Modulation of CFTR gene expression in HT-29 cells by extracellular hyperosmolarity

Hypertonicity has pleiotropic effects on cell function, including activatio n of transporters and regulation of gene expression. It is important to inv estigate the action of hypertonicity on cystic fibrosis gene expression bec ause cystic fibrosis transmembrane conductance regulator (CFTR), the cAMP-r egulated Cl- channel, regulates ion transport across the secretory epitheli a, which are often in a hypertonic environment. We found that adding >150 m osmol/l NaCl, urea, or mannitol to the culture medium reduced the amount of CFTR mRNA in colon-derived HT-29 cells in a time-dependent manner. Studies with inhibitors of various kinases [H-89 (protein kinase A inhibitor), bis indolylmaleimide (protein kinase C inhibitor), staurosporine (serine/threon ine kinase inhibitor) and herbimycin A (tyrosine kinase inhibitor), SE-2035 80 and PD-098059 (mitogen-activated protein kinase inhibitors)] showed that CFTR gene expression and its decrease by added NaCl required p38 kinase ca scade activity. The CFTR gene activity is regulated at the transcriptional level, since adding NaCl diminished the luciferase activity of HeLa cells t ransiently transfected with the CFTR promoter. This regulation requires pro tein synthesis. The complexity of the reactions involved in blocking CFTR g ene transcription by NaCl strongly suggests that the decrease in CFTR mRNA is part of a general cell response to hyperosmolar stress.