Abstract
Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic. The molecular factors that govern tissue specificity of glucocorticoids, however, are poorly understood. The actions of glucocorticoids are mediated by the glucocorticoid receptor (GR). To discover new proteins that interact with GR and modulate its function, we performed a yeast two-hybrid assay. The MyoD family inhibitor domain-containing protein (MDFIC) was identified as a binding partner for GR. MDFIC associated with GR in the cytoplasm of cells, and treatment with glucocorticoids resulted in the dissociation of the GR-MDFIC complex. To investigate the function of the GR-MDFIC interaction, we performed a genome-wide microarray in intact and MDFIC-deficient A549 cells that were treated with glucocorticoids. A large cohort of genes was differentially regulated by GR depending on the presence or absence of MDFIC. These gene changes were strongly associated with inflammation, and glucocorticoid regulation of the inflammatory response was altered in MDFIC-deficient cells. At a molecular level, the interaction of MDFIC with GR altered the phosphorylation status of the receptor. We demonstrate in COS-1 cells that changes in receptor phosphorylation underlie the ability of MDFIC to regulate the transcriptional activity of GR. Finally, we show that GR directly represses the MDFIC gene, revealing a negative feedback loop by which glucocorticoids limit MDFIC activity. These findings identify a new binding partner for cytoplasmic GR that modulates the receptor transcriptome and contributes to the tissue-specific actions of glucocorticoids.
Highlights
Glucocorticoids are primary stress hormones that regulate many physiological processes, and synthetic derivatives of these molecules are widely used in the clinic
B, A549 cells were treated with 100 nM Dex for the indicated times, and MyoD family inhibitor domaincontaining protein (MDFIC) mRNA was measured by RT-PCR. con, vehicle
C, A549 cells were treated with 100 nM Dex for 12 h, and MDFIC levels were evaluated by immunoblot
Summary
To discover new proteins that interact with GR, we performed a yeast two-hybrid screen using as bait a region of human GR spanning the hinge domain (Fig. 1A). Co-immunoprecipitation experiments were performed in COS-1 cells expressing GR and either full-length MDFIC or a truncated version missing the entire cysteine-rich carboxyl-terminal domain (MDFIC[1–164]). Similar to our findings for MDFIC, MDFI co-immunoprecipitated with GR in untreated cells, and the addition of 100 nM Dex promoted the dissociation of the complex (Fig. 2F) These data indicate that the cysteine-rich carboxyl-terminal domain of MDFIC and MDFI mediates their interaction with GR. Dex treatment resulted in the dissociation of the complex as the amount of co-immunoprecipitated GR was reduced in the glucocorticoid-treated cells (Fig. 4D). GR translocated into the nucleus, whereas MDFIC remained in the cytoplasm (Fig. 4E)
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