Posttranscriptional and Posttranslational Regulation of C/EBPdelta in G0 Growth-arrested Mammary Epithelial Cells*

Lawrence R. DearthDagger and James DeWille§||

From the Dagger  Molecular, Cellular, and Developmental Biology Graduate Program, § Department of Veterinary Biosciences,  Comprehensive Cancer Center, the Ohio State University, Columbus, Ohio, 43210-1093

Received for publication, August 5, 2002, and in revised form, January 2, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous work from our laboratory demonstrated that CCAAT/enhancer-binding protein delta  (C/EBPdelta ) functions in the initiation and maintenance of G0 growth arrest in mouse mammary epithelial cells (MECs). In this report, we investigated the posttranscriptional and posttranslational regulation of C/EBPdelta in G0 growth-arrested mouse MECs. The results of transcriptional inhibitor studies demonstrated that the C/EBPdelta mRNA exhibits a relatively short half-life in G0 growth-arrested mouse MECs (t1/2 ~35 min). In contrast, C/EBPdelta mRNA has a longer half-life in G0 growth-arrested mouse fibroblast cells (t1/2 >100 min). Oligo/RNase H cleavage analysis and rapid amplification of cDNA ends-poly(A) test both confirmed the short C/EBPdelta mRNA half-life observed in MECs and demonstrated that the C/EBPdelta mRNA poly(A) tail is relatively short (~100 nucleotides). In addition, the poly(A) tail length was not shortened during C/EBPdelta mRNA degradation, which suggested a deadenylation-independent pathway. The C/EBPdelta protein also exhibited a relatively short half-life in G0 growth-arrested mouse MECs (t1/2 ~120 min). The C/EBPdelta protein was degraded in a ubiquitin-dependent manner, primarily in the nucleus, during G0 growth arrest. In conclusion, these studies indicated that the C/EBPdelta mRNA and protein content are under tight regulation in G0 growth-arrested mouse MECs, despite the general concept that G0 growth arrest is associated with a decrease in cellular activity.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

It is well documented that global gene transcription and translation dramatically decrease as cells exit the cell cycle and enter the quiescent G0 growth arrest state (1, 2). However, a small subset of genes become activated, and these gene products function in the initiation and maintenance of G0 growth arrest (3, 4). Currently, little is known about the regulation and function of G0 growth arrest-specific genes in cell biology. Recently, an increase in expression and activity of the retinoblastoma (Rb) family member p130 was observed in the initiation and maintenance of G0 growth arrest (5). Formation of the p130-E2F complex sequesters members of the E2F family to repress the expression of genes necessary for cellular proliferation. Alterations in the structure and function of G0 growth arrest genes are linked to some types of cancers. For example, germ line mutations found within the von Hippel-Lindau tumor suppressor gene have been linked to hemangioblastomas of the retina and central nervous system and renal carcinogenesis (6). The von Hippel-Lindau protein functions in cell cycle control in a variety of ways, including up-regulating the cyclin-dependent kinase inhibitor p27 (7).

Previous reports from our laboratory (8-13) demonstrate that CCAAT/enhancer-binding protein delta  (C/EBPdelta )1 functions in the initiation and maintenance of G0 growth arrest in mouse mammary epithelial cells (MECs). C/EBPdelta mRNA, protein, and DNA binding activity increase during mouse MEC G0 growth arrest (11-13). STAT3 activation/phosphorylation is necessary for C/EBPdelta transcription in G0 growth-arrested and cytokine-treated mouse MECs (10). In addition, the C/EBPdelta promoter exhibits autoregulation during G0 growth arrest (12).

C/EBPs are a widely expressed, highly conserved family of leucine zipper (bZIP)-type transcription factors (14, 15). Most C/EBPs are encoded by intronless genes and exhibit a high degree of homology in the basic and bZIP regions (14, 15). To date, six family members are characterized including C/EBPalpha , C/EBPbeta (also called CRP2, NF-IL6, LAP, AGP/EBP, IL6BP, or NF-M), C/EBPdelta (also called CRP3, NF-IL6b, and CELF), C/EBPepsilon , C/EBPgamma , and C/EBP-Homologous Protein10 (GADD153) (15). C/EBPs bind to DNA as homodimers or as heterodimers with other C/EBP family members or other bZIP proteins such as c-Fos and CREB/ATF (14, 15). Functional C/EBP-binding sites are present in the promoters of genes that function in cell growth arrest (gadd45gamma ), cell growth (c-fos), and differentiation (phosphoenolpyruvate carboxykinase and beta -casein) (16-19).

C/EBPs are directly involved in the regulation of cell fate determination (20-26). Early reports (20, 27) demonstrate that the sequential expression of C/EBPdelta , C/EBPbeta , and C/EBPalpha is required for optimal adipocyte differentiation. Further studies (28-30) have identified additional roles for C/EBPalpha in hepatocyte metabolism and granulocyte differentiation. C/EBPbeta also plays an essential role in ovarian granulosa cell biology and the development and differentiation of the mammary gland (22-25, 31). Furthermore, C/EBPepsilon functions in the development and differentiation of neutrophils and eosinophils (32, 33).

Control of gene expression can occur at the transcriptional, posttranscriptional, or posttranslational level (34-37). At the posttranscriptional level, mRNA stability is emerging as a key regulatory mechanism in cell cycle control and DNA damage repair (34, 35, 38-40). For example, the stability of growth arrest and DNA damage-inducible mRNA increases after exposure to DNA-damaging agents or other growth arrest treatments (40). In addition, the growth arrest-specific gene 5 (gas-5) exhibits a marked increase in mRNA stability in density-arrested NIH 3T3 cells versus exponentially growing and differentiating cells (41). Alterations in the posttranscriptional regulation of genes that function in cell growth control and cell cycle progression can play a crucial role in tumorigenesis (42, 43). For example, alterations of trans-acting factors that function in c-myc and c-myb mRNA turnover results in increased c-myc and c-myb mRNA stability, which is linked to acute myeloid leukemia (44). Additionally, an increase in the basic fibroblast growth factor mRNA half-life, due to defects in posttranscriptional regulation, has been implicated in a variety of human tumors (45). Although several reports demonstrate that C/EBPdelta is regulated at the transcriptional level (12, 13), posttranscriptional control of C/EBPdelta has not been investigated extensively.

In addition to posttranscriptional control, several genes that play critical roles in cell cycle control are regulated posttranslationally, at the level of protein degradation (46-49). For example, the rate of p27 protein degradation decreases in response to growth arrest, which results in an accumulation of p27 protein (50, 51). Blocking ubiquitination-dependent protein degradation increases p27 protein half-life and demonstrates that the p27 protein is degraded via the ubiquitin/proteasome pathway (52, 53). In addition, increased p53 protein stability occurs during cellular genotoxic stress (54). This is accomplished by N-terminal phosphorylation of the p53 protein, which decreases the degree of ubiquitination and increases protein stability (54). Accumulating evidence indicates that cellular proteins may be degraded by ubiquitin-mediated mechanisms localized to either the nucleus or cytoplasm (55, 56). Nuclear localized ubiquitin-mediated degradation appears to provide a rapid mechanism for the disposal of nuclear cell cycle regulatory proteins (57). For example, the tumor suppressor protein product, p53, is degraded within the nucleus via a ubiquitin-proteasome pathway during post-stress recovery (57).

The overall goal of this study was to investigate the posttranscriptional and posttranslational regulation of C/EBPdelta in G0 growth-arrested mouse MECs. Our laboratory has reported previously (12) that C/EBPdelta exhibits increased transcription and growth suppressor activity in G0 growth-arrested mouse MECs. However, the posttranscriptional and posttranslational regulation of C/EBPdelta has not been systematically investigated. Previous studies (35) have demonstrated that key cell cycle regulatory proteins are encoded by unstable mRNAs. Because G0 growth arrest is associated with a period of decreased cellular activity, we hypothesized that both the C/EBPdelta mRNA and protein would exhibit extended half-lives in G0 growth-arrested MECs. Unexpectedly, the results demonstrate that C/EBPdelta mRNA exhibited a novel short mRNA half-life in G0 growth-arrested mouse MECs (t1/2 ~35 min) and contained a relatively short poly(A) tail of ~100 nucleotides. In addition, the C/EBPdelta protein also exhibited a short half-life in G0 growth-arrested mouse MECs (t1/2 ~120 min). Furthermore, ubiquitination inhibitor studies indicated that C/EBPdelta protein degradation is ubiquitin-dependent and occurs predominantly within the nucleus. The results of these studies demonstrate that the C/EBPdelta mRNA and protein are under tight regulation in G0 growth-arrested MECs, suggesting that C/EBPdelta plays a key role in mouse MEC growth control.

    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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Cell Culture-- The nontransformed HC11 mouse MEC line (a kind gift from Dr. Wolfgang Doppler, Universitat Innsbruck, Austria) was cultured in complete growth media (CGM) consisting of RPMI 1640 (4.5 g/liter glucose) supplemented with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 µg/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 500 ng/ml Fungizone (Invitrogen). NIH 3T3 mouse fibroblast cells (ATCC, Manassas, VA) were cultured in growth media consisting of Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 10 µg/ml insulin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 500 ng/ml Fungizone (Invitrogen). C/EBPdelta overexpression HC11 MECs were maintained as described previously (13). Selection media included the addition of G418 (350 µg/ml) (Invitrogen). For growth arrest experiments, MECs were grown to 80% confluence, washed with serum-free media, and cultured in growth arrest media (GAM) supplemented with 0.1% FBS.

Inhibitors-- Transcriptional inhibitor studies utilized both actinomycin D (5 µg/ml) and 5,6-dichlorobenzimidazole 1-beta -D-ribofuranoside (DRB) (5 µg/ml) (Sigma), and translational inhibitor studies utilized anisomycin (10 µg/ml) (Sigma). Ubiquitination inhibitor studies utilized MG-132 (5 µg/ml) and N-Acetyl-Leu-Leu-Norleu-Al (LLnL) (50 µg/ml) (Sigma).

Northern Blot Analysis-- Total RNA was isolated using RNAzol B (Tel-Test, Friendswood, TX). Thirty µg of total RNA was analyzed by Northern blot analysis as described previously (11). The following [alpha -32P]dCTP-labeled cDNAs were used as probes: C/EBPdelta , C/EBPbeta (kind gifts from Dr. Steven McKnight, University of Texas Southwestern Medical School, Dallas), gas-1, c-fos (ATCC, Manassas, VA), bovine growth hormone, and cyclophilin (Continental Laboratory Products Inc., San Diego, CA). Membranes were visualized by autoradiography or by PhosphorImager (Amersham Biosciences) analysis. Northern blot quantification was performed with AlphaImager 2000 Documentation & Analysis System software (Alpha Innotech, San Leandro, CA). To determine the mRNA half-life of C/EBPdelta , C/EBPbeta , and gas-1, values obtained from densitometric analysis were converted to a percentage of the control time ("0" min) and graphs were plotted as "% mRNA remaining versus time."

Western Blot Analysis-- MECs were washed twice in cold 1% phosphate-buffered saline, transferred to microcentrifuge tubes, pelleted by centrifugation, and subjected to whole cell protein isolation using a RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM EDTA, 1 mM PMSF, 1× Complete Protease Inhibitors (Roche Molecular Biochemicals). In addition, the following kinase and phosphatase inhibitors were added: 100 mM NaF, 100 mM NaVO3, 100 mM Na2MnO4, and 1 µM okadaic acid. Samples were placed in an Eppendorf shaker for 30 min at 4 °C, and the supernatant was recovered after centrifugation. Equal amounts of protein were subjected to electrophoresis on 12.5% denaturing SDS-polyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA) at 150 V for 2 h. Blots were blocked for 60 min in 1× phosphate-buffered saline and 0.5% Tween 20 (PBST) containing 10% non-fat dry milk. Blots were subsequently incubated for 60 min in PBST containing 5% non-fat dry milk and primary antisera against C/EBPdelta (Santa Cruz Biochemicals, Santa Cruz, CA), p27 (Transduction Laboratories, Lexington, KY), Bcl-x (Santa Cruz Biochemicals), and actin (Santa Cruz Biochemicals) (1:1000). Blots were washed in PBST, incubated with horseradish peroxidase-linked secondary antibodies (1:2000) (New England Biolabs, Beverly, MA), and visualized using enhanced chemiluminescence (ECL) (Amersham Biosciences). For nuclear and cytoplasmic protein isolation, MECs were isolated in Dignam buffers (nuclear buffer: 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 1 mM PMSF, 1× complete protease inhibitors, 100 mM NaF, 100 mM NaVO3, 100 mM Na2MnO4, and 1 µM okadaic acid; cytoplasmic buffer: 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1 mM PMSF, 1× complete protease, 100 mM NaF, 100 mM NaVO3, 100 mM Na2MnO4, and 1 µM okadaic acid). Nuclear and cytoplasmic protein lysates were analyzed as described for whole cell protein preparations. Western blot quantification was performed with AlphaImager 2000 Documentation & Analysis System software (Alpha Innotech). To determine protein half-life, C/EBPdelta values obtained from densitometric analysis were converted to a percentage of the control time (0 min) and graphs were plotted as "% protein remaining versus time."

Rapid Amplification of cDNA Ends-Poly(A) Test (RACE-PAT)-- Five µg of total RNA was used to synthesize C/EBPdelta cDNA using the Superscript First-strand Synthesis for RT-PCR kit (Invitrogen). C/EBPdelta cDNA was subsequently used to set up standard PCR (5 min at 93 °C, followed by 30 cycles of 30 s at 93 °C, 30 s at 60 °C, 1 min at 72 °C, and a 7-min extension at 72 °C) using an end-labeled [gamma -32P]dATP C/EBPdelta upstream primer (5'-CCATTGCAGCTAAGGTACAT-3') and an oligo(dT) anchor downstream primer (5'-GGGGATCCGCGGTTTTTTTTTTTT-3') (58). Labeled PCR products were run on 5% polyacrylamide gels at 4 °C for 5 h at 100 V in 0.5% TBE buffer. After electrophoresis, gels were dried and visualized by autoradiography.

Oligo/RNase H Cleavage Northern Blot Analysis-- Ten µg of total RNA and oligomers complementary to the 3' end of the C/EBPdelta mRNA (5'-CCAAAGAAACTAGCGATTCGGG-3') to the beta -globin mRNA (5'-GATCCACGTGCAGCTTGTCA-3') or the poly(A) tail (poly(dT)12-18) were denatured at 65 °C for 10 min. A digestion mixture (4 µl of 5× buffer (200 mM HEPES (pH 7.9), 50 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol (DTT)), 1 unit of RNase H (Invitrogen) and 1 µl of RNasin (40 units/µl) (Promega, Madison, WI), in 20-µl reactions) was added to each sample and incubated at 37 °C for 30 min (58). Reactions were stopped with 1 µl of 0.5 M EDTA and precipitated with 0.1 volume of 3 M NaOAc and 2.5 volumes of 100% ethanol. Samples were pelleted by centrifugation, resuspended in Northern blot tracking dye, and subsequently analyzed by Northern blot analysis as described previously (11).

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

C/EBPdelta mRNA Exhibits a Short Half-life in G0 Growth-arrested Mouse HC11 MECs-- To investigate the posttranscriptional regulation of the C/EBPdelta mRNA in mouse HC11 MECs, we utilized transcriptional inhibitors followed by Northern blot analysis. Briefly, confluent HC11 MECs were G0 growth-arrested by serum and growth factor withdrawal. After 48 h, HC11 MECs were either maintained in growth arrest medium alone (GAM-) or in GAM with the addition of the transcriptional inhibitor, actinomycin D (GAM+) for the indicated times. mRNA half-life was analyzed for a panel of cellular mRNAs including cyclophilin (cp), which was used to confirm equal loading. Consistent with previous reports from our laboratory, C/EBPdelta mRNA was detected in G0 growth-arrested HC11 MECs (Fig. 1A, lanes 1-4). C/EBPdelta mRNA levels rapidly declined with a half-life of ~35 min (t1/2 ~35 min) following actinomycin D treatment (Fig. 1A, lanes 5-8 and Fig. 1C). After 60 min of actinomycin D treatment, C/EBPdelta mRNA was undetectable (Fig. 1A, lane 7). Similar results were observed with a second transcriptional inhibitor, DRB (Fig. 1B).


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Fig. 1.   C/EBPdelta mRNA exhibits a short half-life in G0 growth-arrested mouse HC11 MECs, actinomycin D (A) and DRB (B) studies. Northern blot analysis was performed with 30 µg of total RNA isolated from untreated and actinomycin D or DRB-treated G0 growth-arrested HC11 MECs at the indicated time points. Northern blots were sequentially probed with [alpha -32P]dCTP-labeled C/EBPdelta , C/EBPbeta , gas1, c-fos, and cp cDNAs. cp was used as a loading control. A, lanes 1-4, RNA from G0 growth-arrested MECs (GAM-); lanes 5-8, RNA from G0 growth-arrested MECs treated with actinomycin D (GAM+). B, lanes 1-4, RNA from G0 growth-arrested MECs; lanes 5-8, RNA from G0 growth-arrested MECs treated with DRB. Results are representative of three independent experiments. C, summary of mRNA half-life data obtained from Northern blot/actinomycin D (Act.D) analysis. Signals were quantified, and the relative amount of each mRNA is expressed as a percentage of the "0-min" control time, which was set at 100%. Graphs are plotted as % mRNA remaining versus time. Filled triangles, RNA from actinomycin D-treated MECs; filled circles, RNA from non-treated MECs.

In addition to C/EBPdelta , we also investigated the mRNA level of another C/EBP family member, C/EBPbeta . Reports from a number of laboratories, including our own, demonstrate that C/EBPbeta plays a significant role in mammary gland growth and differentiation (22, 24, 25, 31). C/EBPbeta mRNA was detected in G0 growth-arrested HC11 MECs, suggesting that C/EBPbeta also plays a role in G0 growth arrest (Fig. 1A, lanes 1-4). C/EBPbeta mRNA levels declined with a half-life of ~45 min following the addition of actinomycin D (Fig. 1A, lanes 5-8, and Fig. 1C).

gas-1 mRNA levels are known to be induced during G0 growth arrest of NIH 3T3 fibroblast cells (59-61). In this report, gas-1 mRNA was also detected in G0 growth-arrested HC11 MECs (Fig. 1A, lanes 1-4). Addition of actinomycin D reduced gas-1 mRNA levels (t1/2 ~75 min) (Fig. 1A, lanes 5-8, and Fig. 1C). Compared with C/EBPdelta , gas-1 mRNA was relatively stable during G0 growth arrest. In agreement with previous work, c-fos, an immediate early gene that is induced at the G0/G1 transition, was undetectable at the mRNA level in G0 growth-arrested HC11 MECs (Fig. 1A, lanes 1-4).

In summary, results of transcriptional inhibitor studies indicate that C/EBPdelta mRNA is highly unstable during G0 growth arrest in HC11 MECs. C/EBPbeta , which has been associated previously with cellular proliferation and differentiation, also exhibits a relatively short mRNA half-life in G0 growth-arrested HC11 MECs. In contrast, gas-1 mRNA is more stable during G0 growth arrest. Overall, the results suggest that the C/EBPdelta mRNA is undergoing rapid turnover despite the general decline in global gene expression and biosynthetic activity during G0 growth arrest.

C/EBPdelta mRNA Half-life during Cell Cycle Re-entry in HC11 MECs-- Cell cycle re-entry requires coordination between the inactivation and/or disposal of G0-specific proteins and the expression of early G1 genes, such as c-fos and c-myc. To investigate the posttranscriptional regulation of C/EBPdelta mRNA during early G1, mRNA levels were analyzed from HC11 MECs upon addition of complete growth media alone (CGM-) or CGM plus actinomycin D (CGM+) at the indicated time points. In agreement with previous reports from our laboratory, C/EBPdelta mRNA levels decline with the onset of G1 and the initiation of the cell cycle (Fig. 2, lanes 1-4) (41). C/EBPdelta mRNA levels also decreased in early G1 following actinomycin D treatment; however, the cell cycle-induced decline of C/EBPdelta mRNA was delayed compared with the decline observed in G0 growth arrest (Fig. 2, lanes 5-8).


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Fig. 2.   C/EBPdelta mRNA stability increases during the G0/G1 transition upon transcriptional inhibitor treatment in HC11 MECs. RNA isolated from untreated and actinomycin D-treated serum and growth factor stimulated (G0/G1 transition) HC11 MECs was analyzed by Northern blot analysis as described in Fig. 1. Lanes 1-4, RNA from serum and growth factor-stimulated (G0/G1 transition) MECs (CGM-); lanes 5-8, RNA from serum and growth factor-stimulated (G0/G1 transition) MECs treated with actinomycin D (CGM+). Results are representative of three independent experiments.

When G0 growth-arrested HC11 MECs were induced to re-enter the cell cycle by CGM addition, C/EBPbeta mRNA levels increased ~10-fold within the first 90 min (Fig. 2, lane 1-4). This induction of C/EBPbeta mRNA levels during early G1 is consistent with a growth-promoting role for C/EBPbeta . The addition of actinomycin D blocked the growth-stimulated induction of C/EBPbeta mRNA, which suggests that C/EBPbeta transcription plays a major role in the increase in C/EBPbeta mRNA levels during early G1 in HC11 MECs (Fig. 2, lanes 5-8).

gas-1 mRNA levels also declined after G0 growth-arrested HC11 MECs were induced to re-enter the cell cycle by refeeding with CGM (Fig. 2, lanes 1-4). Interestingly, addition of CGM and actinomycin D stabilized the gas-1 mRNA, resulting in high levels of gas-1 mRNA even at 90 min (Fig. 2, lanes 5-8). In agreement with previous reports, c-fos mRNA was transiently induced following the addition of CGM and the initiation of the cell cycle (Fig. 2, lanes 1-4). The induction of c-fos mRNA was blocked by actinomycin D treatment, indicating that c-fos gene transcription is required for the increase in c-fos mRNA during early G1 (Fig. 2, lanes 5-8).

The results of the transcriptional inhibitor studies during cell cycle re-entry demonstrate that the C/EBPdelta and gas-1 mRNAs are more stable during the G0/G1 transition compared with G0 growth arrest. This suggests that both C/EBPdelta and gas-1 mRNA degradation during cell cycle re-entry is dependent on the transcription of gene product(s) important for mRNA decay during the G0/G1 transition. In addition, the increase in C/EBPbeta and c-fos mRNA levels during the G0/G1 transition are inhibited by actinomycin D treatment, indicating that the increase in these immediate early mRNAs is transcription-dependent.

C/EBPdelta mRNA Is More Stable in G0 Growth-arrested NIH 3T3 Cells Compared with HC11 MECs-- NIH 3T3 cells have been utilized extensively as a model system to investigate mechanisms of cell growth control. We reported previously (11) that C/EBPdelta mRNA is present in NIH 3T3 cells regardless of growth status. To investigate the posttranscriptional control of the C/EBPdelta mRNA in NIH 3T3 cells, transcriptional inhibitor/Northern blot analysis was performed. In agreement with our previous results, C/EBPdelta mRNA was detected in G0 growth-arrested (GAM-) NIH 3T3 cells (Fig. 3A, lanes 1-4). C/EBPdelta mRNA levels declined following actinomycin D treatment, although the rate of decline is slower than that observed in G0 growth-arrested HC11 MECs (t1/2 >100 min for NIH 3T3 cells versus t1/2 ~35 min for HC11 MECs) (Fig. 3A, lanes 5-8 and Fig. 3B). Following cell cycle re-entry, C/EBPdelta mRNA levels decreased by 90 min (Fig. 3A, lanes 9-12). The addition of actinomycin D stabilized C/EBPdelta mRNA (Fig. 3A, lanes 13-16), paralleling the results from experiments in HC11 MECs.


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Fig. 3.   C/EBPdelta mRNA stability during G0 growth arrest and the G0/G1 transition in NIH 3T3 cells. RNA isolated from untreated and actinomycin D-treated G0 growth-arrested or serum and growth factor-stimulated (G0/G1 transition) NIH 3T3 cells was analyzed by Northern blot as described in Fig. 1. A, lanes 1-4, RNA from G0 growth-arrested MECs (GAM-); lanes 5-8, RNA from G0 growth-arrested MECs treated with actinomycin D (GAM+); lanes 9-12, RNA from serum- and growth factor-stimulated (G0/G1 transition) MECs (CGM-); lanes 13-16, RNA from serum- and growth factor-stimulated (G0/G1 transition) MECs treated with actinomycin D (CGM+). Results are representative of three independent experiments. B, summary of mRNA half-life data obtained from Northern blot/actinomycin D (Act.D) analysis as determine in Fig. 1. Filled triangles, RNA from actinomycin D-treated MECs; filled circles, RNA from non-treated MECs.

Consistent with results from mouse MECs, the C/EBPbeta mRNA was detected in G0 growth-arrested NIH 3T3 cells (Fig. 3A, lanes 1-4). Upon addition of actinomycin D, a steady decline in the C/EBPbeta mRNA content was observed (t1/2 >100 min) (Fig. 3A, lanes 5-8, and Fig. 3B). C/EBPbeta mRNA levels increased (15-fold after 90 min) following cell cycle re-entry (Fig. 3A, lanes 9-12). This cell cycle-induced increase in C/EBPbeta mRNA levels was blocked by actinomycin D treatment (Fig. 3A, lanes 13-16). In agreement with previous reports (59, 62), elevated levels of gas-1 mRNA was detected in G0 growth-arrested NIH 3T3 cells (Fig. 3A, lanes 1-4). Addition of actinomycin D resulted in a decline in gas-1 mRNA levels (t1/2 ~75 min) (Fig. 3A, lanes 5-8, and Fig. 3B). Following the addition of complete growth media and the initiation of the cell cycle, gas-1 mRNA content exhibited a decline by 90 min (Fig. 3A, lanes 9-12). However, the addition of actinomycin D stabilized the gas-1 mRNA (Fig. 3A, lanes 13-16). Finally, c-fos mRNA was undetectable in G0 growth-arrested NIH 3T3 cells at all time points taken (Fig. 3A, lanes 1-8). c-fos mRNA levels rapidly increased following cell cycle re-entry and peaked 60 min after the addition of complete growth media (Fig. 3A, lanes 9-12). Cell cycle-induced increase in the c-fos mRNA level was blocked by actinomycin D treatment, paralleling the results from experiments with HC11 MECs (Fig. 3A, lanes 13-16).

The extended C/EBPdelta mRNA half-life detected in G0 growth-arrested NIH 3T3 cells suggests that C/EBPdelta is under less stringent control in mouse fibroblast-derived cells compared with mouse mammary epithelial-derived cells. In contrast, posttranscriptional regulation of C/EBPdelta mRNA during cell cycle re-entry is similar between HC11 MECs and NIH 3T3 cells. The posttranscriptional regulation of C/EBPbeta , gas-1, and c-fos in both G0 growth arrest and cell cycle re-entry is comparable between HC11 MECs and NIH 3T3 cells.

C/EBPdelta mRNA Contains a Short Poly(A) Tail-- Sequences present within mRNAs influence processing, stability, and transport (34, 35, 39, 63). To investigate the role of the poly(A) tail on C/EBPdelta mRNA stability, we utilized an oligo/RNase H cleavage Northern blot analysis (58). In this analysis, a C/EBPdelta -specific oligomer complimentary to the C/EBPdelta mRNA within the 3'-untranslated region (UTR) (oligo 1193) was used to form a DNA/RNA heteroduplex that is cleaved by RNase H, producing C/EBPdelta 5' and 3' mRNA fragments (Fig. 4A). A [alpha -32P]dCTP-labeled C/EBPdelta 3'-UTR-specific probe that spanned the oligo/RNase H digestion site was used to detect both of the oligo/RNase H-generated C/EBPdelta fragments: the 5' C/EBPdelta cleavage product (~1.4 kb) composed of the C/EBPdelta mRNA 5'-untranslated region (UTR), coding sequence, and partial 3'-UTR and the 3' C/EBPdelta mRNA cleavage product composed of the remaining 3'-UTR (~260 bp) plus the length of the poly(A) tail.


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Fig. 4.   C/EBPdelta mRNA contains a short poly(A) tail in mouse HC11 MECs. Oligo/RNase H cleavage Northern blot analysis was performed on 20 µg of total RNA isolated from untreated and actinomycin D-treated G0 growth-arrested or serum- and growth factor-stimulated (G0/G1 transition) HC11 MECs at the indicated time points. A, schematic of the oligo/RNase H cleavage protocol. RNA samples were incubated with a 3'-UTR C/EBPdelta mRNA-specific oligomer (1193), treated with RNase H, and separated by agarose gel electrophoresis. Both 5' and 3' cleavage fragments were detected by using a [alpha -32P]dCTP-labeled probe that spanned the C/EBPdelta 3'-UTR. B, lanes 1-3, RNA from G0 growth-arrested MECs (GAM-); lanes 4 and 5, RNA from G0 growth-arrested MECs treated with actinomycin D (GAM+); lanes 6-8, RNA from serum- and growth factor-stimulated (G0/G1 transition) MECs (CGM-); lanes 9-11, RNA from serum- and growth factor-stimulated (G0/G1 transition) MECs treated with actinomycin D (CGM+); lane 12, RNA from G0 growth-arrested MECs treated with oligo(dT) and RNase H; lane 13, untreated full-length C/EBPdelta mRNA from G0 growth-arrested MECs. Results are representative of three independent experiments.

Initially, we performed the oligo/RNase H cleavage analysis on RNA from G0 growth-arrested (GAM-) HC11 MECs, and Northern blot analysis detected two cleavage products of ~1.4 kb and 370 bp (Fig. 4B, lanes 1-3). The 3'-UTR cleavage product contains 260 bp from the 3'-UTR and reveals a poly(A) tail of ~100 nucleotides. To investigate the mechanism of C/EBPdelta mRNA degradation, mRNA was isolated from actinomycin D-treated G0 growth-arrested (GAM+) HC11 MECs. After 30 min of actinomycin D treatment, both cleavage products were detected (Fig. 4B, lane 4). However, after 60 min of actinomycin D treatment only the 3' oligo/RNase H C/EBPdelta mRNA cleavage product was detected (Fig. 4B, lane 5). These results confirm the short half-life of the C/EBPdelta mRNA in G0 growth-arrested HC11 MECs.

We next investigated the C/EBPdelta mRNA poly(A) tail length during the G0/G1 transition. G0 growth-arrested HC11 MECs were induced to re-enter the cell cycle by the addition of CGM alone (CGM-) or CGM plus actinomycin D (CGM+). Upon addition of CGM, the reduction of C/EBPdelta mRNA oligo/RNase H cleavage products was observed, consistent with a decrease in the C/EBPdelta mRNA content upon the onset of early G1 (Fig. 4B, lanes 6-8). After addition of actinomycin D, the reduction of C/EBPdelta mRNA oligo/RNase H cleavage products was slightly delayed compared with GAM+, and a more complex pattern of C/EBPdelta mRNA degradation was detected (Fig. 4B, lanes 9-11). The results of the CGM and actinomycin D experiment suggest that transcription of gene products important for mRNA decay is required for efficient C/EBPdelta mRNA degradation during cell cycle re-entry. Finally, the estimated size of all the C/EBPdelta 3' oligo/RNase H cleavage products is consistent with a poly(A) tail length of ~100 nucleotides. To confirm the length of the poly(A) tail, we utilized an oligo(dT) in the oligo/RNase H experiment, which generates a C/EBPdelta mRNA product lacking a poly(A) tail (Fig. 4B, lane 12). The results reveal that the mobility of this mRNA product compared with full-length C/EBPdelta mRNA (Fig. 4B, lane 13) is consistent with a poly(A) tail length of ~100 nucleotides. Overall, the results demonstrate that the C/EBPdelta mRNA contains a relatively short poly(A) tail that is not shortened during mRNA degradation in HC11 G0 growth arrest and cell cycle re-entry.

RACE-PAT Analysis Confirms C/EBPdelta mRNA Poly(A) Tail Length-- To investigate further the C/EBPdelta mRNA poly(A) tail length, we utilized a rapid amplification of cDNA ends-poly(A) test (RACE-PAT) (58). Initially, mRNA was obtained from G0 growth-arrested HC11 MECs; cDNA was synthesized, and PCR was performed utilizing a radiolabeled C/EBPdelta 3'-UTR upstream-specific primer and an oligo(dT) downstream primer. The PCR produced multiple products varying in length from 170 to 270 bp (Fig. 5, lanes 1-3), which is consistent with a C/EBPdelta poly(A) tail length of ~100 nucleotides. Following addition of actinomycin D, C/EBPdelta mRNA levels declined rapidly as observed previously in the transcriptional inhibitor/Northern blot analysis (Fig. 5, lanes 4-6). This decline is reflected in the decrease in the amount of RACE-PAT product obtained. RACE-PAT analysis was also performed on mRNA obtained from HC11 MECs refed with CGM, which also indicated that the C/EBPdelta mRNA poly(A) length is approximately ~100 nucleotides (Fig. 5, lanes 7-9). Additionally, actinomycin D treatment resulted in a more stable C/EBPdelta mRNA but had no apparent effect on poly(A) tail length (Fig. 5, lanes 10-12). The results confirm that the C/EBPdelta mRNA contains a poly(A) tail of ~100 nucleotides in G0 growth arrest and during the G0/G1 transition and parallel previous mRNA half-life results from experiments in HC11 MECs (Figs. 1A and 2A).


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Fig. 5.   The short C/EBPdelta mRNA poly(A) tail is confirmed by RACE-PAT analysis. RACE-PAT was performed on 5 µg of total RNA isolated from untreated and actinomycin D-treated G0 growth-arrested or serum- and growth factor-stimulated (G0/G1 transition) HC11 MECs at the indicated time points. cDNA was synthesized and subjected to PCR, and products were visualized by PAGE and autoradiography. Lanes 1-3, G0 growth-arrested MECs (GAM-); lanes 4-6, G0 growth-arrested MECs treated with actinomycin D (GAM+); lanes 7-9, serum and growth factor stimulated (G0/G1 transition) MECs (CGM-); lanes 10-12, serum and growth factor stimulated (G0/G1 transition) MECs treated with actinomycin D (CGM+). Results are representative of three independent experiments.

The 3'-Untranslated Region Influences C/EBPdelta mRNA Stability-- Numerous studies (34, 35, 39) have demonstrated that specific sequences within the 3'-UTR regulate mRNA stability. To investigate the potential role of the C/EBPdelta 3'-UTR in mRNA stability, transcriptional inhibitor/Northern blot analysis was repeated utilizing a stably transfected C/EBPdelta overexpression HC11 MEC line previously developed in our laboratory (13). This HC11 MEC line expresses an exogenous C/EBPdelta mRNA that contains a bovine growth hormone (BGH) 3'-UTR in place of the C/EBPdelta 3'-UTR. The cells were growth-arrested for 48 h and maintained in GAM in the presence or absence of actinomycin D. The C/EBPdelta /BGH 3'-UTR mRNA levels were compared with the endogenous C/EBPdelta mRNA levels. Consistent with previous results, the endogenous C/EBPdelta mRNA levels were detected in G0 growth-arrested cells (Fig. 6, lanes 1-4). Similarly, the C/EBPdelta /BGH 3'-UTR mRNA was also detected in G0 growth-arrested HC11 MECs at all indicated time points (Fig. 6, lanes 1-4). Consistent with previous results, the addition of actinomycin D resulted in a rapid reduction of endogenous C/EBPdelta mRNA levels (t1/2 ~35 min) (Fig. 6, lanes 5-7). Interestingly, the C/EBPdelta /BGH 3'-UTR mRNA levels did not decline after actinomycin D treatment (Fig. 6, lanes 5-7). These results indicate that the presence of the BGH 3'-UTR downstream of the C/EBPdelta coding region stabilizes the C/EBPdelta mRNA compared with the endogenous C/EBPdelta mRNA and suggests that the C/EBPdelta 3'-UTR plays a role in mRNA stability during G0 growth arrest.


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Fig. 6.   The C/EBPdelta 3'-UTR regulates C/EBPdelta mRNA stability in G0 growth-arrested HC11 MECs. RNA isolated from untreated and actinomycin D-treated G0 growth-arrested C/EBPdelta overexpression HC11 MECs was analyzed by Northern blot using either a BGH 3'-UTR, C/EBPdelta 3'-UTR, or a cp probe as described in Fig. 1. Lanes 1-4, RNA from G0 growth-arrested MECs (GAM-); lanes 5-7, RNA from G0 growth-arrested MECs treated with actinomycin D (GAM+). Results are representative of three independent experiments.

C/EBPdelta Protein Exhibits a Short Half-life in G0 Growth-arrested Mouse MECs-- Posttranslational control is a major mechanism by which cells regulate the level of cell cycle control proteins (46-49). To investigate the posttranslational regulation of the C/EBPdelta protein, HC11 MECs were G0 growth-arrested for 48 h and maintained in GAM in the presence or absence of the translational inhibitor anisomycin. Protein half-life was analyzed for a panel of cellular proteins by Western blot analysis including actin, which was used to confirm equal loading. Consistent with previous reports from our laboratory (11-13), C/EBPdelta protein was detected in G0 growth-arrested HC11 MECs (Fig. 7A, lanes 1-6). Following anisomycin treatment, C/EBPdelta protein levels declined with a half-life of ~120 min (Fig. 7A, lanes 7-12, and Fig. 7B). As a control, the stability of p27, a growth arrest-specific protein that is regulated predominantly at the posttranslational level, was assessed (50, 51). As expected, p27 protein was detected during G0 growth arrest of HC11 MECs (Fig. 7A, lanes 1-6). In contrast to the C/EBPdelta protein decay kinetics, the p27 protein is relatively stable in G0 growth-arrested HC11 MECs even after treatment with anisomycin (Fig. 7A, lanes 7-12). The results demonstrate that the C/EBPdelta protein exhibits a shorter half-life compared with p27, which suggests that C/EBPdelta protein is tightly regulated in G0 growth-arrested mouse MECs.


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Fig. 7.   C/EBPdelta protein exhibits a short half-life in G0 growth-arrested HC11 MECs. Western blot analysis was performed on 50 µg of whole cell protein isolated from untreated and anisomycin-treated G0 growth-arrested HC11 MECs at the indicated time points. Western blots were sequentially probed with C/EBPdelta , p27, and actin antibodies. Actin was used as a loading control. A, lanes 1-6, protein from growth-arrested MECs (GAM-); lanes 7-12, protein from growth-arrested MECs treated with anisomycin (GAM+). Results are representative of three independent experiments. B, summary of protein half-life data obtained from Western blot/anisomycin analysis. Signals were quantified, and the relative amount of each protein is expressed as a percentage of the 0-min control time, which was set at 100%. Graphs are plotted as % protein remaining versus time. Filled triangles, protein from anisomycin-treated MECs; filled circles, protein from non-treated MECs.

C/EBPdelta Protein Is Degraded via a Ubiquitin-dependent Pathway in G0 Growth-arrested Mouse MECs-- To investigate the protein degradation pathway utilized by the C/EBPdelta protein in G0 growth-arrested mouse MECs, we used the ubiquitination inhibitor, MG-132. HC11 MECs were growth-arrested by addition of GAM for 48 h and subsequently maintained in either growth arrest media alone (control), GAM plus a vehicle control (+Me2SO), or GAM plus the ubiquitination inhibitor (+MG-132). C/EBPdelta protein was detected in both G0 growth-arrested control and Me2SO samples (Fig. 8A, lanes 1-4 and 5-7, respectively). Interestingly, C/EBPdelta protein content dramatically increased by 60 min of MG-132 treatment and continued to be elevated up to 180 min (Fig. 8A, lanes 8-10). Similar results were obtained with a second ubiquitination inhibitor, LLnL, in G0 growth-arrested HC11 MECs (Fig. 8B).


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Fig. 8.   C/EBPdelta protein undergoes ubiquitination in G0 growth-arrested MECs. Western blot analysis was performed on 50 µg of whole cell protein isolated from untreated and MG-132 or LLnL-treated G0 growth-arrested mouse MECs at the indicated time points as described in Fig. 7. A, mouse HC11 MECs treated with MG-132. Lanes 1-4, protein from G0 growth-arrested MECs (control); lanes 5-7, protein from G0 growth-arrested MECs treated with vehicle (+DMSO); lanes 8-10, protein from G0 growth-arrested MECs treated with ubiquitination inhibitor (+MG-132). B, mouse HC11 MECs treated with LLnL. Lanes 1-4, protein from G0 growth-arrested MECs (control); lanes 5-7, protein from G0 growth-arrested MECs treated with vehicle (+DMSO); lanes 8-10, protein from G0 growth-arrested MECs treated with ubiquitination inhibitor (+LLnL). Results are representative of three independent experiments.

As a control, p27 protein levels were monitored in both mouse and human MECs after MG-132 treatment. Previous work (52, 53) in vitro and in vivo demonstrates that the ubiquitin-proteasome pathway regulates the p27 protein level. p27 protein was detected in both G0 growth-arrested control and Me2SO samples (Fig. 8, A and B). As expected, a modest increase in p27 protein level is detected after MG-132 treatment in HC11 MECs (Fig. 8A, lanes 8-10). Taken together, these results demonstrate that the C/EBPdelta protein is degraded via a ubiquitin-proteasome-dependent pathway that is conserved in both mouse MECs.

Ubiquitination of the C/EBPdelta Protein Is Localized Predominantly to the Nuclear Compartment-- To determine whether C/EBPdelta protein degradation occurs in the nucleus and/or cytoplasm, nuclear and cytoplasmic protein was analyzed from G0 growth-arrested HC11 MECs. HC11 MECs were growth-arrested by addition of GAM for 48 h and subsequently maintained in either growth arrest media alone (control), GAM plus a vehicle control (+Me2SO), or GAM plus the ubiquitination inhibitor (+MG-132). Nuclear and cytoplasmic protein fractions were isolated at the indicated times. C/EBPdelta protein was detected in the nuclear protein fraction but not the cytoplasmic protein fraction in both the G0 growth-arrested control and Me2SO samples (Fig. 9, lanes 1-6 and lanes 7-10, respectively). In agreement with previous results (Fig. 8), C/EBPdelta protein content increased after MG-132 treatment (Fig. 9, lanes 11-14). Interestingly, the increase in C/EBPdelta protein is restricted to the nuclear compartment (Fig. 9, lanes 11 and 13).


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Fig. 9.   C/EBPdelta protein ubiquitination is localized to the nuclear compartment. Western blot analysis was performed on 25 µg of nuclear and cytoplasmic protein isolated from untreated or MG-132-treated G0 growth-arrested mouse HC11 MECs at the indicated time points as described in Fig. 7. Lanes 1, 3, and 5, nuclear protein from G0 growth-arrested MECs (control); lanes 2, 4, and 6, cytoplasmic protein from G0 growth-arrested MECs (control); lanes 7 and 9, nuclear protein from G0 growth-arrested MECs treated with vehicle (+DMSO); lanes 8 and 10, cytoplasmic protein from G0 growth-arrested MECs treated with vehicle (+DMSO); lanes 11 and 13, nuclear protein from G0 growth-arrested MECs treated with ubiquitination inhibitor (+MG-132); lanes 12 and 14, cytoplasmic protein from G0 growth-arrested MECs treated with ubiquitination inhibitor (+MG-132). Results are representative of three independent experiments.

Nuclear and cytoplasmic p27 protein levels were also monitored in MG-132-treated G0 growth-arrested HC11 MECs. Similar to the previous study, p27 protein was detected in G0 growth-arrested control and Me2SO samples (Fig. 9, lanes 1-6 and lanes 7-10, respectively). In contrast to C/EBPdelta protein subcellular localization, p27 protein was found in both the nuclear and cytoplasmic compartments. Detection of the p27 protein increased slightly in both compartments after MG-132 treatment (Fig. 9, lanes 11-14).

As a control for our nuclear and cytoplasmic protein fractionation, we monitored the subcellular localization of Bcl-x, which is known to be localized to the cytoplasmic compartment (64). As expected, the majority of the Bcl-x protein content was localized within the cytoplasmic compartment in both G0 growth-arrested control and Me2SO samples (Fig. 9, lanes 1-6 and lanes 7-10, respectively). Importantly, upon MG-132 treatment, no change in Bcl-x localization was detected (Fig. 9, lanes 11-14). Taken together, these results demonstrate that the C/EBPdelta protein is absent from the cytoplasmic compartment, which suggests a nuclear localized ubiquitin-mediated degradation pathway.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although most cells in the adult animal exist in a G0 growth arrest state, little is known about the regulation and function of genes expressed during G0 (1-4). This study investigated the posttranscriptional and posttranslational regulation of C/EBPdelta in G0 growth-arrested mouse MECs in vitro. Previous reports from our laboratory (11-13) have shown that C/EBPdelta gene expression and DNA binding activity increase in G0 growth-arrested MECs. The G0-specific increase in C/EBPdelta gene expression is STAT3-dependent (10). In addition, overexpression of C/EBPdelta in MECs accelerated G0 growth arrest and apoptosis in response to serum and growth factor withdrawal (13). In contrast, reducing C/EBPdelta levels by antisense RNA delayed MEC G0 growth arrest and apoptosis after serum and growth factor withdrawal (13). In this report, we demonstrated that C/EBPdelta mRNA exhibits a novel short half-life during G0 growth arrest in mouse MECs (t1/2 ~35 min). Interestingly, mRNAs encoding several important cell cycle control proteins, growth factors, lymphokines, cytokines, and proto-oncogenes also exhibit short half-lives (35). For example, the cytokine interleukin 6, which is important in the inflammatory response, has an mRNA half-life of ~20 min (65). We suggest that the short C/EBPdelta mRNA half-life in G0 growth-arrested MECs allows the cells to respond rapidly to potential growth stimuli. Interestingly, the short half-life of C/EBPdelta mRNA observed in mouse MECs appears to be a property of mammary epithelial derived cell lines. For example, in G0 growth-arrested NIH 3T3 cells, the C/EBPdelta mRNA half-life is ~2-3-fold longer. This suggests that tight regulation of the C/EBPdelta mRNA is important in the initiation and maintenance of G0 growth arrest in mouse MECs. Similar to our studies in mouse HC11 MECs, the C/EBPdelta mRNA exhibited a relatively short half-life of ~40 min in G0 growth-arrested human MCF-12A MECs (data not shown). This suggests a conservation of mRNA decay kinetics for C/EBPdelta in both mouse and human MEC systems.

Cell cycle re-entry (G0/G1 transition) is associated with dramatic changes in gene expression. Transcription of growth arrest genes is known to decrease with cell cycle re-entry, although mRNAs encoding growth arrest-specific proteins could persist and may delay or interfere with cell cycle re-entry. The disposal of G0-specific mRNAs and proteins during MEC cell cycle re-entry is not well characterized. This study sought to determine whether or not the decay kinetics of C/EBPdelta mRNA were similar between G0 growth arrest and the G0/G1 transition. Results of transcriptional inhibitor/Northern blot studies upon cycle re-entry demonstrate that C/EBPdelta mRNA has a longer half-life during the G0/G1 transition compared with G0 growth-arrested HC11 MECs. In fact, both C/EBPdelta and gas-1 mRNAs exhibited stabilization during cell cycle re-entry in response to transcriptional inhibitors in two mouse MEC lines, HC11 and COMMA D (later data not shown). Posttranscriptional control is known to play a major role in the regulation of gas family members in many cell types (61, 66, 67). Our results parallel previous work that shows an increase in gas-1 and gas-6 mRNA stability after cell cycle re-entry and treatment with actinomycin D of fibroblastic cell lines (61, 66, 67). Furthermore, actinomycin D treatment of Schwann cells during cell cycle re-entry stabilized the gas-3 mRNA (68). The difference in C/EBPdelta mRNA half-life in G0 growth arrest and the G0/G1 transition suggests that there is a specific mRNA degradation pathway for C/EBPdelta during the G0/G1 transition that differs from G0 growth arrest. In addition, increased stabilization of the C/EBPdelta mRNA suggests that the synthesis of a trans-acting factor(s) or RNA is required to degrade the C/EBPdelta mRNA upon cell cycle re-entry.

The mechanism underlying C/EBPdelta mRNA degradation is currently not known, although numerous studies have demonstrated that the length of the poly(A) tail is a major factor in the stability of eukaryotic mRNAs (i.e. a decrease in poly(A) tail length results in an decrease in mRNA stability) (34, 35, 39, 63). In this report, analysis of poly(A) tail length by oligo/RNase H cleavage and RACE-PAT demonstrated that the C/EBPdelta mRNA has a short poly(A) tail of ~100 nucleotides. This is somewhat shorter than the average eukaryotic mRNA that contains a poly(A) tail of ~200 nucleotides (69).

Structural elements found within the 5'-UTR, coding region, and the 3'-UTR are known to be involved in regulating mRNA stability (34, 35, 39). For example, the 3'-UTR of many labile mRNAs, such as cytokine and oncoprotein mRNAs, contain multiple copies of A/U-rich elements (AREs) (34, 35, 39). These cis-acting elements interact with trans-acting factors to destabilize the mRNA. Analysis of ARE sequences from 12 transcription factor-encoding mRNAs that exhibit early G1 instability classified two distinct groups of mRNAs: 1) mRNAs with 3'-UTRs that contain one or more copies of the well recognized "AUUUA" sequence and 2) mRNAs with 3'-UTRs that contain one or more copies of a "non-AUUUA" sequence (70). An example of a non-AUUUA mRNA is c-jun, which contains "U"-rich regions that confer G1 instability (70). Interestingly, analysis of the C/EBPdelta 3'-UTR revealed a single AUUUA element and two U-rich regions (region 1, 18 uracils/32 nucleotides; region 2, 17 uracils/26 nucleotides). This indicates that the C/EBPdelta mRNA has characteristics of both AUUUA and non-AUUUA AREs. Mutational analysis is ongoing to characterize further the role of these instability elements in C/EBPdelta mRNA decay.

Analysis of another C/EBP family member, C/EBPbeta , demonstrated similar mRNA decay kinetics as C/EBPdelta during mouse MEC G0 growth arrest. Like C/EBPdelta , C/EBPbeta mRNA displayed a short half-life of ~45 min. The results suggest a conserved mRNA decay pathway shared between C/EBPdelta and C/EBPbeta in G0 growth-arrested mouse MECs. Although the homology between the C/EBPdelta and C/EBPbeta 3'-UTRs is ~30%, both 3'-UTR sequences contain multiple U-rich elements that may regulate mRNA degradation.

Because the C/EBPdelta mRNA was shown to have a short half-life in G0 growth-arrested MECs, we hypothesized that the C/EBPdelta protein would exhibit a similar short biological half-life (39). A yeast genome-wide analysis has demonstrated that unstable mRNAs encode for unstable proteins (71). Examples include translation initiation factors, termination factors, and proteins of the mating pheromone signal transduction pathway (71). The results in this report established that the half-life of the C/EBPdelta protein is shorter (t1/2 ~120 min) than the tumor suppressor, p27 (t1/2 >150 min). The short half-life of the C/EBPdelta protein in G0 growth-arrested MECs suggests that C/EBPdelta function is tightly regulated during MEC quiescence, which may allow MECs to respond rapidly to growth signals and re-enter the cell cycle when necessary.

The ubiquitin-proteasome pathway is a major selective decay mechanism of short-lived regulatory proteins (49). Cell cycle regulatory proteins that are degraded by the ubiquitin-proteasome pathway include the tumor suppressors, p21 (t1/2 ~30 min) (72), p53 (t1/2 ~20 min) (73), and p27 (t1/2 ~150 min) (52). The results in this report established that the C/EBPdelta protein is also degraded via the ubiquitin-proteasome pathway in growth-arrested MECs (t1/2 ~120 min). It has yet to be determined whether phosphorylation of the C/EBPdelta protein precedes ubiquitination, which has been observed in the regulation of p27 protein decay.

It has been known for sometime that mammalian proteasome complexes are localized throughout the cell including the nucleus, cytoplasm, and within the endoplasmic reticulum membrane network (55, 56). Proteasomes localized within the nucleus have been shown to be responsible for the turnover of short lived proteins important for many critical cellular processes. Some proteins that undergo ubiquitination within the nuclear compartment include the large subunit of RNA polymerase II (74), the progesterone receptor (75), the Xenopus laevis kinase inhibitor, p27Xic1 (76), and the p53 protein (57). It is speculated that cells are able to rid themselves of nuclear proteins that are no longer necessary by ubiquitination within the nucleus (57). Results of this study demonstrate that C/EBPdelta protein ubiquitination is localized to the nucleus. We speculate that nuclear protein degradation provides a mechanistic explanation for the relatively short half-life of the C/EBPdelta protein during MEC G0 growth arrest and allows for proper cell cycle progression during the G0/G1 transition.

In summary, the data presented establish that the C/EBPdelta mRNA has a short half-life in G0 growth-arrested MECs. The C/EBPdelta mRNA has a relatively short poly(A) tail (~100 nucleotides) that does not vary in length during decay in G0 growth arrest or the G0/G1 transition. It is proposed that the C/EBPdelta mRNA is degraded by a mechanism involving endonucleolytic cleavage during G0 growth arrest. Additionally, the C/EBPdelta protein has a relatively short half-life in G0 growth-arrested MECs and is degraded by the ubiquitin-proteasome pathway within the nuclear compartment. This study suggests that despite the decrease in cellular activity during G0 growth arrest, C/EBPdelta mRNA and protein are tightly regulated in MECs. We predict that this tight regulation allows G0 growth-arrested MECs to proliferate in response to growth stimuli. Studies investigating possible instability elements in the C/EBPdelta mRNA 3'-UTR and characterization of trans-acting factors important in C/EBPdelta mRNA degradation are currently underway.

    ACKNOWLEDGEMENTS

We thank Tim Vojt (Veterinary Technology Services) for figure preparation and Stacey Hull for critical reading of the manuscript. The Comprehensive Cancer Center at the Ohio State University, Columbus, OH, was the recipient of NCI Grant P30CA16058 from the National Institutes of Health.

    FOOTNOTES

* This work was supported in part by NCI Grant CA57607 (to J. D.) from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: Veterinary Biosciences, the Ohio State University, 1900 Coffey Rd., Columbus, OH 43210. Tel.: 614-292-4261; Fax: 614-292-7463; E-mail: dewille.1@osu.edu.

Published, JBC Papers in Press, January 27, 2003, DOI 10.1074/jbc.M207930200

    ABBREVIATIONS

The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; MEC, mammary epithelial cell; gas, growth arrest-specific; STAT, signal transducer and activator of transcription; ARE, A/U-rich element; UTR, untranslated region; bZIP, leucine zipper; RACE-PAT, rapid amplification of cDNA ends-poly(A) test; GAM, growth arrest medium; CGM, complete growth medium; LLnL, N-acetyl-Leu-Leu-Norleu-Al; Me2SO, dimethyl sulfoxide; PMSF, phenylmethylsulfonyl fluoride; DTT, dithiothreitol; DRB, 5,6-dichlorobenzimidazole 1-beta -D-ribofuranoside; cp, cyclophilin; BGH, bovine growth hormone.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Johnson, L. F., Levis, R., Abelson, H. T., Green, H., and Penman, S. (1976) J. Cell Biol. 71, 933-938[Abstract]
2. Johnson, L. F., Williams, J. G., Abelson, H. T., Green, H., and Penman, S. (1975) Cell 4, 69-75[Medline] [Order article via Infotrieve]
3. Hanahan, D., and Weinberg, R. A. (2000) Cell 100, 57-70[Medline] [Order article via Infotrieve]
4. Zhu, L., and Skoultchi, A. I. (2001) Curr. Opin. Genet. & Dev. 11, 91-97[CrossRef][Medline] [Order article via Infotrieve]
5. Smith, E. J., Leone, G., DeGregori, J., Jakoi, L., and Nevins, J. R. (1996) Mol. Cell. Biol. 16, 6965-6976[Abstract]
6. Pause, A., Lee, S., Lonergan, K. M., and Klausner, R. D. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 993-998[Abstract/Free Full Text]
7. Davidowitz, E. J., Schoenfeld, A. R., and Burk, R. D. (2001) Mol. Cell. Biol. 21, 865-874[Abstract/Free Full Text]
8. Gigliotti, A. P., and DeWille, J. W. (1999) Breast Cancer Res. Treat. 58, 57-63[CrossRef][Medline] [Order article via Infotrieve]
9. Gigliotti, A. P., and DeWille, J. W. (1998) J. Cell. Physiol. 174, 232-239[CrossRef][Medline] [Order article via Infotrieve]
10. Hutt, J. A., O'Rourke, J. P., and DeWille, J. (2000) J. Biol. Chem. 275, 29123-29131[Abstract/Free Full Text]
11. O'Rourke, J., Yuan, R., and DeWille, J. (1997) J. Biol. Chem. 272, 6291-6296[Abstract/Free Full Text]
12. O'Rourke, J. P., Hutt, J. A., and DeWille, J. (1999) Biochem. Biophys. Res. Commun. 262, 696-701[CrossRef][Medline] [Order article via Infotrieve]
13. O'Rourke, J. P., Newbound, G. C., Hutt, J. A., and DeWille, J. (1999) J. Biol. Chem. 274, 16582-16589[Abstract/Free Full Text]
14. Hurst, H. C. (1994) Protein Profile 1, 123-168[Medline] [Order article via Infotrieve]
15. Lekstrom-Himes, J., and Xanthopoulos, K. G. (1998) J. Biol. Chem. 273, 28545-28548[Abstract/Free Full Text]
16. Croniger, C., Leahy, P., Reshef, L., and Hanson, R. W. (1998) J. Biol. Chem. 273, 31629-31632[Free Full Text]
17. Sealy, L., Malone, D., and Pawlak, M. (1997) Mol. Cell. Biol. 17, 1744-1755[Abstract]
18. Jung, N., Yi, Y. W., Kim, D., Shong, M., Hong, S. S., Lee, H. S., and Bae, I. (2000) Eur. J. Biochem. 267, 6180-6187[Abstract/Free Full Text]
19. Doppler, W., Welte, T., and Philipp, S. (1995) J. Biol. Chem. 270, 17962-17969[Abstract/Free Full Text]
20. Cao, Z., Umek, R. M., and McKnight, S. L. (1991) Genes Dev. 5, 1538-1552[Abstract]
21. Ron, D., and Habener, J. F. (1992) Genes Dev. 6, 439-453[Abstract]
22. Robinson, G. W., Johnson, P. F., Hennighausen, L., and Sterneck, E. (1998) Genes Dev. 12, 1907-1916[Abstract/Free Full Text]
23. Sterneck, E., Tessarollo, L., and Johnson, P. F. (1997) Genes Dev. 11, 2153-2162[Abstract/Free Full Text]
24. Seagroves, T. N., Lydon, J. P., Hovey, R. C., Vonderhaar, B. K., and Rosen, J. M. (2000) Mol. Endocrinol. 14, 359-368[Abstract/Free Full Text]
25. Seagroves, T. N., Krnacik, S., Raught, B., Gay, J., Burgess-Beusse, B., Darlington, G. J., and Rosen, J. M. (1998) Genes Dev. 12, 1917-1928[Abstract/Free Full Text]
26. Ramji, D. P., and Foka, P. (2002) Biochem. J. 365, 561-575[Medline] [Order article via Infotrieve]
27. Yeh, W. C., Cao, Z., Classon, M., and McKnight, S. L. (1995) Genes Dev. 9, 168-181[Abstract]
28. Ferrini, J. B., Rodrigues, E., Dulic, V., Pichard-Garcia, L., Fabr, J. M., Blanc, P., and Maurel, P. (2001) J. Hepatol. 35, 170-177[CrossRef][Medline] [Order article via Infotrieve]
29. Wang, N. D., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112[Medline] [Order article via Infotrieve]
30. Zhang, P., Iwama, A., Datta, M. W., Darlington, G. J., Link, D. C., and Tenen, D. G. (1998) J. Exp. Med. 188, 1173-1184[Abstract/Free Full Text]
31. Dearth, L. R., Hutt, J., Sattler, A., Gigliotti, A., and DeWille, J. (2001) J. Cell. Biochem. 82, 357-370[CrossRef][Medline] [Order article via Infotrieve]
32. Yamanaka, R., Barlow, C., Lekstrom-Himes, J., Castilla, L. H., Liu, P. P., Eckhaus, M., Decker, T., Wynshaw-Boris, A., and Xanthopoulos, K. G. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13187-13192[Abstract/Free Full Text]
33. Nakajima, H., and Ihle, J. N. (2001) Blood 98, 897-905[Abstract/Free Full Text]
34. Ross, J. (1996) Trends Genet. 12, 171-175[CrossRef][Medline] [Order article via Infotrieve]
35. Guhaniyogi, J., and Brewer, G. (2001) Gene (Amst.) 265, 11-23[CrossRef][Medline] [Order article via Infotrieve]
36. Dice, J. F. (1987) FASEB J. 1, 349-357[Abstract/Free Full Text]
37. Wilkinson, K. D. (2000) Semin. Cell Dev. Biol. 11, 141-148[CrossRef][Medline] [Order article via Infotrieve]
38. Mitchell, P., and Tollervey, D. (2000) Curr. Opin. Genet. & Dev. 10, 193-198[CrossRef][Medline] [Order article via Infotrieve]
39. Ross, J. (1995) Microbiol. Rev. 59, 423-450[Abstract]
40. Jackman, J., Alamo, I., Jr., and Fornace, A. J., Jr. (1994) Cancer Res. 54, 5656-5662[Abstract]
41. Coccia, E. M., Cicala, C., Charlesworth, A., Ciccarelli, C., Rossi, G. B., Philipson, L., and Sorrentino, V. (1992) Mol. Cell. Biol. 12, 3514-3521[Abstract]
42. Dixon, D. A., Tolley, N. D., King, P. H., Nabors, L. B., McIntyre, T. M., Zimmerman, G. A., and Prescott, S. M. (2001) J. Clin. Invest. 108, 1657-1665[Abstract/Free Full Text]
43. Mendell, J. T., and Dietz, H. C. (2001) Cell 107, 411-414[Medline] [Order article via Infotrieve]
44. Baer, M. R., Augustinos, P., and Kinniburgh, A. J. (1992) Blood 79, 1319-1326[Abstract]
45. Touriol, C., Morillon, A., Gensac, M. C., Prats, H., and Prats, A. C. (1999) J. Biol. Chem. 274, 21402-21408[Abstract/Free Full Text]
46. Hochstrasser, M. (1995) Curr. Opin. Cell Biol. 7, 215-223[CrossRef][Medline] [Order article via Infotrieve]
47. Nakayama, K. I., Hatakeyama, S., and Nakayama, K. (2001) Biochem. Biophys. Res. Commun. 282, 853-860[CrossRef][Medline] [Order article via Infotrieve]
48. Pagano, M. (1997) FASEB J. 11, 1067-1075[Abstract/Free Full Text]
49. Conaway, R. C., Brower, C. S., and Conaway, J. W. (2002) Science 296, 1254-1258[Abstract/Free Full Text]
50. Hengst, L., and Reed, S. I. (1996) Science 271, 1861-1864[Abstract]
51. Shirane, M., Harumiya, Y., Ishida, N., Hirai, A., Miyamoto, C., Hatakeyama, S., Nakayama, K., and Kitagawa, M. (1999) J. Biol. Chem. 274, 13886-13893[Abstract/Free Full Text]
52. Pagano, M., Tam, S. W., Theodoras, A. M., Beer-Romero, P., Del Sal, G., Chau, V., Yew, P. R., Draetta, G. F., and Rolfe, M. (1995) Science 269, 682-685[Medline] [Order article via Infotrieve]
53. Tsvetkov, L. M., Yeh, K. H., Lee, S. J., Sun, H., and Zhang, H. (1999) Curr. Biol. 9, 661-664[CrossRef][Medline] [Order article via Infotrieve]
54. Appella, E., and Anderson, C. W. (2001) Eur. J. Biochem. 268, 2764-2772[Abstract/Free Full Text]
55. Tanaka, K., Yoshimura, T., Tamura, T., Fujiwara, T., Kumatori, A., and Ichihara, A. (1990) FEBS Lett. 271, 41-46[CrossRef][Medline] [Order article via Infotrieve]
56. Rivett, A. J. (1998) Curr. Opin. Immunol. 10, 110-114[CrossRef][Medline] [Order article via Infotrieve]
57. Shirangi, T. R., Zaika, A., and Moll, U. M. (2002) FASEB J. 16, 420-422[Abstract/Free Full Text]
58. Salles, F. J., Richards, W. G., and Strickland, S. (1999) Methods (Orlando) 17, 38-45[CrossRef]
59. Del Sal, G., Ruaro, M. E., Philipson, L., and Schneider, C. (1992) Cell 70, 595-607[Medline] [Order article via Infotrieve]
60. Schneider, C., King, R. M., and Philipson, L. (1988) Cell 54, 787-793[Medline] [Order article via Infotrieve]
61. Ciccarelli, C., Philipson, L., and Sorrentino, V. (1990) Mol. Cell. Biol. 10, 1525-1529[Medline] [Order article via Infotrieve]
62. Evdokiou, A., and Cowled, P. A. (1998) Exp. Cell Res. 240, 359-367[CrossRef][Medline] [Order article via Infotrieve]
63. Wilusz, C. J., Wormington, M., and Peltz, S. W. (2001) Nat. Rev. Mol. Cell. Biol. 2, 237-246[CrossRef][Medline] [Order article via Infotrieve]
64. Hausmann, G., O'Reilly, L. A., van Driel, R., Beaumont, J. G., Strasser, A., Adams, J. M., and Huang, D. C. (2000) J. Cell Biol. 149, 623-634[Abstract/Free Full Text]
65. Winzen, R., Kracht, M., Ritter, B., Wilhelm, A., Chen, C. Y., Shyu, A. B., Muller, M., Gaestel, M., Resch, K., and Holtmann, H. (1999) EMBO J. 18, 4969-4980[Abstract/Free Full Text]
66. Gonos, E. S. (1998) Ann. N. Y. Acad. Sci. 851, 466-469[Free Full Text]
67. Ferrero, M., Desiderio, M. A., Martinotti, A., Melani, C., Bernelli-Zazzera, A., Colombo, M. P., and Cairo, G. (1994) J. Cell. Physiol. 158, 263-269[Medline] [Order article via Infotrieve]
68. Bosse, F., Brodbeck, J., and Muller, H. W. (1999) J. Neurosci. Res. 55, 164-177[CrossRef][Medline] [Order article via Infotrieve]
69. Gu, H., Das, G. J., and Schoenberg, D. R. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 8943-8948[Abstract/Free Full Text]
70. Chen, C. Y., and Shyu, A. B. (1995) Trends Biochem. Sci. 20, 465-470[CrossRef][Medline] [Order article via Infotrieve]
71. Wang, Y., Liu, C. L., Storey, J. D., Tibshirani, R. J., Herschlag, D., and Brown, P. O. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5860-5865[Abstract/Free Full Text]
72. Sheaff, R. J., Singer, J. D., Swanger, J., Smitherman, M., Roberts, J. M., and Clurman, B. E. (2000) Mol. Cell 5, 403-410[Medline] [Order article via Infotrieve]
73. Zilfou, J. T., Hoffman, W. H., Sank, M., George, D. L., and Murphy, M. (2001) Mol. Cell. Biol. 21, 3974-3985[Abstract/Free Full Text]
74. Lee, K. B., Wang, D., Lippard, S. J., and Sharp, P. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 4239-4244[Abstract/Free Full Text]
75. Dennis, A. P., Haq, R. U., and Nawaz, Z. (2001) Front. Biosci. 6, D954-D959[Medline] [Order article via Infotrieve]
76. Chuang, L. C., and Yew, P. R. (2001) J. Biol. Chem. 276, 1610-1617[Abstract/Free Full Text]


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