Abstract
A dominant negative inhibitor of the cAMP-dependent protein kinase has been shown to inhibit the basal expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in the human colon carcinoma cell line, T84. A functional cAMP response element (CRE) was localized at -48 in the CFTR promoter, and we have analyzed the interactions of this regulatory region with transcription factors. An adjacent inverted CCAAT element (Y box) at position -60 was also investigated. Mutation of the CRE or the Y box decreases the activity of the promoter in transient transfections of T84 or JEG-3 cells. Electrophoretic mobility shift assays demonstrate that CRE-binding protein (CREB) binds to the CFTR CRE with high affinity and independently of the adjacent Y box and that the CFTR CRE binds CREB and activating transcription factor-1 in nuclear extracts of T84 and CaLu-3 cells. In transient transfections of JEG-3 cells, activation of the CFTR promoter is blocked by a dominant negative CREB mutant. The CFTR CRE will also drive cAMP-mediated expression when placed upstream of a heterologous basal promoter. These results demonstrate that CFTR is a bona fide CRE-dependent gene, and we suggest that CFTR expression levels in vivo may be responsive to hormones or drugs that activate the cAMP-dependent protein kinase system.
Highlights
Many genes are induced by cAMP, including those coding for hormones, metabolic enzymes, structural proteins, and transcription factors
The CFTR promoter contains a variant cAMP response element (CRE) (TGACaTCA) at position Ϫ48 that we have shown to have all of the properties expected of a functional CRE
The CFTR CRE binds cAMP response element-binding protein (CREB) and activating transcription factor (ATF) family members with high affinity, and nuclear extracts from CFTR-expressing cell lines contain CREB/ATF family members that bind this sequence in electrophoretic mobility shift assays
Summary
Many genes are induced by cAMP, including those coding for hormones, metabolic enzymes, structural proteins, and transcription factors. A variant CRE sequence (TGACaTCA) is present in the promoter for the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and this element has recently been implicated in basal and PKA-mediated responsiveness of the promoter. Mutation of the variant CRE sequence in the CFTR promoter decreases both basal and PKA-stimulated CFTR-luc activity in transient transfections of T84 cells [13], suggesting that this element may function as a CRE to confer cAMP regulation to CFTR gene expression. These investigators identified several potential regulatory elements by sequence homology, including the variant CRE One of these studies demonstrated that the CFTR promoter was down-regulated by phorbol ester [15], which is consistent with the effects of phorbol ester on CFTR mRNA [16]. This lack of CFTR at the plasma membrane prevents the activation of chloride efflux and inhibition of sodium influx that is necessary for proper exocrine fluid homeostasis, resulting in inspissated secretions that lead to organ failure and
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