ABSTRACT
Glucocorticoids (GCs) are critical modulators of the immune system. The hypothalamic-pituitary-adrenal (HPA) axis regulates circulating GC levels and is stimulated by endotoxins. Lymphoid organs also produce GCs; however, it is not known how lymphoid GC levels are regulated in response to endotoxins. We assessed whether an acute challenge of lipopolysaccharide (LPS) increases lymphoid levels of progesterone and GCs, and expression of steroidogenic enzymes and key HPA axis components (eg, corticotropin-releasing hormone [CRH], adrenocorticotropic hormone [ACTH]). We administered LPS (50 µg/kg intraperitoneally) or vehicle control to male and female C57BL/6J neonatal (postnatal day [PND] 5) and adult (PND90) mice and collected blood, bone marrow, thymus, and spleen 4 hours later. We measured progesterone, 11-deoxycorticosterone, corticosterone, and 11-dehydrocorticosterone via liquid chromatography-tandem mass spectrometry. We measured gene expression of key steroidogenic enzymes (Cyp11b1, Hsd11b1, and Hsd11b2) and HPA axis components (Crh, Crhr1, Pomc, and Mc2r) via quantitative polymerase chain reaction. At PND5, LPS induced greater increases in steroid levels in lymphoid organs than in blood. In contrast, at PND90, LPS induced greater increases in steroid levels in blood than in lymphoid organs. Steroidogenic enzyme transcripts were present in all lymphoid organs, and LPS altered steroidogenic enzyme expression predominantly in the spleen. Lastly, we detected transcripts of key HPA axis components in all lymphoid organs, and there was an effect of LPS in the spleen. Taken together, these data suggest that LPS regulates GC production by lymphoid organs, similar to its effects on the adrenal glands, and the effects of LPS might be mediated by local expression of CRH and ACTH.
Subject(s)
Bone Marrow/metabolism , Glucocorticoids/biosynthesis , Lipopolysaccharides/pharmacology , Spleen/metabolism , Thymus Gland/metabolism , 11-beta-Hydroxysteroid Dehydrogenase Type 1/genetics , 11-beta-Hydroxysteroid Dehydrogenase Type 2/genetics , Animals , Animals, Newborn/metabolism , Bone Marrow/drug effects , Bone Marrow/enzymology , Corticosterone/analysis , Corticosterone/blood , Female , Glucocorticoids/blood , Hypothalamo-Hypophyseal System/drug effects , Immunity, Innate/drug effects , Male , Mice , Mice, Inbred C57BL , Pituitary-Adrenal System/drug effects , RNA, Messenger/analysis , Receptors, Corticotropin-Releasing Hormone/genetics , Spleen/drug effects , Spleen/enzymology , Steroid 11-beta-Hydroxylase/genetics , Thymus Gland/drug effects , Thymus Gland/enzymologyABSTRACT
We have recently reported that activation of Brd4 is associated with the presence of autophagy in NPMc+ and MLL AML cells. In order to determine the mechanisms underlying this relationship, we have examined the role of Brd4 in regulating the expression of several genes that are central to the process of autophagy. We found that Brd4 binds to the promoters of ATG 3, 7 and CEBPß, and expression of these genes is markedly reduced by inhibitors of Brd4, as well as by Brd4-shRNA and depletion of CEBPß. Inhibitors of Brd4 also dramatically suppress the transcription of Keap1, thereby increasing the expression of anti-oxidant genes through the Nrf2 pathway and reducing the cytotoxicity induced by Brd4 inhibitors. Elimination of ATG3 or KEAP1 expression using CRISPR-cas9 mediated genomic editing markedly reduced autophagy. We conclude that Brd4 plays a significant role in autophagy activation through the direct transcriptional regulation of genes essential for it, as well as through the Keap1-Nrf2 axis in NPMc+ and MLL-fusion AML cells.
