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24-Hour Rhythm of Aquaporin-3 Function in the Epidermis Is Regulated by Molecular Clocks  Naoya Matsunaga, Kazufumi Itcho, Kengo Hamamura, Eriko Ikeda,

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Presentation on theme: "24-Hour Rhythm of Aquaporin-3 Function in the Epidermis Is Regulated by Molecular Clocks  Naoya Matsunaga, Kazufumi Itcho, Kengo Hamamura, Eriko Ikeda,"— Presentation transcript:

1 24-Hour Rhythm of Aquaporin-3 Function in the Epidermis Is Regulated by Molecular Clocks 
Naoya Matsunaga, Kazufumi Itcho, Kengo Hamamura, Eriko Ikeda, Hisako Ikeyama, Yoko Furuichi, Miyako Watanabe, Satoru Koyanagi, Shigehiro Ohdo  Journal of Investigative Dermatology  Volume 134, Issue 6, Pages (June 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Time-dependent skin physiological functions in mice are regulated by the mAqp3 gene. (a) Temporal expression profile of mAqp3 mRNA in mouse epidermis of wild-type and Clk/Clk mice. Levels of mAqp3 mRNA significantly varied over 24 hours in wild-type (P<0.05; analysis of variance (ANOVA)) but not in Clk/Clk mice. (b) Temporal expression profile of mAQP3 protein in mouse epidermis of wild-type and Clk/Clk mice. Levels of mAqp3 protein significantly varied over 24 hours in wild-type (P<0.05; ANOVA) but not in Clk/Clk mice. (c) Temporal stratum corneum hydration profiles of wild-type and Clk/Clk mice using a Corneometer (CM). Stratum corneum hydration is indicated by CM units. Levels of stratum corneum hydration significantly varied over 24 hours in wild-type (P<0.05; ANOVA) but not in Clk/Clk mice. Values are the means±SEM (n=3–8). *P<0.05; **P<0.01. (b) *P<0.05; **P<0.01 compared with wild-type mice. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Transcriptional control of mAqp3 by the circadian regulators D-site-binding protein (DBP) and E4 promoter-binding protein 4 (E4BP4). (a) Computer-aided analysis identified clock gene response elements (E-box, D-site, retinoic orphan receptor response element (RORE), and cAMP response element (CRE)) in the promoter region of the mAqp3 gene. (b) mAqp3 (-1,532 bp) luciferase reporter activity induced by DBP. (c) Temporal expression profiles of mAqp3 in NIH3T3 cells after serum shock. DBP siRNA and E4BP4 siRNA (DBP siRNA/E4BP4 siRNA) were transfected at the time of plating. There was a significant time-dependent variation in cells transfected with control siRNA (P<0.05, analysis of variance (ANOVA)). (d) Temporal expression profile of mDbp mRNA in mouse epidermis of wild-type and Clk/Clk mice. Levels of mDbp mRNA significantly varied over 24 hours in wild-type mice (P<0.05; ANOVA). Values are the means±SEM (n=3–6). *P<0.05; **P<0.01. (c, d) *P<0.05 compared with the indicated time points. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Schematic representation of the molecular mechanisms regulating the circadian expression of the AQP3 gene. (a) In the mAqp3 gene, the D-site-binding protein, DBP, periodically binds to response elements in the mAqp3 promoter to activate its transcription. E4 promoter-binding protein 4 (E4BP4) suppresses its transcription at other times of day, thereby generating 24-hour rhythms. (b) In the hAQP3 gene, CLOCK and BMAL1 periodically bind to the response element in the hAQP3 promoter to activate its transcription. Once the PER and CRY proteins have reached critical concentrations, they attenuate CLOCK/BMAL1 transactivation, thereby generating 24-hour rhythms. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Time-dependent glycerol uptake in HaCaT cells is regulated by the hAQP3 gene. (a) Temporal expression profiles of hAQP3 mRNA in HaCaT cells after serum shock (closed circle) or non-serum shock (open circle). (b) Temporal expression profiles of protein levels of human aquaporin 3 (hAQP3) in HaCaT cells after serum shock. (c) Radiolabeled-glycerol uptake profiles in HaCaT cells after serum shock. Levels of hAQP3 mRNA (a), protein (b), and glycerol uptake (c) significantly varied over 24 hours after serum shock (P<0.05; analysis of variance). Values are the means±SEM (n=3–6). CRE, cAMP response element; DBP, D-site-binding protein; RORE, retinoic orphan receptor response element. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Transcriptional control of hAQP3 by the circadian regulators CLOCK and BMAL1. (a) Computer-aided analysis identified clock gene response elements (E-box, D-site, retinoic orphan receptor response element (RORE), and cAMP response element (CRE)) in the promoter region of the hAQP3 gene. (b) hAQP3 (-1,580 bp) luciferase reporter activity induced by CLOCK and BMAL1. (c) hAQP3 (-1,580 bp) luciferase reporter activity induced by CLOCK/BMAL1 was inhibited by transfection with PER2 or CRY1. The presence (+) or absence (-) of expression plasmids is denoted. Values are the means±SEM (n=3–6). **P<0.01. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 The CLOCK/BMAL1 response region located at -785 bp is critical for the transcriptional control of hAQP3. (a) Deletion analysis of the hAQP3 promoter region. (b) Chromatin immunoprecipitation (IP) analysis of the hAQP3 promoter in HaCaT cells at 32 or 44 hours after serum treatment. Photographs show representative electrophoretic images of PCR products of CLOCK/BMAL1 binding and input DNA. (c) Temporal expression profiles of hAQP3 in HaCaT cells after serum shock. The CLOCK small interfering RNA (siRNA) was transfected at the time of plating. There was a significant time-dependent variation in cells transfected with control siRNA (P<0.05, ANOVA). (d) Radiolabeled-glycerol uptake in cells treated with CLOCK siRNA in HaCaT cells after serum shock. There was a significant time-dependent variation of hAQP3 expression levels in cells transfected with control siRNA (P<0.05, ANOVA). Values are the means±SEM (n=3–6). *P<0.05; **P<0.01. (c, d) *P<0.05; **P<0.01 compared with the indicated time points. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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