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

  • Ángel G. ValdiviesoEmail author
  • Mariángeles Clauzure
  • María M. Massip-Copiz
  • Carla E. Cancio
  • Cristian J. A. Asensio
  • Consuelo Mori
  • Tomás A. Santa-ColomaEmail author
Original Article


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 HCO3 transport 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.


CFTR Cystic fibrosis Lactate Oxamate Extracellular pH IL-1β Inflammation 



Airway surface liquid


Cystic fibrosis


Cystic fibrosis transmembrane conductance regulator


CFTR knockdown


Dimethyl sulfoxide




JUN N-terminal kinase


Lactate dehydrogenase


Mitochondrial Complex I


Extracellular pH


Short hairpin RNA



We thank Professor Diego Battiato and Romina D’Agostino for administrative assistance, and María de los Angeles Aguilar for technical assistance. We also thank Dr. Lutz Birnbaumer for his continuous support to our work and very valuable criticisms. This work was supported by National Agency for the Promotion of Science and Technology (ANPCYT) [grant numbers PICT 2012-1278 to TASC and PICT-2015-1031 to AGV]; National Scientific and Technical Research Council of Argentina (CONICET) [grants PIP-2016 112201-501002-27 and PUE-2016 22920160100129CO to TASC]; and Pontifical Catholic University of Argentina (UCA) to TASC. Fellowships from CONICET to MMC, CM, and MC.


  1. 1.
    Riordan JR et al (1989) Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 245(4922):1066–1073CrossRefGoogle Scholar
  2. 2.
    Sheppard DN, Welsh MJ (1999) Structure and function of the CFTR chloride channel. Physiol Rev 79(1 Suppl):S23–S45CrossRefGoogle Scholar
  3. 3.
    Rommens JM et al (1991) cAMP-inducible chloride conductance in mouse fibroblast lines stably expressing the human cystic fibrosis transmembrane conductance regulator. Proc Natl Acad Sci USA 88(17):7500–7504CrossRefGoogle Scholar
  4. 4.
    Althaus M (2013) ENaC inhibitors and airway re-hydration in cystic fibrosis: state of the art. Curr Mol Pharmacol 6(1):3–12CrossRefGoogle Scholar
  5. 5.
    Chen JH et al (2010) Loss of anion transport without increased sodium absorption characterizes newborn porcine cystic fibrosis airway epithelia. Cell 143(6):911–923CrossRefGoogle Scholar
  6. 6.
    Rogers CS et al (2008) Disruption of the CFTR gene produces a model of cystic fibrosis in newborn pigs. Science 321(5897):1837–1841CrossRefGoogle Scholar
  7. 7.
    Pezzulo AA et al (2012) Reduced airway surface pH impairs bacterial killing in the porcine cystic fibrosis lung. Nature 487(7405):109–113CrossRefGoogle Scholar
  8. 8.
    McShane D et al (2003) Airway surface pH in subjects with cystic fibrosis. Eur Respir J 21(1):37–42CrossRefGoogle Scholar
  9. 9.
    Schultz A et al (2017) Airway surface liquid pH is not acidic in children with cystic fibrosis. Nat Commun 8(1):1409CrossRefGoogle Scholar
  10. 10.
    Massip-Copiz MM, Santa-Coloma TA (2018) Extracellular pH and lung infections in cystic fibrosis. Eur J Cell Biol 97:402–410CrossRefGoogle Scholar
  11. 11.
    Figueira MF, Webster MJ, Tarran R (2018) CrossTalk proposal: mucosal acidification drives early progressive lung disease in cystic fibrosis. J Physiol 596(16):3433–3437CrossRefGoogle Scholar
  12. 12.
    González Guerrico AM et al (1999) Abstract M238 [El gen c-src es un posible marcador funcional del canal de cloruro afectado en fibrosis quística: CFTR]. In: 35th Annual meeting of the argentine society for biochemistry and molecular biology research (SAIB), November 9-12, Mendoza, Argentina (abstracts book)Google Scholar
  13. 13.
    Cafferata EG et al (1995) Abstract M99 [Identificación mediante “differential display” de genes específicamente regulados por diferentes factores que afectan la expresión del CFTR (canal de cloro afectado en Fibrosis Quística)] Abstracts of the 31th annual meeting of the Argentine Society for biochemistry and molecular biology research (SAIB), 15–18 November, Villa Giardino, Córdoba (abstracts book)Google Scholar
  14. 14.
    Gonzalez-Guerrico AM et al (2002) Tyrosine kinase c-Src constitutes a bridge between cystic fibrosis transmembrane regulator channel failure and MUC1 overexpression in cystic fibrosis. J Biol Chem 277(19):17239–17247CrossRefGoogle Scholar
  15. 15.
    Estell K et al (2003) Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif. Mol Cell Biol 23(2):594–606CrossRefGoogle Scholar
  16. 16.
    Valdivieso AG et al (2007) The expression of the mitochondrial gene MT-ND4 is downregulated in cystic fibrosis. Biochem Biophys Res Commun 356(3):805–809CrossRefGoogle Scholar
  17. 17.
    Taminelli GL et al (2008) CISD1 codifies a mitochondrial protein upregulated by the CFTR channel. Biochem Biophys Res Commun 365(4):856–862CrossRefGoogle Scholar
  18. 18.
    Hofhaus G, Attardi G (1993) Lack of assembly of mitochondrial DNA-encoded subunits of respiratory NADH dehydrogenase and loss of enzyme activity in a human cell mutant lacking the mitochondrial ND4 gene product. EMBO J 12(8):3043–3048CrossRefGoogle Scholar
  19. 19.
    Valdivieso AG, Santa-Coloma TA (2013) CFTR activity and mitochondrial function. Redox Biol 1(1):190–202CrossRefGoogle Scholar
  20. 20.
    Valdivieso AG et al (2012) The mitochondrial complex I activity is reduced in cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function. PLoS One 7(11):e48059CrossRefGoogle Scholar
  21. 21.
    Clauzure M et al (2014) Disruption of interleukin-1beta autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function. PLoS One 9(6):e99257CrossRefGoogle Scholar
  22. 22.
    Warburg O (1956) On the origin of cancer cells. Science 123(3191):309–314CrossRefGoogle Scholar
  23. 23.
    Alfarouk KO et al (2014) Glycolysis, tumor metabolism, cancer growth and dissemination. A new pH-based etiopathogenic perspective and therapeutic approach to an old cancer question. Oncoscience 1(12):777–802CrossRefGoogle Scholar
  24. 24.
    Zhou M et al (2010) Warburg effect in chemosensitivity: targeting lactate dehydrogenase-A re-sensitizes taxol-resistant cancer cells to taxol. Mol Cancer 9:33CrossRefGoogle Scholar
  25. 25.
    Robinson BH (2006) Lactic acidemia and mitochondrial disease. Mol Genet Metab 89(1–2):3–13CrossRefGoogle Scholar
  26. 26.
    Massip-Copiz MM et al (2017) CFTR impairment upregulates c-Src activity through IL-1beta autocrine signaling. Arch Biochem Biophys 616:1–12CrossRefGoogle Scholar
  27. 27.
    Zeitlin PL et al (1991) A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection. Am J Respir Cell Mol Biol 4(4):313–319CrossRefGoogle Scholar
  28. 28.
    Bargon J et al (1992) Expression of the cystic fibrosis transmembrane conductance regulator gene can be regulated by protein kinase C. J Biol Chem 267(23):16056–16060PubMedGoogle Scholar
  29. 29.
    Shoshani T et al (1992) Association of a nonsense mutation (W1282X), the most common mutation in the Ashkenazi Jewish cystic fibrosis patients in Israel, with presentation of severe disease. Am J Hum Genet 50(1):222–228PubMedPubMedCentralGoogle Scholar
  30. 30.
    Zeitlin PL et al (1992) CFTR protein expression in primary and cultured epithelia. Proc Natl Acad Sci USA 89(1):344–347CrossRefGoogle Scholar
  31. 31.
    Flotte TR et al (1993) Stable in vivo expression of the cystic fibrosis transmembrane conductance regulator with an adeno-associated virus vector. Proc Natl Acad Sci USA 90(22):10613–10617CrossRefGoogle Scholar
  32. 32.
    Flotte TR et al (1993) Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter. J Biol Chem 268(5):3781–3790PubMedGoogle Scholar
  33. 33.
    Purkey HE et al (2016) Cell active hydroxylactam inhibitors of human lactate dehydrogenase with oral bioavailability in mice. ACS Med Chem Lett 7(10):896–901CrossRefGoogle Scholar
  34. 34.
    Brighenti E et al (2017) The inhibition of lactate dehydrogenase A hinders the transcription of histone 2B gene independently from the block of aerobic glycolysis. Biochem Biophys Res Commun 485(4):742–745CrossRefGoogle Scholar
  35. 35.
    Chen EP et al (1989) Inactivation of lactate dehydrogenase by UV radiation in the 300 nm wavelength region. Radiat Environ Biophys 28(3):185–191CrossRefGoogle Scholar
  36. 36.
    Schmid I, Uittenbogaart CH, Giorgi JV (1991) A gentle fixation and permeabilization method for combined cell surface and intracellular staining with improved precision in DNA quantification. Cytometry 12(3):279–285CrossRefGoogle Scholar
  37. 37.
    Brady KD et al (1994) Relationships between amplitudes and kinetics of rapid cytosolic free calcium fluctuations in GH4C1 rat pituitary cells: roles for diffusion and calcium-induced calcium release. Biophys J 66(5):1697–1705CrossRefGoogle Scholar
  38. 38.
    Levene H (1960) Robust tests for equality of variances in contributions to probability and statistics: essays in honor of Harold Hotelling, I. Olkin, et al., editors. Stanford University Press, Palo Alto, pp 278–292Google Scholar
  39. 39.
    Shapiro SS, Wilk MB (1965) An analysis of variance test for normality (complete samples). Biometrika 52(3–4):591–611CrossRefGoogle Scholar
  40. 40.
    Kruskal WH, Wallis WA (1952) Use of ranks in one-criterion variance analysis. J Am Stat Assoc 47(260):583–621CrossRefGoogle Scholar
  41. 41.
    Mann HB, Whitney DR (1947) On a test of whether one of two random variables is stochastically larger than the other. Ann Math Stat 18(1):50–60CrossRefGoogle Scholar
  42. 42.
    Student (Gossett W.S.) (1907) On the error of counting with a haemacytometer. Biometrika 5(3):351–360CrossRefGoogle Scholar
  43. 43.
    Counillon L et al (2016) Na(+)/H(+) antiporter (NHE1) and lactate/H(+) symporters (MCTs) in pH homeostasis and cancer metabolism. Biochim Biophys Acta 1863(10):2465–2480CrossRefGoogle Scholar
  44. 44.
    Halestrap AP (2013) The SLC16 gene family—structure, role and regulation in health and disease. Mol Aspects Med 34(2–3):337–349CrossRefGoogle Scholar
  45. 45.
    Halestrap AP (2013) Monocarboxylic acid transport. Compr Physiol 3(4):1611–1643CrossRefGoogle Scholar
  46. 46.
    Massip-Copiz M et al (2018) Epiregulin (EREG) is upregulated through an IL-1beta autocrine loop in Caco-2 epithelial cells with reduced CFTR function. J Cell Biochem 119(3):2911–2922CrossRefGoogle Scholar
  47. 47.
    Valdivieso AG et al (2017) CFTR modulates RPS27 gene expression using chloride anion as signaling effector. Arch Biochem Biophys 633:103–109CrossRefGoogle Scholar
  48. 48.
    Clauzure M et al (2017) Intracellular chloride concentration changes modulate IL-1beta expression and secretion in human bronchial epithelial cultured cells. J Cell Biochem 118(8):2131–2140CrossRefGoogle Scholar
  49. 49.
    Valdivieso AG et al (2016) The chloride anion acts as a second messenger in mammalian cells—modifying the expression of specific genes. Cell Physiol Biochem 38(1):49–64CrossRefGoogle Scholar
  50. 50.
    Bardon A (1987) Cystic fibrosis. Carbohydrate metabolism in CF and in animal models for CF. Acta Paediatr Scand Suppl 332:1–30PubMedGoogle Scholar
  51. 51.
    Bardon A, Ceder O, Kollberg H (1986) Increased activity of four glycolytic enzymes in cultured fibroblasts from cystic fibrosis patients. Res Commun Chem Pathol Pharmacol 51(3):405–408PubMedGoogle Scholar
  52. 52.
    Shapiro BL (1988) Mitochondrial dysfunction, energy expenditure, and cystic fibrosis. Lancet 2(8605):289CrossRefGoogle Scholar
  53. 53.
    Shapiro BL, Feigal RJ, Lam LF (1979) Mitrochondrial NADH dehydrogenase in cystic fibrosis. Proc Natl Acad Sci USA 76(6):2979–2983CrossRefGoogle Scholar
  54. 54.
    Quinton PM (2007) Cystic fibrosis: lessons from the sweat gland. Physiology (Bethesda) 22:212–225Google Scholar
  55. 55.
    Abcouwer SF et al (2008) Effect of IL-1beta on survival and energy metabolism of R28 and RGC-5 retinal neurons. Invest Ophthalmol Vis Sci 49(12):5581–5592CrossRefGoogle Scholar
  56. 56.
    Manerba M et al (2017) Lactate dehydrogenase inhibitors can reverse inflammation induced changes in colon cancer cells. Eur J Pharm Sci 96:37–44CrossRefGoogle Scholar
  57. 57.
    Wetmore DR et al (2010) Metabolomic profiling reveals biochemical pathways and biomarkers associated with pathogenesis in cystic fibrosis cells. J Biol Chem 285(40):30516–30522CrossRefGoogle Scholar
  58. 58.
    Bensel T et al (2011) Lactate in cystic fibrosis sputum. J Cyst Fibros 10(1):37–44CrossRefGoogle Scholar
  59. 59.
    Kottmann RM et al (2012) Lactic acid is elevated in idiopathic pulmonary fibrosis and induces myofibroblast differentiation via pH-dependent activation of transforming growth factor-beta. Am J Respir Crit Care Med 186(8):740–751CrossRefGoogle Scholar
  60. 60.
    Ye X, Lotan R (2008) Potential misinterpretation of data on differential gene expression in normal and malignant cells in vitro. Brief Funct Genom Proteom 7(4):322–326CrossRefGoogle Scholar
  61. 61.
    Bijman J, Quinton PM (1987) Lactate and bicarbonate uptake in the sweat duct of cystic fibrosis and normal subjects. Pediatr Res 21(1):79–82CrossRefGoogle Scholar
  62. 62.
    Ishiguro H et al (2012) Physiology and pathophysiology of bicarbonate secretion by pancreatic duct epithelium. Nagoya J Med Sci 74(1–2):1–18PubMedPubMedCentralGoogle Scholar
  63. 63.
    Garnett JP et al (2016) Hyperglycaemia and Pseudomonas aeruginosa acidify cystic fibrosis airway surface liquid by elevating epithelial monocarboxylate transporter 2 dependent lactate-H + secretion. Sci Rep 6:37955CrossRefGoogle Scholar
  64. 64.
    Lin YC et al (2018) The pseudomonas aeruginosa complement of lactate dehydrogenases enables use of d- and l-lactate and metabolic cross-feeding. MBio 9(5):e00961-18CrossRefGoogle Scholar
  65. 65.
    Silva IN et al (2017) Regulator LdhR and d-lactate dehydrogenase LdhA of burkholderia multivorans play roles in carbon overflow and in planktonic cellular aggregate formation. Appl Environ Microbiol 83(19):e01343-17CrossRefGoogle Scholar
  66. 66.
    Miskimins WK et al (2014) Synergistic anti-cancer effect of phenformin and oxamate. PLoS One 9(1):e85576CrossRefGoogle Scholar
  67. 67.
    Zhao Z et al (2015) Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: involvement of the Akt-mTOR signaling pathway. Cancer Lett 358(1):17–26CrossRefGoogle Scholar
  68. 68.
    Massip Copiz MM, Santa Coloma TA (2016) c- Src and its role in cystic fibrosis. Eur J Cell Biol 95(10):401–413CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Ángel G. Valdivieso
    • 1
    Email author
  • Mariángeles Clauzure
    • 1
  • María M. Massip-Copiz
    • 1
  • Carla E. Cancio
    • 1
  • Cristian J. A. Asensio
    • 1
  • Consuelo Mori
    • 1
  • Tomás A. Santa-Coloma
    • 1
    Email author
  1. 1.Laboratory of Cellular and Molecular Biology, Institute for Biomedical Research (BIOMED), School of Medical SciencesPontifical Catholic University of Argentina (UCA), and The National Scientific and Technical Research Council of Argentina (CONICET)Buenos AiresArgentina

Personalised recommendations