Glutamine and KGF each regulate extracellular thiol/disulfide redox and enhance proliferation in Caco-2 cells

Departments of 1 Medicine and 4 Biochemistry, 2 Graduate Program in Molecular and Systems Pharmacology and 5 Center for Clinical and Molecular Nutrition, Emory University School of Medicine, Atlanta, Georgia 30322; and 3 Department of Surgery, University of Rochester Medical Center, Rochester, New York 14642 Submitted 13 November 2002 ; accepted in final form 27 August 2003 Glutamine (Gln) and keratinocyte growth factor (KGF) each stimulate intestinal epithelial cell growth, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-2 cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (E h ) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 µg/l) did not alter BrdU incorporation at reducing E h (-131 to -150 mV), but significantly increased incorporation at more oxidizing E h (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) E h was unaffected by Gln, KGF, or variations in extracellular Cys/CySS E h . Control cells largely maintained extracellular E h at initial values after 24 h (-36 to -136 mV). However, extracellular E h shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -92 mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-2 cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-2 cells to modulate extremes of extracellular redox. cysteine; glutathione; intestine; oxidation-reduction; keratinocyte growth factor Address for reprint requests and other correspondence: T. R. Ziegler, Suite GG-23, General Clinical Research Center, Emory Univ. Hospital, 1364 Clifton Rd., Atlanta, GA 30322 (E-mail: tzieg01{at}emory.edu ).