Woloshin P I, Walton K M, Rehfuss R P, Goodman R H, Cone R D
Vollum Institute for Advanced Biomedical Research, Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland 92701-3098.
Mol Endocrinol. 1992 Oct;6(10):1725-33. doi: 10.1210/mend.6.10.1333055.
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 tranfected with wild type CREB.(ABSTRACT TRUNCATED AT 250 WORDS)
甲状腺滤泡细胞需要升高的环磷酸腺苷（cAMP）水平以实现正常生长和分化表型的最佳表达。最近对cAMP调节增强子结合（CREB）蛋白的发现促使我们以FRTL5甲状腺细胞系作为模型系统，分析这些转录因子在控制甲状腺细胞生长和分化表型中的可能作用。用含有野生型CREB（WTCREB）基因或CREB的显性负性突变形式（称为KCREB）的表达载体稳定转染FRTL5细胞，KCREB与内源性CREB二聚化并使其失活。发现转染的克隆表达转染的KCREB和WTCREB mRNA的水平高于内源性CREB mRNA。在这些克隆中生长抑素-氯霉素乙酰转移酶融合基因的瞬时表达表明，在KCREB转染的克隆中环磷酸腺苷调节增强子依赖性转录活性降低了60%，而在WTCREB转染的克隆中活性为野生型水平。然后分析转染克隆中的生长参数（DNA合成和生长速率）和分化参数（碘摄取和甲状腺球蛋白mRNA水平）。与未转染的细胞相比，WTCREB的转染对所检测的任何参数均无影响，推测是因为CREB在正常FRTL5细胞中已经以最大水平组成性表达。然而，与野生型细胞或转染野生型CREB的细胞相比，表达KCREB的细胞在促甲状腺激素刺激下的胸苷掺入减少了18 - 40%，细胞周期长度增加了31%，促甲状腺激素刺激的碘摄取减少了4倍。（摘要截短于250字）
