Characterization of Glucocorticoid-Induced Changes in Gene Expression in the Embryonic Pituitary Gland
Jenkins, Sultan Ali
Porter, Tom E
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We have previously shown that corticosterone (CORT) can induce premature differentiation of somatotrophs in the chicken embryo. CORT induction of GH mRNA is indirect, in that protein synthesis inhibition blocks its inducing effect. In this study, we used a custom chicken microarray to analyze global gene expression in the embryonic pituitary gland and to identify potential direct targets of CORT that may regulate its induction of somatotroph differentiation. Dispersed embryonic (e) day 11 pituitary cells were pretreated with cycloheximide then with CORT or treated with CORT alone for 24 hrs. Amplified RNA from these samples was then used on our microarray to analyze gene expression and in quantitative real-time PCR (qRT-PCR) analysis to determine relative gene expression levels. qRT-PCR from these samples showed that GH was induced within 1.5 hrs and continued to significantly increase until 3 hrs. Our microarray analysis revealed 25 direct early targets of CORT. From these 25 we chose 3 genes, Dexras1, Ras-dva, and Prostaglandin-D Synthase to transfect into e11 pituitary cells and measure their effect on GH mRNA. Neither of these genes had a direct effect on GH mRNA; however Dexras1 acted synergistically with CORT to induce GH mRNA. Dexras1 was discovered in murine AtT-20 corticotroph cells because its expression was rapidly induced in response to glucocorticoids. We report here a chicken Dexras1 cDNA that is 1631 bp in length and encodes a protein of 278 amino acid residues. Comparison of the consensus chicken Dexras1 amino and nucleic acid sequence with those of human, mouse, and rat Dexras1 showed high homology among the species. Expression of Dexras1 mRNA was detected in various tissues by Northern analysis, but was highest in the pituitary. RT-PCR analysis showed expression of Dexras1 only in the pituitary. The precise role of DexRas1 is unidentified at the present time; however, its distribution in a range of tissues suggests a possible role in glucocorticoid action within a variety of systems.