Boudreau F, Blais S, Asselin C
Département d'Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Exp Cell Res. 1996 Jan 10;222(1):1-9. doi: 10.1006/excr.1996.0001.
Members of the CCAAT/enhancer binding protein (C/EBP) gene family are expressed in murine intestine. However, little is known about their regulation in intestinal epithelial cells. In an attempt to determine regulatory mechanisms involved in their expression, we examined C/EBP alpha, beta, and delta isoform expression in response to serum and glucocorticoids in the rat intestinal epithelial crypt-derived cell line IEC-6, by Northern blot, transcription run-on assays, indirect immunofluorescence, Western blot, and electrophoretic mobility shift assays. Serum leads to rapid and transient increases in C/EBP alpha and beta mRNA and protein levels by posttranscriptional regulatory mechanisms, without affecting transcriptional initiation. However, C/EBP-specific DNA binding capacity is not affected by serum. Whereas C/EBP alpha expression is not regulated by glucocorticoids, C/EBP beta and delta mRNA and protein levels are rapidly induced. These inductions result from both increased transcription rates and posttranscriptional regulatory mechanisms as well. Moreover, C/EBP beta and delta containing DNA binding complexes are increased by glucocorticoids as determined by supershift assays, in contrast to C/EBP alpha containing complexes. Immunofluorescence studies show cytoplasmic and nuclear localization for C/EBP alpha, in contrast to a restricted nuclear localization for both C/EBP beta and C/EBP delta. These results confirm C/EBP isoforms expression in intestinal epithelial cells. Differential regulation by serum and glucocorticoids as well as different localization of three C/EBP isoforms suggest a role for this class of transcription factors in the control of gene expression in intestinal epithelial cells.
CCAAT/增强子结合蛋白（C/EBP）基因家族的成员在小鼠肠道中表达。然而，它们在肠上皮细胞中的调控机制却鲜为人知。为了确定其表达所涉及的调控机制，我们通过Northern印迹、转录延伸分析、间接免疫荧光、Western印迹和电泳迁移率变动分析，检测了大鼠肠上皮隐窝来源的细胞系IEC-6中C/EBPα、β和δ异构体对血清和糖皮质激素的反应。血清通过转录后调控机制导致C/EBPα和β的mRNA及蛋白水平迅速短暂升高，而不影响转录起始。然而，血清不影响C/EBP特异性DNA结合能力。虽然C/EBPα的表达不受糖皮质激素调控，但C/EBPβ和δ的mRNA及蛋白水平迅速被诱导。这些诱导是由转录速率增加和转录后调控机制共同导致的。此外，与含C/EBPα的复合物相反，超迁移分析表明糖皮质激素可增加含C/EBPβ和δ的DNA结合复合物。免疫荧光研究显示，C/EBPα定位于细胞质和细胞核，而C/EBPβ和C/EBPδ仅定位于细胞核。这些结果证实了C/EBP异构体在肠上皮细胞中的表达。血清和糖皮质激素的差异调控以及三种C/EBP异构体的不同定位表明，这类转录因子在肠上皮细胞基因表达控制中发挥作用。
