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

Abstract

Mutations in the gene encoding the CFTR chloride channel produce cystic fibrosis (CF). CF patients are more susceptible to bacterial infections in lungs. The most accepted hypothesis sustains that a reduction in the airway surface liquid (ASL) volume favor infections. Alternatively, it was postulated that a reduced HCO3transport through CFTR leads to a decreased ASL pH, favoring bacterial colonization. The issue is controversial since recent data from cultured primary cells and CF children showed normal pH values in the ASL. We have reported previously a decreased mitochondrial Complex I (mCx-I) activity in cultured cells with impaired CFTR activity. Thus, we hypothesized that the reduced mCx-I activity could lead to increased lactic acid production (Warburg-like effect) and reduced extracellular pH (pHe). In agreement with this idea, we report here that cells with impaired CFTR function (intestinal Caco-2/pRS26, transfected with an shRNA-CFTR, and lung IB3-1 CF cells) have a decreased pHe. These cells showed increased lactate dehydrogenase (LDH) activity, LDH-A expression, and lactate secretion. Similar effects were reproduced in control cells stimulated with recombinant IL-1β. The c-Src and JNK inhibitors PP2 and SP600125 were able to increase the pHe, although the differences between control and CFTR-impaired cells were not fully compensated. Noteworthy, the LDH inhibitor oxamate completely restored the pHe of the intestinal Caco-2/pRS26 cells and have a significant effect in lung IB3-1 cells; therefore, an increased lactic acid secretion seems to be the key factor that determine a reduced pHe in these epithelial cells.

The chloride anion as a signalling effector

“…Interestingly, TMEM16A also has effects on endosomal pH acidification and renal proximal protein reabsorption; TMEM16A currents were activated by acidic pH . CFTR failure induces reduced extracellular pH in cultured epithelial cells due to the overexpression and increased activity of lactate dehydrogenase (LDH), partially through SRC proto-oncogene, non-receptor tyrosine kinase (c-Src) and JNK signalling Valdivieso et al, 2019).…”

“…The chloride anion (Cl − ), the most abundant anion in nature (Turekian, ; Bonifacie et al ., ; Berend, van Hulsteijn & Gans, ), is involved in general biophysical processes such as osmotic equilibrium (Houssay, Lewis & Orias, ; Willumsen, Davis & Boucher, ; West, ; Barrett et al ., ) and the transport of water and salt through epithelia (Jentsch, Maritzen & Zdebik, ; Hubner & Jentsch, ; Lee et al ., ). However, in addition to these general functions, Cl − has other specific roles, acting as a signalling effector or modulator of specific cell functions, including cell volume (Treharne, Crawford & Mehta, ; Pedersen, Hoffmann & Novak, ), membrane potential (Liang et al ., ; Funabashi et al ., ; Crutzen et al ., ), intracellular pH (Paredes et al ., ) and extracellular pH (Massip‐Copiz & Santa‐Coloma, ; Valdivieso et al ., ). It also affects the function of endosomes (Miller Jr. et al ., ; Matsuda et al ., ), phagosomes (Painter et al ., ; Riazanski et al ., ), endoplasmic reticulum (ER) (Barro‐Soria et al ., ), and mitochondria (Tomaskova & Ondrias, ; Nunes et al ., ).…”

“…The chloride anion (Cl − ), the most abundant anion in nature (Turekian, 1968;Bonifacie et al, 2008;Berend, van Hulsteijn & Gans, 2012), is involved in general biophysical processes general functions, Cl − has other specific roles, acting as a signalling effector or modulator of specific cell functions, including cell volume (Treharne, Crawford & Mehta, 2006;Pedersen, Hoffmann & Novak, 2013), membrane potential (Liang et al, 2009;Funabashi et al, 2010;Crutzen et al, 2016), intracellular pH (Paredes et al, 2016) and extracellular pH Valdivieso et al, 2019). It also affects the function of endosomes (Miller Jr. et al, 2007;Matsuda et al, 2010), phagosomes (Painter et al, 2010;Riazanski et al, 2015), endoplasmic reticulum (ER) (Barro- Soria et al, 2010), and mitochondria (Tomaskova & Ondrias, 2010;Nunes et al, 2015).…”

Human airway epithelial cell pH regulatory proteins in cystic fibrosis: Differential expression in nasal and bronchial airway cells

“…Hv1 mediates H + e ux from cells under steady-state conditions and has therefore been described as an 'acid relief valve' (35). In addition to altering the driving force for H + movement through Hv1 and other channels, changes in the pH gradient (ΔpH = pH o -pH i ) are a physiologically important regulator of Hv1 channel gating (3,34,36) The increase in LDHa and LDHd, which are mainly localized in the cytosol, we observed in CF cells may alter pHi buffering by shifting the relative intracellular balances of lactate and pyruvate compared to healthy cells (37). ATP12a has previously been shown to be increased in CF airway epithelial cells and to deplete the ASL of potassium (38, 39).…”

CFTR dysfunction leads to defective bacterial eradication on cystic fibrosis airways

“…Previous studies ( Pezzulo et al, 2012 ; Shah et al, 2016a ; Li et al, 2016 ; Tang et al, 2016 ) indicate that the ASL pH of cultured CF pig airway epithelia is more acidic than that of non-CF. Using a pH microelectrode, other studies demonstrate that the apical pH of cultured epithelia or the extracellular pH of epithelial cells from different human cell lines, such as CFBE41o− ( Simonin et al, 2019 ), Calu-3 ( Shan et al, 2012 ), IB3-1 vs. C38 ( Valdivieso et al, 2019 ) and CuFi-1 vs. NuLi cells ( Muraglia et al, 2019 ) or from human bronchial epithelial cells ( Coakley et al, 2003 ; Haggie et al, 2016 ; Scudieri et al, 2018 ; Simonin et al, 2019 ; Gianotti et al, 2020 ) is consistently more acidic in CF than that in non-CF. The pH difference (∆pH) between CF and non-CF among these studies is about 0.2–0.65 ( Coakley et al, 2003 ; Pezzulo et al, 2012 ; Shan et al, 2012 ; Haggie et al, 2016 ; Scudieri et al, 2018 ; Simonin et al, 2019 ; Gianotti et al, 2020 ), whereas well-differentiated epithelia with better epithelial polarization and more CFTR expressed in the apical membrane than those of less differentiated epithelia ( Sheppard et al, 1994 ), exhibit larger ∆pH (∼0.5) decreases in CF than non-CF epithelia ( Scudieri et al, 2018 ; Gianotti et al, 2020 ).…”