Epidermal growth factor receptor activity upregulates lactate dehydrogenase A expression, lactate dehydrogenase activity, and lactate secretion in cultured IB3-1 cystic fibrosis lung epithelial cells.

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It has been postulated that reduced HCO3- transport through CFTR may lead to a decreased airway surface liquid pH. In contrast, others have reported no changes in the extracellular pH (pHe). We have recently reported that in carcinoma Caco-2/pRS26 cells (transfected with short hairpin RNA for CFTR) or CF lung epithelial IB3-1 cells, the mutation in CFTR decreased mitochondrial complex I activity and increased lactic acid production, owing to an autocrine IL-1ß loop. The secreted lactate accounted for the reduced pHe, because oxamate fully restored the pHe. These effects were attributed to the IL-1ß autocrine loop and the downstream signaling kinases c-Src and JNK. Here we show that the pHe of IB3-1 cells can be restored to normal values (∼7.4) by incubation with the epidermal growth factor receptor (EGFR, HER1, ErbB1) inhibitors AG1478 and PD168393. PD168393 fully restored the pHe values of IB3-1 cells, suggesting that the reduced pHe is mainly due to increased EGFR activity and lactate. Also, in IB3-1 cells, lactate dehydrogenase A mRNA, protein expression, and activity are downregulated when EGFR is inhibited. Thus, a constitutive EGFR activation seems to be responsible for the reduced pHe in IB3-1 cells.

Extracellular pH and lung infections in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by CFTR mutations. It is characterized by high NaCl concentration in sweat and the production of a thick and sticky mucus, occluding secretory ducts, intestine and airways, accompanied by chronic inflammation and infections of the lungs. This causes a progressive and lethal decline in lung function. Therefore, finding the mechanisms driving the high susceptibility to lung infections has been a key issue. For decades the prevalent hypothesis was that a reduced airway surface liquid (ASL) volume and composition, and the consequent increased mucus concentration (dehydration), create an environment favoring infections. However, a few years ago, in a pig model of CF, the Na+/K+ concentrations and the ASL volume were found intact. Immediately a different hypothesis arose, postulating a reduced ASL pH as the cause for the increased susceptibility to infections, due to a diminished bicarbonate secretion through CFTR. Noteworthy, a recent report found normal ASL pH values in CF children and in cultured primary airway cells, challenging the ASL pH hypothesis. On the other hand, recent evidences revitalized the hypothesis of a reduced ASL secretion. Thus, the role of the ASL pH in the CF is still a controversial matter. In this review we discuss the basis that sustain the role of CFTR in modulating the extracellular pH, and the recent results sustaining the different points of view. Finding the mechanisms of CFTR signaling that determine the susceptibility to infections is crucial to understand the pathophysiology of CF and related lung diseases.

CFTR impairment upregulates c-Src activity through IL-1ß autocrine signaling.

Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to 'CFTR-corrected' S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1ß receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1ß→c-Src and IL-1ß→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1ß constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity.

c- Src and its role in cystic fibrosis.

Cystic fibrosis (CF) is a lethal inherited disease produced by mutations in the gene encoding the CFTR chloride channel. Loss of function in the CFTR gene is associated with a not much noticed increased expression and activity of the non-receptor protein-tyrosine kinase c-Src. CF is therefore the result from the loss of CFTR chloride transport function and its consequences, including a chronic and excessive c-Src signaling. On the other hand, c-Src, encoded by the SRC gene, is involved in diverse signaling mechanisms that regulate key cellular functions such as cell proliferation, apoptosis, oxidative stress, inflammation, and innate immunity. These c-Src-regulated cellular functions are also affected in CF; however, studies exploring a direct role of c-Src in the regulation of these cellular functions in CF are yet scarce and often controversial. Here we describe the c-Src regulation and functions, with emphasis in those altered in CF, and describe the role of CFTR as a "signaling molecule" that negatively modulates c-Src expression and activity. It is also discussed the emerging role of intracellular Cl- and IL-1ß as intermediate signaling effectors between CFTR and c-Src.