Inflammatory Gene Changes in Spleens from WT and Rescued GR-C3 Knockin Mice Treated with LPS

Glucocorticoids are primary stress hormones and their synthetic derivatives are widely used clinically. The therapeutic efficacy of these steroids is limited by severe side effects and glucocorticoid resistance. Multiple glucocorticoid receptor (GR) isoforms are produced by alternative translation initiation; however, the role individual isoforms play in controlling tissue-specific responses to glucocorticoids in vivo is unknown. We have generated knockin mice that exclusively express the most active receptor isoform, GR-C3. The GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid administration. Rescued GR-C3 mice exhibited alterations in circadian rhythm in a sexually dimorphic manner and in sensitivity to lipopolysaccharide (LPS)-induced endotoxemia. To evaluate the ability of glucocorticoids to protect against LPS-induced inflammation, we measured gene expression in spleens from WT and rescued GR-C3 knockin mice that had been treated with vehicle or LPS for 3 and 24 hours. The GR-C3 isoform was found to be deficient in its ability to repress a large cohort of immune and inflammatory genes. GR-C3 knockin mice were generated by standard gene targeting procedures. In brief, ES cells were generated in which exon 2 of the NR3C1 gene was replaced by a neomycin cassette. An exchange vector was generated containing a hygromycin cassette and a mutant NR3C1 exon 2 in which the ATG start codon for GR-C3 was left intact but mutated to ATC for each of the other seven translational isoforms. The exchange vector and Cre-recombinase were transfected into the targeted ES cells by electroporation, and positive ES cells were selected using G418 (sensitive) and hygromycin (resistant). A GR-C3 positive ES cell clone was injected into C57Bl/6 blastocysts to create chimeric mice. Chimeric males were bred with C57Bl/6 females for germline transmission, and the hygromycin cassette was deleted using Flp-deleter mice. The resulting mice were maintained on a C57Bl/6 background and intercrossed to produce WT, heterozygous GR-C3 knockin, and homozygous GR-C3 knockin mice. Homozygous GR-C3 knockin mice die at birth due to respiratory distress but can be completely rescued by antenatal glucocorticoid treatment. Adult male WT and rescued homozygous GR-C3 knockin mice were injected intraperitoneally with PBS or LPS (10μg/g body weight). Mice were sacrificed 3 hours and 24 hours after LPS administration and the spleen was removed. Total RNA was isolated from the spleens (n=4 per treatment group) using TRIzol reagent according to the manufacturer's instructions.