24-Hour Rhythm of Aquaporin-3 Function in the Epidermis Is Regulated by Molecular Clocks

Abstract

Aquaporin 3 (AQP3) is located in the basal layer of the epidermis and regulates biological functions of skin such as water content and trans-epidermal water loss. A recent study showed that the biological function of skin exhibits a 24-hour rhythm, but the molecular mechanism of the variation remains poorly understood. Here we show that mice mutated in the core clock component CLOCK (Clk/Clk) show decreased stratum corneum hydration. An extensive search for the underlying cause led us to identify AQP3 as a new regulator to control the 24-hour variation in biological functions of skin. In mouse epidermis of wild-type mice, mAqp3 exhibits circadian rhythms; however, these are significantly decreased in Clk/Clk. Luciferase reporter gene analysis revealed that transcription of mAqp3 is activated by D-site-binding protein, a clock gene. A human homolog, hAQP3, also exhibited significant oscillation in human keratinocyte (HaCaT) cells synchronized with medium containing 50% serum, and this rhythm was regulated by the endogenous CLOCK/BMAL1 heterodimer. These data indicate that although the molecular mechanisms underlying the rhythmic expression of mAqp3 and hAQP3 are different, clock genes are involved in time-dependent skin hydration. Our current findings provide a molecular link between the circadian clock and AQP3 function in mouse dorsal skin and HaCaT cells.

Tumor necrosis factorαdecreases aquaporin 3 expression in intestinal epithelial cells through inhibition of constitutive transcription

“…; Matsunaga et al. ) that would be expected to correlate with AQP3 protein levels. We observed a time‐dependent linear increase in 1,2,3‐ 3 H‐glycerol uptake that was significantly dampened in cells expressing AQP3 shRNA, suggesting that AQP3 protein was expressed in HT‐29 cells and its downregulation could be assessed using this approach (Fig.…”

“…We experienced significant challenges in detecting AQP3 protein expression in HT29 cells using all commercially available antibodies (data not shown). To circumvent these technical challenges, we measured the uptake of radiolabeled 1,2,3-3 H-glycerol which has previously been used by others to assess functional changes in AQP3 membrane expression (Jiang et al 2011;Matsunaga et al 2014) that would be expected to correlate with AQP3 protein levels. We observed a time-dependent linear increase in 1,2,3-3 H-glycerol uptake that was significantly dampened in cells expressing AQP3 shRNA, suggesting that AQP3 protein was expressed in HT-29 cells and its downregulation could be assessed using this approach (Fig.…”

“…; Matsunaga et al. ). In order to assess the proportion of radiolabeled glycerol uptake mediated by AQP3, human AQP3 shRNA lentiviral particles (Cat#sc‐29713‐V, Santa Cruz, Dallas, TX) were used to stably transfect and downregulate AQP3 expression in HT‐29 cells.…”

“…Uptake of radiolabeled glycerol to assess AQP3 function has been previously described (Ma et al 1994(Ma et al , 2002Yang and Verkman 1997;Jiang et al 2011;Matsunaga et al 2014). In order to assess the proportion of radiolabeled glycerol uptake mediated by AQP3, human AQP3 shRNA lentiviral particles (Cat#sc-29713-V, Santa Cruz, Dallas, TX) were used to stably transfect and downregulate AQP3 expression in HT-29 cells.…”

Clockwork allergy: How the circadian clock underpins allergic reactions

“…5,36 In the epidermis of wild-type mice, expression of aquaporin 3 (Aqp3), a regulator of water content and TEWL, exhibits circadian rhythms, which become arrhythmic in Clock-mutated mice. 37 Specifically, mouse Aqp3 and the human homolog AQP3 exhibit daily oscillation in mouse skin or human keratinocytes. The oscillation is regulated by molecular clocks.…”