3′,5′-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line

Paul I. Woloshin, Kevin M. Walton, Robert P. Rehfuss, Richard Goodman, Roger D. Cone

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Abstract

The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells transfected with wild type CREB. These experiments demonstrate that the CREB family of transcription factors is involved in both the control of growth and differentiated phenotype in the thyroid follicular cell. The regulation of cell growth by CREB appears to be a tissue-specific event since neither CREB nor KCREB had any effect on BALB/c 3T3 fibroblast cell growth. In contrast to its action on iodide uptake, KREB did not alter TSH-stimulated thyroglobulin mRNA levels, demonstrating that while cAMP regulates most aspects of the differentiated thyroid phenotype, only some are controlled specifically by the CREB family of transcription factors.

Original languageEnglish (US)
Pages (from-to)1725-1733
Number of pages9
JournalMolecular Endocrinology
Volume6
Issue number10
StatePublished - 1992

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Cyclic AMP
Phenotype
Cell Line
Growth
Clone Cells
Iodides
Messenger RNA
Thyroid Gland
Transcription Factors
Thyroglobulin
BALB 3T3 Cells
Thyroid Epithelial Cells
Chloramphenicol O-Acetyltransferase
Gene Fusion
Somatostatin
Thymidine
Transfection
Cell Cycle
Carrier Proteins
Fibroblasts

ASJC Scopus subject areas

  • Molecular Biology
  • Endocrinology, Diabetes and Metabolism

Cite this

3′,5′-cyclic adenosine monophosphate-regulated enhancer binding (CREB) activity is required for normal growth and differentiated phenotype in the FRTL5 thyroid follicular cell line. / Woloshin, Paul I.; Walton, Kevin M.; Rehfuss, Robert P.; Goodman, Richard; Cone, Roger D.

In: Molecular Endocrinology, Vol. 6, No. 10, 1992, p. 1725-1733.

Research output: Contribution to journalArticle

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abstract = "The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60{\%} reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40{\%} reduction in TSH-stimulated thymidine incorporation, a 31{\%} increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells transfected with wild type CREB. These experiments demonstrate that the CREB family of transcription factors is involved in both the control of growth and differentiated phenotype in the thyroid follicular cell. The regulation of cell growth by CREB appears to be a tissue-specific event since neither CREB nor KCREB had any effect on BALB/c 3T3 fibroblast cell growth. In contrast to its action on iodide uptake, KREB did not alter TSH-stimulated thyroglobulin mRNA levels, demonstrating that while cAMP regulates most aspects of the differentiated thyroid phenotype, only some are controlled specifically by the CREB family of transcription factors.",
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AU - Walton, Kevin M.

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AU - Cone, Roger D.

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N2 - The thyroid follicular cell requires elevated levels of cAMP for normal growth and optimal expression of the differentiated phenotype. The recent discovery of cAMP-regulated enhancer binding (CREB) proteins prompted us to analyze the possible role of these transcription factors in controlling thyroid cell growth and differentiated phenotype using the FRTL5 thyroid cell line as a model system. FRTL5 cells were stably transfected with an expression vector containing either the gene for wild type CREB (WTCREB) or a dominant negative mutant form of CREB, termed KCREB, which dimerizes with and inactivates endogenous CREB. Transfected clones were found to express the transfected KCREB and WTCREB mRNAs at higher levels than the endogenous CREB mRNA. Transient expression of a somatostatin-chloramphenicol acetyltransferase fusion gene in these clones demonstrated a 60% reduction of cAMP-regulated enhancer-dependent transcriptional activity in the KCREB transfected clones and wild type levels of activity in the WTCREB transfected clones. Parameters of growth (DNA synthesis and growth rate) and differentiation (iodide uptake and thyroglobulin mRNA levels) were then analyzed in the transfected clones. Transfection of WTCREB had no effect on any of the parameters examined in comparison to untransfected cells, presumably because CREB is already constitutively expressed at maximal levels in normal FRTL5 cells. However, cells expressing KCREB showed an 18-40% reduction in TSH-stimulated thymidine incorporation, a 31% increase in the length of the cell cycle, and a 4-fold reduction in TSH-stimulated iodide uptake in comparison with wild type cells or cells transfected with wild type CREB. These experiments demonstrate that the CREB family of transcription factors is involved in both the control of growth and differentiated phenotype in the thyroid follicular cell. The regulation of cell growth by CREB appears to be a tissue-specific event since neither CREB nor KCREB had any effect on BALB/c 3T3 fibroblast cell growth. In contrast to its action on iodide uptake, KREB did not alter TSH-stimulated thyroglobulin mRNA levels, demonstrating that while cAMP regulates most aspects of the differentiated thyroid phenotype, only some are controlled specifically by the CREB family of transcription factors.

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