Abstract
The unfolded protein response (UPR) aids cellular recovery by increasing the capacity and decreasing the protein load of the endoplasmic reticulum (ER). Although the main pathways of the UPR are known, the mechanisms of UPR-associated transcriptional repression have not been explored in mammalian cells. Previous studies indicate that endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency decrease when the UPR is activated. In the present study, we demonstrate that inhibition of CFTR expression under ER stress leads to reduced cAMP-activated chloride secretion in epithelial monolayers, an indication of diminished CFTR function. Moreover, ER stress and the UPR obliterate endogenous DeltaF508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant DeltaF508 CFTR mRNA levels or mRNA half-life. These results emphasize that transcriptional repression of CFTR under ER stress, in concert with decreased CFTR maturation efficiency, leads to diminished function. Using human CFTR promoter reporter constructs, we confined the ER stress-associated CFTR transcriptional repression to the minimal promoter. Chromatin immunoprecipitation assays established the binding of the UPR-activated ATF6 transcription factor to this region during ER stress, which links the repression to the UPR. Methylation-specific PCR (MSP) revealed hypermethylation of CpG sites inside and in the vicinity of the MAZ transcription factor binding region of CFTR, demonstrating methylation-dependent repression. Using pharmacological inhibitors, we show that both DNA methylation and histone deacetylation contribute to CFTR transcriptional inhibition. These studies provide novel insight into the mechanism of gene repression during the mammalian UPR.
Highlights
From the Departments of ‡Cell Biology and ¶Genetics and §Cystic Fibrosis Research Center, University of Alabama, Birmingham, In eukaryotic cells, the endoplasmic reticulum (ER)3 is the site of protein folding and assembly
We tested the functionality of this construct as follows: HeLa above for the 1-kb promoter vector, we measured the effects of cells were transfected ER stress and activation of the unfolded protein response (UPR) on the luciferase expression with pCFTR-pLuc, and ER stress was induced using TM or pro- driven by these promoter constructs
Understanding cellular stress responses is critical for progress against a growing number of human disorders associated with cellular stress [73]
Summary
(expressing endogenous ⌬F508 CFTR) [27], and HeLa cells were obtained from the ATCC. CFBE41o⌬F (expressing recombinant ⌬F508 CFTR) was produced as previously described [28]. Total CFTR mRNA levels at each time point were measured as described previously [35]. The molecular pathology is not fully understood for any of these disorders, but several approaches that rescue the foldingdeficient ⌬F508 CFTR mutant from ERAD have been reported to deliver functional CFTR protein to the cell surface Such approaches include proteasome inhibition, ER Ca2⫹ transport blockers, and depletion of ER Ca2⫹ [22,23,24,25]. In addition to enhancing our knowledge of cellular mechanisms behind CF and many other pulmonary diseases, the results presented provide novel information regarding UPR-associated transcriptional regulation of membrane protein expression in mammalian cells. RNA was isolated, and CFTR mRNA levels were measured in real-time RT-PCR experiments, as previously described [17]. Statistical significance among means was determined using the Student’s t test (paired and unpaired samples)
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