Epiregulin (EREG) is upregulated through an IL-1ß autocrine loop in Caco-2 epithelial cells with reduced CFTR function.

CFTR is a cAMP-regulated chloride channel, whose mutations produce cystic fibrosis. The impairment of CFTR activity increases the intracellular Cl- concentration, which in turn produces an increased interleukin-1ß (IL-1ß) secretion. The secreted IL-1ß then induces an autocrine positive feedback loop, further stimulating IL-1ß priming and secretion. Since IL-1ß can transactivate the epidermal growth factor receptor (EGFR), we study here the levels of expression for different EGFR ligands in Caco-2/pRS26 cells (expressing shRNA against CFTR resulting in a reduced CFTR expression and activity). The epiregulin (EREG), amphiregulin (AREG), and heparin binding EGF like growth factor (HBEGF) mRNAs, were found overexpressed in Caco-2/pRS26 cells. The EREG mRNA had the highest differential expression and was further characterized. In agreement with its mRNA levels, Western blots (WB) showed increased EREG levels in CFTR-impaired cells. In addition, EREG mRNA and protein levels were stimulated by incubation with exogenous IL-1ß and inhibited by the Interleukin 1 receptor type I (IL1R1) antagonist IL1RN, suggesting that the overexpression of EREG is a consequence of the autocrine IL-1ß loop previously described for these cells. In addition, the JNK inhibitor SP600125, and the EGFR inhibitors AG1478 and PD168393, also had an inhibitory effect on EREG expression, suggesting that EGFR, activated in Caco-2/pRS26 cells, is involved in the observed EREG upregulation. In conclusion, in Caco-2 CFTR-shRNA cells, the EGFR ligand EREG is overexpressed due to an active IL-1ß autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl- , and IL-1ß.

CFTR modulates RPS27 gene expression using chloride anion as signaling effector.

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl- concentrations ([Cl-]i), we observed several Cl--dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl- might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl-]i (using the Cl- fluorescent probe SPQ). The [Cl-]i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl- accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl- behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1ß receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1ß and JNK signaling downstream of Cl- in RPS27 modulation.

The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.