Jondal M 2008 Age-related synthesis of glucocorticoids in thymocytes
The report by Qiao et al. () in the current issue addresses the regulation of the thymic GC synthesis in an innovative way. Adrenalectomy results in the lack of feedback inhibition of CRH and ACTH production by GC and a dramatic increase in plasma ACTH. One might predict that this increase in ACTH would stimulate thymocyte steroidogenesis, as it does in the adrenal gland and, if the system were functional, would cause thymus involution. However, as was known, thymus size increased significantly, suggesting that GC synthesis in the thymus does not have a significant regulatory role in thymocyte function. They found that stimulation with ACTH and cAMP, the second messenger in the ACTH signal cascade, produced a significant down-regulation of the CYP11B1 mRNA expression and GC synthesis in thymocytes. This effect was a direct result of ACTH, because thymocyte numbers were not increased by adrenalectomy in IL-1β/ IL18 double-KO mice in which the increase in plasma ACTH is significantly less than in wild-type mice. Administration of ACTH to IL-1β/IL-18 double-KO mice increased thymocyte numbers, indicating a direct effect of ACTH in thymocytes either mediated by the down-regulation of the steroidogenic enzymes or a direct effect of ACTH unrelated to steroidogenesis. In contrast, ACTH stimulates both the expression of steroidogenic enzymes and proliferation of the steroidogenic adrenal cells. The explanation of this paradox is not clear. Both MC2R and MC5R are expressed in adrenal steroidogenic cells and thymocytes. Although MC2R is the predominant ACTH receptor in the adrenal gland (,), the relative expression and function of each of these receptors in thymocytes is unknown.
Age-related synthesis of glucocorticoids in ..
Cyanoketone treatment (corticosterone synthesis inhibition) of rats virtually eliminates the decrease in white blood cell (WBC) and lymphocyte numbers observed during acute stress, and significantly enhances the increase in neutrophil numbers observed after the cessation of stress. Changes in WBC , lymphocyte , and neutrophil numbers during 2‐h stress (restraint), and following recovery 3 h after the cessation of stress are shown ( = 5/group). The percentage of change in leukocyte numbers after 2‐h stress relative to baseline (0 h) is indicated. Statistically significant differences are indicated: * test); ° test).
For each array sample, the RNA was prepared from the purified thymocytes. The thymocytes were processed by using the RNEasy Mini Kit (Qiagen, Valencia, CA). Quality and quantity of total RNA samples was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). This total RNA was used to generate fluorescent cRNA for use with Agilent's oligonucleotide microarrays. The RNA was amplified and labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit following manufactures protocols. In Short: Between 0.5μg to 2μg of total RNA was used to generate first and second strands of cDNA containing a T7 RNA polymerase promoter. Then cRNA was synthesized using T7 RNA polymerase which simultaneously incorporates cyanine 3- or cyanine 5- labeled CTP (Perkin Elmer, Wellesley, MA). Qiagen RNeasy columns (Qiagen Valencia, CA) were used to purify the labeled cRNA and the final concentration was assessed using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). 750 ng of Cy3-labeled cRNA and 750 ng of Cy5-labeled control sample were combined with spiked in control probes specific for targets on the arrays and hybridized over night at 600C to Agilent Mouse Whole Genome 44K Oligo Microarrays (Agilent Technologies, Palo Alto, CA). The arrays were washed at room temperature 6X SSC with 0.005% Triton X-102 for 10 minutes and 0.01x SSC with 0.005% Triton X-102 at 40C for 5 minutes. The slides were then dried in a nitrogen stream and scanned at 10 micron resolution using an Agilent Microarray scanner G2565BA. Data was extracted using Agilent Feature Extractor Software (v7.1).
Glucocorticoid Production and Regulation in Thymus: ..
Androgens contribute to the involution process of the aging thymus gland. However, molecular mechanisms behind this effect remain largely unknown. We have investigated the influence of testosterone on the ectopic synthesis of glucocorticoids (GCs) in thymocytes, an activity recently shown by us to be important for the homeostatic regulation of these cells. Castration, which leads to a strong increase in thymus tissue and function, was associated with a reduced GC release from thymocytes caused by down-regulated expression of several enzymes involved in GC synthesis, without affecting GC synthesis in the adrenals. Testosterone treatment of castrated male mice reversed these effects, also without affecting adrenal GC synthesis. The effects of testosterone in castrated mice on thymocyte homeostasis and GC release were strongly reduced in mice pretreated with the CYP11B1 enzyme inhibitor metyrapone, acting on the last step in the corticosterone synthesis. The androgen-induced thymic involution was dependent on GC action, because this was completely absent in mice lacking GC receptor (GR) expression specifically in thymocytes. We provide here an unrecognized mechanism how androgens contribute to thymic involution by stimulating local synthesis and release of GCs in the thymus.—Chen, Y., Qiao, S., Tuckermann, J., Okret, S., Jondal, M. Thymus-derived glucocorticoids mediate androgen effects on thymocyte homeostasis.
the inherent synthesis of GC by thymocytes was not certain
GCs are primarily synthesized in the adrenal glands, but an ectopic de novo synthesis has also been demonstrated in the brain, gastrointestinal tract, skin, and thymus (). In the mouse the major GC is corticosterone (CS), derived from cholesterol through the sequential action of steroidogenic acute regulatory protein StAR and the steroidogenic enzymes CYP11A1, 3β-HSD, CYP21, and CYP11B1 (). The tissue expression of 11β-HSD1 and 11β-HSD2 enzymes, which shuttle the hormone between an active (CS) and an inactive form (11-dehydrocorticosterone), is also important for setting the local GC concentration (). The initial demonstration of GC synthesis in the thymus was found to occur in TECs and suggested to be important for the T-cell selection process that occurs during thymocyte differentiation (). More recently, we have shown that thymocytes also synthesize GCs starting from around 4 wk after birth, in contrast to the GC synthesis in TECs, which decline with age (). We have also found that GC synthesis in thymocytes is under the regulation of ACTH and important for the homeostatic regulation of these cells (). Importantly, by using transgenic mice with an inducible increase in GC sensitivity selectively in thymocytes, we have clearly demonstrated that thymus-derived GCs are important in the regulation of thymocyte homeostasis ().