Indoleamine 2,3-Dioxygenase Overexpression Causes Kynurenine-modification of Proteins, Fiber Cell Apoptosis and Cataract Formation in the Mouse Lens

Maneesh Mailankot; Magdalena M. Staniszewska; Heather Butler; Moonkyung H. Caprara; Scott Howell; Benlian Wang; Catherine Doller; Lixing W. Reneker; Ram H. Nagaraj

Disclosures

Lab Invest. 2009;89(5):498-512. 

In This Article

Abstract and Introduction

Abstract

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the kynurenine pathway. The kynurenines formed in this pathway chemically modify proteins and cause apoptosis in cells. Evidence suggests that kynurenines and their protein modifications are involved in cataract formation, but this has yet to be directly demonstrated. We generated transgenic (Tg) mouse lines that overexpress human IDO in the lens. Homozygous Tg (homTg) lenses had higher IDO immunoreactivity, ~4.5 times greater IDO mRNA, and ~8 times higher IDO activity compared to lenses from hemizygous Tg (hemTg) animals. The kynurenine content was threefold higher in homTg than in hemTg but was not detected in wild-type (Wt) lenses. Kynurenine modifications were ~2.6 times greater in homTg than in hemTg or Wt. HomTg lenses had vacuoles in the epithelium and cortical fiber cells. Kynurenine modifications coincided with apoptosis in the secondary fiber cells of homTg lenses. Caspase-3 and caspase-9 activities were markedly higher in homTg than in hemTg and Wt. The glutathione content was ~36% lower in homTg compared to hemTg and Wt lenses. HomTg animals also developed bilateral cataracts within 3 months of birth. Together these data demonstrate that IDO-mediated production of kynurenines results in defects in fiber cell differentiation and their apoptosis and suggest that IDO activity is kept low in the lens to prevent deleterious effects by kynurenines.

Introduction

The kynurenine (KYN) pathway is the primary route of L-tryptophan catabolism and a source of NAD.[1] In extrahepatic tissues, the KYN pathway is initiated by indoleamine 2,3-dioxygenase (IDO). IDO is a heme-containing dioxygenase; it oxidizes L-tryptophan to N-formylkynurenine (NFK). Further catalysis of NFK leads to the formation of NAD.[2] IDO synthesis is induced by inflammatory stimuli, especially interferon-γ. Enhanced synthesis of IDO appears to deplete tryptophan and restrict viral and bacterial growth and infection.[3] IDO has been implicated in the protection of fetal allografts from maternal T-cell-mediated immunity, possibly through KYN-mediated apoptosis of thymocytes.[4,5] Inhibition of T-cell-mediated immunity by IDO is also involved in engraftment of skin substitutes[6] and transplantation of other organs.[7] IDO secreted by tumor cells starves T cells by depleting tryptophan,[8] and inhibition of IDO promotes tumor regression.[9] Thus, IDO appears to have important functions in infection, inflammation, and T-cell-mediated immunity.

Although these effects are defensive strategies to cope with infection and inflammation, they may have unintended consequences because KYNs formed during IDO-mediated degradation of tryptophan can chemically modify proteins and have been shown to be cytotoxic.[10,11] In coronary heart disease, inflammation and immune activation are associated with increased blood levels of KYN,[12] possibly through interferon-γ-mediated activation of IDO. In experimental chronic renal failure, activation of IDO leads to increased blood levels of KYNs,[13] and in uremic patients KYN-modified proteins are present in urine.[14] Further, renal IDO expression may be deleterious during inflammation because it enhances tubular cell injury.[15] KYNs and IDO also are implicated in the pathophysiology of neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, and KYNs have been shown to be neurotoxic.[10,16,17,18,19] Tryptophan degradation increases in rheumatoid arthritis[20] and development of collagen-induced arthritis in mice is associated with increased IDO activity and enhanced tryptophan catabolism.[21] These observations suggest that KYN may contribute to various neurological, renal, and inflammatory pathologies. Chronic inflammation in these diseases may be an underlying factor for IDO overexpression.

Several recent studies, especially those investigating the eye lens, shed light on how KYNs might chemically alter protein structure. In the human lens, IDO activity is present mainly in the anterior epithelium.[22] Several KYNs, such as KYN, 3-hydroxykynurenine, and 3-hydroxykynurenine glucoside, are present in the lens.[23] They are thought to protect the retina by absorbing UV light and therefore are commonly referred to as UV filters. However, several recent studies show that KYNs are prone to deamination and oxidation.[24] The deamination results in formation of α,ß-unsaturated ketones that chemically react and modify lens proteins. Such reactions occur mostly at cysteinyl, histidinyl, and lysyl residues.[25] The predominant modifications appear to be Michael adducts; however, other more stable and protein-cross-linking adducts also are likely, but their structures are not fully understood.

In the normal human lens, KYN modifications increase after 50 years of age,[26] and in cataractous lenses they decrease due to degradation.[27] Our studies, using highly specific monoclonal antibodies (mAbs), have provided additional support for the presence of Michael adducts in proteins of the human lens.[28,29] These observations suggest that KYN-mediated modification could contribute to the lens protein modifications during aging and cataractogenesis. They may also reduce the chaperone function of α-crystallin, which is necessary for maintaining lens transparency.[30] Although these observations link KYN formation to aging and cataract formation, whether KYN formation is directly involved in lens protein modification and cataract development has yet to be demonstrated. To address this issue, we have generated transgenic mouse lines that express high levels of IDO in the lens. Using this model, we show that IDO overexpression adversely affects lens development, causes massive apoptosis of fiber cells, and leads to poorly differentiated fiber cells and cataract formation.

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