FORUM ORIGINAL RESEARCH COMMUNICATION
Characterization of Transgenic Gfrp Knock-In Mice:
Implications for Tetrahydrobiopterin in Modulation
of Normal Tissue Radiation Responses
Rupak Pathak,1Snehalata A. Pawar,1Qiang Fu,1Prem K. Gupta,2Maaike Berbe ´e,1Sarita Garg,1
Vijayalakshmi Sridharan,1Wenze Wang,1Prabath G. Biju,1Kimberly J. Krager,1Marjan Boerma,1
Sanchita P. Ghosh,3Amrita K. Cheema,4Howard P. Hendrickson,2
Nukhet Aykin-Burns,1and Martin Hauer-Jensen1,5
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 triphos-
phate cyclohydrolase I (GTPCH1), which is regulated by the inhibitory GTP cyclohydrolase I feedback regu-
latory 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. Antioxid. Redox Signal. 00, 0000–0000.
may switch from producing NO to becoming an important
source of superoxide and peroxynitrite, a process termed
enzymatic ‘‘uncoupling.’’ Inadequate availability of the
redox-sensitive NOS cofactor
(BH4), as a result of rapid oxidation of BH4 to 7,8-dihy-
drobiopterin (BH2), is believed to be an important cause of
NOS uncoupling (28). Moreover, BH4 has been reported to
after radiation exposure, nitric oxide synthase (NOS)
have reactive oxygen species (ROS)-scavenging activity (21).
BH4 has been shown to play a critical role in the pathogenesis
of several conditions characterized by increased oxidative
stress, for example, diabetes, hypertension, and arterioscle-
rosis, as well as in radiation injury (3).
The de novo synthesis of BH4 is under strict control by the
(GTPCH1) (28). The catalytic activity of GTPCH1 is regulated
by the inhibitory GTP cyclohydrolase I feedback regulatory
protein (GFRP) (13). GFRP inhibits the catalytic activity of
GTPCH1 by negative feedback (33) and thereby limits
1Division of Radiation Health and2Department of Pharmaceutical Sciences, University of Arkansas for Medical Sciences, Little Rock,
3Armed Forces Radiobiology Research Institute, Uniformed Services University of the Health Sciences, Bethesda, Maryland.
4Department of Oncology, Georgetown University Medical Center, Washington, District of Columbia.
5Surgical Service, Central Arkansas Veterans Healthcare System, Little Rock, Arkansas.
ANTIOXIDANTS & REDOX SIGNALING
Volume 00, Number 00, 2013
ª Mary Ann Liebert, Inc.
excessive BH4 biosynthesis under normal conditions. GFRP
may be dysregulated under conditions of oxidative stress
(11,16,17). For example, hydrogen peroxide-induced oxida-
tive stress increases Gfrp mRNA expression in endothelial
cells, leading to a decline in BH4 levels (17). These in vitro data
suggest that cellular redox homeostasis plays a critical role in
GFRP expression and hence may regulate BH4 bioavailability
in vivo after exposure to ionizing radiation (IR).
While the significance of GFRP in the regulation of
(18), there is a paucity of studies validating these concepts
in vivo. To obtain further insight into the role of in vivo GFRP
overexpression in the BH4 biosynthetic pathway and BH4
levels during the radiation response in vivo, a novel GFRP
overexpressing Cre-Lox-regulated transgenic mouse model
was generated. Our results demonstrate that in vivo over-
expression of Gfrp induces changes in GTPCH1–GFRP inter-
action, decreases tissue BH4 levels, increases parameters
of oxidative stress, and induces changes in mitochondrial
bioenergetic functions. Moreover, exposure to radiation is
associated with differential effects in redox homeostasis in
transgenic mice and littermate controls. The novel Gfrp
transgenic mouse model will likely become a valuable tool to
study the involvement of BH4 in whole body radiation and is
also suitable for studies of tissue-specific radiation responses
and other conditions associated with oxidative stress.
Generation and analysis of Gfrp transgenic mice
To determine the role of in vivo Gfrp overexpression on
the BH4 biosynthetic pathway, a Gfrp transgenic mouse
model was generated. Gfrp is a single exon gene and the 84
amino acid long GFRP’s molecular weight is *10kDa. Figure
1a illustrates the gene targeting strategy to generate Gfrp
transgenic mice and the subsequent scheme of transgene ex-
pression used in this study. The targeting construct was de-
livered by pronuclear microinjection. Microinjections were
stop vector containing Gfrp cDNA was used to generate the
Gfrp transgenic ‘‘knock-in’’ founder lines in a C57BL/6
background. Upstream of the Gfrp cDNA and downstream of
CAG promoter, a STOP cassette (neo cassette flanked by two
loxP sites) was positioned to prevent transcription. Transgene
expression was achieved upon Cre-mediated deletion of the
Gfrp transgenic mice were generated to investigate
the effect of in vivo Gfrp overexpression on 5,6,7,8-
tetrahydrobiopterin (BH4) biosynthesis and on radiation re-
sponse. This study showed that increased in vivo Gfrp ex-
pression in Gfrp+/Cre+ mice was accompanied by reduced
BH4 levels, increased oxidative stress, differential mitochon-
drial functions, increased radiation-induced peroxynitrite
formation, and decreased white blood cell counts compared
with control mice. Moreover, irradiated control mice ex-
triphosphate cyclohydrolase I feedback regulatory protein
tightly regulates biosynthesis of BH4, which plays a critical
role in cardiovascular, neurological, and oxidative stress-
to understand the roles of BH4 in health and disease.
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
Generation of guanosine triphosphate cyclohydrolase I feedback regulatory protein (Gfrp) knock-in mouse
2 PATHAK ET AL.
STOP cassette allowing the promoter to transcribe the trans-
gene constitutively in Gfrp overexpressing transgenic mice
(Gfrp+/Cre+); while littermates that do not carry the Gfrp
transgene were used as control (Gfrp-/Cre+).
Polymerase chain reaction (PCR) analysis initially identified
six different founder lines, which showed transmission of the
Gfrp transgene tothe germline.The germline transmission of the
Gfrp transgene was assessed by genotyping (Fig. 1b) using
transgene specific primers. Functionality of the transgene was
tested by breeding all the transgenic founder male animals with
EIIa Cre female mice expressing Cre recombinase to delete the
tissues was measured by quantitative reverse transcription PCR
(qRT-PCR) and western blot analysis. Two transgenic founder
lines (706 and 712) were identified that exhibited particularly
high Gfrp expression in all tissues examined and were used to
develop the mouse colonies. The ‘‘706’’ transgenic founder line
was used for this entire study. The Gfrp+/Cre+ mice and the
control littermates were normal in terms of gross morphological
features, development, and fertility (data not shown). No dis-
cernible difference in hematocrit values was observed between
Gfrp+/Cre+ mice and Gfrp-/Cre+ littermates except for gran-
ulocyte percentage. Gfrp+/Cre+ mice had slightly lower gran-
ulocyte percentage (without a difference in total granulocyte
count) compared with control littermates (Fig. 1c).
Next, the transgene copy number was estimated using a
duplex TaqMan PCR-based method. A custom-made ampli-
con spanning part of the Gfrp transgene and part of the in-
sertion vector was designed so that it amplified only the Gfrp
transgene under the specific experimental setup without
amplifying the endogenous Gfrp gene. TaqMan assays for the
Gfrp transgene and the internal control were performed to-
gether for eachanimal inquadruplet todetermine thenumber
of transgene copies integrated in the genome. A significantly
greater Gfrp transgene copy number was observed in Gfrp+/
Cre+ mice, while Gfrp-/Cre+ exhibited similar transgene
copy numbers as those of C57BL/6 mice. Approximately 35
copies of the Gfrp transgene were integrated in Gfrp+/Cre+
mice as estimated from the standard curve (Fig. 1d).
Increased GFRP expression in tissues
of Gfrp+/Cre+ mice
Gfrp expression at the mRNA level was measured in mul-
Gfrp mRNA expression was significantly higher in Gfrp+/Cre+
mice than in control littermates in every organ evaluated.
However, variability in the level of Gfrp transgene expression
was observed in different tissues of Gfrp+/Cre+ mice. Gfrp ex-
pression was highest in thymus followed by spleen, lung, in-
testine, kidney, and liver in Gfrp+/Cre+ mice. In the same
tissues, no statistically significant change in Gtpch1 expression
levels was found (Supplementary Fig. S1; Supplementary Data
are available online at www.liebertpub.com/ars).
We also measured GFRP protein expression in six different
tissues from the Gfrp+/Cre+ mice and Gfrp-/Cre+ littermates.
GFRP was detected in all tissues of Gfrp+/Cre+ mice that were
investigated. As expected, the level of GFRP expression was
significantly higher in Gfrp+/Cre+ mice compared with con-
liver tissue. Similarly, basal level of GFRP in control mice was
relatively high in liver compared with other tissues. Im-
munohistochemical analysis further confirmed the presence of
Cre+ mice compared with the control group (Fig. 2c and
Supplementary Figs. S2–S5). These data are consistent with
electron microscopy studies performed by others (6).
Reduced BH4 levels and increased GTPCH1–GFRP
interaction in Gfrp+/Cre+mice
Since GFRP is known to inhibit GTPCH1 catalytic activity
by protein–protein interaction, we next investigated the effect
GFRP protein in tissues. Re-
change in liver, kidney, in-
thymus as detected by quan-
titative reverse transcription
polymerase chain reaction in
male Gfrp-Tg (n=4) mice and
control littermates (n=4) (a).
Expression of GFRP (10kDa)
was detected by western blot-
ting in Gfrp+/Cre+ mice (n=2)
and Gfrp-/Cre+ control litter-
(42kDa) as internal control (b).
chemical staining of GFRP in
tissues from Gfrp+/Cre+ mice
and Gfrp-/Cre+ control litter-
mates, original magnification
20· (c). All data presented as
mean. NS, not statistically sig-
Gfrp mRNA and
GFRP MODULATES BH4 SYNTHESIS AND OXIDATIVE STRESS3
of in vivo GFRP overexpression on the de novo BH4 biosyn-
thetic pathway. BH4 level in lung sample of Gfrp+/Cre+ and
control littermates wasestimated by aliquid chromatography
with tandem mass spectrometry (LC-MS/MS) method. The
reason for selecting lung tissue in this study is that lung has
maximum endothelial cellsper unit areacompared withother
tissues, and that BH4 plays a particularly critical role in reg-
ulating endothelial function by modulating eNOS activity.
Gfrp+/Cre+ mice exhibited significantly less BH4 level com-
pared with control littermates (Fig. 3a). In addition, we ob-
served significantly less BH2 level in transgenic mice, and, as
a result, no significant difference in BH4/BH2 ratio was ob-
served between two genotypes (Fig. 3a).
To determine the reason for the lower BH4 levels in Gfrp+/
Cre+ mice, we hypothesized that the interaction of GFRP
with its protein partner GTPCH1 would be increased in
Gfrp+/Cre+ mice compared with control littermates, thereby
inhibiting GTPCH1 activity. To address this, we determined
the interaction between these two proteins by co-immuno-
precipitation (IP) assay. Significantly increased GTPCH1–
GFRP interaction in Gfrp+/Cre+ mice compared with control
littermates was observed (Fig. 3band Supplementary Fig. S6).
Increased oxidative stress and elevated reserve
respiratory capacity in Gfrp+/Cre+mice
Decreased BH4 bioavailability may lead to increased oxida-
determine the status of cellular oxidative stress, total glutathi-
one (GSH) and oxidized glutathione (GSSG) levels were mea-
sured in peripheral blood obtained from Gfrp+/Cre+ mice and
Gfrp-/Cre+ littermates. Significantly lower total GSH levels
and higher percentage GSSG, an endpoint indicative of oxida-
tive stress, were found in peripheral blood samples of Gfrp+/
Cre+ mice compared with Gfrp-/Cre+ littermates (Fig. 4a, b).
Because increased oxidative/nitrosative stress is strongly
correlated with mitochondrial dysfunction, we next investi-
gated the bioenergetic profile of mitochondria isolated from
primary thymocytes of Gfrp+/Cre+ mice and their Gfrp-/
Cre+ littermates. The function of individual components of
the respiratory chain was examined by sequentially adding
chemical inhibitors to primary thymocytes (Fig. 4c, d). Com-
pared with Gfrp-/Cre+ controls, Gfrp+/Cre+ mice demon-
strated increased basal oxygen consumption rate (OCR).
Adenosine triphosphate (ATP)-linked OCR and proton-
leaked OCR were also determined in thymocytes of Gfrp+/
Cre+ andcontrolGfrp-/Cre+ micebyoligomycinadditionto
inhibit ATP synthase (Complex V). Although OCR decreased
after oligomycin addition in both groups, increased ATP-
linked OCR and proton-leaked OCR were observed in Gfrp+/
Cre+ mice. To determine the maximal OCR, primary thymo-
cytes were next treated with proton ionophore (uncoupler)
(FCCP). As expected, OCR increased in both groups after
FCCP treatment as mitochondrial inner membrane became
permeable to protons; however, the maximal OCR and reserve
capacity was higher in Gfrp+/Cre+ mice. Finally, to determine
non-mitochondrial OCR, a mixture of rotenone and antimycin
A was injected to inhibit electron flux through complexes I and
III causing drastic suppression of OCR. No difference in non-
mitochondrial OCR was observed in either group.
Increased Gfrp mRNA expression in irradiated
To determine the effect of IR on in vivo Gfrp expression,
C57BL/6 male mice were exposed to 8.5 Gy of total body
irradiation (TBI). Gfrp expression at the mRNA level in lung
samples was measured by qRT-PCR at 24h and 3.5 days after
irradiation. As shown in Figure 5, Gfrp expression in lung
samples increased after radiation. Gfrp expression increased
*2-fold and 1.5-fold at 24h and 3.5 days after radiation
exposure, respectively. These data indicate that radiation-
induced oxidative stress modulates in vivo Gfrp expression,
which may play a role in de novo BH4 biosynthesis.
Decreased BH4/BH2 ratio in irradiated
Next, we investigated the effect of IR on the BH4 level, BH2
level, and BH4/BH2 ratio in Gfrp+/Cre+ and Gfrp-/Cre+
cyclohydrolase I (GTPCH1)-GFRP interaction in unirradiated mice. BH4 level, BH2 level and BH4/BH2 ratio were esti-
mated 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-immunoprecipi-
tation 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
Estimation of 5,6,7,8-tetrahydrobiopterin (BH4) level, 7,8-dihydrobiopterin (BH2) level, BH4/BH2 ratio, and GTP
4 PATHAK ET AL.
mice. Although we did not observe significant difference in
BH4 and BH2 level between Gfrp+/Cre+ and Gfrp-/Cre+
mice after irradiation, however, significantly low BH4/BH2
ratio was observed in irradiated Gfrp+/Cre+ mice compared
with irradiated Gfrp-/Cre+ (Fig. 6a). We also observed sig-
nificant decrease in BH4 level and BH4/BH2 ratio, while in-
crease in BH2level in both Gfrp+/Cre+ and Gfrp-/Cre+ mice
after radiation exposure compared with unirradiated group.
These data are consistent with the notion that oxidative stress
including IR has a critical role in BH4-BH2 homeostasis and
irradiation causes further decrease in BH4/BH2 ratio in
Gfrp+/Cre+ mice compared with the Gfrp-/Cre+ group.
Increased peroxynitrite formation and decreased white
blood cell counts in irradiated Gfrp+/Cre+mice
Next, wedetermined theeffectofIRonnitrosative stressby
measuring peroxynitrite formation in Gfrp+/Cre+
Gfrp-/Cre+ mice. Peroxynitrite formation, a marker of oxi-
24h and 3.5 day after TBI. Vascular peroxynitrite formation
significantly increased after irradiation compared with un-
irradiated groups and significantly higher peroxynitrite for-
mation was observed in Gfrp+/Cre+ mice compared with
Gfrp-/Cre+ littermates at day 3.5 after exposure to 8.5 (Fig.
6b). Ontheotherhand, nostatistically significantdifference in
peroxynitrite formation between two groups was observed at
24h after irradiation (data not shown). These results indicate
that Gfrp+/Cre+ mice are more prone to radiation-induced
oxidative/nitrosative stress, probably due to GFRP over-
expression mediated BH4 unavailability.
Blood cell counts were also assessed in Gfrp+/Cre+ mice
and its Gfrp-/Cre+ littermates at 24h and 3.5 days after ex-
posure to 8.5 Gy of TBI. No significant decrease in red blood
cell counts and hemoglobin levels were observed at 24h,
while both the parameters were significantly decreased at 3.5
days; however, no statistically significant differences between
Gfrp+/Cre+ mice and Gfrp-/Cre+ control group were ob-
served (data not shown). White blood cell (WBC) count was
also significantly decreased (p<0.001) at 24h in irradiated
groups compared with unirradiated groups. Notably, signif-
icant decrease in WBC counts were observed in Gfrp+/Cre+
mice after irradiation compared with irradiated Gfrp-/Cre+
littermates as shown in Figure 6c. The counts of all WBC
subtypes were also significantly lower in irradiated Gfrp+/
Cre+ mice compared with irradiated controls (Fig. 6c). WBC
Relative Gfrp mRNA fold change in lung tissue samples
from wild-type C57BL/6 mice (n=4) at different time inter-
vals 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).
Gfrp mRNA expression in irradiated lung tissue.
glutathione (GSH) level and
functions. Total GSH (a) and
oxidized glutathione (GSSG)
percent (b) estimated in blood
samples of Gfrp+/Cre+ trans-
genic mice (n=6) and Gfrp-/
Cre+ control littermates (n=6).
(OCR) in primary thymocytes
from Gfrp+/Cre+ transgenic
mice (n=4) and Gfrp-/Cre+
control littermates (n=4) (c).
Individual mitochondrial func-
tional parameters in primary
thymocytes from Gfrp+/Cre+
transgenic mice (n=4) and
Gfrp-/Cre+ control littermates
To see this illustration in color,
version of this article at www
Estimation of blood
GFRP MODULATES BH4 SYNTHESIS AND OXIDATIVE STRESS5
were undetectable at 3.5 days after irradiation, both in Gfrp+/
Cre+ mice and Gfrp-/Cre+ controls. Consistent with these
results, limited TBI experiments, while not reaching statistical
significance, showed 30 day survival in Gfrp+/Cre+ mice to
be 0%, compared with 25% in Gfrp-/Cre+ littermates (Sup-
plementary Fig. S7).
This study reports the generation and initial characteriza-
tion of a novel Cre-Lox-driven Gfrp overexpressing knock-in
transgenic mouse model. A key role for GFRP in the mainte-
nance of BH4 and redox homeostasis in vivo was revealed.
Moreover, we showed that in vivo Gfrp overexpression is an
important factor in modulating aspects of radiation-induced
oxidative damage, thus lending further support to the notion
that BH4 is critically involved in radiation toxicity in normal
Although GTPCH1 is ubiquitously expressed in most eu-
karyotic tissues, GFRP expression is more tissue specific, with
the level of expression and interaction with GTPCH1 varying
from organ to organ (9,20). Consistent with previous studies
from other laboratories (20), our study showed higher basal
level of Gfrp expression in liver.
The role of GFRP in the regulation of BH4 biosynthesis
in vivo is not known and the in vitro data are somewhat con-
troversial. Numerous studies have contributed strong evi-
dence that GFRP inhibits GTPCH1 activity in an allosteric
manner resulting in reduced BH4 biosynthesis (22,31,32).
Moreover, several in vitro studies have revealed that GFRP
overexpression significantly decreases BH4 biosynthesis,
while lowering GFRP expression with lipopolysaccharide
increases BH4 level in endothelial cells (17,22). While these
data are consistent with the notion that GFRP negatively
modulates BH4 biosynthesis, other investigators report no
role for GFRP in the regulation of BH4 biosynthesis (27). We
examined theroleofinvivo Gfrpoverexpression onthedenovo
BH4 biosynthesis and observed a reduction in tissue BH4
levels in Gfrp overexpressing transgenic mice. The GFRP-
mediated BH4 downregulation could be due to increased in-
teraction of GFRP with GTPCH1 in transgenic mice. Indeed,
our pull-down assay confirmedincreased interaction ofGFRP
with GTPCH1 in different tissues of Gfrp+/Cre+ mice com-
pared with control littermates.
Maintenance of BH4 homeostasis is critical for the preser-
vation of cellular redox balance (26). Decreased BH4 bio-
availability results in NOS uncoupling, leading to generation
of more superoxide compared with NO, which in turn oxi-
an imbalance in cellular redox homeostasis. The current study
revealed reduced levels of BH4 and increased oxidative stress
in Gfrp+/Cre+ mice, consistent with a role for BH4 in re-
ducing oxidative stress. Moreover, oxidative and/or ni-
trosative stress have a profound effect on mitochondrial
function (8). Assessing various aspects of mitochondrial
function, we observed increased basal, ATP-linked, and
proton-leaked OCR in primary thymocytes from Gfrp+/Cre+
mice, suggesting elevated oxidative stress in these animals
compared with control littermates. Consistent with our ob-
servation, Dranka et al. (2011) also found that basal, ATP-
linked and proton-leaked OCR was higher in neonatal rat
ventricular astrocytes after hydrogen peroxide-induced
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) 24h 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), ir-
radiated 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) 24h after exposure
to 8.5 Gy TBI (c). All data presented as mean–standard error of mean.
Estimation of BH4 to BH2 ratio, peroxynitrite formation, and blood cell counts in irradiated mice. BH4 level, BH2
6 PATHAK ET AL.
oxidative stress (7). Moreover, in our study, we observed in-
creased maximal OCR and reserve capacity in transgenic
mice, suggesting that the mitochondria in thymocytes ob-
tained from Gfrp+/Cre+ mice still maintain their membrane
potential and might be responding to a stress-induced in-
crease in energy demand. Notably, these changes occur
without an increase in non-mitochondrial OCR, indicating
that non-mitochondrial respiration is negligible in both
groups of animals.
Oxidative stress, including radiation-induced oxidative
stress, significantly alters GTPCH1 and GFRP expression re-
sulting in alteration in BH4 bioavailability (14,16,17,22,29).
We have recently shown that radiation causes a temporary,
significant decrease inlung BH4level (3),but therole ofGFRP
in this regard is unknown. The present study revealed a time-
dependent increase in Gfrp mRNA expression in irradiated
control mice, which may repress GTPCH1 activity and result
in a relative BH4 deficiency. Like radiation-induced oxidative
been shown to induce Gfrp mRNA expression in a time-
dependent manner in human endothelial cells (17). These
in vivo and in vitro results clearly indicate that Gfrp expression
is susceptible to oxidant signaling and that it negatively im-
pacts BH4 bioavailability. On the other hand, Ishi et al. (16)
reported decreased GFRP expression after oxidative stress
for these contradictory results may be the use of different cell
types and different concentrations of hydrogen peroxide by
the two groups.
Irradiation causes immediate production of ROS, with
formation of superoxide being particularly detrimental be-
cause of its high reactive potential (24). Superoxide may
irradiation, to form the powerful toxic oxidant peroxynitrite.
Recently, we showed that increased vascular peroxynitrite
formation inirradiatedCD2F1micewassignificantly reduced
by gamma tocotrienol treatment, and by BH4 supplementa-
tion (3). Peroxynitrite formed after radiation exposure or ra-
diation-induced ROS can oxidize BH4 to form BH2 resulting
indecreased cellular BH4/BH2ratio, which is criticalfor NOS
uncoupling. Our data revealed significantly lower BH4/BH2
ratio and increased formation of peroxynitrite in irradiated
Gfrp+/Cre+ mice compared with control littermates, consis-
tent with the notion that irradiation induces more oxidative
damage in transgenic mice.
Irradiation, by affecting the bone marrow, has a profound
effect on the levels of circulating blood cells. It has been re-
ported that WBC are more susceptible to radiation in mutant
mouse models of oxidative stress. For example, p53 knockout
mice exhibit a marked decrease in WBC counts compared
with control mice after exposure to sublethal dose of IR (30).
Similarly, we also observed a significant decrease in WBC
counts in Gfrp overexpressing transgenic mice.
The profound alteration of mitochondrial activity caused
by GFRP overexpression was a striking finding. The most
likely explanation is that GFRP overexpression alters mito-
chondrial bioenergetics indirectly via redox processes and
reduced levels of BH4. This is consistent with studies dem-
onstrating that oxidative stress changes the mitochondrial
bioenergetic profile (8) and that BH4 deficiency is linked to
mitochondrial dysfunction (2). The first very simple expla-
nation for why GFRP overexpression (accompanied by de-
creased BH4 bioavailability, NOS uncoupling, and decreased
NOproduction) leads to changes in mitochondrial respiration
is that NO reversibly binds to the oxygen-binding site of cy-
with oxygen in complex IV is limited, thus likely causing an
increase in mitochondrial respiration. Similar responses (in-
creased oxygen consumption) was demonstrated when NO
levels were decreased using L-NAME (a NOS inhibitor) in
other studies (19). Another possibility to explain the altered
mediated stressresponse. Hence,oxidative damage to proteins
in the mitochondrial electron transport chain might lead to al-
terations in the rate of electron flux through electron transport
chain and hence, changes in mitochondrial respiration.
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 ofthe 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
Schematic model showing effect of GFRP overexpression on cellular homeostasis and endothelial function. Genetic
GFRP MODULATES BH4 SYNTHESIS AND OXIDATIVE STRESS7
In conclusion, this is, to our knowledge, the first demon-
stration that in vivo Gfrp overexpression increases the inter-
action of GFRP with GTPCH1 to negatively regulate BH4
biosynthesis and increase the level of oxidative/nitrosative
stress induced by IR. A schema summarizing the findings
from the present study is shown in Figure 7. Further research
is required to investigate theexact relationships among invivo
GFRP overexpression, NOS uncoupling, and oxidative/ni-
trosative stress. The novel Gfrp+/Cre+ transgenic mouse
model presented here is a useful new tool for studies into the
role of BH4 in health and disease and, because of the Cre-Lox
technology used, provides opportunities for tissue-specific
Materials and Methods
Male mice with an average body weight of 23–26g were
used. The experimental protocols were reviewed and ap-
proved by the Central Arkansas Veterans Healthcare System
Institutional Animal Care and Use Committee (IACUC) and
by the IACUC at the University of Arkansas for Medical
C57BL/6J (stock No. 000664) and EIIa Cre (stock No.
003724) mice were obtained from The Jackson Laboratory.
The Gfrp transgenic mice were developed on a C57BL/6
background in the Transgenic Core Facility of University of
Arkansas for Medical Sciences.
Plasmid construction for transgenic mice
Briefly, total cDNA was generated from mRNA of CD2F1
bone marrow cells by RT-PCR using the oligo dT primers
(Universal Riboclone cDNA synthesis system; Promega). The
Gfrp gene specific primers (pQFLNL-gfrp forward primer
and pQFLNL -gfrp reverse primer sequence 5¢-GCGGCCGC
GCTCAAGCAGCTCATTC-3¢) were used to amplify a 300bp
respectively from the total cDNA. This product was gel pu-
rified, subcloned in vector, and verified by sequencing. The
GFP gene in pCALNL-GFP was replaced with the 0.3kb Gfrp
cDNA by digesting with Bcl I/BstB I. In the pCALNL-Gfrp
construct, the Gfrp cDNA was flanked by loxP sites on either
sides of a stop codon, downstream of a CAG enhancer in the
pCALNL vector (Addgene). Cre-mediated excision ofthe stop
codon will allow for the expression of the mouse Gfrp trans-
gene. The resulting plasmid, pQFLNL-Gfrp was again con-
firmed by DNA sequencing using standard techniques and
used to create the Gfrp transgenic founder lines.
Gfrp positive male founders were crossed with female
C57BL/6J mice to maintain the founder lines. To generate
Gfrp overexpressing transgenic
founders were crossed with EIIa Cre females and pups were
genotyped to determine the presence of the Gfrp transgene.
The littermates that had not received the Gfrp transgene after
crossing with EIIa Cre were used as control mice (Gfrp-/
Cre+). All experiments were carried out with the mice ob-
first filial generation mice, irrespective of their Gfrp status.
The presence of the Gfrp transgene was determined with
PCR on tail DNA. Mouse tail was lysed in DirectPCR (Tail)
lysis reagent (Viagen Biotech) with Proteinase-K (Applied
of lysate was used for PCR.
The forward primer (5¢GTTATCTCGAGTCGCTCGGT
ACG 3¢) in the vector construct and the reverse primer
(5¢CATGGTGGGGCCCACCTCCATAC 3¢) in the transgene
were designed to particularly amplify transgene. Gfrp PCR
amplification was performed in a 25ll reaction mixture con-
taining 1· Green Go Taq reaction buffer (Promega), 1.25mM
magnesium chloride (MgCl2; Invitrogen), 200lM deox-
ynucleoside triphosphate (Thermo Scientific), and 0.04U of
Go Taq DNA polymerase (Promega). The thermal conditions
for Gfrp PCRswereasfollows: initialdenaturation for 5min at
94?C, followed by 30 cycles of 94?C for 1min, 58?C for 45s,
and 72?C for 1min, and finally 1 extension cycle of 10min at
The concentration of MgCl2was 1.5mM for Cre PCR. The
forward primer sequence was 5¢GCG GTC TGG CAG TAA
AAA CTA TC 3¢, while the reverse primer sequence was
5¢GTG AAA CAG CAT TGC TGT CAC TT 3¢. The Cre am-
plification parameters were as follows: after an initial dena-
turation at 94?C for 3min, amplification was performed
for 35 cycles at 94?C for 30s, 51?C for 1min, and 72?C for
1min, followed by a final extension step at 72?C for 2min. All
the primers were obtained from Integrated DNA Technolo-
Isolation of genomic DNA from mouse tails
DNA was extracted from mouse tail biopsies using a
DNeasy Blood & Tissue Kit (Qiagen) as per manufacturer’s
protocol. Genomic DNA samples were quantified by Nano-
Drop (Thermo Scientific).
Custom-made TaqMan-based Copy Number Assay
Custom plus TaqMan?Copy Number Assay was designed
using online software (Applied Biosystems) to determine
copy number as previously described (15). The amplicon
was designed spanning the vector construct and the
transgene to avoid endogenous Gfrp amplification. Each
PCR mix contained 10ll of 2·TaqMan?Universal Master
Mix, 1ll of the TaqMan Copy Number target assay (RP-
GFRP), and 1ll of the TaqMan Copy Number reference
assay (Tfrc), which is known to exist only in two copies in a
diploid genome, 4ll of Nuclease-free water, and 4ll of tail
DNA (20ng). All the reagents were procured from Applied
Biosystems. The reactions were processed in an ABI 7500
Fast Real-Time PCR System. The copy number of transgene
was calculated using the standard curve method as previ-
ously described (15).
RNA extraction and qRT-PCR
Total RNA was purified from frozen tissue using RNeasy
Plus Mini Kit (Qiagen) as instructed by manufacturer after
homogenizing the samples in TRIzol?Reagent (Life Tech-
nologies). cDNA was synthesized using a cDNA reverse
transcription kit (Applied Biosystems) after treating with RQ-
DNase I (Promega). Predesigned TaqMan assay (Applied
8PATHAK ET AL.
Biosystems) for mouse gene: Gfrp, Mm00622819_m1; Gtpch1,
Mm01322973_m1; and 18s rRNA, Hs99999901_s1 was used.
The mRNA levels were normalized to eukaryotic 18s rRNA
and calculated relative to control mice, using the standard
Western blotting analysis
GFRP (Proteintech), GTPCH1 (Santa Cruz Biotechnology,
Inc.), and b-actin (Cell Signaling Technology) was used at
1:1000 ratio, while secondary antibody was used at a ratio of
Immunohistochemical staining for GFRP was performed
elsewhere (25). Images were captured at 20· magnification.
BH4 and BH2 estimation
Frozen tissue was homogenized for 1min in 500ll of ho-
mogenizing buffer, consisted of Tris-HCl (pH 7.3), 10mM di-
thiothreitol, 1mM ethylenediaminetetraacetic acid (EDTA)
solution, and 166ng/ml internal standard mix [(5-15N)-biopter-
in, (5-15N)-dihydrobiopterin, and (5-15N)-tetrahydrobiopterin].
Biopterins were purchased from Schircks Laboratories. The ho-
(250ll) of supernatant was added to 100ll of a mixture con-
and gently mixed. Perchloric acid (38ll) was added and incu-
bated on ice for 5 min; the samples were then centrifuged. The
supernatant (250ll) was added to 25ll of a 2.5 M potassium
bicarbonate and mixed. Samples were placed on ice for 10min
of an ammoniumformate buffer (500mM, pH 2.7). The samples
Extractedbiopterins wereseparated onaBetabasic 3lmC8
analytical column (150mm·2.1mm), which was maintained
at 45?C. The linear binary gradient consisted of solvent A;
5mM ammonium formate buffer (pH 2.7), 0.1% per-
fluoroheptanoic acid, and 10% acetonitrile and solvent B; 99%
acetonitrile, 5mM ammonium formate buffer (pH 2.7), and
0.1% perfluoroheptanoic acid. The sample (8ll) was injected
onto a 20-ll loop. The initial flow rate and %B was 0.3ml/min
and 10%. The %B was increased over the next 4min to 30%.
From 4 to 4.5min the gradient was increased to 90% B and the
flow rate increased to 0.5ml/min. The gradient was held at
90% for 2min and then returned to initial conditions (6.5–
7.0min). The total run time was 10min.
Positive ions were generated using electrospray ionization
at a capillary voltage of 2.5kV. The cone voltage (35 V) was
adjusted to optimize the precursor ion for each compound.
The mass analyzer, a Quattro Premier triple quadrupole
(Waters) was operated in multiple-reaction-monitoring mode
using argon at a pressure of 3.9·10-3bar. Biopterin, (5-15N)-
biopterin, dihydrobiopterin, (5-15N)-dihydrobiopterin, tetra-
hydrobiopterin, and (5-15N)-tetrahydrobiopterin, were detected
using transitions 237.9>219.9, 238.9>220.9, 239.9>167.9,
240.9>165.9, 241.9>165.9, 243.3>167.0, respectively.
Frozen lung and liver tissue was rapidly homogenized in
500ll buffer (50mM Tris-HCl, 150mM sodium chloride,
1mM ethyleneglycoltetraacetic acid (EGTA), 1mM EDTA,
and 1%Triton·100). One milligram of protein was incubated
1h on a rotary shaker at 4?C. After incubation, protein G-
magnetic beads (Life Technologies) were added for an addi-
tional hour, and then washed with fresh buffer, and the im-
munoprecipitates were eluted, boiled for 5min in laemmli
sodium dodecyl sulfate (SDS) sample buffer, and frozen until
used for western blotting.
Blood samples were collected from the retro-orbital plexus,
immediately lysed in 5% sulfosalicylic acid and centrifuged.
The 5,5¢-dithiobis-2-nitrobenzoic acid recycling assay was
performed to measure GSH and GSSG levels in the superna-
tants, as described previously (12,23). The data were nor-
malized to protein content, determined by the bicinchoninic
acid (BCA) protein assay, as per the manufacturer’s instruc-
tions (Pierce Biotechnology).
Measurement of mitochondrial function in primary
thymocytes using the XF96-extracellular flux analyzer
OCR was measured at 37?C using an XF96 extracellular an-
alyzer (Seahorse Bioscience) as previously described (10). Brief-
ly, 175,000 freshly isolated thymocytes per well were plated in
CellTak-coated plates, changed to unbuffered Dulbecco’s Mod-
ified Eagle Medium (DMEM) supplemented with 4mM gluta-
baseline measurements were acquired before injection of mito-
chondrial inhibitors or uncouplers. Readings were taken after
sequential addition of oligomycin (4lM), FCCP (4lM), and
rotenone/antimycin A (10lM). OCR was calculated by the
Seahorse XF-96 software and represents an average of 40–80
day). The OCR was normalized to cell number.
TBI was performed as described before (4). The average
dose rate was 1.21 Gy/min.
Blood cell counts
At 24h and 3.5 day after exposure to 8.5 Gy of TBI, whole
blood was collected into EDTA-coated tubes (Fisher Scien-
tific). Mouse peripheral blood cell counts were obtained using
a veterinary hemocytometer (Hematrue System; Heska Cor-
poration) according to the manufacturer’s instructions.
Vascularperoxynitrite production wasmeasuredat3.5day
after 8.5 Gy of TBI as described earlier (3). Protein concen-
tration in the supernatant was measured using BCA protein
assay kit. Fluorescence was expressed per mg protein.
Results were expressed as mean–standard error of mean.
Data were analyzed using Prism software (version 4.0;
GFRP MODULATES BH4 SYNTHESIS AND OXIDATIVE STRESS9
GraphPad). Comparison among multiple means was per-
formed by analysis of variance and pairwise comparison of
means with the Student’s t-test. A two-sided value of p<0.05
was considered statistically significant.
We would like to thank Dr. Charles A. O’Brien of the
University of Arkansas for Medical Sciences Transgenic
Mouse Facility for helping in generation of Gfrp over-
expressing transgenic mice. Financial support was received
and the Veterans Administration.
Author Disclosure Statement
The authors declare that no competing financial interests
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Address correspondence to:
Dr. Martin Hauer-Jensen
University of Arkansas for Medical Sciences
4301 West Markham Street, Slot 522-10
Little Rock, AR 72205
of final revised submission, February 22, 2013; date of ac-
ceptance, March 22, 2013.
FCCP¼carbonyl cyanide 4-(trifluoromethoxy)
GFRP¼GTP cyclohydrolase I feedback
GTPCH1¼GTP cyclohydrolase I
LC-MS/MS¼liquid chromatography and tandem
NOS¼nitric oxide synthase
OCR¼oxygen consumption rate
PCR¼polymerase chain reaction
qRT-PCR¼quantitative reverse transcription PCR
ROS¼reactive oxygen species
TBI¼total body irradiation
WBC¼white blood cell
GFRP MODULATES BH4 SYNTHESIS AND OXIDATIVE STRESS11