Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of non-epithelial origin

Abstract

Consistent with the fact that the clinical disorder cystic fibrosis (CF) is manifested on epithelial surfaces, active transcription of the CF transmembrane conductance regulator (CFTR) gene and CFTR mRNA transcripts are detectable in a variety of epithelial cells, suggesting CFTR gene expression might be epithelial cell-specific. However, analysis of the CFTR gene promoter suggests it is a housekeeping gene, implying more widespread expression than only in epithelial cells. To evaluate the latter hypothesis, various human cells of non-epithelial origin, including lung fibroblasts, U-937 histiocytic lymphoma cells, K-562 erythroleukemia cells, HL-60 promyelocytic leukemia cells as well as freshly isolated blood lymphocytes, neutrophils, monocytes, and alveolar macrophages were examined for CFTR gene expression. Although Northern analysis failed to show CFTR mRNA transcripts in these cells, amplification of mRNA (after conversion to cDNA) by polymerase chain reaction combined with Southern analysis demonstrated the presence of CFTR mRNA transcripts at low levels in all cells evaluated except HL-60 cells. Comparative quantitative analysis showed fibroblasts contained 200-400 fold less CFTR mRNA transcripts than the T84 and HT-29 colon carcinoma epithelial cell lines, but had similar levels of CFTR transcripts to those of other epithelial cell lines. Nuclear transcription run-on analyses demonstrated very low level CFTR gene transcription in fibroblasts and U-937 cells, similar to that of other epithelial cells, but lower than the T84 and HT-29 colon carcinoma cell lines. Interestingly, while chromatin DNA of fibroblasts had no DNase I hypersensitivity sites in the 5' flanking region of the CFTR gene, HT-29 chromatin DNA exhibited four DNase I accessible sites in the same region, suggesting that these sites may be related to more active transcription of the CFTR gene in the intestinal epithelial cells than in fibroblasts.

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation

“…Using real-time PCR we could again identify CFTR mRNA in normal and monocyte-depleted human neutrophil preparations from healthy volunteers only at very high (.45) cycle numbers; message in T84 cells was 50,000-100,000 fold more abundant than in neutrophils (data not shown). CFTR protein has been found in cells other than epithelia [12], including lymphocytes [29] and erythrocytes [30], but the expression of functional CFTR protein in the neutrophil is uncertain [13,14]. Despite identifying low level CFTR mRNA we were unable to detect CFTR protein either in neutrophil lysates or by immunoprecipitation of whole cells or membrane-enriched fractions (all samples derived from healthy non-CF individuals; fig.…”

“…However, in many of these studies the patient groups were heterogeneous in terms of age, genetic defect, infection status, colonising organism or medication, all variables that may modulate neutrophil responsiveness. While mRNA encoding CFTR has been reported to be present at low copy number from preparations of human neutrophils [12], it is unclear whether this message originates from the neutrophils themselves or from other cells, such as monocytes or lymphocytes present in small but significant numbers in conventional neutrophil isolates. Likewise, there are conflicting reports as to whether CFTR is expressed by neutrophils at the protein level [13,14].…”

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

“…Despite more than 20 y of study (42), the mechanisms governing CFTR transcription remain incompletely understood. The CFTR promoter has features of a "housekeeping" gene (43,44), and several transactivating factors and regulatory elements have been characterized (43,(45)(46)(47). Given that SIN3A-mediated transcriptional silencing involves associated histone deacetylases (35), identifying an interaction between SIN3A and CTCF on the CFTR promoter at the −20.9 kb DHS improves our understanding of how CFTR is transcriptionally regulated.…”

Interactions of connexins with other membrane channels and transporters

“…The gene product, a cAMP and ATP regulated Cl − channel, is mostly expressed in the apical plasma membrane of secretory and reabsorptive epithelia of affected organs, allowing the transepithelial movement of water and solutes. Other observations indicate a more widespread CFTR gene expression than in epithelial cells and suggest that this channel protein may function in most human cells to help maintain cellular homeostasis (Yoshimura et al, 1991;Abraham et al, 2001;Assef et al, 2003Assef et al, , 2005. The first suggestion for a role of GJIC in CF came from experiments aimed at correcting the CF defect by insertion of wild-type CFTR gene (Johnson et al, 1992;Zabner et al, 1994).…”