Role of Increased Guanosine Triphosphate Cyclohydrolase-1 Expression and Tetrahydrobiopterin Levels upon T Cell Activation

Wei Chen,Li Li,Torben Brod,Omar Saeed,Salim Thabet,Thomas Jansen,Sergey Dikalov,Cornelia Weyand,Jorg Goronzy,David G Harrison

doi:10.1074/jbc.m110.191023

Abstract

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.

Highlights

The nitric-oxide synthase (NOS)2 enzymes were initially identified in neuronal cells, macrophages, and the endothelium
Our studies show that resting T cells have virtually no NOS, guanosine triphosphate cyclohydrolase-1 (GTPCH-1), or BH4
Effect of T Cell Activation on NOS Expression and Pterin Content—In initial experiments, we found that resting T cells express minimal levels of either NOS2 or NOS3 protein; stimulation with anti-CD3 for 48 h robustly stimulated expression of both of these (Fig. 1, A and B)

Summary

Introduction

The nitric-oxide synthase (NOS)2 enzymes were initially identified in neuronal cells (nNOS or NOS1), macrophages (iNOS or NOS2), and the endothelium (eNOS or NOS3). Because the NOS enzymes require BH4 as an essential co-factor for production of NO, we examined levels of BH4 in resting and stimulated T cells. Levels of BH4 and its oxidized products were nearly undetectable in resting T cells, but were markedly increased by exposure to anti-CD3-coated plates for 48 h (Fig. 1C).

Results

Conclusion