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.

Parkinson's disease (PD) is a common neurodegenerative disorder that is characterized by the degeneration of dopaminergic neurons in the substantia nigra and dopamine depletion in the striatum. Although the motor symptoms are still regarded as the main problem, non-motor symptoms in PD also markedly impair the quality of life. Several non-motor symptoms, such as sleep disturbances and depression, are suggested to be implicated in the alteration in circadian clock function. In this study, we investigated circadian disruption and the mechanism in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. MPTP-treated mice exhibited altered 24-h rhythms in body temperature and locomotor activity. In addition, MPTP treatment also affected the circadian clock system at the genetic level. The exposure of human neuroblastoma cells (SH-SY5Y) to 1-metyl-4-phenylpyridinium (MPP(+)) increased or decreased the mRNA levels of several clock genes in a dose-dependent manner. MPP(+)-induced changes in clock genes expression were reversed by Compound C, an inhibitor of AMP-activated protein kinase (AMPK). Most importantly, addition of ATP to the drinking water of MPTP-treated mice attenuated neurodegeneration in dopaminergic neurons, suppressed AMPK activation and prevented circadian disruption. The present findings suggest that the activation of AMPK caused circadian dysfunction, and ATP may be a novel therapeutic strategy based on the molecular clock in PD.

We recently reported that Δ9-tetrahydrocannabinol (Δ9-THC), a major cannabinoid component in Cannabis Sativa (marijuana), significantly stimulated the expression of fatty acid 2 hydroxylase (FA2H) in human breast cancer MDA-MB-231 cells. Peroxisome proliferator-activated receptor α (PPARα) was previously implicated in this induction. However, the mechanisms mediating this induction have not been elucidated in detail. We performed a DNA microarray analysis of Δ9-THC treated samples and showed the selective up-regulation of the PPARα isoform coupled with the induction of FA2H over the other isoforms (β and γ). Δ-THC itself had no binding/activation potential to/on PPARα, and palmitic acid (PA), a PPARα ligand, exhibited no stimulatory effects on FA2H in MDA MB 231 cells; thus, we hypothesized that the levels of PPARα induced were involved in the Δ9-THC mediated increase in FA2H. In support of this hypothesis, we herein demonstrated that; (i) Δ9 THC activated the basal transcriptional activity of PPARα in a concentration-dependent manner, (ii) the concomitant up-regulation of PPARα/FA2H was caused by Δ9-THC, (iii) PA could activate PPARα after the PPARα expression plasmid was introduced, and (iv) the Δ9-THC induced up regulation of FA2H was further stimulated by the co-treatment with L-663,536 (a known PPARα inducer). Taken together, these results support the concept that the induced levels of PPARα may be involved in the Δ9 THC up-regulation of FA2H in MDA-MB-231 cells.

The dopamine D3 receptor (DRD3) in the ventral striatum is thought to influence motivation and motor functions. Although the expression of DRD3 in the ventral striatum has been shown to exhibit 24-hour variations, the mechanisms underlying the variation remain obscure. Here, we demonstrated that molecular components of the circadian clock act as regulators that control the 24-hour variation in the expression of DRD3. The transcription of DRD3 was enhanced by the retinoic acid-related orphan receptor a (RORa), and its activation was inhibited by the orphan receptor REV-ERBa, an endogenous antagonist of RORa. The serum or dexamethasone-induced oscillation in the expression of DRD3 in cells was abrogated by the downregulation or overexpression of REV-ERBa, suggesting that REV-ERBa functions as a regulator of DRD3 oscillations in the cellular autonomous clock. Chromatin immunoprecipitation assays of the DRD3 promoter indicated that the binding of the REVERBa protein to the DRD3 promoter increased in the early dark phase. DRD3 protein expression varied with higher levels during the dark phase. Moreover, the effects of the DRD3 agonist 7-hydroxy-N,N-dipropyl-2-aminotetralin (7-OH-DPAT)-induced locomotor hypoactivity were significantly increased when DRD3 proteins were abundant. These results suggest that RORa and REV-ERBa consist of a reciprocating mechanism wherein RORa upregulates the expression of DRD3, whereas REV-ERBa periodically suppresses the expression at the time of day when REV-ERBa is abundant. Our present findings revealed that a molecular link between the circadian clock and the function of DRD3 in the ventral striatum acts as a modulator of the pharmacological actions of DRD3 agonists/antagonists.

-Metastases are known to be responsible for approximately 90% of breast cancer-related deaths. Cyclooxygenase-2 (COX-2) is involved not only in inflammatory processes, but also in the metastasis of cancer cells; it is expressed in 40% of human invasive breast cancers. To comprehensively analyze the effects of cannabidiolic acid (CBDA), a selective COX-2 inhibitor found in the fiber-type cannabis plant (Takeda et al., 2008), on COX-2 expression and the genes involved in metastasis, we performed a DNA microarray analysis of human breast cancer MDA-MB-231 cells, which are invasive breast cancer cells that express high levels of COX-2, treated with CBDA for 48 hr at 25 μM. The results obtained revealed that COX-2 and Id-1, a positive regulator of breast cancer metastasis, were down-regulated (0.19-fold and 0.52-fold, respectively), while SHARP1 (or BHLHE41), a suppressor of breast cancer metastasis, was up-regulated (1.72-fold) and CHIP (or STUB1) was unaffected (1.03-fold). These changes were confirmed by real-time RT-PCR analyses. Taken together, the results obtained here demonstrated that i) CBDA had dual inhibitory effects on COX-2 through down-regulation and enzyme inhibition, and ii) CBDA may possess the ability to suppress genes that are positively involved in the metastasis of cancer cells in vitro.