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Comparative Study
. 2013 Oct;170(3):581-91.
doi: 10.1111/bph.12309.

Epigenetic control of skin differentiation genes by phytocannabinoids

Mariangela Pucci  1 , Cinzia Rapino, Andrea Di Francesco, Enrico Dainese, Claudio D'Addario, Mauro Maccarrone
Affiliations
Comparative Study

Epigenetic control of skin differentiation genes by phytocannabinoids

Mariangela Pucci et al. Br J Pharmacol. 2013 Oct.

Abstract

Background and purpose: Endocannabinoid signalling has been shown to have a role in the control of epidermal physiology, whereby anandamide is able to regulate the expression of skin differentiation genes through DNA methylation. Here, we investigated the possible epigenetic regulation of these genes by several phytocannabinoids, plant-derived cannabinoids that have the potential to be novel therapeutics for various human diseases.

Experimental approach: The effects of cannabidiol, cannabigerol and cannabidivarin on the expression of skin differentiation genes keratins 1 and 10, involucrin and transglutaminase 5, as well as on DNA methylation of keratin 10 gene, were investigated in human keratinocytes (HaCaT cells). The effects of these phytocannabinoids on global DNA methylation and the activity and expression of four major DNA methyltransferases (DNMT1, 3a, 3b and 3L) were also examined.

Key results: Cannabidiol and cannabigerol significantly reduced the expression of all the genes tested in differentiated HaCaT cells, by increasing DNA methylation of keratin 10 gene, but cannabidivarin was ineffective. Remarkably, cannabidiol reduced keratin 10 mRNA through a type-1 cannabinoid (CB1 ) receptor-dependent mechanism, whereas cannabigerol did not affect either CB1 or CB2 receptors of HaCaT cells. In addition, cannabidiol, but not cannabigerol, increased global DNA methylation levels by selectively enhancing DNMT1 expression, without affecting DNMT 3a, 3b or 3L.

Conclusions and implications: These findings show that the phytocannabinoids cannabidiol and cannabigerol are transcriptional repressors that can control cell proliferation and differentiation. This indicates that they (especially cannabidiol) have the potential to be lead compounds for the development of novel therapeutics for skin diseases.

Keywords: DNA methylation; endocannabinoid system; gene expression; phytocannabinoids; skin.

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Figures

Figure 1
Figure 1
Expression of K10 gene in HaCaT cells. Keratinocytes were induced to differentiate by treatment with TPA plus calcium for 5 days. Differentiated HaCaT cells were treated with 1 μM AEA and different amounts (0.1–0.5–1.0 μM) of CBD, CBG and CBDV. K10 was detected by quantitative RT-PCR, under different conditions and with primers, as described in the Methods section. For the quantification of gene expression, β-actin was used as a housekeeping gene. The results are shown as fold induction over proliferating cells of three independent experiments. Prol, proliferating cells; Ctrl, differentiated cells. ***P < 0.001 versus Prol; ###P < 0.001 versus Ctrl; ##P < 0.01 versus Ctrl; #P < 0.05 versus Ctrl.
Figure 2
Figure 2
Expression of epidermal differentiation-related genes in HaCaT cells. Differentiated HaCaT cells were treated with 1 μM AEA, 0.5 μM CBD, 0.5 μM CBG or 1.0 μM CBDV. K10 (A), involucrin (B), TGase5 (C) and K1 (D) were detected by quantitative RT-PCR, under condition and with primers described in the Methods section. The results are shown as fold induction over proliferating cells of three independent experiments. Prol, proliferating cells; Diff, differentiated cells. ***P < 0.001 versus Prol; ###P < 0.001 versus Diff; ##P < 0.01 versus Diff; #P < 0.05 versus Diff.
Figure 3
Figure 3
Effect of AEA (1.0 μM), CBD and CBG (both used at 0.5 μM), alone or in the presence of 0.05 μM SR141716, 0.05 μM SR144528 or 0.5 μM capsazepine (CPZ), on K10 gene expression in HaCaT cells. SR141716, SR144528 and CPZ were ineffective when used alone. Prol, proliferating cells; Diff, differentiated cells. ***P < 0.001 versus Prol; ###P < 0.001 versus Diff; #P < 0.05 versus Diff; $$P < 0.01 versus Diff + AEA; &P < 0.05 versus Diff + CBD.
Figure 4
Figure 4
Methylation-specific primed PCR. DNA methylation levels of K10 gene in differentiated HaCaT cells treated with CBD and CBG (both used at 0.5 μM), alone or in the presence of SR141716 (0.05 μM). SR141716 was ineffective when used alone. The methylation status of the K10 gene was analysed as described in the Methods section. Prol, proliferating cells; Diff, differentiated cells. ***P < 0.001 versus Prol; ###P < 0.001 versus Diff; #P < 0.05 versus Diff; &P < 0.05 versus Diff + CBD.
Figure 5
Figure 5
(A) Methylation levels of genomic DNA were measured by methyl-accepting assay with CpG methylase SssI, in the presence of S-adenosyl-L-[methyl-3H]-methyonine (see Methods for details). Higher levels of [3H]methyl group incorporated into DNA indicated lower level of genomic DNA methylation. Prol, proliferating cells; Diff, differentiated cells. *P < 0.05 versus Prol; ##P < 0.01 versus Diff; #P < 0.05 versus Diff; &P < 0.05 versus Diff + CBD. (B) Proliferating and differentiated keratinocytes treated with 1 μM AEA, 0.5 μM CBD or 0.5 μM CBG were lysed, and DNA methyltransferase activity was measured as described in the Methods section. Prol, proliferating cells; Diff, differentiated cells. **P < 0.01 versus Prol.
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