Cell Cycle Arrest by Kynurenine in Lens Epithelial Cells

Abstract

Indolemine 2,3-dioxygenase (IDO)-mediated oxidation of tryptophan produces kynurenines (KYNs), which may play a role in cataract formation. The molecular mechanisms by which KYNs cause cellular changes are poorly understood. The effects of KYNs on mouse lens epithelial cells by overexpression of human IDO were investigated. Lens epithelial cells (mLECs) derived from human IDO-overexpressing hemizygous transgenic (hemTg) and wild-type (Wt) mice were used. IDO activity was measured by quantifying kynurenine (KYN) by HPLC. KYN-mediated protein modifications were detected by immunocytochemistry and measured by ELISA. Cell proliferation and apoptosis were measured with commercially available kits. Cell distribution between cell cycle phases was examined with flow cytometric analysis. Immunoprecipitation followed by LC/MS was used to identify kynurenine-modified proteins. mLECs derived from hemTg animals exhibited considerable IDO immunoreactivity and enzyme activity, which were barely detectable in Wt mLECs. KYN and KYN-mediated protein modification were detected in hemTg but not in Wt mLECs; the modified proteins were myosin II and alpha/gamma-actin. HemTg mLECs displayed reduced viability and proliferation. Cell cycle analysis of hemTg mLEC cultures showed approximately a twofold increase in cells at G(2)/M or in both phases, relative to Wt mLECs. Blocking IDO activity with 1-methyl-d,l-tryptophan in hemTg mLECs prevented KYN formation, KYN-mediated protein modification, and G(2)/M arrest. Excess IDO activity in mLECs results in KYN production, KYN-mediated modification of myosin II and alpha/gamma-actin, and cell cycle perturbation. Modification of myosin II and gamma-actin by KYN may interfere with cytokinesis, leading to defective epithelial cell division and thus a decreased number of fiber cells.