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. 2000 Aug 1;14(15):1920-32.

Translational control of C/EBPalpha and C/EBPbeta isoform expression

Affiliations

Translational control of C/EBPalpha and C/EBPbeta isoform expression

C F Calkhoven et al. Genes Dev. .

Abstract

Transcription factors derived from CCAAT/enhancer binding protein (C/EBP)alpha and C/EBPbeta genes control differentiation and proliferation in a number of cell types. Various C/EBP isoforms arise from unique C/EBPbeta and C/EBPalpha mRNAs by differential initiation of translation. These isoforms retain different parts of the amino terminus and therefore display different functions in gene regulation and proliferation control. We show that PKR and mTOR signaling pathways control the ratio of C/EBP isoform expression through the eukaryotic translation initiation factors eIF-2alpha and eIF-4E, respectively. An evolutionary conserved upstream open reading frame in C/EBPalpha and C/EBPbeta mRNAs is a prerequisite for regulated initiation from the different translation initiation sites and integrates translation factor activity. Deregulated translational control leading to aberrant C/EBPalpha and C/EBPbeta isoform expression or ectopic expression of truncated isoforms disrupts terminal differentiation and induces a transformed phenotype in 3T3-L1 cells. Our results demonstrate that the translational controlled ratio of C/EBPalpha and C/EBPbeta isoform expression determines cell fate.

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Figures

Figure 1
Figure 1
Representation of the vertebrate C/EBPα and C/EBPβ mRNA structure and comparison of the potential translation initiation sites. (Top) Shaded areas indicate the C/EBP coding region. The potential translation initiation sites are designated A, B1, B2, and C. The solid box represents the small uORF initiated at site D, which is out of frame with respect to the C/EBP reading frame. (Below) Shading indicates critical nucleotides at position −3 and +4 corresponding to the optimal Kozak translation initiation consensus sequence (bottom).
Figure 2
Figure 2
Identification of C/EBPα and C/EBPβ translation initiation sites. Schematic representations of the C/EBP mRNAs with the initiation sites indicated in relation to the protein bands are shown at left. Wild-type cDNA (wt), constructs that lack distinct translation initiation codons (ΔA, ΔB1, ΔB2, ΔC, and ΔD) or an amino-terminal deletion construct (C) were transiently transfected in COS-1 cells. C/EBP protein expression was analyzed by immunoblotting of total cell extracts: (a) rC/EBPα; (b) cC/EBPα; (c) rC/EBPβ; (d) cC/EBPβ.
Figure 3
Figure 3
Translation of truncated C/EBPα and C/EBPβ proteins from downstream initiation sites depends on the uORF. Schematic representations of the mRNAs with potential initiation sites indicated in relation to the protein bands are shown at left. COS-1 cells were transiently transfected with C/EBP wild-type (wt) and mutant constructs and analyzed for C/EBP protein expression by immunoblotting. In ΔD mutants, the uORF translation initiation site is mutated to a noninitiation site. In the Aopt mutant, the sequence context of site A is mutated to an optimal Kozak sequence. The X mutants harbor an additional, optimal translation initiation site upstream of site C. (a) rC/EBPα; (b) cC/EBPα; (c) rC/EBPβ; (d) cC/EBPβ.
Figure 4
Figure 4
The uORF regulates translation from downstream initiation sites. Schematic representations of the mRNAs are shown at left. (a) The C/EBPα isoform ratio is modified by the efficiency of the uORF translation. The wild-type suboptimal sequence context of site D (wt) was placed in an optimal Kozak consensus sequence (Dopt) or in a non-Kozak context (d). Initiation site sequences are shown at right. (b) cC/EBPα and C/EBPβ 5′ UTRs were replaced by a 50-bp sequence of the β-globin leader, and the 3′ UTRs were deleted. The uORF translation initiation site D was left intact (–) or was mutated to a noninitiation site (ΔD). (c) The Flag-tagged MyoD coding region was placed downstream of a 5′ β-globin leader sequence and the rC/EBPα uORF sequence (D) or the uORF initiation site mutant (ΔD) as indicated. All constructs were transiently transfected in COS-1 cells. C/EBP or MyoD protein expression was analyzed by immunoblotting.
Figure 5
Figure 5
The ratio of C/EBPα and C/EBPβ protein isoform expression is modulated by translation initiation factor activity. (a) 3T3-L1 cells ectopically expressing PKRΔ6, eIF-2αS52A, eIF-4E, and control cells (empty vector) were induced to undergo adipogenesis and analyzed for expression of endogenous C/EBPα and C/EBPβ isoforms at day 4 of the differentiation protocol by immunoblotting. (Fl) Full-length isoform; (Tr) truncated isoform. Ratios of full-length vs. truncated C/EBP isoforms of three independent experiments are depicted below; immunoblots of experiment 1 are shown. (b) PKRΔ6, eIF-2αSA, and eIF-4E transgene expression were analyzed by immunoblotting in transgene (+) and control (–) cells. (c) 3T3-L1 cells at day 4 of differentiation were treated with 5 mm 2-aminopurine (+2-AP) or 1 μm rapamycin (+Rap) for 12 hr and compared with control cultures for rC/EBPα protein expression. Bottom panels show protein expression of endogenous PKR and 4E-BP1. The respective hyperphosphorylated forms are indicated (-P).
Figure 6
Figure 6
The uORF is essential for the modulation of C/EBP isoform expression through eIF activity in 3T3-L1 cells. (a) Undifferentiated 3T3-L1 control cells and cells expressing eIF-2αS52A or eIF-4E were transiently transfected with wild-type rC/EBPα expression constructs (wt) or constructs with mutated uORF initiation site (ΔD). Cell extracts were analyzed for rC/EBPα expression by immunoblotting. (Fl) Full-length isoform; (Tr) truncated isoform. Ratios of full-length vs. truncated C/EBP isoforms of two independent experiments are depicted below; immunoblots of experiment 1 are shown. (b) A construct expressing a uORF–rC/EBPα fusion protein was cotransfected with an eIF-4E expression construct or empty vector (–) in COS-1 cells. uORF–rC/EBPα fusion protein expression was analyzed by immunoblotting of total cell extracts. A schematic representation of the mRNA is shown at left.
Figure 7
Figure 7
Constitutive ectopic expression of truncated C/EBPα or C/EBPβ induces cellular transformation in differentiating 3T3-L1 cells. (a) 3T3-L1 control cells and 3T3-L1 ectopically expressing truncated rC/EBPα or truncated rC/EBPβ were induced to undergo adipogenesis. At day 8 of differentiation cells were fixed and stained with Oil Red O (lipid staining). Microscopic pictures of different magnifications (as indicated) are shown. In parallel experiments the cells were incubated for 8 hr with BrdU, fixed and stained by immunohistochemistry (right). (b) Ectopic and endogenous expression of C/EBP protein isoforms was analyzed by immunoblotting. (Fl) Full-length isoform; (Tr) truncated isoform.
Figure 8
Figure 8
Translation initiation factor activity determines cell fate through modulation of the C/EBP isoform ratio.

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