Cortisol Awakening Response and Nighttime Salivary Cortisol Levels in Healthy Working Korean Subjects

∙ The authors have no financial conflicts of interest. Purpose: Cortisol awakening response (CAR) and nighttime cortisol levels have been used as indices of adrenocortical activity. However, population-based statistical information regarding these indices has not been provided in healthy subjects. This study was carried out to provide basic statistical information regarding these indices. Materials and Methods: Cortisol levels were measured in saliva samples collected immediately upon awakening (0 min), 30 min after awakening and in the nighttime on two consecutive days in 133 healthy subjects. Results: We determined the mean [standard deviation (SD)], median (interquartile range) and 5th-95th percentile range for each measure and auxiliary indices for CAR, i.e., the secreted cortisol concentration within 30 min of awakening (CARscc) and absolute and relative increases in cortisol level within 30 min of awakening (CARi and CARi%, respectively). We also determined these values for auxiliary indices derived from nighttime cortisol level, i.e., the ratio of cortisol level 30 min afte

IMPROVEMENT OF ALOPECIA AND DECREASES IN HAIR CORTISOL AND DIHYDROTESTOSTERONE CONCENTRATIONS AFTER THE TOPICAL USE OF PLANT EXTRACTS IN MEN WITH ANDROGENETIC ALOPECIA

Objectives: Plant extracts possessing specific constituents with anti-inflammatory, antimicrobial, antioxidant, or 5α-reductase inhibitory properties are known to provide benefits against androgenetic alopecia (AGA) in men. A solid shampoo was formulated, and it contained a mixture of six different plant extracts that possess these beneficial properties against AGA. The improvement in AGA and changes in steroid concentrations were assessed after 4 months of formulated shampoo use. Methods: This study was conducted based on a randomized, placebo-controlled, and single-blind design. Hair-related variables and hair and saliva samples were collected bi-monthly in the treatment (n=48) and placebo (n=52) groups and at a single time point in the hairy controls (n=50). Results: The formulated shampoo was more effective on AGA than the placebo based on the hair shaft thickness and hair density in the receding hairline. The baseline hair cortisol and dihydrotestosterone (DHT) concentrations were significantly higher in the treatment and placebo groups than in the hairy controls. After 4 months, the hair steroid concentrations in the treatment group were reduced to those observed in the hairy controls, although the main effect of time on hair steroid concentrations was negligible in the placebo group. Salivary cortisol and DHT levels during the post-awakening period were comparable among the groups or assessment time points. Conclusion: The constituents of plant extracts included in the formulated shampoo would prevent hair loss, increase hair growth effects, and reduce hair cortisol and DHT concentrations without changes in the post-awakening salivary steroid levels in men with AGA

Transforming growth factor-β1 signaling represses testicular steroidogenesis through cross-talk with orphan nuclear receptor Nur77.

Transforming growth factor- β1 (TGF-β1) has been reported to inhibit luteinizing hormone (LH) mediated-steroidogenesis in testicular Leydig cells. However, the mechanism by which TGF-β1 controls the steroidogenesis in Leydig cells is not well understood. Here, we investigated the possibility that TGF-β1 represses steroidogenesis through cross-talk with the orphan nuclear receptor Nur77. Nur77, which is induced by LH/cAMP signaling, is one of major transcription factors that regulate the expression of steroidogenic genes in Leydig cells. TGF-β1 signaling inhibited cAMP-induced testosterone production and the expression of steroidogenic genes such as P450c17, StAR and 3β-HSD in mouse Leydig cells. Further, TGF-β1/ALK5 signaling repressed cAMP-induced and Nur77-activated promoter activity of steroidogenic genes. In addition, TGF-β1/ALK5-activated Smad3 repressed Nur77 transactivation of steroidogenic gene promoters by interfering with Nur77 binding to DNA. In primary Leydig cells isolated from Tgfbr2flox/flox Cyp17iCre mice, TGF-β1-mediated repression of cAMP-induced steroidogenic gene expression was significantly less than that in primary Leydig cells from Tgfbr2flox/flox mice. Taken together, these results suggest that TGF-β1/ALK5/Smad3 signaling represses the expression of steroidogenic genes via the suppression of Nur77 transactivation in testicular Leydig cells. These findings may provide a molecular mechanism involved in the TGF-β1-mediated repression of testicular steroidogenesis

Smad3 physically interacts with Nur77.

<p>(<b>A</b>) mKG_N-MC-NLS-Smad3 and mKG_C-MC-Nur77 were transfected into HeLa cells for 24 hours. Interaction between Nur77 and NLS-Smad3 yielded fluorescent green signals in the nucleus. The single fusion protein alone (mKG_N-MC-NLS-Smad3 or mKG_C-MC-Nur77) and another pair (mKG_N-MC-NLS-Smad3 and mKG_C-MN-Nur77) gave no fluorescent signal. The scale bars represent 25 µm. (<b>B</b>) [³⁵S] methionine-labeled Smad3 produced by <i>in vitro</i> translation was incubated with the GST-Nur77 fusion protein and its deletion mutants. Coomassie blue staining shows the protein level of the purified GST, GST-Nur77 and GST-Nur77 deletion mutant (bottom). (<b>C</b>) [³⁵S] methionine-labeled Smad3 deletion mutants were incubated with GST-Nur77 fusion protein. The data are representative of three independent experiments.</p

TGF-β1/ALK5 signaling represses cAMP-induced steroidogenic gene expression in Leydig cells.

<p>(<b>A and B</b>) The culture medium of purified mouse primary Leydig cells treated with 300 µM of 8-Br-cAMP and 5 ng/ml of TGF-β1 (A) and R2C cells treated with vehicle or 5 ng/ml of TGF-β1 (B) for 24 hours was collected for the measurement of testosterone levels by RIA. (<b>C and D</b>) The expression levels of steroidogenic genes in primary Leydig cells (C), which were treated with 300 µM of 8-Br-cAMP, 2.5 ng/ml of TGF-β1 and 10 µM SB431542 for 24 hours, and R2C cells (D), which were treated with 5 ng/ml of TGF-β1 for 24 hours, were analyzed by qRT-PCR. (<b>E</b>) The expression level of Tgfbr2 and Tgfbr1 was analyzed using total RNAs from primary Leydig, R2C and MA-10 cells by RT-PCR. (<b>F</b>) MA-10 cells were transiently transfected with the ALK5 (TD; constitutively active form) expression plasmid, along with an indicated reporter of the natural promoter, in medium containing 5% charcoal stripped FBS. Twenty four hours after transfection, the cells were treated with 300 µM of 8-Br-cAMP for 24 hours and harvested for luciferase assay. The pSV-β-gal expression plasmid was used as a control for transfection efficiency. The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

ALK5 signaling inhibits Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A and B</b>) MA-10 cells were transiently transfected with the ALK5 WT (wild type), ALK5 mutant (TD; constitutively active form or KR; inactive form), and Nur77 expression plasmids, along with the indicated reporter. The CMVβ expression plasmid was used as a control for transfection efficiency. (<b>C</b>) Whole cell extracts and subcellular fractions of primary Leydig cells, which were treated with 300 µM of 8-Br-cAMP and 2.5 ng/ml of TGF-β1 for 4 hours, were analyzed by western blot analysis with anti-Nur77, anti-pSmad3, anti-α-Tubulin (cytoplasmic marker) and anti-Lamin B (nuclear marker) antibodies. (<b>D</b>) MA-10 cells were transiently transfected with scrambled or Nur77 siRNA, ALK5 (TD) expression plasmid and P450c17 promoter reporter (top). Silenced Nur77 protein levels in HEK293T cells, which were transiently transfected with scrambled or Nur77 siRNA, Flag-Nur77 and CMVβ expression vector for 48 hours, were determined by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. **, P<0.01; ***, P<0.001; ns, not significant.</p

ALK5-activated Smad3 represses Nur77 transactivation of steroidogenic gene promoters.

<p>(<b>A</b>) MA-10 cells were transiently transfected with siRNA, Nur77, ALK5 (TD) and an indicated reporter for 48 hours and were harvested for luciferase assay. The CMVβ expression plasmid was used as a control for transfection efficiency (bottom). The silencing efficiencies of Smad2 and Smad3 siRNA were determined by western blot analysis (top). (<b>B</b>) MA-10 cells were transiently transfected with Nur77, increasing amounts of Smad (60 and 150 ng) expression plasmids and the NBRE reporter construct. (<b>C–E</b>) MA-10 cells were transiently transfected with ALK5 WT, ALK5 mutant (TD or KR), Smad3 and Nur77 expression plasmids, along with the indicated reporter construct. (<b>F</b>) MA-10 cells were transiently transfected with expression plasmids of Nur77, ALK5 (TD), Flag-Smad3 (WT) or a phosphorylation mutant (S3A or S3D), and NurRE-luc reporter construct (top). A similar amount of expressed protein was confirmed by western blot analysis (bottom). The data are presented as the mean ± SEM of at least three independent experiments. *, P<0.5; **, P<0.01; ***, P<0.001; ns, not significant.</p

TGF-β1 signaling regulates steroidogenic gene expression, affecting testicular testosterone levels in mice.

<p>(<b>A</b>) Decreased Tgfbr2^fl°^x allele in purified primary Leydig cells isolated from mice harboring the Cyp17iCre transgene. The genomic DNA isolated from primary Leydig cells of Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was amplified for Tgfbr2 intron region containing the LoxP site. A pair of β-actin primers was used as the control for the amount of genomic DNA. (<b>B</b>) Decreased TGF-β1-mediated repression of steroidogenic gene expression with Tgfbr2 silencing. Purified primary Leydig cells from the testes of 12-week-old Tgfbr2^flox/flox (n = 6) and Tgfbr2^flox/flox Cyp17iCre (n = 6) mice were treated with 300 µM of 8-Br-cAMP and 2 ng/ml of TGF-β1 for 24 hours, and mRNA expression levels were measured using qRT-PCR. β-actin expression was used as a loading control. The data are presented as the mean ± SEM. **, P<0.01; ***, P<0.01. (<b>C</b>) Testicular testosterone levels were measured by RIA in the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice. (<b>D</b>) Total protein (100 µg) from the testes of 5 week-old Tgfbr2^flox/flox and Tgfbr2^flox/flox Cyp17iCre mice was subjected to western blot analysis for protein levels of steroidogenic genes. The relative level of each protein/GAPDH was quantified by densitometric analysis using Image J software. In panels C and D, the data are presented as the mean ± SD (n = 10). **, P<0.01.</p