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CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

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Cells were transfected with the control expression vector (Sham), the transcriptionally active C/EBPα-GFP expression vector or the transcriptionally inactive GFP-C/EBPα expression vector and treated one day later with A, nocodazole or B, mimosine. Cells from the transfections with the C/EBP-GFP and GFP-C/EBP expression vectors were separated into cells with green fluorescence above background levels (C/EBP-GFP and GFP-C/EBP) or at background (No C/EBP). The proportion of cells in the G1, S and G2/M phases were plotted as the mean +/- standard deviation from A, six or B, three independent experiments. Statistically significant differences in the proportion of cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (one-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Figure 4: Cells were transfected with the control expression vector (Sham), the transcriptionally active C/EBPα-GFP expression vector or the transcriptionally inactive GFP-C/EBPα expression vector and treated one day later with A, nocodazole or B, mimosine. Cells from the transfections with the C/EBP-GFP and GFP-C/EBP expression vectors were separated into cells with green fluorescence above background levels (C/EBP-GFP and GFP-C/EBP) or at background (No C/EBP). The proportion of cells in the G1, S and G2/M phases were plotted as the mean +/- standard deviation from A, six or B, three independent experiments. Statistically significant differences in the proportion of cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (one-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).

Mentions: We determined if either C/EBPα-GFP or GFP-C/EBPα altered the distribution of GHFT1-5 cells in the G1, S or G2/M phases of the cell cycle. GHFT1-5 cells were transfected with the expression vectors for C/EBPα-GFP or GFP-C/EBPα, or with the sham expression vector not containing the C/EBPα cDNA. The cells were incubated for one day to allow time for C/EBPα expression. Cells were then synchronized in M-phase or in G1/S by 20-hour incubations with nocodazole (Fig. 4A) or mimosine (Fig. 4B). Cells were collected and stained with propidium iodide. Flow cytometry was used to measure DNA content in 1) cells transfected with the sham expression vector (Fig. 4, Sham), 2) green fluorescent cells expressing GFP-tagged C/EBPα (Fig. 4, C/EBPα-GFP or GFP-C/EBPα) or 3) the subpopulation of cells from the C/EBPα-GFP or GFP-C/EBPα transfections that did not express measurable amounts of green fluorescent C/EBPα (Fig. 4, No C/EBP). The "No C/EBP" cells represent an internal control for cells not expressing C/EBPα collected simultaneously with cells expressing C/EBPα. The No C/EBP controls also indicated the extent to which green fluorescent, C/EBPα-expressing cells were distinguished from non-expressing cells by flow cytometry. For all our experiments, the distribution of cells in the G1, S and G2/M phases was similar for the "No C/EBPα " and sham-transfected cells.


CCAAT/enhancer binding protein alpha uses distinct domains to prolong pituitary cells in the growth 1 and DNA synthesis phases of the cell cycle.

Liu W, Enwright JF, Hyun W, Day RN, Schaufele F - BMC Cell Biol. (2002)

Cells were transfected with the control expression vector (Sham), the transcriptionally active C/EBPα-GFP expression vector or the transcriptionally inactive GFP-C/EBPα expression vector and treated one day later with A, nocodazole or B, mimosine. Cells from the transfections with the C/EBP-GFP and GFP-C/EBP expression vectors were separated into cells with green fluorescence above background levels (C/EBP-GFP and GFP-C/EBP) or at background (No C/EBP). The proportion of cells in the G1, S and G2/M phases were plotted as the mean +/- standard deviation from A, six or B, three independent experiments. Statistically significant differences in the proportion of cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (one-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
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Related In: Results  -  Collection

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Figure 4: Cells were transfected with the control expression vector (Sham), the transcriptionally active C/EBPα-GFP expression vector or the transcriptionally inactive GFP-C/EBPα expression vector and treated one day later with A, nocodazole or B, mimosine. Cells from the transfections with the C/EBP-GFP and GFP-C/EBP expression vectors were separated into cells with green fluorescence above background levels (C/EBP-GFP and GFP-C/EBP) or at background (No C/EBP). The proportion of cells in the G1, S and G2/M phases were plotted as the mean +/- standard deviation from A, six or B, three independent experiments. Statistically significant differences in the proportion of cells in G1, S or G2/M, relative to the proportions determined for the sham-transfected cells, are indicated (one-way ANOVA: **, p < 0.01; *p < 0.05; -, no difference).
Mentions: We determined if either C/EBPα-GFP or GFP-C/EBPα altered the distribution of GHFT1-5 cells in the G1, S or G2/M phases of the cell cycle. GHFT1-5 cells were transfected with the expression vectors for C/EBPα-GFP or GFP-C/EBPα, or with the sham expression vector not containing the C/EBPα cDNA. The cells were incubated for one day to allow time for C/EBPα expression. Cells were then synchronized in M-phase or in G1/S by 20-hour incubations with nocodazole (Fig. 4A) or mimosine (Fig. 4B). Cells were collected and stained with propidium iodide. Flow cytometry was used to measure DNA content in 1) cells transfected with the sham expression vector (Fig. 4, Sham), 2) green fluorescent cells expressing GFP-tagged C/EBPα (Fig. 4, C/EBPα-GFP or GFP-C/EBPα) or 3) the subpopulation of cells from the C/EBPα-GFP or GFP-C/EBPα transfections that did not express measurable amounts of green fluorescent C/EBPα (Fig. 4, No C/EBP). The "No C/EBP" cells represent an internal control for cells not expressing C/EBPα collected simultaneously with cells expressing C/EBPα. The No C/EBP controls also indicated the extent to which green fluorescent, C/EBPα-expressing cells were distinguished from non-expressing cells by flow cytometry. For all our experiments, the distribution of cells in the G1, S and G2/M phases was similar for the "No C/EBPα " and sham-transfected cells.

Bottom Line: C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells.Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Metabolic Research Unit, Diabetes Research Center and Department of Medicine, University of California, San Francisco, CA 94143-0540, USA. liu_weiqun@hotmail.com

ABSTRACT

Background: A number of transcription factors coordinate differentiation by simultaneously regulating gene expression and cell proliferation. CCAAT/enhancer binding protein alpha (C/EBPalpha) is a basic/leucine zipper transcription factor that integrates transcription with proliferation to regulate the differentiation of tissues involved in energy balance. In the pituitary, C/EBPalpha regulates the transcription of a key metabolic regulator, growth hormone.

Results: We examined the consequences of C/EBPalpha expression on proliferation of the transformed, mouse GHFT1-5 pituitary progenitor cell line. In contrast to mature pituitary cells, GHFT1-5 cells do not contain C/EBPalpha. Ectopic expression of C/EBPalpha in the progenitor cells resulted in prolongation of both growth 1 (G1) and the DNA synthesis (S) phases of the cell cycle. Transcription activation domain 1 and 2 of C/EBPalpha were required for prolongation of G1, but not of S. Some transcriptionally inactive derivatives of C/EBPalpha remained competent for G1 and S phase prolongation. C/EBPalpha deleted of its leucine zipper dimerization functions was as effective as full-length C/EBPalpha in prolonging G1 and S.

Conclusion: We found that C/EBPalpha utilizes mechanistically distinct activities to prolong the cell cycle in G1 and S in pituitary progenitor cells. G1 and S phase prolongation did not require that C/EBPalpha remained transcriptionally active or retained the ability to dimerize via the leucine zipper. G1, but not S, arrest required a domain overlapping with C/EBPalpha transcription activation functions 1 and 2. Separation of mechanisms governing proliferation and transcription permits C/EBPalpha to regulate gene expression independently of its effects on proliferation.

Show MeSH
Related in: MedlinePlus