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

Tetrahydrobiopterin (BH4) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O2̇̄) rather than NO. The rate-limiting enzyme for BH4 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 BH4 levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH4. 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 BH4 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 BH4. Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH4 accumulation, reduced NO production, and increased T cell O2̇̄ production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH2 polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH4 in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH4 levels, NO production, and modulating T cell cytokine production.

[1]  L. Davis,et al.  Oxidative Stress Promotes Polarization of Human T Cell Differentiation Toward a T Helper 2 Phenotype1 , 2006, The Journal of Immunology.

[2]  J. Esplugues,et al.  Endothelial nitric oxide synthase regulates N-Ras activation on the Golgi complex of antigen-stimulated T cells , 2008, Proceedings of the National Academy of Sciences.

[3]  S. Gross,et al.  Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. , 1992, The Journal of biological chemistry.

[4]  R. V. van Breemen,et al.  Mechanism of inactivation of inducible nitric oxide synthase by amidines. Irreversible enzyme inactivation without inactivator modification. , 2005, Journal of the American Chemical Society.

[5]  D. Harrison,et al.  GTP Cyclohydrolase I Phosphorylation and Interaction With GTP Cyclohydrolase Feedback Regulatory Protein Provide Novel Regulation of Endothelial Tetrahydrobiopterin and Nitric Oxide , 2010, Circulation research.

[6]  F. Murad,et al.  Nitric Oxide and Cyclic GMP in Cell Signaling and Drug Development , 2006 .

[7]  T. Yoneyama,et al.  Decameric GTP Cyclohydrolase I Forms Complexes with Two Pentameric GTP Cyclohydrolase I Feedback Regulatory Proteins in the Presence of Phenylalanine or of a Combination of Tetrahydrobiopterin and GTP* , 1998, The Journal of Biological Chemistry.

[8]  Nicole M. Chapman,et al.  The T cell receptor‐mediated phosphorylation of Pyk2 tyrosines 402 and 580 occurs via a distinct mechanism than other receptor systems , 2010, Journal of leukocyte biology.

[9]  F. Liew,et al.  Nitric oxide induces CD4+CD25+ Foxp3− regulatory T cells from CD4+CD25− T cells via p53, IL-2, and OX40 , 2007, Proceedings of the National Academy of Sciences.

[10]  N. S. Thomas,et al.  Lck is a key target of imatinib and dasatinib in T-cell activation , 2010, Leukemia.

[11]  J. Kwon,et al.  T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation , 2004, Nature Immunology.

[12]  Qianqian Wang,et al.  Rapid Kinetic Studies Link Tetrahydrobiopterin Radical Formation to Heme-dioxy Reduction and Arginine Hydroxylation in Inducible Nitric-oxide Synthase* , 2001, The Journal of Biological Chemistry.

[13]  Shu Chien,et al.  Activation of integrins in endothelial cells by fluid shear stress mediates Rho‐dependent cytoskeletal alignment , 2001, The EMBO journal.

[14]  D. Harrison,et al.  Regulation of Tetrahydrobiopterin Biosynthesis by Shear Stress , 2007, Circulation research.

[15]  M. Sahin-Tóth,et al.  Activation and dimerization of type III nitric oxide synthase by submicromolar concentrations of tetrahydrobiopterin in microsomal preparations from human primordial placenta. , 1997, Placenta.

[16]  B. Crane,et al.  Tetrahydrobiopterin radical enzymology. , 2003, Chemical reviews.

[17]  S. Davies,et al.  Selectivity of 4,5,6,7‐tetrabromobenzotriazole, an ATP site‐directed inhibitor of protein kinase CK2 (‘casein kinase‐2’) , 2001, FEBS letters.

[18]  J. Houtman,et al.  T cell receptor activation leads to two distinct phases of Pyk2 activation and actin cytoskeletal rearrangement in human T cells. , 2010, Molecular immunology.

[19]  S. Esnault,et al.  Differential regulation of IFN‐γ, IL‐10 and inducible nitric oxide synthase in human T cells by cyclic AMP‐dependent signal transduction pathway , 1997, Immunology.

[20]  Inhibition of Pyk2 blocks airway inflammation and hyperresponsiveness in a mouse model of asthma. , 2010, American journal of respiratory cell and molecular biology.

[21]  Nelson Jm,et al.  Biopterin. III. Purification and characterization of enzymes involved in the cerebral synthesis of 7,8-dihydrobiopterin. , 1978 .

[22]  D. Stuehr.  Structure-function aspects in the nitric oxide synthases. , 1997, Annual review of pharmacology and toxicology.

[23]  T. Rabelink,et al.  Tetrahydrobiopterin regulates superoxide and nitric oxide generation by recombinant endothelial nitric oxide synthase. , 1997, Biochemical and biophysical research communications.

[24]  J. Esplugues,et al.  Endothelial nitric oxide synthase regulates T cell receptor signaling at the immunological synapse. , 2006, Immunity.

[25]  P. Klatt,et al.  Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. , 1992, The Biochemical journal.

[26]  D. Harrison,et al.  Measurement of reactive oxygen species in cardiovascular studies. , 2007, Hypertension.

[27]  C. Loddenkemper,et al.  Self-Limitation of Th1-Mediated Inflammation by IFN-γ1 , 2006, The Journal of Immunology.

[28]  S. Snyder,et al.  Generation of superoxide by purified brain nitric oxide synthase. , 1992, The Journal of biological chemistry.

[29]  F. Liew,et al.  Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor β2 expression via cGMP , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[30]  W. Heath,et al.  Defective TCR expression in transgenic mice constructed using cDNA‐based α‐ and β‐chain genes under the control of heterologous regulatory elements , 1998, Immunology and cell biology.

[31]  J. Abe,et al.  Fluid Shear Stress Activates Proline-Rich Tyrosine Kinase via Reactive Oxygen Species–Dependent Pathway , 2002, Arteriosclerosis, thrombosis, and vascular biology.

[32]  K. Pahan,et al.  Suppression of Regulatory T Cells by IL-12p40 Homodimer via Nitric Oxide1 , 2009, The Journal of Immunology.

[33]  I. Megson,et al.  Inducible nitric oxide synthase‐derived superoxide contributes to hypereactivity in small mesenteric arteries from a rat model of chronic heart failure , 2000, British journal of pharmacology.

[34]  Zhi‐qiang Wang,et al.  Catalytic Reduction of a Tetrahydrobiopterin Radical within Nitric-oxide Synthase* , 2008, Journal of Biological Chemistry.

[35]  K. Channon.  Tetrahydrobiopterin: regulator of endothelial nitric oxide synthase in vascular disease. , 2004, Trends in cardiovascular medicine.

[36]  M. Stratford,et al.  Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. , 2002, Free radical biology & medicine.