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. 2013 Aug;33(16):3242-58.
doi: 10.1128/MCB.01674-12. Epub 2013 Jun 17.

C/EBPγ suppresses senescence and inflammatory gene expression by heterodimerizing with C/EBPβ

Christopher J Huggins  1 , Radek Malik, Sook Lee, Jacqueline Salotti, Sara Thomas, Nancy Martin, Octavio A Quiñones, W Gregory Alvord, Mary E Olanich, Jonathan R Keller, Peter F Johnson
Affiliations

VSports手机版 - C/EBPγ suppresses senescence and inflammatory gene expression by heterodimerizing with C/EBPβ

Christopher J Huggins et al. Mol Cell Biol. 2013 Aug.

Abstract

C/EBPβ is an important regulator of oncogene-induced senescence (OIS). Here, we show that C/EBPγ, a heterodimeric partner of C/EBPβ whose biological functions are not well understood, inhibits cellular senescence VSports手机版. Cebpg(-/-) mouse embryonic fibroblasts (MEFs) proliferated poorly, entered senescence prematurely, and expressed a proinflammatory gene signature, including elevated levels of senescence-associated secretory phenotype (SASP) genes whose induction by oncogenic stress requires C/EBPβ. The senescence-suppressing activity of C/EBPγ required its ability to heterodimerize with C/EBPβ. Covalently linked C/EBPβ homodimers (β∼β) inhibited the proliferation and tumorigenicity of Ras(V12)-transformed NIH 3T3 cells, activated SASP gene expression, and recruited the CBP coactivator in a Ras-dependent manner, whereas γ∼β heterodimers lacked these capabilities and efficiently rescued proliferation of Cebpg(-/-) MEFs. C/EBPβ depletion partially restored growth of C/EBPγ-deficient cells, indicating that the increased levels of C/EBPβ homodimers in Cebpg(-/-) MEFs inhibit proliferation. The proliferative functions of C/EBPγ are not restricted to fibroblasts, as hematopoietic progenitors from Cebpg(-/-) bone marrow also displayed impaired growth. Furthermore, high CEBPG expression correlated with poorer clinical prognoses in several human cancers, and C/EBPγ depletion decreased proliferation and induced senescence in lung tumor cells. Our findings demonstrate that C/EBPγ neutralizes the cytostatic activity of C/EBPβ through heterodimerization, which prevents senescence and suppresses basal transcription of SASP genes. .

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Figures

Fig 1
Fig 1
CEBPγ is required for efficient cellular proliferation. (A) Expression of C/EBPβ but not C/EBPγ increases G1 arrest in HepG2 cells and L929 fibroblasts. Cells were transfected with CD20 expression vector and C/EBPβ, C/EBPγ, or C/EBPβ and C/EBPγ, harvested after 24 h, and stained with propidium iodide. CD20-positive cells were analyzed for DNA content. Graphs (left panels) show the effects of C/EBPβ overexpression. Average percent changes are shown on the right; data are mean values ± SEM from five experiments. (B) WT, Cebpg−/−, and Cebpb−/− fibroblasts (MEFs) were isolated from E13.5 embryos and analyzed for colony growth. C/EBPβ and C/EBPγ protein levels were determined (right). Bands corresponding to the three C/EBPβ translational isoforms (LAP*, LAP, and LIP) are indicated. (C) Proliferation of WT, Cebpg+/−, and Cebpg−/− MEFs was measured over 6 days. The data are mean values ± SEM from at least 10 independent MEF preparations, each assayed in triplicate. (D) Proliferation rates for WT and Cebpg−/− MEFs, without or with ectopic CEBPγ. The data are mean values ± SEM from at least 5 MEF isolates, assayed in triplicate. Right, C/EBPγ Western blot. Recombinant C/EBPγ is HA tagged and therefore migrates more slowly. (E) Long-term proliferative capacity of WT, Cebpg+/−, and Cebpg−/− MEFs. Cells were propagated on a 3T3 protocol over 8 passages. The data are mean values ± SEM from 4 isolates of each genotype assayed in triplicate.
Fig 2
Fig 2
C/EBPγ is necessary for normal cell cycle progression. (A) WT and Cebpg−/− MEFs were synchronized by serum starvation, stimulated with serum, and labeled with BrdU. The percentage of cells in S phase after 18 h is shown. (B) Time course of cell cycle entry in WT and Cebpg−/− MEFs. Cells were serum starved, restimulated, and pulse-labeled with BrdU at the indicated times. Data are averages ± SEM from two experiments. (C) Expression of C/EBPγ or HPV oncoprotein E7 partially rescues delayed S-phase entry in Cebpg−/− MEFs. C/EBPγ and E7 were expressed in WT or Cebpg−/− MEFs, and the cells were analyzed as described above. Data are averages ± SEM from two experiments. (D) Cyclins D1, A, and E are reduced in Cebpg−/− MEFs. Lysates from serum-stimulated MEFs analyzed by Western blotting for CDKs (Cdk1/2/3/6), cyclins (A/B1/D1/E), and CDK inhibitors (p21/p27). (E to G) E7 and, to a lesser extent, cyclin D1 and c-Myc rescue growth of Cebpg−/− MEFs. WT and Cebpg−/− MEFs were transduced with vectors for E7 (E), cyclin D1 (F), or c-Myc (G) and proliferation was measured. Data are averages ± SEM from at least two experiments performed in triplicate.
Fig 3
Fig 3
Increased senescence in Cebpg−/− MEFs. (A) Morphology of WT and Cebpg−/− cells 6 days after being plated. (B) Increased SA–β-Gal expression in Cebpg−/− MEFs. The frequency of SA–β-Gal-positive cells is shown. Data are the mean values ± SEM from 2 experiments. (C) Expression of cell cycle inhibitors in WT and Cebpg−/− MEFs. Left, p16Ink4a and p19Arf protein levels at passages 2 to 4. A lysate from p16−/−/p19−/− MEFs is shown as a negative control (lane 1). Right, p15Ink4b and p18Ink4c protein levels in WT, Cebpb−/−, and Cebpg−/− MEFs at passage 2. The apparent increase in p18Ink4c expression in Cebpg−/− cells was not consistently observed.
Fig 4
Fig 4
Cebpg−/− MEFs display a proinflammatory gene signature and enhanced expression of SASP genes. (A) Analysis of genes positively regulated by C/EBPβ and negatively regulated by C/EBPγ. Gene lists were generated from expression array comparisons: Cebpg−/− versus WT MEFs (increased expression; >+1.5-fold; P < 0.05), Cebpb−/−/RasV12 versus WT/RasV12 MEFs (decreased expression; <−1.5-fold; P = 0.05), and C/EBPβ-3T3RasV12 cells versus 3T3RasV12 cells (increased expression; >+1.5-fold; P < 0.05). NIH 3T3 data are from Basu et al. (8). Venn diagram depicts the intersections of these groups. The triple intersect represents genes that are positively regulated by C/EBPβ and inhibited by C/EBPγ. (B) GeneGo pathway and disease associations of the tri-intersect gene set. (C) Expression of representative SASP genes in WT and Cebpg−/− MEFs. mRNA levels were determined by qPCR; data are mean values ± SEM from 2 triplicate measurements from 2 independent MEF isolates. (D) SASP gene expression is induced by oncogenic RasV12 in WT but not Cebpb−/− MEFs. RNA isolated from control and RasV12-expressing MEFs was analyzed for SASP gene expression by qPCR. The data are mean values ± SEM from 2 triplicate measurements from 2 samples each. (E) Effect of C/EBPγ deficiency on expression of C/EBPβ target genes involved in tumor suppression or cell proliferation (8). The indicated genes were analyzed in WT and Cebpg−/− MEFs. Data are the means ± SEM from two experiments, assayed in triplicate.
Fig 5
Fig 5
C/EBPγ stimulates cell proliferation by heterodimerizing with C/EBPβ. (A) EMSA was performed using a C/EBP consensus site probe and nuclear extracts from WT and Cebpg−/− MEFs (lanes 1 to 7). Antibody supershifts were performed to identify specific dimeric complexes. EMSAs were compared to those generated from cells overexpressing the indicated C/EBP proteins (lanes 8 to 14). The identities of C/EBP complexes are indicated. UT, untransfected cells; NRS, normal rabbit serum. (B) Domain structures of CEBPβ (LAP and LIP), C/EBPγ, and C/EBPγ-GLZ. LAP is the major C/EBPβ isoform and is expressed from the second in-frame AUG codon; LIP is a truncated translational isoform (34). C/EBPγ-GLZ contains the GCN4 leucine zipper. TAD, transactivation domain; REG, regulatory domain; BR, basic region; LZ, leucine zipper. (C) Proliferation assays of Cebpg−/− MEFs expressing C/EBPγ, LIP, or C/EBPγ-GLZ. The data are mean values ± SEM from at least 4 experiments assayed in triplicate. (D) Colony assays. The cells described in the legend to panel C were plated at a density of 5 × 104 cells/dish and stained after 2 weeks. (E) The same populations were analyzed for senescent (SA–β-Gal-positive) cells (±standard deviation [SD]) from a representative experiment (>400 cells analyzed).
Fig 6
Fig 6
Opposing effects of C/EBPβ (LAP) homodimers and C/EBPγ:C/EBPβ heterodimers on cell proliferation. (A) Diagram of β∼β, γ∼β, and γ∼γ tethered dimers. Proteins are HA tagged, and internal in-frame AUG codons were deleted. (B) DNA binding of tethered dimers. Proteins were expressed in 293T cells ± RasV12 and cell extracts were subjected to EMSA. Protein levels were determined by HA Western blot. (C) Effects of linked dimers on proliferation of MEFs. Cebpb−/− or Cebpb−/−/RasV12 MEFs were infected with retroviruses encoding the indicated proteins, and cell proliferation was assayed. Data are the mean values ± SEM from 2 experiments assayed in triplicate. Right, C/EBP protein levels in transduced Cebpb−/−/RasV12 MEFs. The blot was probed with an HA antibody. The low levels of monomeric C/EBPβ and C/EBPγ in cells expressing tethered dimers result from partial proteolytic cleavage.
Fig 7
Fig 7
C/EBPβ homodimers inhibit growth and RasV12-induced transformation in NIH 3T3 cells. (A) Proliferation rates were determined for cells expressing the indicated C/EBP proteins. Right, C/EBP protein levels. The cells were plated for focus assays (B) or soft agar colony growth assays (C). Data are the mean values ± SEM from 3 experiments, each assayed in duplicate. (D) SASP gene expression in 3T3RasV12 cells expressing C/EBPβ, C/EBPγ, or linked dimers. Data are the mean values ± SEM from 2 measurements (performed in triplicate) from 2 independent samples.
Fig 8
Fig 8
C/EBPβ homodimers recruit the CBP coactivator. (A) C/EBPβ homodimers but not γ∼β heterodimers bind to CBP in a RasV12-dependent manner. C/EBP proteins were coexpressed with a Flag-tagged C-terminal fragment of CBP containing the TAZ2 domain (CBP1680-2441) in 293T cells, without or with RasV12. Cell lysates were immunoprecipitated with Flag antibody and analyzed for C/EBPβ. (B) Association of C/EBPβ, C/EBPγ, and CBP with SASP gene promoters in MEFs. ChIP assays were performed using control and RasV12-expressing cells; qPCR was performed with primers spanning C/EBP binding sites in the Il6, Il1a, and Il1b promoters (see materials and methods in the supplemental material). Binding was normalized to nonspecific (NS) antibody values. Data are from a representative experiment assayed in triplicate. (C) Increased binding of C/EBPβ to SASP gene promoters in Cebpg−/− MEFs. ChIP was performed as described for panel B. (D) RasV12-induced recruitment of CBP to SASP gene promoters requires C/EBPβ. CBP ChIP was performed on WT, Cebpb−/−, and Cebpg−/− MEFs. For each gene, the values are normalized to CBP binding in WT control cells (without RasV12). RasV12-expressing Cebpg−/− cells were not included in this experiment since they are undergoing spontaneous senescence, and the objective was to explain increased basal expression of SASP genes in the mutant cells.
Fig 9
Fig 9
C/EBPβ depletion partially rescues the proliferation defect in Cebpg−/− MEFs. (A) Proliferation assays of Cebpb; Cebpg double-knockout MEFs. MEFs from embryos representing all 9 possible genotypes were seeded at 25,000 cells/well, and proliferation was measured over 6 days. Cebpb−/−; Cebpg−/− cells underwent rapid apoptosis after plating, and nearly all cells died during growth assays. (B) Partial depletion of C/EBPβ restores growth of Cebpg−/− MEFs. WT and mutant cells were infected with two different C/EBPβ shRNA retroviruses. Cells were seeded at 106 cells/plate, and proliferation was assessed by staining after 7 days. Bottom, C/EBPβ Western blot. (C) C/EBPβ knockdown abrogates the increased senescence seen in Cebpg−/− MEFs. SA–β-Gal assays were performed on the cells described for panel B. The data are mean values ± SD (>400 cells were analyzed). (D) Elevated expression of Cxcl1 and Cxcl2 in Cebpg−/− cells requires C/EBPβ. Expression was analyzed by qPCR; data are the mean values ± SEM from 2 replicate measurements assayed in triplicate.
Fig 10
Fig 10
Expression of the γ∼β dimer rescues proliferation and inhibits senescence of Cebpg−/− MEFs. WT and Cebpg−/− cells were infected with retroviruses expressing C/EBPγ or γ∼β and analyzed for proliferation (A), colony formation (B), and senescence (SA–β-Gal staining) (C). Proliferation data are mean values ± SD from a representative experiment assayed in triplicate. (D) Ectopic C/EBPγ and γ∼β expression in Cebpg−/− MEFs. The graph shows Cebpg reverse transcription qPCR assays of RNA from retrovirally infected Cebpg−/− cells. A weak signal is observed in control Cebpg−/− cells because the primers span the Neo insert in the knockout allele (16). (E) Ectopic C/EBP protein expression in WT and Cebpg−/− MEFs. Top, EMSA of nuclear extracts from the cells described for panel D and for Fig. 5C. LIP DNA-binding activity is not observed in Cebpg−/− cells (lane 9), supporting observations from our laboratory that LIP is autoinhibited (25) and can be activated by coexpression of C/EBPγ (unpublished data). Bottom, C/EBPβ Western blot (WB) of the same extracts. The LIP species detected in lanes 3 and 8 is apparently a proteolytic product from the γ∼β dimer. CRM, cross-reacting material.
Fig 11
Fig 11
C/EBPγ is required for proliferation of murine hematopoietic cells and human cancer cells. (A, B) Cell proliferation and CFU-c assays of WT and Cebpg−/− bone marrow cells. Bone marrow was isolated from adult mice on a mixed C57BL/6-129Sv background. The cells were incubated with the indicated combinations of hematopoietic growth factors and evaluated for proliferation (EdU incorporation assays) (A) and colony growth (1.5 × 104 cells/plate) (B). (C) C/EBPγ expression is negatively associated with relapse-free survival of lung adenocarcinoma patients. Graph shows Kaplan-Meier relapse-free survival from a study of lung adenocarcinoma patients (47) stratified according to high (n = 130) and low (n = 74) CEBPG expression in tumors. Dotted lines indicate 95% confidence intervals. Meta-analysis of microarray data are from PrognoScan (26). (D) C/EBPγ depletion impairs proliferation of human A549 lung adenocarcinoma cells. C/EBPγ was ablated by two independent shRNAs (1 and 2), and colony growth was analyzed. Data are the mean values ± SEM from 2 experiments. C/EBPγ mRNA levels are shown in graphs on the right. (E) Increased senescence in C/EBPγ-depleted A549 cells. Four days after plating, cells were stained for SA–β-Gal, and senescent cells were visualized (upper panels) and counted (bottom panels). Data are mean values ± SEM from 2 experiments. (F) Differential regulatory properties of C/EBPβ homo- and heterodimers. In proliferating cells and in the absence of an activating signal such as oncogenic Ras, C/EBPβ occurs predominantly as a heterodimer with C/EBPγ. β:γ heterodimers bind to target C/EBP sites in SASP gene promoters but are transcriptionally inactive. In cells undergoing Ras-induced senescence, p90RSK-mediated phosphorylation on Ser273 in the C/EBPβ leucine zipper stabilizes the LZ interaction and promotes formation of β:β homodimers (15). Activated homodimers are capable of binding p300/CBP, inducing cell cycle arrest, and activating transcription of SASP genes. In contrast, β:γ heterodimers do not recruit p300/CBP and promote cell proliferation and cancer.
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