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PNAS 109 (37): 2457-2465

Copyright © 2012 by the National Academy of Sciences.

Core circadian protein CLOCK is a positive regulator of NF-{kappa}B–mediated transcription

Mary L. Spenglera, Karen K. Kuropatwinskia, Maria Comasa, Alexander V. Gasparianb,1, Natalia Fedtsovac, Anatoli S. Gleibermanb, Ilya I. Gitlinc, Natalia M. Artemichevac,2, Krysta A. Delucaa,3, Andrei V. Gudkovc, and Marina P. Antocha,4

Departments of aMolecular and Cellular Biology and cCell Stress Biology, Roswell Park Cancer Institute, Buffalo, NY 14263; and b Cleveland BioLabs, Inc., Buffalo, NY 14263


Figure 01
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  		Fig. 1.. Daily variations in response to immune challenge correlate with variations in the activation of NF-{kappa}B transcription factor. (A) LPS-induced toxicity depends on the time of immunostimulation. Kaplan–Meier survival curves of BALB/c mice maintained on a 12:12-h LD cycle and challenged with 20 mg/kg LPS at ZT6 (red line) or ZT18 (blue line) (n = 10 per group). Animals treated in the middle of the active phase of their daily cycle (ZT18) showed higher tolerance (P = 0.018, log-rank test). (B) The activation of the NF-{kappa}B–responsive promoter in vivo depends on the time of immunostimulation. Six BALB/C-Tg(I{kappa}Bα-Luc) reporter mice received 1 μg of CBLB502 at ZT6 or ZT18. Animals were injected i.p. with D-luciferin 1, 2, and 4 h later, and the luciferase signal was monitored in live animals using the Xenogen IVIS 50 system. (C) Quantitation of the luciferase signal shown in B. Closed squares represent animals injected at ZT6; open squares represent animals injected at ZT18. Values are mean of three animals ± SD. (D) Induction of IL-1α mRNA in livers of IkB-Luc reporter mice 20, 40, and 60 min after CBLB502 administration at ZT6 (closed squares) or ZT18 (open squares). (E) Daily variations in NF-{kappa}B activation. Eighteen BALB/C-Tg(I{kappa}Bα-Luc) male mice that were maintained on a 12:12-h LD cycle were injected s.c. with 1 ug CBLB502 at different times of the day. Three hours later animals were injected i.p. with D-luciferin, and luciferase signal was monitored in live animals using the Xenogen IVIS 50 system. Shown are representative images for each time group. Maximum I{kappa}B promoter activation is detected in animals treated at ZT6. (F) Quantitative analysis of in vivo images. Values are mean of three animals ± SD. There is a statistically significant difference in I{kappa}B-Luc activation in animals treated at ZT6 and animals treated between ZT10–ZT18 (P < 0.001; one-way ANOVA with post hoc Tukey’s test).

 

Figure 02
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  		Fig. 2.. CLOCK enhances NF-{kappa}B activation. (A) Increasing CLOCK expression correlates with NF-{kappa}B activation. HEK-293T cells were transfected with 10 ng {kappa}B-Luc reporter plasmid, various combinations of p65-, MYC-Clock–, and HA-Bmal1–expressing plasmids, and pcDNA empty vector to equalize the total amount of DNA used in transfection. Bars represent relative luciferase signal normalized for efficiency of transfection using the β-gal assay. Experiments were performed at least three times in duplicate. Values are mean ± SD. The expression of 25 ng of p65-expressing plasmid was used as a positive control to show I{kappa}B-Luc reporter gene activation mimicking activation of the NF-{kappa}B pathway by stimuli. The effect of higher CLOCK expression on the activation of the reporter was determined to be statistically greater than the effect of p65 expression alone (P = 0.01; Student’s t test). Overexpression of BMAL1 blocks CLOCK-mediated up-regulation of the NF-{kappa}B–responsive promoter (P = 0.05). (B) CLOCK-mediated up-regulation of NF-{kappa}B does not require BMAL1. MEFs isolated from Bmal1–/– mice were transfected with 10 ng of {kappa}B-Luc reporter, 25 ng of p65-expressing plasmid, and either 100 ng MYC-CLOCK– or 40 ng HA-BMAL1–expressing plasmid. Ectopic CLOCK significantly up-regulates the {kappa}B-Luc reporter (P = 0.02). Bars represent mean values ± SD. (C) CLOCK enhances NF-{kappa}B activation in response to TNF-α. HEK-293T cells were transfected with the {kappa}B-Luc reporter gene with either pcDNA- or MYC-CLOCK–expressing plasmid. The following morning cells were treated with 2 ng/mL of TNF-α for 6 h. Ectopic CLOCK significantly enhances TNF-α–mediated activation of the {kappa}B-Luc reporter (P < 0.01; Student’s t test). (D) CLOCK-dependent up-regulation of {kappa}B-Luc reporter in response to TNF-α correlates with an increase in the active phosphorylated form of p65. HEK-293T cells were transfected with increasing concentrations of CLOCK-expressing plasmid; 24 h posttransfection cells were treated with 2 ng/mL TNF-α for 5 h, and transcriptionally active forms of p65 were visualized by Western blot with antibodies against CLOCK, total p65, pSer536-p65, and Actin for loading control. (E) Quantitative analysis of Western blot presented in D. The increase in the ratio of pSer536-p65 to total p65 correlates with an increase in CLOCK abundance. Experiments were repeated three times with similar results.

 

Figure 03
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  		Fig. 3.. CLOCK is detected in a protein complex with p65. (A) Endogenous p65 pulls down ectopic CLOCK. HEK-293T cells were transfected with expression plasmids for CLOCK and BMAL1 either together or separately. Cells lysates were immunoprecipitated with anti-p65 antibody (IP p65) and analyzed for CLOCK by Western blot. (B) HEK-293T cells were transfected with CLOCK-expressing plasmid. The cell lysates were divided into two equal portions, and anti-CLOCK and nonspecific GAL4 antibody (ns-Ab) were used to pull down immunoprecipitates which were analyzed by Western blot with anti-p65 antibody. (C) Endogenous CLOCK–p65 interaction. L929 cells, which are high in CLOCK protein, were harvested in lysis buffer and immunoprecipitated with control nonimmune serum or with specific antibodies directed against either p65 or CLOCK. The immunoprecipitates were resolved in SDS/PAGE and analyzed for p65 expression by Western blot. (D) CLOCK interacts with p65 independently of BMAL1. Bmal1-deficient MEFs were transfected with expression plasmids of CLOCK and BMAL1 as indicated. The whole-cell extracts of the transfected cells and the corresponding anti-p65 immunoprecipitates were analyzed for ectopic CLOCK and BMAL1 proteins by anti-HA/MYC Western blot. WCE, whole-cell extract.

 

Figure 04
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  		Fig. 4.. NF-{kappa}B activation is reduced in Clock-deficient MEFs. (A) WT (black bars) and Clock-deficient (gray bars) MEFs stably expressing {kappa}B-Luc reporter were treated with 2 ng/mL TNF-α for 5 h, and luciferase activity was measured in cell lysates. (B) Clock-deficient cells show reduced levels of nuclear phospho-active p65 after TNF-α–mediated NF-{kappa}B activation. WT and Clock-deficient MEF cells were treated with 2 ng/mL of TNF-α for the indicated times and were used to prepare nuclear extracts, which were analyzed by Western blots with antibodies against total p65 and its pSer536 phosphorylated form. (C) Quantitative analysis of the Western blot shown in B. ACTIN was used as a loading control. Black bars, WT; gray bars, Clock-deficient MEFs. (D) Deficiency in Clock has no effect on total levels of p65. WT and Clock-deficient MEFs were treated with 2 ng/mL of TNF-α for the indicated times. Total cell lysates were analyzed by Western blot as in B. (E) Impaired induction of NF-{kappa}B DNA binding in Clock–/– MEF cells treated with TNF-α. WT and Clock-deficient MEFs were treated with 1 ng/mL TNF-α for the indicated times. (F) Quantitative analysis of intensity of the band corresponding to DNA-bound p65/p50 dimer. The amount of DNA-bound complex in Clock-deficient MEFs (gray bars) is reduced compared with WT MEFs (black bars); the decrease is particularly pronounced after 45 min of TNF-α treatment, suggesting that the overall amount of active NF-{kappa}B is diminished in Clock-deficient MEFs. (G) CLOCK has no effect on the DNA-binding properties of p65. HEK-293T cells were cotransfected with a luciferase reporter gene encoding a GAL4-binding element and plasmid encoding a p65 fusion of the GAL4 DNA-binding domain. The CLOCK- and BMAL1-expressing plasmids were transfected as indicated to show CLOCK’s dose-dependent effect on p65 activation and BMAL1’s capacity to counter this effect (Left). The GAL4 luciferase reporter gene also was transfected with the CLOCK-expressing plasmid to show that CLOCK did not drive expression of the reporter gene (Right).

 

Figure 05
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  		Fig. 5.. NF-{kappa}B activation in response to CBLB502 is reduced in liver and primary hepatocytes of Clock-deficient mice. (A) BALB/C-Tg(I{kappa}Bα-Luc) mice were crossed to C57BL/6J WT or Clock-deficient mice (C57BL/6J background). The progeny (WT or Clock+/–) received 1 ug of CBLB502 at ZT06 and were monitored for luciferase expression in vivo 2 h later. Representative images for each genotype are shown. (B) Quantitative analysis of luciferase expression in livers of WT (n = 3) and Clock+/– (n = 3) mice measured 2 h post CBLB502 administration. CBLB502-mediated NF-{kappa}B activation is reduced in Clock+/– mice (P < 0.05). UT, untreated (C) CBLB502-mediated induction of IL-6 correlates with the scale of NF-{kappa}B activation. Two WT and two Clock–/– mice received a single injection of CBLB502 at the times indicated. Blood was collected 2 h later, and the plasma concentration of IL-6 was measured by ELISA. (D) Impaired induction of NF-{kappa}B DNA binding in primary hepatocytes of Clock–/– mice treated with CBLB502 in vitro. Primary hepatocytes isolated from age-matched WT and Clock–/– mice were treated with 100 ng/mL of CBLB502 for the indicated times. (E) Quantitative analysis of the intensity of the band corresponding to DNA-bound p65/p50 dimer. The amount of DNA-bound complex in Clock-deficient hepatocytes (gray bars) is reduced significantly compared with WT hepatocytes (black bars).

 

Figure 06
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  		Fig. 6.. CLOCK-dependent modulation of NF-{kappa}B activation is distinct from its transactivation function on circadian promoters. (A) The CLOCK-{Delta}19 mutant modulates NF-{kappa}B–responsive promoters as well as WT CLOCK. HEK-293T cells were cotransfected with the {kappa}B-Luc reporter in combination with Clock- or Clock-{Delta}19 –expressing plasmids. Twenty-four hours posttransfection cells were treated with 2 ng/mL of TNF-α for 5 h. Both constructs modulate NF-{kappa}B (P < 0.05). (B) The Clock-{Delta}19 mutation has no effect on the amount of DNA-bound p65/p50 complex. Primary hepatocytes obtained from WT and Clock-{Delta}19–mutant mice were treated with CBLB502 (100 ng/mL) for the times indicated. (C) Quantitative analysis of intensity of the band corresponding to DNA-bound p65/p50 dimer shows no difference in the strength and kinetics of CBLB502-induced NF-{kappa}B DNA binding in Clock-{Delta}19 (gray bars) and WT (black bars) cells. Values are the average of two samples run on the same gel ± SD. (D) The circadian repressor CRY1 has no effect on CLOCK’s ability to up-regulate NF-{kappa}B–responsive promoters. HEK-293T cells were transfected with 10 ng of Per1-Luc or kB-Luc reporters and various expression plasmids of clock proteins. CRY1 represses CLOCK/BMAL1-dependent transactivation on the Per1 promoter but has no effect on CLOCK-dependent up-regulation of the {kappa}B promoter in response to TNF-α.

 

Figure 07
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  		Fig. 7.. CLOCK cooperates with NF-{kappa}B coactivators. (A) Overexpression of CLOCK facilitates acetylation of p65 at Lys-310. HEK-293T cells were transfected with either the control pcDNA vector or CLOCK-expressing plasmid. Cells were harvested in Nonidet P-40 buffer 24 h posttransfection, and lysates were immunoprecipitated with anti-p65. The precipitates were analyzed by Western blot using p65-specific Ac-Lysine 310 antibody. An anti-p65 Western blot was used to normalize for loading. (B) Intrinsic HAT activity is not important for CLOCK-dependent up-regulation of NF-{kappa}B–responsive promoters. HEK-293T cells were cotransfected with a luciferase reporter gene driven by either the Per1 promoter (Left) or the NF-{kappa}B–responsive promoter (Right) and the combination of the BMAL1-expressing plasmid and a plasmid expressing either WT CLOCK or HAT-deficient CLOCK as indicated. TNF-α was added at 2 ng/mL 24 h posttransfection. (C) CLOCK cooperates with CBP in activating the {kappa}B-responsive promoter. 293T cells were cotransfected with the kB-Luc reporter and combinations of plasmids expressing CLOCK, BMAL1, and CBP. TNF-α was added at 2 ng/mL 24 h posttransfection. The coexpression of CLOCK and CBP results in higher NF-{kappa}B activation (P < 0.05).

 

Figure 01
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  		Fig. P1.. Two mechanisms underlying circadian control of NF-{kappa}B response. (A) CLOCK/BMAL1-regulated circadian oscillation in the expression of genes encoding components of the NF-{kappa}B pathway. (B) CLOCK-dependent up-regulation of the transcriptional activity of NF-{kappa}B. Daily oscillations in the abundance and functional state of free (not bound by BMAL1) CLOCK result in corresponding variations in the scale of NF-{kappa}B activation.

 

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