Characterization of transgenic Gfrp knock-in mice: implications for tetrahydrobiopterin in modulation of normal tissue radiation responses

Abstract

Aims: The free radical scavenger and nitric oxide synthase cofactor, 5,6,7,8-tetrahydrobiopterin (BH4), plays a well-documented role in many disorders associated with oxidative stress, including normal tissue radiation responses. Radiation exposure is associated with decreased BH4 levels, while BH4 supplementation attenuates aspects of radiation toxicity. The endogenous synthesis of BH4 is catalyzed by the enzyme guanosine triphosphate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regulatory protein (GFRP). We here report and characterize a novel, Cre-Lox-driven, transgenic mouse model that overexpresses Gfrp.

Results: Compared to control littermates, transgenic mice exhibited high transgene copy numbers, increased Gfrp mRNA and GFRP expression, enhanced GFRP-GTPCH1 interaction, reduced BH4 levels, and low glutathione (GSH) levels and differential mitochondrial bioenergetic profiles. After exposure to total body irradiation, transgenic mice showed decreased BH4/7,8-dihydrobiopterin ratios, increased vascular oxidative stress, and reduced white blood cell counts compared with controls.

Innovation and conclusion: This novel Gfrp knock-in transgenic mouse model allows elucidation of the role of GFRP in the regulation of BH4 biosynthesis. This model is a valuable tool to study the involvement of BH4 in whole body and tissue-specific radiation responses and other conditions associated with oxidative stress.

FIG. 1.

Generation of guanosine triphosphate cyclohydrolase I feedback regulatory protein (Gfrp) knock-in mouse model. Schematic diagram of Gfrp vector construct showing the CAG promoter region, a neomycin-resistant gene coding region flanked by two loxP sites, followed by the EGFP gene, which was replaced with the gene of interest, Gfrp. Transgene expression was achieved by Cre recombinase-mediated deletion of the Neo cassette (a). Polymerase chain reaction gel showing an intense band of the Gfrp transgene in Gfrp+/Cre+ mice, no band was detected in control littermates (Gfrp−/Cre+) (b). Granulocyte percentage in peripheral blood of Gfrp+/Cre+ mice (n=3) and control littermates (n=4) (c). Number of transgene copies integrated in C57BL/6 (n=7), Gfrp−/Cre+ mice (n=8), and control littermates (n=8) as detected by custom-made copy number assay (d). All data presented as mean±standard error of mean. NS, not statistically significant. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

Gfrp mRNA and GFRP protein in tissues. Relative Gfrp mRNA fold change in liver, kidney, intestine, lung, spleen, and thymus as detected by quantitative reverse transcription polymerase chain reaction in male Gfrp-Tg (n=4) mice and control littermates (n=4) (a). Expression of GFRP (10 kDa) was detected by western blotting in Gfrp+/Cre+ mice (n=2) and Gfrp−/Cre+ control littermates (n=2) with β-actin (42 kDa) as internal control (b). Representative immunohistochemical staining of GFRP in tissues from Gfrp+/Cre+ mice and Gfrp−/Cre+ control littermates, original magnification 20× (c). All data presented as mean±standard error of mean. NS, not statistically significant.

FIG. 3.

Estimation of 5,6,7,8-tetrahydrobiopterin (BH4) level, 7,8-dihydrobiopterin (BH2) level, BH4/BH2 ratio, and GTP cyclohydrolase I (GTPCH1)-GFRP interaction in unirradiated mice. BH4 level, BH2 level and BH4/BH2 ratio were estimated in lung tissue samples of unirradiated Gfrp+/Cre+ mice (n=5) and Gfrp−/Cre+ control littermates (n=5). Transgenic mice (Gfrp+/Cre+) had significantly lower levels of BH4 and BH2, but no difference in BH4/BH2 ratio compared with control mice (Gfrp−/Cre+) (a). A representative blot showing interaction of GFRP with GTPCH1 in lung and liver tissue samples from Gfrp+/Cre+ transgenic mice and Gfrp−/Cre+ control littermates. For each of the tissues, co-immunoprecipitation was performed three biological replicates from each genotype (b). All data presented as mean±standard error of mean. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

Estimation of blood glutathione (GSH) level and mitochondrial bioenergetic functions. Total GSH (a) and oxidized glutathione (GSSG) percent (b) estimated in blood samples of Gfrp+/Cre+ transgenic mice (n=6) and Gfrp−/Cre+ control littermates (n=6). Oxygen consumption rate (OCR) in primary thymocytes from Gfrp+/Cre+ transgenic mice (n=4) and Gfrp−/Cre+ control littermates (n=4) (c). Individual mitochondrial functional parameters in primary thymocytes from Gfrp+/Cre+ transgenic mice (n=4) and Gfrp−/Cre+ control littermates (n=4) (d). All data presented as mean±standard error of mean. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 5.

Gfrp mRNA expression in irradiated lung tissue. Relative Gfrp mRNA fold change in lung tissue samples from wild-type C57BL/6 mice (n=4) at different time intervals after exposure to 8.5 Gy of total body irradiation. All data were represented as mean±standard deviation (SD) (error bars are too small to be visualized).

FIG. 6.

Estimation of BH4 to BH2 ratio, peroxynitrite formation, and blood cell counts in irradiated mice. BH4 level, BH2 level, and BH4/BH2 ratio were estimated in lung tissue samples from irradiated Gfrp+/Cre+ transgenic mice (n=7) and irradiated Gfrp−/Cre+ control mice (n=8) 24 h after exposure to 8.5 Gy total body irradiation (TBI) (a). Aortal peroxynitrite formation measured in unirradiated Gfrp+/Cre+ transgenic mice (n=4), unirradiated Gfrp−/Cre+ control mice (n=4), irradiated Gfrp+/Cre+ transgenic mice (n=7), and irradiated Gfrp−/Cre+ control mice (n=6) 3.5 days after exposure to 8.5 Gy of TBI (b). White blood cell (WBC), lymphocyte, monocyte, and granulocyte count in unirradiated Gfrp−/Cre+ control (n=4), unirradiated Gfrp+/Cre+ (n=4), irradiated Gfrp−/Cre+ control (n=6), and irradiated Gfrp+/Cre+ (n=7) 24 h after exposure to 8.5 Gy TBI (c). All data presented as mean±standard error of mean.

FIG. 7.

Schematic model showing effect of GFRP overexpression on cellular homeostasis and endothelial function. Genetic modification or radiation-induced GFRP overexpression causes increased interaction of GFRP with GTPCH1, thereby inhibiting the catalytic activity of GTPCH1 (the rate limiting enzyme in the endogenous BH4 synthesis pathway). Inhibition of GTPCH1 leads to decrease in BH4 biosynthesis and BH4/BH2 ratio. The altered BH4/BH2 ratio causes nitric oxide synthase (NOS) uncoupling, resulting in increased production of superoxide and decreased NO production. Superoxide can directly induce oxidative stress in cells by changing the reduced to oxidized ratio of the cellular thiol pool. Superoxide may also react with NO to form peroxynitrite, thus inducing nitrosative stress. Peroxynitrite also induces oxidative stress by further decreasing the cellular BH4 pool. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars