transcription and were incubated for 2 hours at 16 °C to synthesize second strand cDNA. RNA, primers, enzymes and salts that would inhibit in vitro transcription were removed through cDNA filter cartridges (part of the amplification kit). In vitro transcription was performed and biotinylated cRNA was synthesized by 14-hour amplification with dNTP mix containing biotin-dUTP and T7 RNA polymerase. Amplified cRNA was subsequently purified and concentration was measured by NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies, DE). An aliquot of 1.5 micrograms of amplified products were loaded onto Illumina Sentrix Beadchip Array Mouse-6 arrays, hybridized at 55°C in an Illumina Hybridization Oven (Illumina, Cat# 198361) for 17 hours, washed and incubated with straptavidin-Cy3 to detect biotin-labeled cRNA on the arrays. Arrays were dried and scanned with BeadArray Reader (Illumina, CA). Data were analyzed using BeadStudio software (Illumina, CA). Clustering and pathway analysis were performed with BeadStudio and Ingenuity Pathway Analysis (Ingenuity Systems, Inc.) softwares respectively.

Chromatin-immunoprecipitation (chip) assay

The chromatin immunoprecipitation assay was performed using the ChIP-IT kit (Active Motif). The chromatin solutions were isolated from 3T3-L1 and NIH3T3 cells, and DNA was sheared by enzymatic digestion according to the instruction manual. The size of DNA fragments produced averaged between 500–1000 bp in length. Antibodies against C/EBPα were added to each aliquot of pre-cleared chromatin and incubated for overnight. Protein G beads were added and incubated for 1.5h at 4 °C. After reversing the crosslinking, DNA was isolated and used for PCR reactions with primers to the ARL6IP5 promoter. The sequences of the primers are as follows. F: 5′-GCACGGGTCGCTAGTTGCTTTCTGTCATTTC-3′; R: 5′ –GAAGATCTTCTCAGCTTTGCGTTCTTCG-3′. PCR mixtures were amplified for 1 cycle of 95°C for 5 min, annealing temperature for primers (55°C) for 5 min, and 72°C for 2 min. Then PCR mixtures were amplified for 34 cycles of 95°C for 1 min, annealing temperature for 2 min, and 72°C for 1.5 min. PCR products were separated by 4% PAGE.

RESULTS

Ectopic expression of C/EBPα in NIH3T3 cells causes a massive death of the cells

Since biological activities of C/EBPα are regulated by cell-type specific mechanisms, we have performed studies of C/EBPα growth arrest in four cell lines, Hep3B2, HEK293, 3T3-L1 and NIH3T3. In the course of these studies, we have found that the response of NIH3T3 cells to C/EBPα expression differs dramatically from that observed in 3T3-L1, HEK293 and Hep3B2 cells. We have also realized that the investigation of NIH3T3 cells requires a number of C/EBPα mutants which are shown in Fig 1A. We have surprisingly observed that NIH3T3 cells die shortly after transfections with wild type C/EBPα, while C/EBPα does not cause death in Hep3B2, HEK293 and 3T3-L1 cells. To compare survived cells in these tissue culture cell lines, we have counted number of colonies which survived after several days after transfections. Figure 1B shows a typical result of these studies. As one can see, 50% of transfected NIH3T3 cells died at day 2 and less than 10% cells survived at day 3. Note that even at day 1 the number of green transfected with C/EBPα cells was 2–3-fold lower than that in cells transfected with an empty vector. WT and C/EBPα-S193A mutant possess identical capabilities to cause cell death in NIH3T3 cells. Under the same conditions, transfections of 3T3-L1, HEK293 and Hep3B2 cells with WT C/EBPα do not show reduction in the number of colonies within 3 days; a small reduction is observed at days 4–5. Examination of C/EBPα in protein extracts isolated from the experimental plates showed the lack of NIH3T3 cells expressing WT C/EBPα (Fig. 1C) confirming that C/EBPα causes cell death in NIH3T3 cells. Since NIH3T3 cells die shortly after transfections with WT C/EBPα, we have performed studies to determine regions of C/EBPα responsible for the death of NIH3T3 cells and to find conditions under which mechanisms of C/EBPα-mediated inhibition of NIH3T3 cells are possible to examine.

DNA binding activity of C/EBPα is required for cell death in NIH3T3 cells

We have first examined if C/EBPα initiates cell death in NIH3T3 cells using DNA laddering assay. Figure 2A shows that the apoptotic laddering is detectable in NIH3T3 cells at 8 hours after transfections with WT C/EBPα. Previous studies mapped several single amino acids of C/EBPα which are involved in the regulation of biological activities of C/EBPα. Mutation of one of these residues, S193 to Ala (which blocks growth inhibitory activity of C/EBPα in the liver and 3T3-L1 cells), does not block C/EBPα-mediated apoptosis (Fig 1). Therefore, we have tested if DNA binding activity of C/EBPα is required for the apoptosis. Since the mutation of R290 to Ala abolishes DNA binding activity of C/EBPα [1], we have transfected NIH3T3 cells with WT C/EBPα and with R290A mutant and counted survived cells. Parallel examinations of C/EBPα binding activity and protein levels by EMSA and Western blotting using nuclear extracts isolated at day 1 after transfections showed that the R290A mutant does not bind to DNA despite high levels of expression, while WT C/EBPα interacts with DNA (Fig 2B and C). We have found that C/EBPα–R290A mutant does not cause death in NIH3T3 cells. The survival curve for NIH3T3 cells transfected with the C/EBPα-R290A mutant is identical to that of cells transfected with an empty vector (Fig. 2D). Thus, these studies revealed that DNA binding activity of C/EBPα is required for the initiation of apoptosis in NIH3T3 cells.

Figure 2
DNA binding activity of C/EBPα is required for initiation of apoptosis in NIH3T3 cells

C/EBPα activates apoptotic pathways and diminishes expression of p16 family in NIH3T3 cells

Given quite different effects of C/EBPα on cell fate in NIH3T3 cells, we have next examined pathways by which C/EBPα causes death of NIH3T3 cells. Since DNA binding activity of C/EBPα is required for the initiation of apoptosis, we have focused our search on the transcriptional targets of C/EBPα which might be involved in the initiation of apoptosis. For this goal, we have compared C/EBPα-mediated activation of genes in NIH3T3 cells and in 3T3-L1 cells since both these cell lines are derived from disaggregated day 17 Swiss NIH mouse embryos. WT C/EBPα was delivered into these cells using adenovirus vectors; RNA was isolated and used for the global micro array analysis. Comparison of the up-regulated genes showed that C/EBPα increases mRNA levels of 71 and 122 genes in NIH3T3 and 3T3-L1 cells correspondingly. Only 14 identical genes (12–28%) are up-regulated by C/EBPα in these cells (Fig. 3A). Similar examination of the down-regulated genes also showed that only small portion (5–7%) of identical genes is down-regulated in the NIH3T3 and 3T3-L1 cells (Fig 4B). We have further compared differentially up and down-regulated genes in NIH3T3 and 3T3-L1 cells. Among several changes in alterations of signal transduction pathways, most significant differences were found for apoptotic signaling and for expression of cell cycle genes. The list of selected genes with highest differences is shown in the table (Fig. 3C). Four pro-apoptotic genes, ARL6IP, calpain 5, Ras and Fas, were increased in NIH-3T3 cells up to 10–26 fold compared to the levels of these proteins in 3T3-L1 cells (Fig 3C). We have also observed a down-regulation of certain apoptotic proteins; however, this down-regulation is within 1.7–2.3 folds. Analysis of the differences in the expression of cell cycle genes revealed dramatic differences in the expression of inhibitors of cyclin dependent kinases, p14INK4, p15INK4, p16INK4 and p19ARF. These inhibitors are required for cell cycle arrest. Despite C/EBPα is a strong inhibitor of cell proliferation, surprisingly micro array studies showed that C/EBPα inhibits expression of this family of proteins (Fig 3C). Interestingly, C/EBPα also up-regulates three members of HDAC family HDAC1, HDAC4 and HDAC5 in NIH3T3 cells, but not in 3T3-L1 cells. These findings suggested that WT C/EBPα might not inhibit proliferation of NIH3T3 cells due to these alterations in cell cycle proteins (see below). Taken together, global micro array search showed quite different patterns of the C/EBPα-mediated regulation of genes in NIH3T3 and 3T3-L1 cells and has identified a dramatic activation of pro-apoptotic proteins in NIH3T3 cells.

Figure 3
Differential activation of gene expression by C/EBPα in NIH3T3 and 3T3-L1 cells

C/EBPα increases expression of a pro-apoptotic protein ARL6IP5 via direct activation of the ARL6IP5 promoter

Among several pro-apoptotic proteins activated in NIH3T3 cells, the high level of activation by C/EBPα was observed for ARL6IP5 protein which is also known as DER11, GTRP3-18, HSPC127, JWA and hp22. In addition to our analyses, a paper by Mao et al has shown that all-trans retinoic acid inhibits proliferation and causes apoptosis through the activation of ARL6IP5 and that this activation is mediated by C/EBPα through direct binding to the promoter of ARL6IP5 [21]. Therefore, we suggested that the ARL6IP5 gene might be a key protein which mediates pro-apoptotic activity of C/EBPα in NIH3T3 cells and have investigated the role of ARL6IP5 in pro-apoptotic activity of C/EBPα. We have cloned a short region (214 bp) of the mouse ARL6IP5 promoter and examined the interactions of C/EBPα with the promoter. The nucleotide sequence of the ARL6IP5 promoter is shown in Fig. 4A. In addition to previously described two C/EBP binding sites (sites 1 and 2, ref #22) we have found a new C/EBPα consensus (site 3). Therefore, we have examined interactions of C/EBPα with these three sites. Gelshift assay with corresponding oligomers revealed that C/EBPα binds to all three sites (Fig 4B). The strongest interaction is observed for site 3, while the interaction with sites 1 and 2 is weaker but well detectable. Thus these studies showed that C/EBPα interacts with the ARL6IP5 promoter and that this interaction involves at least three regions of the promoter.

We have next examined the effects of C/EBPα on the ARL6IP5 promoter. The ARL6IP5 promoter was linked to the luciferase reporter construct and co-transfected with WT C/EBPα and C/EBPα-R290A into different cultured cells. Figure 4C shows a typical result of these experiments. In 3T3-L1 cells, the ARL6IP5 promoter has a weak activity and C/EBPα does not activate this promoter in these cells. Although the ARLP6IP5 promoter is active in both Hep3B2 and NIH3T3 cells, the effects of C/EBPα on the ARL6IP5 promoter are quite different. In Hep3B2 cells, C/EBPα inhibits the activity of the ARL6IP5 promoter; while in NIH3T3 cells, the expression of C/EBPα leads to a 7–10 fold activation of the promoter. These C/EBPα effects are mediated through a direct binding of C/EBPα to the promoter since the R290A mutant does not affect activity of the ARL6IP5 promoter in the transfected cells. The activation of the ARL6IP5 promoter by WT C/EBPα, but not by R290A mutant, correlates with the ability of WT C/EBPα to cause apoptosis and with a lack of the apoptosis in cells transfected with the R290A mutant. We next examined if the transfections of C/EBPα into NIH3T3 cells activate expression of endogenous ARL6IP5 gene. WT and R290A mutant C/EBPα proteins were transfected into the cells using adenovirus-based delivery, mRNAs and proteins were isolated in 8 hours after transfections and ARL6IP5 expression was determined by RT-PCR and Western blotting. Figure 4D shows that WT C/EBPα increases ARL6IP5 mRNA up to 15–20 folds; while R290A mutant has a minor (1.5 fold) induction of ARL6IP5 mRNA. Consistent with these data, examination of protein levels of ARL6IP5 showed a dramatic elevation of ARL6IP5 in NIH3T3 cells by WT C/EBPα and a weak induction by the R290A mutant (Fig 4E). A re-probe of the membrane with antibodies to C/EBPα and to β-actin showed that both WT and R290A mutant are expressed in the cells. To further determine if C/EBPα increases expression of ARL6IP5 in NIH3T3 cells via activation of the promoter, we have performed chromatin immunoprecipitation assay (chip) with NIH3T3 and 3T3-L1 cells. Chromatin solutions were prepared from cells transfected with empty vector, with WT C/EBPα and with C/EBPα-R290A mutant. C/EBPα was immunoprecipitated from these solutions and PCR was performed with primers which cover three C/EBPα sites. The results of these studies are shown in Fig 4F. As one can see, C/EBPα is not bound to the ARL6IP5 promoter in 3T3-L1 cells, but it is associated with the promoter in NIH3T3 cells. Consistent with failure of C/EBPα-R290A mutant to increase levels of ARLP6IP5 mRNA, association of this mutant with the promoter is not detectable. Taken together, these studies demonstrated that C/EBPα increases expression of pro-apoptotic protein ARL6IP5 via interaction with the promoter of ARL6IP5 gene and that this activation is specific and takes place only in NIH3T3 cells, but it is not detectable in 3T3-L1 cells.

ARL6IP5 is required for the C/EBPα-mediated apoptosis in NIH3T3 cells

We next asked if the C/EBPα-mediated activation of ARL6IP5 is involved in the apoptosis. To address this question, we have inhibited expression of ARL6IP5 by siRNA (25 nucleotides RNA oligomer, position +80 – +104) in cells transfected with WT C/EBPα and examined number of survival cells. Figure 5A shows that siRNA to ARL6IP5 reduces mRNA levels in un-transfected cells as well as in cells transfected with C/EBPα to the 15–20% of the original levels. Most important, the siRNA completely blocked the C/EBPα-mediated induction of ARL6IP5 mRNA. Consistent with the inhibition of ARL6IP5 mRNA, Western blotting showed that siRNA also inhibits expression of the protein (Fig 5B). This inhibition is specific and does not involve neutralization of C/EBPα activities since EMSA assay with the site 3 of the ARL6IP5 promoter showed a strong DNA binding activity of C/EBPα in cells transfected with siRNA to ARL6IP5 (Fig 5C). We next co-transfected siRNA to ARL6IP5 with C/EBPα and examined survived cells within 5 days after transfection. We have observed that siRNA-mediated inhibition of ARL6IP5 in NIH3T3 cells almost completely blocked pro-apoptotic activities of C/EBPα; while more than 90% cells transfected with WT C/EBPα alone died at day three (Fig 5D). These results showed that ARL6IP5 is a key mediator of C/EBPα apoptotic activities in NIH3T3 cells. Taken together, investigations of the mechanisms of C/EBPα-mediated apoptosis showed that, in NIH3T3 cells, C/EBPα activates the promoter of ARL6IP5, increases mRNA and protein levels of ARL6IP5 and that this activation causes the apoptosis.

Figure 5
Inhibition of ARL6IP5 abolishes apoptotic activities of C/EBPα

C/EBPα-R290A mutant fails to inhibit proliferation of NIH3T3 cells

Given established conditions for the surviving NIH3T3 cells after transfections with C/EBPα, we have next examined the growth inhibitory activities of the C/EBPα-R290A mutant in NIH3T3 cells. Parallel experiments were performed with 3T3-L1 and HEK293 cells. Figure 6A–B show results of these studies. Consistent with previous finding that DNA binding activity of C/EBPα is not required for growth arrest, C/EBPα-R290A mutant inhibits proliferation of HEK293 and 3T3-L1 cells. On the contrary, no inhibition of NIH3T3 cells was observed with this C/EBPα mutant. Although these initial studies suggested that DNA binding activity of C/EBPα (which is blocked by R290A mutation) might be required for growth arrest in NIH3T3 cells, further experiments revealed that transcriptional activity of C/EBPα is also not required to inhibit NIH3T3 cells since the double R290A-S193D mutant inhibits proliferation of these cells (see below). Thus, these studies suggested that NIH3T3 cells have developed a mechanism which blocks growth inhibitory activity of C/EBPα Since micro-array studies showed a significant increase in the expression of HDAC family and a down-regulation of the p16INK4 inhibitors by WT C/EBPα, we have examined if the R290A mutant is able to produce these alterations. Micro-array studies were performed with 3T3-L1 and NIH3T3 cells transfected with the R290A mutant. We have found that, although the R290A C/EBPα mutant does not activate HDAC4 and does not inhibit p14, it still up-regulates HDAC1 and HDAC5 and reduces expression of p15, p16 and p19 to the levels observed in cells transfected with WT C/EBPα (see Fig 3). To verify data of micro array analysis, we have determined protein levels of HDAC1, p16 and p21 in NIH3T3 cells transfected with C/EBPα. These studies revealed that HDAC1 and p21 are up-regulated; while p16 is inhibited by both WT C/EBPα and C/EBPα-R290A mutant (Fig 6D). Since the C/EBPα-R290 mutant does not bind to DNA, these data suggest that the differences in the C/EBPα-dependent expression of HDAC4 and p14 are mediated by transcriptional activity of C/EBPα, while alterations of other cell cycle proteins are mediated by protein-protein interactions of C/EBPα.

Figure 6
A. C/EBPα does not inhibit proliferation of NIH3T3 cells. Bar graphs show colony growth assay of NIH3T3, 3T3-L1 and HEK293 cells transfected with WT, S193D and R290A C/EBP proteins. B. Images show typical pictures of transfected cells at day 3 ...

Low levels of cyclin D3 in NIH3T3 cells are not sufficient to phosphoryate C/EBPα and to support cdk2 and Brm dependent pathways of growth arrest

We have previously shown that cyclin D3 supports growth inhibitory activity of C/EBPα in the liver [12]. Although down-regulation of p16 family proteins in NIH3T3 cells might partially explain the lack of growth arrest, we have examined if cyclin D3 pathways might be also changed in NIH3T3 compared with HEK293, Hep3B2 and 3T3-L1 cells. Figure 7A shows results of these studies. In 3T3-L1 and HEK293 cells, expression of cyclin D3 is relatively high; while levels of cyclin D3 are very low in Hep3B2 and in NIH3T3 cells. This observation suggests that the levels of cyclin D3 in NIH3T3 cells might be not sufficient to support the growth inhibitory activity of C/EBPα. To examine this hypothesis, we have compared the interaction of cyclin D3 with C/EBPα and the formation of growth inhibitory C/EBPα-cdk2 and C/EBPα-Brm complexes in 3T3-L1 and NIH3T3 cells. We have first examined kinase activity of cyclin D3-cdk4 complexes in these cells. Cyclin D3 and cdk4 were immunoprecipitated from nuclear extracts of 3T3-L1 and NIH3T3 cells and examined in an in vitro kinase assay with GST-Rb substrate. As can be seen in Figure 7B, both cyclin D3 and cdk4 IPs from 3T3-L1 cells display a significant kinase activity; while cyclin D3 and cdk4 IPs from NIH3T3 cells have much lower activities against Rb. Fig 7B shows results with 2 hours exposure of the film, under which the kinase activity of cdk4 and cyclin D3 IPs from NIH3T3 cells is not detectable. After overnight exposure; however, the activity of these enzymes in NIH 3T3 cells could be detected (data not shown). We next examined the interactions of C/EBPα with cdk2 and Brm in NIH3T3 and in 3T3-L1 cells. WT and mutant R290A C/EBPα proteins were transfected into these cells, nuclear extracts were isolated 16 hours after transfections and C/EBPα was immunoprecipitated. C/EBPα IPs were then examined by Western blotting with antibodies to which specifically recognize Ser193-phosphorylated isoform of C/EBPα, cyclin D3, cdk2 and Brm. Figure 7C shows that C/EBPα is associated with and phosphorylated by cyclin D3 in 3T3-L1 cells, while no interaction of C/EBPα with cyclin D3 and no phosphorylation of C/EBPα are detected in NIH3T3 cells. Consistent with the role of cyclin D3-mediated phosphorylation of C/EBPα in the formation of growth inhibitory complexes, cdk2 and Brm are observed in complexes with C/EBPα in 3T3-L1 cells, but these proteins are not detectable in C/EBPα IPs from NIH3T3 cells. Thus, these studies showed that the failure of C/EBPα to inhibit NIH3T3 cells correlates with the lack of phosphorylation at Ser193 and with the lack of formation of the C/EBPα-Brm and C/EBPα cdk2 complexes.

Figure 7
The growth arrest of NIH3T3 cells by C/EBPα requires high levels of cyclin D3

Ectopic expression of cyclin D3 and the mutation of Ser193 to aspartate restore growth inhibitory activity of C/EBPα in NIH3T3 cells

To examine if the phosphorylation of C/EBPα in NIH3T3 cells by cyclin D3 might restore inhibitory activity of C/EBPα, cyclin D3 was co-transfected with C/EBPα-R290A into NIH3T3 cells. Examination of green colonies at day 3 after transfections showed that cyclin D3 partially restores growth inhibitory activity of C/EBPα. However, a portion of NIH3T3 cells still proliferate (Fig 7D). Western blotting with protein extracts isolated from experimental plates showed that levels of cyclin D3 are only slightly increased in NIH3T3 cells transfected with pAdTrack cyclin D3 plasmid (see Fig 7D). Although the restoration of growth inhibitory activity of C/EBPα was not complete, these studies clearly demonstrated that cooperation of cyclin D3 and C/EBPα is required for the inhibition of proliferation of NIH3T3 cells. To confirm this conclusion, we have generated a double mutant of C/EBPα (R290A + S193D) which contains negatively charged residue in position of 193 and mimics cyclin D3/cdk4-mediated phosphorylation of C/EBPα. Examination of growth inhibitory activity of this mutant in NIH3T3 cells revealed quite different results which are shown in Fig 7E. Around 80% of NIH3T3 cells are arrested by the double C/EBPα-R290A-S193D mutant; while C/EBPα-R290A mutant does not inhibit proliferation of NIH3T3 cells. Thus, these studies demonstrated that NIH3T3 cells are resistant to C/EBPα growth arrest perhaps due to a low level of cyclin D3 in these cells.

Cyclin D3 cooperates with C/EBPα in the inhibition of HEK293 cells

Given a critical role of cyclin D3 in the growth inhibitory activity of C/EBPα in NIH3T3 cells, we have examined if cyclin D3 is required for growth inhibitory activity of C/EBPα in HEK293 cells. Since under certain settings cyclin D3 promotes proliferation, we have initially examined the effects of overexpression of cyclin D3 on the proliferation of HEK293 cells. For this goal, we have transfected HEK293 cells with cyclin D3-pAdTrack plasmid and examined the formation of single and multi-cells colonies. Western blotting with antibodies to cyclin D3 showed a 5–7 fold elevation of cyclin D3 in cells transfected with cyclin D3 relatively to cells transfected with empty vector (Fig 8A). Colony growth assay showed that cyclin D3 does not inhibit proliferation of C/EBPα-negative HEK293 cells (Fig 8A bottom). Moreover, under our conditions, overexpression of cyclin D3 in C/EBPα-negative HEK293 cells leads to the formation of a larger number (up to 55–60%) of multi-cell colonies, while the amounts of multi-cell colonies in cells transfected with empty vector are less than 8% (Fig. 8A). These observations suggest that cyclin D3 promotes proliferation of C/EBPα-negative HEK293 cells and are consistent with previous observations showing that ectopic expression of cyclin D3 accelerates progression of rat fibroblasts through G1 phase [22].

Figure 8
Cyclin D3 and C/EBPα cooperate in the inhibition of HEK293 and Hep3B2 cells

We have next examined if cyclin D3 is required for C/EBPα-mediated inhibition of HEK293 cells. For this goal, we have inhibited endogenous cyclin D3 in HEK293 cells by siRNA and examined C/EBPα growth arrest in cells with low levels of cyclin D3. C/EBPα (pAdTrack-C/EBPα plasmid) was co-transfected with siRNA to cyclin D3 into HEK293 cells and proliferation of these cells was examined by colony formation assay. A parallel examination of cyclin D3 levels after transfection showed that siRNA significantly reduces levels of cyclin D3, which represents 10–15% of levels in control cells (Fig 8B, Western). This inhibition of cyclin D3 leads to a significant reduction of C/EBPα growth arrest. Only 35–40% of HEK293 cells transfected with siRNA to cyclin D3 are arrested by C/EBPα. We suggest that the remaining activity of C/EBPα might be because of a non-complete inhibition of cyclin D3 or due to a partial redundancy of D-type cyclins. Taken together, these studies revealed that cyclin D3-C/EBPα pathway is required for the inhibition of cell proliferation.

Cyclin D3 inhibits proliferation of hepatoma cells by activating endogenous C/EBPα

The low levels of cyclin D3 in Hep3B2 cells suggested that this reduction might be important event in the loss of a negative control of proliferation. To examine if cyclin D3 might inhibit proliferation of Hep3B2 cells, pAdTrack-cyclin D3 was transfected into Hep3B2 cells and proliferation of these cells was investigated. Western blotting with antibodies to cyclin D3 revealed a 4.2–4.5 fold inductions of cyclin D3 levels relative to levels of endogenous cyclin D3 (Fig. 8C). Since Hep3B2 cells express endogenous C/EBPα, we have next established conditions for the inhibition of C/EBPα in these cells by siRNA. Western blotting (Fig. 8D) showed that, under our conditions, protein levels of C/EBPα are not detectable in cells transfected with siRNA. We next examined the effects of cyclin D3 on proliferation of Hep3B2 cells which express endogenous C/EBPα and cells in which C/EBPα is inhibited by siRNA as described in our previous papers [1, 11]. Single cells (growth arrested) and colonies of two-three cluster cells were counted at day 3 after transfections with pAdTrack-cyclin D3 plasmid. As one can see in Fig 8E, around 80% of Hep3B2 cells are arrested by cyclin D3. These studies demonstrated that cyclin D3 inhibits proliferation of Hep3B2 cells. Results of the colony growth arrest assay with Hep3B2 cells treated with siRNA to C/EBPα demonstrated that cyclin D3 is not able to arrest proliferation of Hep3B2 cells in which C/EBPα is inhibited by siRNA (Fig 8E). Interestingly, the inhibition of C/EBPα in cells transfected with cyclin D3 resulted in acceleration of cell proliferation. This result confirmed data obtained in C/EBPα-negative HEK293 cells and emphasized that biological activities of cyclin D3 are dependent on C/EBPα. Taken together, these studies show that the elevation of cyclin D3 levels in hepatoma 3B2 cells inhibits proliferation and that this inhibition requires endogenous C/EBPα. Previous studies revealed that cyclin D3-cdk4-mediated phosphorylation of C/EBPα at Ser193 is critical for C/EBPα inhibitory activity and that this phosphorylation activates two pathways of C/EBPα: the inhibition of cdk2 and the inhibition of E2F targets in the complexes with Brm [1, 11]. Therefore, we determined if the ectopic expression of cyclin D3 is sufficient to phosphorylate C/EBPα and to initiate formation of growth inhibitory C/EBPα-cdk2 and C/EBPα-Brm complexes. C/EBPα was immunoprecipitated from cells transfected with empty vector and with cyclin D3 and these IPs were analyzed by Western blotting with antibodies to phosphorylated isoform of C/EBPα (ph-Ser193), cdk2, and Brm. As one can see in Fig. 8F, elevation of cyclin D3 leads to the phosphorylation of C/EBPα at Ser193 and to formation of complexes with cdk2 and Brm. Taken together, these studies revealed that cyclin D3 inhibits proliferation of hepatoma cells via activating C/EBPα-cdk2 and C/EBPα Brm pathways.

DISCUSSION

Role of cyclin D3-C/EBPα pathway in inhibition of cell proliferation

The major function of D-type cyclins is the activation of cdk4/cdk6 and promotion of cell proliferation. On the contrary to cyclins D1 and D2 which strongly promote cell growth, cyclin D3 is expressed in certain growth arrested cells and is involved in growth arrest and differentiation [1217, 19]. Data in this paper show that the biological functions of cyclin D3 depend on the cellular environment. Experiments with C/EBPα-positive Hep3B2 and C/EBPα-negative HEK293 cells revealed that cyclin D3 inhibits proliferation of cells which express C/EBPα and promotes proliferation of cells that lack C/EBPα. The cyclin D3 mediated inhibition of Hep3B2 cells involves activation of C/EBPα and formation of growth inhibitory complexes of C/EBPα with cdk2 and Brm. Examination of NIH3T3 cells showed that, similar to Hep3B2 cells, NIH3T3 cells express very low levels of cyclin D3. As the result, C/EBPα is not able to form growth inhibitory complexes with cdk2 and Brm in NIH3T3 cells. These data are consistent with recently published observations showing that, in NIH3T3 cells, C/EBPα does not interact with cdk2 [23]. The low levels of cyclin D3 in NIH3T3 cells perhaps are the result of a rapid degradation or inhibition of translation of cyclin D3 since the expression of cyclin D3 from a transfected plasmid does not produce protein above the levels of endogenous cyclin D3.

Apoptotic activities of C/EBPα

Among four cell types examined in our experiments, NIH3T3 cells showed quite different responses to the expression of C/EBPα. In these cells, we have observed a strong apoptotic activity of C/EBPα Several previous studies described C/EBPα-mediated apoptosis in myeloid lineages at days 3–4 after transfections with C/EBPα [2426]. We have surprisingly found that the C/EBPα initiates apoptosis in NIH3T3 cells at 4–8 hours after transfections and that less than 5% of NIH3T3 cells survived at days 4–5 after transfections. This apoptotic activity was observed in experiments with low density cells; while NIH3T3 cells at high density were less sensitive to the apoptotic activity of C/EBPα. Since growth arrest studies require delivery of the C/EBPα into low density proliferating cells, this situation prevents investigations of growth inhibitory activity of WT C/EBPα in these cells. Therefore, we have investigated in details how C/EBPα causes apoptosis and generated mutants, in which apoptotic and growth inhibitory activities of C/EBPα were separated. Interestingly, several previous publications have examined C/EBPα inhibitory activities in NIH3T3 cells using approaches with a selection of C/EBPα expressing cells for resistance to antibiotics during 2–3 weeks [23, 27, 28]. Because C/EBPα causes a massive cell death in NIH3T3 cells, it is possible that these investigations have examined the survival cells which have developed a resistance to the apoptotic activities of C/EBPα. It is also possible that some of the C/EBPα mutants, previously determined in NIH3T3 cells as defective in growth arrest, might lack apoptotic activity, but possessed growth inhibitory activity which has not been displayed in these cells due to low levels of cyclin D3. In summary, given the C/EBPα apoptotic activity (data in this paper and in other publications [2426], we think that the growth inhibitory activities of C/EBPα in cultured cells should be examined within first 2–3 days after transfections and/or should be carefully separated from apoptotic activity of C/EBPα if a longer time is required.

The pro-apoptotic activity of C/EBPα in hematopoietic cells has been initially shown by a work from Tenen’s lab [25]; however, pathways by which C/EBPα causes apoptosis have not been elucidated. Searching for the mechanisms of the apoptotic activities of C/EBPα, we have performed careful analyses of gene expression in NIH 3T3 cells and in 3T3-L1 cells after transfections with WT C/EBPα. We have found quite different alterations in the patterns of gene expression after transfections with C/EBPα. The most dramatic differences in the C/EBPα-mediated alterations of gene expression were observed for the apoptotic signaling and for the cell cycle proteins. We have also identified a direct target of C/EBPα which is critical for C/EBPα-mediated apoptosis in NIH3T3 cells. This target is the ARL6IP5 protein which has been previously characterized as an inducer of apoptosis in cultured cells. The promoter region of the ARL6IP5 gene contains two binding sites for C/EBP proteins and potentially might be regulated by C/EBP proteins [21]. Given the ubiquitous expression of another member of C/EBP family, C/EBPα, one would assume that the ARL6IP5 promoter might be active in many cell types. However, the basal activity of the ARL6IP5 promoter is cell type-dependent with maximum activity in NIH3T3 cells and with a very low activity in 3T3-L1 cells. The C/EBPα-mediated up-regulation of the ARL6IP5 promoter is also cell type specific and takes place in NIH3T3 cells, but not in Hep3B2 cells.

The major result of this paper is that the cellular environment significantly changes biological activities of C/EBPα and that C/EBPα has different targets in NIH3T3 and in 3T3-L1 cells. In addition to known targets of C/EBPα within cell cycle, we have identified genes which are specifically affected only in NIH 3T3 cells. In these cells, C/EBPα increases mRNAs of several members of HDAC family and down-regulates mRNAs of p16INK4 and p19ARF. Interestingly, these new targets of C/EBPα are likely to be controlled through protein-protein interactions since the mutant C/EBPα-R290A (which does not bind to DNA, Fig 2) changes expression of majority of these proteins to the levels observed in transfections with WT C/EBPα. We think that the cell type specific down-regulation of p16INK4 and p19ARF proteins by C/EBPα and low levels of cyclin D3 contribute to the inability of C/EBPα to arrest proliferation of NIH 3T3 cells. The experiments with overexpression of cyclin D3 and with double mutant C/EBPα-S193D-R290A; however, suggest that the reduction of cyclin D3 is the major reason for the lack of growth inhibitory activity of C/EBPα in NIH 3T3 cells. Taken together, data in this paper show that biological functions of C/EBPα are controlled by a complex network of pathways which operate up-stream and downstream of C/EBPα and that these pathways differ in different cell types.

Acknowledgments

This work was supported by National Institutes of Health Grants CA10070, GM55188 and AG025477 (NAT).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

1. Wang GL, Iakova P, Wilde M, Awad SS, Timchenko NA. Liver tumors escape negative control of proliferation via PI3K/Akt-mediated block of C/EBPα growth inhibitory activity. Gen & Dev. 2004;18:912–925. [PMC free article] [PubMed]
2. Tan EH, Hooi SC, Laban M, Wong E, Ponniah AW, Wang N. CCAAT/Enhancer Binding Protein α Knock-in Mice Exhibit Early Liver Glycogene Storage and Reduced Susceptibility to Hepatocellular Carcinoma. Cancer Res. 2005;65:10330–10337. [PubMed]
3. Johnson PE. Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors. J Cell Science. 2005;118:2545–2455. [PubMed]
4. Birkenmeier EB, Gwynn B, Howard S, Jerry J, Gordon JI, Landschulz WH, McKnight SL. Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein. Genes & Dev. 1989;3:1146–1156. [PubMed]
5. Timchenko NA, Wilde M, Darlington GJ. C/EBPα regulates formation of S-phase specific E2F/p107 complexes in livers of newborn mice. Mol Cell Biol. 1999;19:2936–2945. [PMC free article] [PubMed]
6. Sato Y, Miyake K, Kaneoka H, Iijima S. Sumoylation of CCAAT/Enhancer Binding Protein alpha and its functional Role in Hepatocyte Differentiation. J Biol Chem. 2006;281:21629–21639. [PubMed]
7. Timchenko NA, Harris TE, Wilde M, Bilyeu TA, Burgess-Beusse BL, Finegold MJ, Darlington GJ. CCAAT/enhancer binding protein α regulates p21 protein and hepatocyte proliferation in newborn mice. Mol Cell Biol. 1997;17:7353–7361. [PMC free article] [PubMed]
8. Flodby P, Barlow C, Kylefjord H, Ahrlund-Richter L, Xanthopoulos KG. Increased Hepatic Cell Proliferation and Lung Abnormalities in Mice Deficient in CCAAT/Enhancer Binding Protein α J Biol Chem. 1996;271:42753–42760. [PubMed]
9. Yamasaki H, Sada A, Iwata T, Niwa T, Tomizawa M, Xanthopoulos KG, Koike T, Shiojiri N. Suppression of C/EBPα expression in periportal hepatoblasts may stimulate biliary cell differentiation through increased Hnf6 and Hnf1b expression. Development. 2006;133:4233–4243. [PubMed]
10. Wang H, Iakova P, Wilde M, Welm A, Goode T, Roesler WJ, Timchenko NA. C/EBPα arrests cell proliferation through direct inhibition of cdk2 and cdk4. Molecular Cell. 2001;8:817–828. [PubMed]
11. Iakova P, Awad SS, Timchenko NA. Aging reduces proliferative capacities of liver by switching pathways of C/EBPα growth arrest. Cell. 2003;113:495–506. [PubMed]
12. Wang GL, Shi X, Salisbury E, Sun Y, Albrecht JH, Smith RG, Timchenko NA. Cyclin D3 maintains growth inhibitory activity of C/EBPα by stabilizing C/EBPα-cdk2 and C/EBPα-Brm complexes. Mol Cell Biol. 2006;26:2570–2582. [PMC free article] [PubMed]
13. Bartkova J, Lukas J, Strauss M, Bartek J. Cyclin D3: requirement for G1/S transition and high abundance in quiescent tissues suggest a dual role in proliferation and differentiation. Oncogene. 1998;17:1027–1037. [PubMed]
14. Cenciarelli C, De Santa F, Puri PL, Mattei E, Ricci L, Bucci F, Felsani A, Caruso M. Critical role played by cyclin D3 in the MyoD-mediated arrest of cell cycle during myoblasts differentiation. Mol Cell Biol. 1999;19:5203–5217. [PMC free article] [PubMed]
15. Doglioni C, Chiarelli C, Macri E, Dei Tos A-P, Meggiolaro E, Dalla Palma P, Barbaresch M. Cyclin D3 expression in normal, reactive and neoplastic tissues. J Pathol. 1998;185:159–166. [PubMed]
16. de La Serna IL, Roy K, Carlson KA, Imbalzano AN. MyoD Can Induce Cell Cycle Arrest but Not Muscle Differentiation in the Presence of Dominant Negative SWI/SNF Chromatin Remodeling Enzymes. J Biol Chem. 2001;276:41486–41491. [PubMed]
17. Reichert M, Eick D. Analysis of cell cycle arrest in adipocyte differentiation. Oncogene. 1999;18:459–466. [PubMed]
18. Sarruf DA, Iankova I, Abella A, Assou S, Miard S, Fajas L. Cyclin D3 promotes adipogenesis through activation of peroxisome proliferator-activated receptor γ Mol Cell Biol. 2005;25:9985–9995. [PMC free article] [PubMed]
19. Rickheim DG, Nelsen CJ, Fassett JT, Timchenko NA, Hansen LK, Albrecht JH. Differential regulation of cyclins D1 and D3 in hepatocyte proliferation. Hepatology. 2002;36:30–38. [PubMed]
20. Wang GL, Timchenko NA. Dephosphorylated C/EBPα accelerates cell proliferation through sequestering retinoblastoma protein. Mol Cell Biol. 2005;25:1325–1338. [PMC free article] [PubMed]
21. Mao WG, Liu ZL, Li AP, Zhou JW. JWA is required for the antiproliferative and pro-apoptotic effects of all-trans retinoic acid in Hela cells. Clin Exp Pharm Physiol. 2006;33:816–824. [PubMed]
22. Herzinger T, Reed SI. Cyclin D3 Is Rate-limiting for the G1/S Phase Transition in Fibroblasts. J Biol Chem. 1998;273:14958–14961. [PubMed]
23. Porse BT, Pederson TA, Haseman MS, Schuster MB, Kirsteter P, Luedde T, Damgaard I, Kurtz E, Schjerling XK, Nerlov C. The proline-histidine rich cdk2/cdk4 interaction region of C/EBPα is dispensable for C/EBPα-mediated growth regulation in vivo. Moll Cell Biol. 2006;26:1028–1037. [PMC free article] [PubMed]
24. Keeshan K, Santilli G, Corradini F, Perrotui D, Calabretta B. Transcriptional activation function of C/EBPα is required for induction of granulocytic differentiation. Blood. 2003;102:1267–1275. [PubMed]
25. D’Alo F, Johansen LM, Nelson EA, Radomska HS, Evans EK, Zhang P, Nerlov C, Tenen DG. The amino terminal and E2F interaction domains are critical for C/EBPα-mediated induction of granulopoietic development of hematopoietic cells. Blood. 2003;102:3163–3171. [PubMed]
26. Wang QF, Cleaves R, Kummalue T, Nerlov C, Friedman AD. Cell cycle inhibition by the outer surface of the C/EBPα basic region is required but not sufficient for granulopoiesis. Oncogene. 2003;22:2548–2557. [PubMed]
27. Porse BT, Pederson TA, Xu X, Lindbergh B, Wewer UM, Fris-Hansen L, Nerlov C. E2F repression by C/EBPα is required for adipogenesis and granulopoiesis in vivo. Cell. 2001;107:247–258. [PubMed]
28. Muller K, Calkhoven CF, Sha X, Leutz A. The CCAAT/Enhancer binding protein α (C/EBPα) requires a SWI/SNF complex for proliferation arrest. J Biol Chem. 2004;297:7353–7358. [PubMed]
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...