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,1
* Department of Veterinary and Biomedical Science,
Department of Animal Sciences,
Center for Biotechnology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA
1Correspondence: Department of Veterinary and Biomedical Science, University of Nebraska-Lincoln, E117 Beadle Center, 1901 Vine St., Lincoln, NE 68588-0665, USA. E-mail: yzhou2{at}unl.edu
SPECIFIC AIMS
The specific aims of this study were to characterize a polymorphic form of glucocorticoid receptor (GR) found with high distribution frequency in a mouse line developed through genetic selection for altered stress response and to investigate the phenotypic properties of this polymorphic GR in neuroendocrine and behavioral response to stress.
PRINCIPAL FINDINGS
1. Two mouse lines have marked genetic difference in corticosterone response to stress
We developed two mouse lines (high=SH and low=SL) through divergent genetic selection for altered corticosterone response to stress, starting from separate base populations of two lines of mice already characterized by genetic difference in heat loss, locomotor activity, and expression of hypothalamic genes. The SH mice exhibited significantly higher serum corticosterone levels in response to restraint stress (overall difference, F=68.82; P<0.0001).
2. A polymorphic form of GR with an expanded track of polyglutamines has a much higher frequency in the SL line of mice
Using RT-polymerase chain reaction (RT-PCR), we discovered the existence of a GR polymorphism with a marked difference in distribution frequency between the SH and SL lines (Fig. 1
A). Molecular and biochemical characterization (Fig. 1B
) revealed that the polymorphic GR (GRQn) has eight extra CAGs or glutamines expanded from the existing track of CAG repeats/polyglutamines in Exon 2 of the wild-type (WT) form of GR (GRwt, the mouse NR3C1 gene; mRNA Sequence ID: NM-008173). Using additional primers based on the published sequences, we cloned full-length cDNA of both forms of GR and sequenced several clones obtained from independent RT-PCR using different mouse brain RNA. Sequence data indicated that both forms of GR have the same initiation site. However, the GRQn has two silent mutations in contrast to GRwt: position 222, A 
G and position, 468, T C (444 for the WT GR). The two GR isoforms were further confirmed in protein level by immunoprecipitation followed by Western blot analysis using GR-specific antibodies, which revealed the GR isoforms with different molecular mass (Fig. 1C
).
We screened all G4 mice by genomic polymerase chain reaction (PCR) using the same primers covering the coding region containing the CAG repeats of the GR gene. Analysis of the PCR products of the GR isoforms revealed that frequency of the GRQn form was much greater in SL line (total 68 mice; GRQn frequency=0.84, GRwt frequency=0.16; none with GRwt/wt), whereas frequency of the GRwt form was much higher in SH mice (total 78 mice; GRwt frequency=0.91, GRQn frequency=0.09).
3. The distribution pattern of the two forms of GR suggests an association of GRQn with low corticosterone response to stress
We measured serum corticosterone in response to a 30 min restraint among three GR genotypes of fourth selection generation male mice: GRwt/wt (from SH line), GRQn/Qn (from SL line), and GRwt/Qn (from SL line). While no difference in levels of serum corticosterone concentration was seen in control groups of the three GR genotypes, GRwt/wt mice showed significantly greater corticosterone response at both given time points than the GRQn/Qn and GRwt/Qn mice exposed to restraint (F=70.43, P<0.0001). However, no difference in corticosterone levels was found in the latter two GR genotypes in response to stress. This indicates that the GRQn is probably involved, if not directly, in altered corticosterone response to restraint stress.
4. The GRwt/Qn and GRQn/Qn mice exhibit hypoactive and anxiety-type behavior
Using the same mice that were used for corticosterone assays as described above, we performed behavioral assessment by means of open field and elevated plus maze tests.
Open field assessment
Both the GRQn/Qn and GRwt/Qn mice had similar velocity, which was lower than the GRwt/wt mice (overall difference: F=13.4, P=0.001). The 30 min restraint caused a decrease in velocity in both the GRQn/Qn and GRwt/Qn mice, which was much lower than that of GRwt/wt mice (F=7.97, P=0.008). When compared with the WT mice both the GRQn/Qn and GRwt/Qn traveled less (overall difference: F=14.21, P=0.0007), especially the ones exposed to the restraint stress, which had the shortest distance traveled among all the groups in day 1 open field test. Again, the GRQn had a dominant effect on behavioral response to stress (F=4.97, P=0.033). The differences in distance traveled between either the GRQn/Qn or GRwt/Qn mice and the GRwt/wt were not only due to their lower locomotor activity, but also the duration they stayed in the corner zones without much movement, compared with the WT (overall difference: F=17.08, P=0.0003), especially those exposed to restraint (
60–80% of time remaining in corner zone). None of the control GRwt/Qn mice visited the center zone when exposed to the open field for the second time (day 2). The restraint stress resulted in a prolonged time of stay of the GRwt/Qn and GRQn/Qn, but not the WT mice, in the starting corner. We observed that most of the GRwt/Qn stayed in the starting corner or moved out of the starting corner for only a few seconds; with only one exception, they never traveled to the center zone. The GRQn/Qn and GRwt/Qn mice had significantly lower center activities (represented as the frequency and time in the center zone) than that of the GRwt/wt mice; but these increased when preexposed to the 30 min restraint.
Elevated plus maze
The lower locomotor activity and increased anxiety-type behaviors in mice with the polymorphic GR were further confirmed by the elevated-plus-maze assessment. The GRQn/Qn and GRwt/Qn mice spent significantly less time than the GRwt/wt mice in the open arms (F=52.15, P<0.0001) and much longer in the closed arms (F=6.7, P=0.016). In addition, the mice with WT GR showed much higher locomotor activity, traveling more than the GRQn/Qn and GRwt/Qn mice (F=75.83, P<0.001). Stronger response to restraint stress was observed in the GRwt/Qn mice, which appeared to have lower mobility and spent more time in the center zone (data not shown), resulting a nonstatistically significant decrease in time in closed or open arms but much less distance moved (F=6.53, P<0.014). Taken together with data from the open field test (time in corner zones, the much lower activity of both the GRQn/Qn and GRwt/Qn mice in the center zones) suggests a possible linkage of this polymorphic form of GR with the hypoactivity and anxiety-like behaviors.
5. Identification of the polymorphic form of GR in commonly used inbred lines of mice
Using the specific GR primers covering the track of CAG repeats, we screened 16 inbred mouse strains by genomic PCR. Five of the examined inbred lines of mice have the polymorphic form of GR (ST/bJ, SWR/J, C57BL/6J, C57BR/cdJ, and C57L/J), while the other 11 strains have the WT form (129x1/SvJ, A/J, AKR/J, BALB/cJ, C3H/HeJ, C58/J, I/LnJ, NOR/Lt, RF/J, PERA/Rk, RIIIS/J). Among the five strains of mice with the GRQn, ST/bJ line has a different origin from the other four strains. The C57BL/6J, C57BR/cdJ, and C57L/J share the same origin with a genetic linkage to Asian Mus musculus, including the SWR strain.
CONCLUSIONS AND SIGNIFICANCE
The GRQn with an expanded track of polyglutamines (PolyQ) and two silent mutations was found in this study, with high frequency in a mouse line (SL line) with lower corticosterone response to stress, suggesting an involvement of this polymorphic form of GR in a mouse population with inhered altered response to stress. Behavioral assessment by open field and elevated plus maze tests indicate that this polymorphic form of GR may also involve in genetic mechanisms leading to anxiety-like behaviors.
It has been shown in vitro in rat that GR polymorphic forms with different lengths of CAG repeats have similar ligand binding affinity to dexamethasone and corticosterone. However, when length of CAG repeats are altered, the mutant form of GR exhibited significantly higher steroid binding affinity, suggesting a possible domain-domain interaction between the polyQ and the ligand binding regions within the GR. In addition, the GR serves not only as the well-known transcription regulator but also as a modulator in mechanisms of aggregation and nuclear localization of other polyQ proteins. It is still unknown whether the mouse GR with expanded PolyQ track we reported here has different efficacies in vivo in the central regulation of neuroendocrine response to stress, or more specifically, whether the polyQ expansion in combination with silent mutations alters the properties of the GR itself in steroid binding, transactivation, translocation, or aggregation in addition to its well-known function in mediating other polyQ-containing proteins.
To our knowledge, this is the first report on characterization of this GR polymorphism in genetically selected mouse lines with differences in metabolic and locomotor activity and altered stress responses. Further molecular and phenotypic characterization of this polymorphic GR, using the mouse model we developed, should provide useful information for better understanding the role of the GR and its polymorphism in the complex genetic and environmental interactions leading to altered neuroendocrine response and anxiety-type behaviors.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5926fje
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