Table 1.
Clinical characteristics and endocrine parameters of the subjects enrolled in the study.
Table 2.
Primer pairs used in real-time PCR mRNA quantitations.
Figure 1.
Concentrations of plasma ACTH and serum cortisol at 8 am and 8 pm in healthy adult volunteers.
Concentrations of plasma ACTH (left panel) and serum cortisol (right panel) at 8 am (Day) and 8 pm (Night) are shown. Bars represent the mean ± S.E. values of serum cortisol and plasma ACTH. **: P<0.01, compared to the conditions indicated (n = 10, m = 10).
Figure 2.
Daily change of CLOCK-related gene mRNA expressions in PBMCs (A) and their response to hydrocortisone in EBV-transformed peripheral lymphocytes (B).
A: Morning and evening mRNA expression of CLOCK-related genes in PBMCs. Relative mRNA expression of Clock, Bmal1, Per1, Cry1 and RORα at 8 am (Day) and 8 pm (Night) in PMBCs obtained from 10 healthy subjects is shown. The measurement was performed in duplicate for each subject. Bars represent mean ± S.E. values of relative mRNA expression of the genes indicated. **: P<0.01, compared to the conditions indicated (n = 10, m = 20). B: The effect of hydrocortisone on the mRNA expression of CLOCK-related genes in EBV-transformed peripheral lymphocytes. EBV-transformed peripheral lymphocytes were incubated with 5×10−7 M of hydrocortisone (HC) for 5 hours and mRNA expression of CLOCK-related genes was evaluated. The measurement was performed in triplicate. Bars represent mean ± S.E. values of hydrocortisone (HC)-induced fold mRNA expression of indicated genes. **: P<0.01, compared to the conditions indicated (m = 3).
Figure 3.
Daily changes of the GR acetylation and its mRNA expression in PBMCs, and response of GR mRNA expression to hydrocortisone in EBV-transformed peripheral lymphocytes.
A: Acetylation of GR protein is high in the morning and low in the evening. Acetylation of GR was examined in whole cell lysates obtained at 8 am (Day) and 8 pm (Night) from PBMCs purified from 5 subjects. Band intensity of acetylated GR obtained in 3 independent experiments was corrected for that of total precipitated GR, and relative GR acetylation was calculated as the mean value of all subjects' measurements corrected values at Night as “1”. Bars in the top panel represent mean ± S.E. values of relative GR acetylation, while representative Western blot images are shown in the bottom panel. **: P<0.01, compared to conditions indicated (n = 5, m = 15). B and C: Daily change of the GR mRNA expression in PBMCs (B) and its response to hydrocortisone in EBV-transformed peripheral lymphocytes (C). Relative GR mRNA expression at 8 am (Day) and at 8 pm (Night) in PBMCs (B) and the effect of hydrocortisone (HC) on GR mRNA expression in EBV-transformed peripheral lymphocytes (C) are shown. EBV-transformed peripheral lymphocytes were incubated with 5×10−7 M of hydrocortisone (HC) for 5 hours. The measurements were performed in duplicate and in triplicate for panel B and C, respectively. Bars represent mean ± S.E. values of relative GR mRNA expression (B) or hydrocortisone (HC)-induced fold GR mRNA expression (C). *: P<0.05, **: P<0.01, compared to the conditions indicated (B: n = 10, m = 20, C: m = 3).
Figure 4.
Response of glucocorticoid-responsive gene mRNA expressions to hydrocortisone in EBV-transformed peripheral lymphocytes and their daily changes in PBMCs.
A: The effect of hydrocortisone on the expression of the mRNAs of known glucocorticoid-responsive genes in EBV-transformed peripheral lymphocytes. Samples obtained as in Figure 3 were used for the evaluation of mRNA expression of the known glucocorticoid-responsive genes indicated. DUSP1 and tristetraprolin are the genes known to be up-regulated by glucocorticoids, while IL-1α and TNFα represent those known to be down-regulated. The measurements were performed in triplicate. Bars represent the mean ± S.E. values of hydrocortisone (HC)-induced fold mRNA expression of indicated genes. **: P<0.01, compared to the conditions indicated (m = 3). B: mRNA expression of the known glucocorticoid-responsive genes in the morning and in the evening. Relative mRNA expression of DUSP1, tristetraprolin, IL-1α and TNFα at 8 am (Day) and at 8 pm (Night) in PMBCs obtained from 10 healthy subjects is shown. The measurements were performed in duplicate for each subject. Bars represent mean ± S.E. values of relative mRNA expression of the genes indicated. **: P<0.01, n.s.: not significant, compared to the conditions indicated (n = 10, m = 20).
Figure 5.
CLOCK regulates mRNA expression of glucocorticoid-responsive genes in a gene-specific fashion ex vivo.
PBMCs obtained at 6 am from 6 healthy subjects were cultured in the medium and were treated with 5×10−7 M of hydrocortisone (HC) for 3 hours at every 6 hours. mRNA levels of Clock and Cry1 (A), acetylation of the GR (B) and mRNA levels of glucocorticoid-responsive genes DUSP1, tristetraprolin, IL-1α and TNFα (C) were determined. The measurements were performed in duplicate for each subject. Circles represent the mean ± S.E. values of hydrocortisone (HC)-induced fold mRNA expression of the genes indicated and fold acetylation of GR. Values obtained in the absence of hydrocortisone (HC) at time “0” were employed as controls for glucocorticoid-responsive genes, while values obtained in the presence of hydrocortisone (HC) at time “0” were used as controls *: p<0.05, **: P<0.01, n.s.: not significant, compared to the values obtained at time “0” in the presence of hydrocortisone (n = 6, m = 12).
Figure 6.
CLOCK regulates mRNA expression of glucocorticoid-responsive genes possibly through acetylation of GR by Clock.
A: Clock and GR are associated in a hydrocortisone-dependent fashion in PBMCs cultured ex vivo. PBMCs obtained at 6 am from 6 healthy subjects were cultured in the absence or presence of 5×10−7 M of hydrocortisone (HC) for 3 hours and co-immunoprecipitation using anti-GR or control antibody was performed. Top panel indicates results of co-immunoprecipitation, while the bottom two panels show expression of Clock and GR in Western blots using their specific antibodies. Representative images of 3 independent experiments are shown. B, C and D: Knockdown of Clock abolishes diurnal fluctuation of DUSP1 and TNFα mRNA expression in PBMCs cultured ex vivo. PBMCs obtained at 6 am from 3 healthy subjects were transfected with Clock or control siRNA and were treated with 5×10−7 M of hydrocortisone (HC) for 3 hours at every 6 hours. mRNA levels of Clock and acetylation of the GR (B), mRNA expression of glucocorticoid-responsive genes, DUSP1, tristetraprolin, IL-1α and TNFα (C and D) were determined. Experiments were performed in duplicate for each subject. Circles represent the mean ± S.E. values of fold mRNA expression of the indicated genes obtained in the absence and presence of hydrocortisone (HC). The values obtained in the absence of hydrocortisone (HC) were employed as controls. *: P<0.05, **: P<0.01, n.s.: not significant, compared to the values obtained in the absence of hydrocortisone (HC) at the same time-point for Clock mRNA expression and GR acetylation, and to the values obtained in the presence of hydrocortisone (HC) at time “0” for mRNA expression of glucocorticoid-responsive genes (n = 3, m = 6).