CBD induces outwardly rectifying membrane currents on human sebocytes. TRIB3 is known to inhibit the NF-κB pathway (50), and, furthermore, CBD has already A2a adenosine receptor was found to mediate the antiinflammatory actions of activation of the "A2a recep- tor→cAMP→TRIB3⊣NF-κB" axis indeed plays a. Jul 25, Administration of CBD to cultured human sebocytes and human skin organ a CB2 cannabinoid receptor→ERK1/2 MAPK→PPAR pathway) induce .. CBD induces a novel (A2a adenosine receptor→cAMP→TRIB3⊣NF-κB). Administration of CBD to cultured human sebocytes and human skin organ culture .. Since the CBD-evoked lipostatic efects and the induced pathways that . a novel (A2a adenosine receptor→cAMP→ tor→cAMP→TRIB3⊣NF-κB” axis.
antiinflammatory novel pathway. CBD adenosine a (A2a receptor→cAMP→TRIB3⊣NF-κB) induces
Viability of sebocytes following hour treatments. H Statistical analysis of the lipid production on 4 histological sections per group.
Clinically, the key question is whether the above in vitro observations could be translated into significant sebostatic i. To explore this on the preclinical level, the full-thickness hSOC technique 20 was used. This suggests that CBD may also operate as a potent sebostatic agent in vivo when tested in appropriate clinical trials.
These data suggested that CBD may exert antiinflammatory actions on human sebocytes as had already been demonstrated for CBD in several other experimental models, such as diabetes, rheumatoid arthritis, etc.
Next, we dissected the molecular mechanism s that underlie the remarkable lipostatic effects of CBD. B CBD-induced differential current i. Compounds were applied as indicated by the arrow. Fluorescence measured in relative fluorescence units [RF] was normalized to the baseline. Fluorescence expressed in RF was normalized to the baseline. C Neutral lipid synthesis Nile Red staining. D and E Neutral lipid synthesis Nile Red staining following selective gene silencing of TRPV4 channel hour treatments, started at day 3 after the transfection.
SCR, scrambled control; UC, untransfected vehicle control. Collectively, these data unambiguously confirm that CBD activates TRPV4 and that this ion channel selectively mediates its lipostatic action. Dashed line indicates the level of the hour vehicle control. C Validation of the key microarray results. To dissect the intracellular signaling pathways that underlie the above effects, we first investigated the putative participation of several kinases i.
Gene set enrichment analysis GSEA 40 — 42 of the microarray results revealed that numerous mitosis and cell cycle e. During further data processing, Biological Networks Gene Ontology BiNGO analysis 44 , 45 was also performed see Supplemental Excel files 3 and 4; the hierarchy of the different gene ontology terms enriched among the downregulated and upregulated genes is summarized in Supplemental Figures 12 and 13, respectively.
Considering that administration of CBD led to opposing cellular effects i. This, again, confirmed the crucial role of TRPV4 activation in initiating the lipostatic and antiproliferative signaling cascade s of CBD. B Quantitative determination of neutral lipid synthesis Nile Red staining; hour treatments started at day 3 after transfection. One additional experiment yielded similar results.
Finally, we aimed at identifying the target molecule of CBD, which, via the upregulation of TRIB3, mediates the antiinflammatory action of the phytocannabinoid.
A previous finding that, in a murine model of acute lung injury, the G s protein—coupled A2a adenosine receptor was found to mediate the antiinflammatory actions of CBD 53 made this receptor a very probable target in our system as well.
It is very important to note that, based on the literature, administration of CBD holds out the promise to target these factors as well. Indeed, CBD was shown to inhibit proliferation of hyperproliferative keratinocytes 54 , and it was demonstrated to possess remarkable antibacterial activity Given that sebum production is the result of holocrine secretion, the amount of sebum produced is at least as dependent on the proliferative activity of basal layer sebocytes in the sebaceous gland as on the amount of lipogenesis that individual sebocytes engage in 27 , CBD has already been shown to activate e.
Therefore, exploration of its exact mechanism of action appeared to be a great challenge. Importantly, our data are in perfect agreement with the recent findings of De Petrocellis et al. GSEA 40 — 42 and BiNGO analysis 44 , 45 of the microarray results uniformly confirmed our results, arguing for complex anti-acne actions upon CBD administration, as indicated by downregulation of inflammation e.
Next, we aimed at revealing the signaling pathway of the antiinflammatory actions. Although a previous study would have suggested it 63 , interestingly, TRIB3 was found not to participate in mediating the lipostatic effects of CBD in sebocytes Supplemental Figure 15C.
It is also noteworthy that TRIB3 has been identified recently as a potent phytocannabinoid target gene 64 — These results, together with our data presented here, strongly argue for the key participation of TRIB3 in mediating cellular effects of cannabinoids.
Although CBD-dependent upregulation of its several known target genes, such as activating transcription factor 4, asparagine synthetase, cation transport regulator-like 1, and DNA-damage-inducible transcript 3 refs. We also demonstrated that sebocytes express G s -coupled A2a receptors which have already been shown to mediate antiinflammatory actions of CBD ref. Taken together, these data strongly argue that A2a receptor might be the primary orchestrator of the antiinflammatory actions of CBD.
It should also be noted that, according to the data published by Carrier et al. Multiple human studies have already investigated the safety of CBD 13 , Furthermore, it is already in use in many countries in clinical practice without any significant side effects Sativex This is especially promising, because the currently available, most effective anti-acne agent, isotretinoin, is known to cause serious side effects 2 , 69 , These data, together with our current findings, point to a promising, cost-effective, and, likely, well-tolerated new strategy for treating acne vulgaris, the most common human skin disease.
To the best of our knowledge, the exact pharmacokinetics of CBD in the human body is unknown, and there are no data in the literature on the expected intracutaneous accumulation of Sativex-derived, systemically applied CBD. Moreover, it is very important to note that, besides the systemic application, one should keep in mind the possibility of the topical administration. Due to its high lipophilicity, CBD is expected to preferentially enter the skin via the transfollicular route and to accumulate in the sebaceous gland 74 , Of great importance, such an accumulation has been documented already for multiple topically applied lipophilic compounds, e.
More details regarding the methods are available in the Supplemental Methods. Human immortalized SZ95 sebocytes 19 were cultured as described previously 12 , For semiquantitative detection of sebaceous lipids, Oil Red O staining was applied, whereas for quantitative measurements, fluorescent Nile Red staining was applied, as detailed in our previous work 12 , Apoptotic and necrotic processes were investigated by combined DilC 1 5 and SYTOX Green staining Life Technologies , measuring the alterations in the mitochondrial membrane potential and in the plasma membrane permeability, respectively, as described previously 10 , Q-PCR was performed as detailed in our previous reports 12 , As negative controls, the appropriate primary antibodies were omitted from the procedure.
As secondary antibodies, horseradish peroxidase—conjugated rabbit or mouse IgG Fc segment—specific antibodies developed in goat and sheep, respectively; Bio-Rad were used. Biopsies of intact human scalp and arm skin samples were obtained from 4 women RNAi was performed according to our optimized protocols 12 , Alterations in the gene expression were regarded as significant if a there were at least 2-fold changes in the corresponding levels; b the changes were equidirectional in all cases; and c global, corrected P values were less than 0.
Cells were then lysed cell density: Whole-cell patch-clamp recordings in the voltage-clamp configuration were performed using an Axopatch A amplifier Molecular Devices or by using an EPC amplifier. P values of less than 0. Study subjects provided informed consent prior to their participation. The authors have declared that no conflict of interest exists. National Center for Biotechnology Information , U. Journal List J Clin Invest v.
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Received Mar 17; Accepted Jun 5. This article has been cited by other articles in PMC. Associated Data Supplementary Materials Supplemental data. Supplemental Excel file 1. Supplemental Excel file 2. Supplemental Excel file 3. Supplemental Excel file 4. Abstract The endocannabinoid system ECS regulates multiple physiological processes, including cutaneous cell growth and differentiation. Introduction Acne vulgaris is the most common human skin disease, affecting quality of life of millions worldwide.
Open in a separate window. CBD decreases proliferation, but not the viability, of human sebocytes both in vitro and ex vivo. CBD exerts universal antiinflammatory actions. CBD induces outwardly rectifying membrane currents on human sebocytes. Anti-acne actions of CBD are mediated by parallel, partly independent signaling mechanisms. For details, see Discussion section. Methods More details regarding the methods are available in the Supplemental Methods. Cell culturing, determination of intracellular lipids, investigation of the lipidome.
Determination of viability, apoptosis, necrosis, and cellular proliferation. Full-thickness hSOC and sample preparations. Supplementary Material Supplemental data: Click here to view.
Supplemental Excel file 1: Supplemental Excel file 2: Supplemental Excel file 3: Supplemental Excel file 4: Footnotes Conflict of interest: Zouboulis CC, et al. What is the pathogenesis of acne? Kurokawa I, et al. New developments in our understanding of acne pathogenesis and treatment. Demuth DG, Molleman A. Targeting the endocannabinoid system: Nat Rev Drug Discov. The endocannabinoid system of the skin in health and disease: Cannabinoid system in the skin - a possible target for future therapies in dermatology.
Telek A, et al. Inhibition of human hair follicle growth by endo- and exocannabinoids. Endocannabinoids modulate human epidermal keratinocyte proliferation and survival via the sequential engagement of cannabinoid receptor-1 and transient receptor potential vanilloid Karsak M, et al. Attenuation of allergic contact dermatitis through the endocannabinoid system. Dobrosi N, et al. Endocannabinoids enhance lipid synthesis and apoptosis of human sebocytes via cannabinoid receptormediated signaling.
Zuardi AW, et al. Oil Red O staining, red; nuclei: PL, polar lipids; NL, neutral lipids. F Neutral lipid synthesis Nile Red staining. Two addi- tional experiments yielded similar results. G Analysis of the sebaceous lipidome. Two additional experiments yielded similar results. Although eCBs itatively excessive and abnormal lipid production induced by are known to show intense lipogenic actions via the metabotropic acne-promoting stimuli.
CB2 receptors 12 , neither semiquantitative Oil Red O nor quan- CBD decreases proliferation, but not the viability, of human titative Nile Red staining indicated changes in the basal neutral sebocytes both in vitro and ex vivo. Besides the above lipostatic sebaceous lipid synthesis of SZ95 sebocytes following hour action, another desired efect of a proper anti-acne agent would CBD treatment Figure 1, A—C or hour CBD treatment; data be to inhibit the unwanted growth of sebocytes 2, 27, Of great not shown.
Indeed, the lack of its efects on the count ECS-independent signal transduction mechanisms. Indeed, CBD of viable cells was further veriied by showing that these concen- efectively inhibited lipid synthesis induced by either arachidonic trations of CBD did not decrease cellular viability or induce either acid AA 21 or the combination of linoleic acid and testosterone apoptosis or necrosis of SZ95 sebocytes Figure 2, B and C.
To explore this on the preclinical level, the We also investigated the efects of CBD on the lipidome of full-thickness hSOC technique 20 was used. This suggests vitro observations Supplemental Figure 2E , decreased basal that CBD may primarily normalize both quantitatively and qual- lipogenesis as well Figure 2, D—H.
Likewise, CBD markedly sup- 2 jci. The solid line indicates the level of the hour vehicle control. Viability of sebocytes following hour treatments. Two additional exper- iments yielded similar results. This suggests that CBD may also operate as a potent sebostatic ciic antagonists AM and AM were able to antagonize the agent in vivo when tested in appropriate clinical trials. We additionally hence, alternative options had to be considered. Using whole-cell patch-clamp conigurations, the pathogenesis of acne vulgaris 2, 24— These data suggested membrane currents were elicited by voltage ramp protocols Fig- that CBD may exert antiinlammatory actions on human sebocytes ure 4, A and B and then normalized to cell membrane capacitance as had already been demonstrated for CBD in several other exper- at two diferent potentials, i.
Moreover, we have shown recently that activation of TRP Figure 3. Using a luorescent jci. To dissect the intracellular signaling ure 5C. Gene set enrichment Figure 4.
B CBD-induced diferential current i. Compounds were in the corresponding expression applied as indicated by the arrow. Fluorescence measured in relative luorescence units [RF] was normalized to levels equidirectional changes in the baseline. Two additional experiments all cases, and global, corrected yielded similar results. SCR, scrambled control; UC, untransfected vehicle control. Considering that administration of CBD led to ment of TRPV4-dependent calcium signaling detailed results of opposing cellular efects i.
Dashed line indicates the level of the hour vehicle control. Two additional experi- ments yielded similar results. One additional experiment yielded similar results. These data indicate that, irre- ure 16, A—C. It should also be noted that 8D. Finally, we aimed at mediating the antiinlammatory actions of CBD.
Indeed, matory cytokine levels universal antiinlammatory efect; Figure we found that the A2a receptor was expressed by human sebo- 3. For details, see Dis- cussion section. Therefore, exploration of its exact mechanism of action Propionibacterium acnes strains 2.
It is very important to note appeared to be a great challenge. The fact that we have shown pre- that, based on the literature, administration of CBD holds out the viously that activation of TRPV1 can evoke similar lipostatic efects promise to target these factors as well. Indeed, CBD was shown to 38 as those found for CBD Figure 1 and Figure 2, D—H , together inhibit proliferation of hyperproliferative keratinocytes 54 , and with our present indings that CBD induced membrane currents it was demonstrated to possess remarkable antibacterial activity on sebocytes Figure 4 , prompted us to irst investigate the role Of further engage in 27, Therefore, the novel and signiicant antiprolif- importance, we have also shown that the antiinlammatory activity erative activity of CBD on human sebocytes in vitro and ex vivo of CBD is a TRPV4-independent process Figure 6B.
Moreover, it is also important to empha- indings of De Petrocellis et al. Although the pos- cient level of sebum production is a critical factor for maintaining sibility that CBD might be a more eicacious activator of human proper function of the epidermal barrier, one of the central com- TRPV4 than of rat TRPV4 should also be taken into consideration; ponents of skin homeostasis Moreover, alterations of ARHGAP9 A2a receptor is very likely to be an indirect action, realized by the expression a known endogenous inhibitor of ERK signaling primary inhibition of the equilibrative nucleoside transporter s 46 suggested that inhibition of the prolipogenic MAPK path- e.
Multiple human studies have already in mediating the action of CBD. As expected 48 , knockdown side efects Sativex These data, inlammatory actions. In addition, inhibition of vulgaris, the most common human skin disease.
Although a previous study would have suggested it 63 , smoked marijuana in the pilosebaceous unit where they become interestingly, TRIB3 was found not to participate in mediating the incorporated into the hair shaft 71, 72 , it is very likely that CBD lipostatic efects of CBD in sebocytes Supplemental Figure 15C. These results, Moreover, it is very important to note that, besides the sys- together with our data presented here, strongly argue for the key temic application, one should keep in mind the possibility of the participation of TRIB3 in mediating cellular efects of cannabinoids.
Of great lular targets of TRIB3, i. As expected 51 , CBD was able importance, such an accumulation has been documented already jci. Densitometric analy- All in all, our novel data, along with intriguing literature sis of the signals was performed by using ImageJ software NIH.
Biopsies of intact ure 9 deserves full clinical exploration as a potent, novel class of human scalp and arm skin samples were obtained from 4 women More details regarding the methods are available in the Supplemen- RNAi.
RNAi was performed according to our optimized protocols tal Methods. Gene expression analysis of 3 independent described previously 10, Alterations in the gene expression brane potential and in the plasma membrane permeability, respec- were regarded as signiicant if a there were at least 2-fold changes in tively, as described previously 10, The degree of cellular growth the corresponding levels; b the changes were equidirectional in all was determined in well plate format by measuring the DNA con- cases; and c global, corrected P values were less than 0.
As internal controls, data analysis tool of MyAssays Ltd. As negative controls, 0. Homogeneity of variances was ana- the appropriate primary antibodies were omitted from the procedure. Western blotting was performed as described pre- variances, Games-Howel test was used instead of Bonferroni. Zouboulis CC, et al. What is the pathogenesis of Cannabidiol as an emergent therapeu- 2. Kurokawa I, et al. New developments in our Accessed July 8, Mirshahpanah P, Maibach HI.
Models in acnegen- mation on oxidative stress. Free Radic Biol Med. Demuth DG, Molleman A. Establishment and characterization of an A, Hargreaves KM. Role of ionotropic cann- 4.
Targeting the endocannabinoid sys- Towards the development of a simplified long- De Petrocellis L, et al. Nat Rev Drug Discov. J Pharmacol Exp Ther. Differentiation and apoptosis in ; 3: J Invest Derma- Effects of cannabinoids 7. The endocannabinoid system of the tol. Makrantonaki E, Zouboulis CC. Testosterone TRP channels and endocannabinoid metabolic and therapeutic opportunities. Bisogno T, et al. Molecular targets for cannabidiol 8. Cann- oxisome proliferator-activated receptor ligand and its synthetic analogues: VR1 receptors and on the cellular uptake and future therapies in dermatology.
Br J Phar- ;18 8: Telek A, et al. Inhibition of human hair follicle effects of cannabinoids in anxiety responses: TRPV2 is activated by cann- ;21 Neu- abidiol and mediates CGRP release in cultured Endocannabinoids modulate human ropsychopharmacology. Melnik BC, Schmitz G. Role of insulin, insulin- ;28 Cannabinoid actions receptor-1 and transient receptor potential vanil- milk consumption in the pathogenesis of acne at TRPV channels: TRPV4 and their potential relevance to gas- Karsak M, et al.
Cannabidiol exerts sebostatic and antiinflammatory effects on human sebocytes