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

Abstract

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-1β (IL-1β) secretion. The secreted IL-1β then induces an autocrine positive feedback loop, further stimulating IL-1β priming and secretion. Since IL-1β 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-1β 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-1β 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-1β autocrine loop that indirectly activates EGFR, constituting new signaling effectors for the CFTR signaling pathway, downstream of CFTR, Cl , and IL-1β.

The chloride anion as a signalling effector

“…Using this strategy, we found several differentially expressed mRNAs and characterized two of these, corresponding to RPS27 and GLRX5 (Valdivieso et al ., ). We also demonstrated that IL‐1β is a Cl − ‐dependent gene (Clauzure et al ., ; Massip‐Copiz et al ., ). Our results demonstrated the existence of numerous Cl − ‐dependent genes, and that changes in [Cl − ] i could, directly or indirectly, modulate the expression of specific genes in mammalian cells.…”

“…We have recently identified additional steps in CFTR signalling mechanisms that involve Cl − (Valdivieso et al ., , ), IL‐1β (Clauzure et al ., ; Massip‐Copiz et al ., , ) and c‐Src (Massip‐Copiz et al ., , ), which may explain the reduced mitochondrial Complex I activity (Valdivieso, ; Valdivieso et al ., ; Clauzure et al ., ) and increased ROS levels found in cultured CF cells (Clauzure et al ., , ; Massip Copiz & Santa Coloma, ; Massip‐Copiz et al ., ). Cl − modulates IL‐1β mRNA and protein expression in a biphasic way, with maximal expression at 75 mM [Cl − ] i in the presence of nigericin and tributyltin (Clauzure et al ., ).…”

Impairment of CFTR activity in cultured epithelial cells upregulates the expression and activity of LDH resulting in lactic acid hypersecretion

“…IL-1β, added to cultured Caco-2/pRSctrl cells, produced a pHe reduction, reaching a similar pHe value compared to Caco2/pRS26 cells (with shRNA-CFTR) when the IL-1β concentration was over 1 ng/ml (Figure 3). This pHe decreased in Caco-2/pRSctrl cells treated with IL-1β was also accompanied by increased LDH activity and lactate secretion, suggesting that the effects seen in these cells are partially due to the previously described IL-1β autocrine loop [21,26,46,48]. Therefore, we decided to study the effects of inhibitors of the IL-1β signaling pathway, including p38, JNK, MEK1/2 and c-Src inhibitors.…”

“…We have reported that the impairment of the CFTR activity results in Claccumulation and IL-1β overexpression and secretion; in turn, the secreted IL-1β creates an autocrine positive feed-back loop that enhances its own signal, increases the oxidative stress and reduces the mCx-I activity [21,26,[46][47][48][49]. Therefore, here we study possible effects of recombinant IL-1β on pHe.…”

CX3CL1 and CX3CR1 Expressing Tendon Cells – A novel Immune Cell Population in the Tendon Core

“…Similarly, for adipose derived mesenchymal stem cells epiregulin has been described to promote migration and chemotaxis ability via mitogen-activated protein kinase signalling pathways (Cao et al, 2018). Further, in Caco-2 epithelial cells EREG mRNA and protein levels have been shown to be increased by incubation with exogenous IL-1β (Massip-Copiz et al, 2018). This finding is well in line with our own data revealing that stimulation of 3D tendonlike constructs with IL-1β, or a combination of IL-1β and TNF-α significantly increased the expression of Ereg both on the gene as well as on the protein level.…”