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Cannabinoid Signaling in the Skin: Therapeutic Potential of the “C(ut)annabinoid” System

Authors:

Abstract and Figures

The endocannabinoid system (ECS) has lately been proven to be an important, multifaceted homeostatic regulator, which influences a wide-variety of physiological processes all over the body. Its members, the endocannabinoids (eCBs; e.g., anandamide), the eCB-responsive receptors (e.g., CB1, CB2), as well as the complex enzyme and transporter apparatus involved in the metabolism of the ligands were shown to be expressed in several tissues, including the skin. Although the best studied functions over the ECS are related to the central nervous system and to immune processes, experimental efforts over the last two decades have unambiguously confirmed that cutaneous cannabinoid (“c[ut]annabinoid”) signaling is deeply involved in the maintenance of skin homeostasis, barrier formation and regeneration, and its dysregulation was implicated to contribute to several highly prevalent diseases and disorders, e.g., atopic dermatitis, psoriasis, scleroderma, acne, hair growth and pigmentation disorders, keratin diseases, various tumors, and itch. The current review aims to give an overview of the available skin-relevant endo- and phytocannabinoid literature with a special emphasis on the putative translational potential, and to highlight promising future research directions as well as existing challenges.
Schematic overview of the (endo)cannabinoid system (ECS) and its putative connections to other signaling systems. Depending on how we choose to limit the definition, the number of the putative ligands as well as that of the possible targets increases dramatically; therefore, on the figure, we only summarize the most important ones. Each ligand possesses a unique molecular fingerprint, i.e., the ability to concentration-dependently activate/antagonize/inhibit a selected group of possible targets. Obviously, all these actions are highly context-dependent (e.g., they are influenced by the relative expression of the potential targets in the given tissue, the concentration of the substance), resulting in characteristic, and in some cases even opposing biological responses. Although the classical, lipophilic eCBs definitely require inter-and intracellular carriers, relatively little is known about these transporter systems. Intracellular eCB transporters may include fatty acid binding proteins (FABPs) and heat shock protein 70 (HSP70), whereas FABP4, albumins, HSP70 and extracellular vesicles [61,62] are likely to be involved in their intercellular transport [63]. With respect to FAAH1 and-2 it is important to note that only scarce evidence is available about the expression and functionality of the latter. Intriguingly, FAAH2 is not expressed in mice and rats, but shares substrate spectrum of FAAH1 (however, it has inferior affinity towards AEA and N-acyl taurines). Conventional FAAH-inhibitors can inhibit its activity [48], and its missense polymorphism (A458S) may lead to psychiatric disorders (anxiety, mild learning disability) [64]. Later in the text, except when stated otherwise, by mentioning "FAAH", we refer to "FAAH1". 5-HT: 5-hydroxytryptamine (serotonin) receptor; A 2A and A 3 : adenosine 2A and 3 receptors; ABDH6 and-12: α/β-hydrolase domain containing 6 and 12; CBC: (−)-cannabichromene; CBD: (−)-cannabidiol; CBDV: (−)-cannabidivarin; CBG: (−)-cannabigerol; CBGV: (−)-cannabigerovarin; CBN: (−)-cannabinol; (−)-cis-PET: (−)-cis-perrottetinene; COX 2 : cyclooxygenase-2; DAGL: diacylglycerol lipase; eCB: endocannabinoid; FAAH: fatty acid amide hydrolase; GPR: G protein-coupled receptor; LOX: lipoxygenase; MAGL: monoacylglycerol lipase; NAAA: N-acylethanolamine hydrolyzing acid amidase; NAPE-PLD: N-acylphosphatidylethanolamine-specific phospholipase D; PPAR: peroxisome proliferator-activated receptor; PTPN22: protein tyrosine phosphatase non-receptor type 22; THC: (−)-trans-∆ 9-tetrahydrocannabinol; THCV: (−)-∆ 9-tetrahydrocannabivarin; TRP: transient receptor potential.
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molecules
Review
Cannabinoid Signaling in the Skin: Therapeutic
Potential of the “C(ut)annabinoid” System
Kinga Fanni Tóth 1, , Dorottya Ádám1, , Tamás Bíró2, 3, and Attila Oláh1,*,‡
1Department of Physiology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary;
toth.kinga.fanni@med.unideb.hu (K.F.T.); adam.dorottya@med.unideb.hu (D.Á.)
2Department of Immunology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary;
biro.tamas@med.unideb.hu
3HCEMM Nonprofit Ltd., 6720 Szeged, Hungary
*Correspondence: olah.attila@med.unideb.hu; Tel.: +36-52-255-575
These authors contributed equally.
These authors contributed equally.
Received: 12 February 2019; Accepted: 1 March 2019; Published: 6 March 2019


Abstract:
The endocannabinoid system (ECS) has lately been proven to be an important, multifaceted
homeostatic regulator, which influences a wide-variety of physiological processes all over the body.
Its members, the endocannabinoids (eCBs; e.g., anandamide), the eCB-responsive receptors (e.g., CB
1
,
CB
2
), as well as the complex enzyme and transporter apparatus involved in the metabolism of
the ligands were shown to be expressed in several tissues, including the skin. Although the
best studied functions over the ECS are related to the central nervous system and to immune
processes, experimental efforts over the last two decades have unambiguously confirmed that
cutaneous cannabinoid (“c[ut]annabinoid”) signaling is deeply involved in the maintenance of skin
homeostasis, barrier formation and regeneration, and its dysregulation was implicated to contribute
to several highly prevalent diseases and disorders, e.g., atopic dermatitis, psoriasis, scleroderma,
acne, hair growth and pigmentation disorders, keratin diseases, various tumors, and itch. The current
review aims to give an overview of the available skin-relevant endo- and phytocannabinoid literature
with a special emphasis on the putative translational potential, and to highlight promising future
research directions as well as existing challenges.
Keywords: acne; atopic dermatitis; cannabinoid; fibrosis; hair growth; inflammation; itch; psoriasis;
skin; tumor; wound healing
1. Introduction
1.1. The Barrier and Beyond: Novel Aspects of Cutaneous (Patho)physiology
The skin is a vital organ that fulfills multiple roles. Besides being a complex protective barrier
against a wide-variety of environmental challenges [
1
3
], it is an active neuroendocrinoimmuno organ,
which produces several hormones, plays an important role in thermoregulation, and is involved in the
detection of various environmental signals, as well as in their translation/transmission to the nervous
and immune systems [
3
5
]. Indeed, functional expression of olfactory [
6
,
7
], photo [
8
,
9
], and taste
receptors [
10
12
]—among others—has recently been proven in different non-neuronal cells of the
integumentary system.
The complex protection provided by the skin is based on a fine-tuned barrier system, which
includes the cutaneous physicochemical, immunological and microbiological barriers. The development
of this complex barrier requires active and tightly regulated cooperation, and therefore appropriate
Molecules 2019,24, 918; doi:10.3390/molecules24050918 www.mdpi.com/journal/molecules
Molecules 2019,24, 918 2 of 56
communication of several cell types, including numerous “professional” immune cells (e.g., Langerhans
cells, dendritic cells, macrophages, mast cells, various T cell populations), and other cell types (e.g.,
keratinocytes, fibroblasts, melanocytes, sebocytes, adipocytes) [
1
3
,
13
18
]. Moreover, cells of the
human skin express a wide-array of pathogen- and danger-associated molecular pattern recognizing
receptors, and are capable of producing several anti-microbial peptides and lipids, as well as pro- and
anti-inflammatory cytokines and chemokines, by which they can initiate and regulate local immune
responses [
1
,
2
,
4
,
16
25
]. Obviously, these interactions are under the tight control of several signaling
systems, among which the current review aims to focus on a remarkably multifaceted one, namely the
cutaneous cannabinoid (“c[ut]annabinoid”) system.
1.2. (Endo)cannabinoid Signaling and its most Important Interactions
The endocannabinoid system (ECS) is a complex, evolutionarily conserved [
26
30
] homeostatic
signaling network. It comprises endogenous ligands (endocannabinoids [eCB], e.g., anandamide
[AEA]), eCB-responsive receptors (e.g., CB
1
and CB
2
cannabinoid receptors), and a complex
enzyme and transporter apparatus. These molecules are involved in the synthesis (e.g., N-acyl
phosphatidylethanolamine-specific phospholipase D [NAPE-PLD], diacylglycerol lipase [DAGL]-
α
and -
β
, protein tyrosine phosphatase non-receptor type 22 [PTPN22]), cellular uptake and release (i.e.,
the putative endocannabinoid membrane transporter(s) [EMT]), inter- and intracellular transport
(e.g., fatty acid binding proteins), and degradation (e.g., fatty acid amide hydrolase [FAAH],
monoacylglycerol lipase [MAGL]) of eCBs (Figure 1) [
31
50
]. Importantly, depending on the definition,
several other endogenous molecules can be classified as “cannabinoid-like” or “cannabinoid-related”
(e.g., palmitoylethanolamine [PEA], oleoylethanolamide [OEA]) beyond the “classical” eCBs [
31
47
,
51
].
Besides eCBs and related endogenous mediators, the Cannabinaceae-derived “classical” (e.g.,
the psychotropic (
)-trans-
9
-tetrahydrocannabinol [THC] or the non-psychotropic (
)-cannabidiol
[CBD]) and other plants-derived “non-classical” (e.g., the CB
2
-selective agonist
β
-caryophyllene, or the
liverwort-derived (
)-cis-perrottetinene [(
)-cis-PET]) phytocannabinoids (pCBs) represent another
important, and ever growing group of cannabinoids [
31
47
,
52
]. To date, more than 500 biologically
active components were identified in the plants of the Cannabis genus, among which more than 100
were classified as pCBs. Moreover, as mentioned above, several other plants were already shown
to produce molecules with cannabinoid activity [
30
,
32
,
47
,
52
]. It is suggested that consumption of
cannabimimetic food components might have played a role in hominid evolution, and production of
cannabimimetic food seems to be a promising future nutraceutical strategy [30].
Depending on their concentration, eCBs and pCBs are able to activate/antagonize/inhibit a
remarkably wide-variety of cellular targets including several metabotropic (e.g., CB
1
or CB
2
), ionotropic
(certain transient receptor potential [TRP] ion channels) and nuclear (peroxisome proliferator-activated
receptors [PPARs]) receptors, various enzymes, and transporters [
31
47
,
53
56
] (Figure 1). Importantly,
each ligand can be characterized by a unique, molecular fingerprint, and in some cases, they can even
exert opposing biological actions on the same target molecule (Figure 2a).
Indeed, it was nicely shown in several biochemical studies that THC was a partial CB
1
agonist,
whereas CBD was an antagonist/inverse agonist of the receptor [
57
]. Keeping this in mind it is easy to
understand why CBD is co-administered with THC in the oromucosal spray Sativex
®
, where the intent
is to prevent the onset of potential psychotropic side effects rooting from the THC-induced activation
of CB
1
expressed in the central nervous system [
58
]. Intriguingly, despite solid experimental and
clinical evidence proving that CBD is able to antagonize CB
1
, it is very important to emphasize that it
can context-dependently behave as a functional CB
1
activator as well. Indeed, by inhibiting FAAH and/or
EMT, its administration can lead to an elevation of the local eCB-tone, and hence to an indirectly
increased CB1activity in certain systems [59,60].
Molecules 2019,24, 918 3 of 56
Molecules 2018, 23, x FOR PEER REVIEW 3 of 55
Figure 1. Schematic overview of the (endo)cannabinoid system (ECS) and its putative connections to
other signaling systems. Depending on how we choose to limit the definition, the number of the
putative ligands as well as that of the possible targets increases dramatically; therefore, on the figure,
we only summarize the most important ones. Each ligand possesses a unique molecular fingerprint,
i.e., the ability to concentration-dependently activate/antagonize/inhibit a selected group of possible
targets. Obviously, all these actions are highly context-dependent (e.g., they are influenced by the
relative expression of the potential targets in the given tissue, the concentration of the substance),
resulting in characteristic, and in some cases even opposing biological responses. Although the
classical, lipophilic eCBs definitely require inter- and intracellular carriers, relatively little is known
about these transporter systems. Intracellular eCB transporters may include fatty acid binding
proteins (FABPs) and heat shock protein 70 (HSP70), whereas FABP4, albumins, HSP70 and
extracellular vesicles [61,62] are likely to be involved in their intercellular transport [63]. With respect
to FAAH1 and -2 it is important to note that only scarce evidence is available about the expression
and functionality of the latter. Intriguingly, FAAH2 is not expressed in mice and rats, but shares
substrate spectrum of FAAH1 (however, it has inferior affinity towards AEA and N-acyl taurines).
Conventional FAAH-inhibitors can inhibit its activity [48], and its missense polymorphism (A458S)
may lead to psychiatric disorders (anxiety, mild learning disability) [64]. Later in the text, except when
stated otherwise, by mentioning “FAAH”, we refer to “FAAH1”. 5-HT: 5-hydroxytryptamine
(serotonin) receptor; A
2A
and A
3
: adenosine 2A and 3 receptors; ABDH6 and -12: α/β-hydrolase
domain containing 6 and 12; CBC: ()-cannabichromene; CBD: ()-cannabidiol; CBDV: ()-
cannabidivarin; CBG: ()-cannabigerol; CBGV: ()-cannabigerovarin; CBN: ()-cannabinol; ()-cis-
PET: ()-cis-perrottetinene; COX
2
: cyclooxygenase-2; DAGL: diacylglycerol lipase; eCB:
endocannabinoid; FAAH: fatty acid amide hydrolase; GPR: G protein-coupled receptor; LOX:
lipoxygenase; MAGL: monoacylglycerol lipase; NAAA: N-acylethanolamine hydrolyzing acid
amidase; NAPE-PLD: N-acylphosphatidylethanolamine-specific phospholipase D; PPAR:
peroxisome proliferator-activated receptor; PTPN22: protein tyrosine phosphatase non-receptor type
22; THC: ()-trans-Δ
9
-tetrahydrocannabinol; THCV: ()-Δ
9
-tetrahydrocannabivarin; TRP: transient
receptor potential.
The high number of possible ligands and cellular targets together with the above context-
dependence already indicate that one has to be very careful when predicting the biological effects of
each cannabinoid based on mere biochemical observations obtained in artificially “clean”
overexpressor systems. Still, use and systematic assessment of such systems is extremely important
because of additional layers of complexity in (endo)cannabinoid signaling, including signaling bias
(i.e., ligand-dependent preference to the second messenger system) [31,32,65–73], receptor
heteromerization [32,74–80], cellular localization (surface membrane, mitochondria [81,82] or
lysosomes [83]), the regulatory role of the membrane lipid microenvironment [58,84] or agonist-
induced down-regulation [85] (Figure 2b). Finally, in some cases, effects of non-conventional
activators should also be taken into consideration, since certain cannabinoid-responsive receptors
(namely CB
1
, CB
2
, and TRPV4) were shown to be activated by UV-irradiation as well [86,87].
Figure 1.
Schematic overview of the (endo)cannabinoid system (ECS) and its putative connections
to other signaling systems. Depending on how we choose to limit the definition, the number
of the putative ligands as well as that of the possible targets increases dramatically; therefore,
on the figure, we only summarize the most important ones. Each ligand possesses a unique
molecular fingerprint, i.e., the ability to concentration-dependently activate/antagonize/inhibit
a selected group of possible targets. Obviously, all these actions are highly context-dependent
(e.g., they are influenced by the relative expression of the potential targets in the given tissue,
the concentration of the substance), resulting in characteristic, and in some cases even opposing
biological responses. Although the classical, lipophilic eCBs definitely require inter- and intracellular
carriers, relatively little is known about these transporter systems. Intracellular eCB transporters
may include fatty acid binding proteins (FABPs) and heat shock protein 70 (HSP70), whereas FABP4,
albumins, HSP70 and extracellular vesicles [
61
,
62
] are likely to be involved in their intercellular
transport [
63
]. With respect to FAAH1 and -2 it is important to note that only scarce evidence is
available about the expression and functionality of the latter. Intriguingly, FAAH2 is not expressed
in mice and rats, but shares substrate spectrum of FAAH1 (however, it has inferior affinity towards
AEA and N-acyl taurines). Conventional FAAH-inhibitors can inhibit its activity [
48
], and its missense
polymorphism (A458S) may lead to psychiatric disorders (anxiety, mild learning disability) [
64
].
Later in the text, except when stated otherwise, by mentioning “FAAH”, we refer to “FAAH1”.
5-HT: 5-hydroxytryptamine (serotonin) receptor; A
2A
and A
3
: adenosine 2A and 3 receptors; ABDH6
and -12:
α
/
β
-hydrolase domain containing 6 and 12; CBC: (
)-cannabichromene; CBD: (
)-cannabidiol;
CBDV: (
)-cannabidivarin; CBG: (
)-cannabigerol; CBGV: (
)-cannabigerovarin; CBN: (
)-cannabinol;
(
)-cis-PET: (
)-cis-perrottetinene; COX
2
: cyclooxygenase-2; DAGL: diacylglycerol lipase;
eCB: endocannabinoid; FAAH: fatty acid amide hydrolase; GPR: G protein-coupled receptor;
LOX: lipoxygenase; MAGL: monoacylglycerol lipase; NAAA: N-acylethanolamine hydrolyzing acid
amidase; NAPE-PLD: N-acylphosphatidylethanolamine-specific phospholipase D; PPAR: peroxisome
proliferator-activated receptor; PTPN22: protein tyrosine phosphatase non-receptor type 22;
THC: (
)-trans-
9
-tetrahydrocannabinol; THCV: (
)-
9
-tetrahydrocannabivarin; TRP: transient
receptor potential.
The high number of possible ligands and cellular targets together with the above
context-dependence already indicate that one has to be very careful when predicting the biological
effects of each cannabinoid based on mere biochemical observations obtained in artificially
“clean” overexpressor systems. Still, use and systematic assessment of such systems is extremely
important because of additional layers of complexity in (endo)cannabinoid signaling, including
signaling bias (i.e., ligand-dependent preference to the second messenger system) [
31
,
32
,
65
73
],
receptor heteromerization [
32
,
74
80
], cellular localization (surface membrane, mitochondria [
81
,
82
] or
lysosomes [
83
]), the regulatory role of the membrane lipid microenvironment [
58
,
84
] or agonist-induced
down-regulation [
85
] (Figure 2b). Finally, in some cases, effects of non-conventional activators should
also be taken into consideration, since certain cannabinoid-responsive receptors (namely CB
1
, CB
2
,
and TRPV4) were shown to be activated by UV-irradiation as well [86,87].
Molecules 2019,24, 918 4 of 56
Molecules 2018, 23, x FOR PEER REVIEW 4 of 55
Figure 2. Examples of the context-dependent complexity of the cannabinoid signaling. (a) Overview
of the most important potential targets of the phytocannabinoids (pCBs), which can be concentration-
dependently activated/antagonized/inhibited by these molecules. Each pCB can be characterized by
a unique molecular fingerprint, and every pCB was found to interact with only a subset of potential
targets shown on panel (a). Importantly, the interactions can even result in opposing outcomes (e.g.,
THC is a partial CB1 agonist, whereas CBD is a CB1 antagonist/inverse agonist), making prediction of
cellular effects of the pCBs even more difficult. (b) The actual biological response, which develops
following the activation of CB1 receptor depends on several additional factors, including biased
agonism [31,32,65–73], possible receptor heteromerization [32,74–80], localization (i.e., cell membrane
vs. mitochondria vs. lysosomes [81–83]), as well as the composition of the lipid microenvironment of
the given membrane [58,84]. Green arrows on panel (b): the most common signaling pathways of CB1.
Note that besides CB1, biased agonism is well-described in case of CB2, GPR18, GPR55 and GPR119
as well, whereas CB2 was proven to heteromerize with, e.g., C-X-C chemokine receptor type 4
chemokine receptor (CXCR4), or GPR55 (for details, see the above references). The question mark
indicates that functional heteromerization of CB1 and GABAB receptors is questionable. AT1:
angiotensin II receptor type 1; CYP: cytochrome P450 enzymes; D2: dopamine receptor 2; EMT(s):
endocannabinoid membrane transporter(s); ENT1: equilibrative nucleoside transporter 1; GABAB: γ-
aminobutyric acid receptor B; LPA1: lysophosphatidic acid receptor 1; Nav: voltage-gated Na+
channels; OX1: orexin 1 receptor; VDAC1: voltage-dependent anion channel 1. The figure was adapted
and modified from [31] originally licensed under CC-BY, version 4.0.
1.3. Cannabinoids in the Skin: Brief Overview of the “c(ut)annabinoid” Signaling
It has recently been shown that abuse of synthetic, hyperpotent cannabinoids (e.g., “Bonsai”,
“fake weed”, “K2”, and “Jamaica”) can result in dermatological disorders, such as premature skin
aging, hair loss and graying, or acne [88], indicating that cannabinoid signaling can profoundly
influence skin biology. Indeed, several lines of evidence demonstrate that both endogenous and
phytocannabinoids can exert various biological effects in the skin, implicating cannabinoid signaling
as a key contributor to cutaneous homeostasis. The presence of different eCBs, cannabinoid receptors,
as well as other members of the ECS has already been shown on many different cell types of the skin,
including, but not limited to epidermal keratinocytes, melanocytes, mast cells, fibroblasts, sebocytes,
sweat gland cells, as well as certain cell populations of hair follicles. Since these data have been
extensively reviewed in excellent recent papers [88–101], besides providing a brief general overview,
our current paper intends to focus on areas which have received less attention in said papers, and to
highlight the mostly neglected therapeutic potential present in the pharmacological modulation of
the “c(ut)annabinoid” signaling. Last, but not least, we intend to discuss the potential limitations and
side effects of such medications as well.
Activation
of β-arrestins
G
i
G
s
G
q
G
12/13
A
2A
μopioid
D
2
GABA
B
(?)
OX
1
Biased agonism
Heteromerization
Modulation of
ion channels
(βγ subunits)
cAMP
PKA
PLC
(IP
3
, DAG,
Ca
2+
, PKC)
Rho
pCBs
CB
1
/CB
2
TRP
channels
PPARs
5-HT receptors
α
2
receptor
A
2A
receptor
(indirect effect via
inhibiting ENT1?)
Opioid
receptors
LOX
COX
FAAH
EMT(s)
CYP enzymes
GPR3/6/12/55 Na
v
channels
VDAC1 δopioid
LPA
1
AT
1
(b)(a)
Activation of various
kinase cascades
Localization (cell membrane vs.
mitochondria vs. lysosomes)
Lipid environment
CB
1
Figure 2.
Examples of the context-dependent complexity of the cannabinoid signaling. (
a
) Overview
of the most important potential targets of the phytocannabinoids (pCBs), which can be
concentration-dependently activated/antagonized/inhibited by these molecules. Each pCB can be
characterized by a unique molecular fingerprint, and every pCB was found to interact with only
a subset of potential targets shown on panel (
a
). Importantly, the interactions can even result in
opposing outcomes (e.g., THC is a partial CB
1
agonist, whereas CBD is a CB
1
antagonist/inverse
agonist), making prediction of cellular effects of the pCBs even more difficult. (
b
) The actual biological
response, which develops following the activation of CB
1
receptor depends on several additional factors,
including biased agonism [
31
,
32
,
65
73
], possible receptor heteromerization [
32
,
74
80
], localization
(i.e., cell membrane vs. mitochondria vs. lysosomes [
81
83
]), as well as the composition of the
lipid microenvironment of the given membrane [
58
,
84
]. Green arrows on panel (b): the most
common signaling pathways of CB
1
. Note that besides CB
1
, biased agonism is well-described in
case of CB
2
, GPR18, GPR55 and GPR119 as well, whereas CB
2
was proven to heteromerize with,
e.g., C-X-C chemokine receptor type 4 chemokine receptor (CXCR4), or GPR55 (for details, see the
above references). The question mark indicates that functional heteromerization of CB
1
and GABA
B
receptors is questionable. AT
1
: angiotensin II receptor type 1; CYP: cytochrome P450 enzymes;
D
2
: dopamine receptor 2; EMT(s): endocannabinoid membrane transporter(s); ENT1: equilibrative
nucleoside transporter 1; GABA
B
:
γ
-aminobutyric acid receptor B; LPA
1
: lysophosphatidic acid
receptor 1; Na
v
: voltage-gated Na
+
channels; OX
1
: orexin 1 receptor; VDAC1: voltage-dependent
anion channel 1. The figure was adapted and modified from [
31
] originally licensed under CC-BY,
version 4.0.
1.3. Cannabinoids in the Skin: Brief Overview of the “c(ut)annabinoid” Signaling
It has recently been shown that abuse of synthetic, hyperpotent cannabinoids (e.g., “Bonsai”,
“fake weed”, “K2”, and “Jamaica”) can result in dermatological disorders, such as premature skin aging,
hair loss and graying, or acne [
88
], indicating that cannabinoid signaling can profoundly influence skin
biology. Indeed, several lines of evidence demonstrate that both endogenous and phytocannabinoids
can exert various biological effects in the skin, implicating cannabinoid signaling as a key contributor
to cutaneous homeostasis. The presence of different eCBs, cannabinoid receptors, as well as other
members of the ECS has already been shown on many different cell types of the skin, including, but not
limited to epidermal keratinocytes, melanocytes, mast cells, fibroblasts, sebocytes, sweat gland cells,
as well as certain cell populations of hair follicles. Since these data have been extensively reviewed
in excellent recent papers [
88
101
], besides providing a brief general overview, our current paper
intends to focus on areas which have received less attention in said papers, and to highlight the mostly
neglected therapeutic potential present in the pharmacological modulation of the “c(ut)annabinoid”
signaling. Last, but not least, we intend to discuss the potential limitations and side effects of such
medications as well.
Molecules 2019,24, 918 5 of 56
2. Translational Potential of the Cutaneous Cannabinoid Signaling
2.1. Sebaceous Gland (SG)-Related Disorders: Acne and Skin Dryness
The most obvious role of sebaceous glands (SG) is the production of lipid-rich sebum, which
contributes to the development of the physicochemical barrier, and, via its acid and anti-microbial
lipid content, also controls the growth of cutaneous microbiota [
102
104
]. SGs have endocrine and
immune regulatory functions as well [
105
109
], and their clinical significance is also very high, since
they are key players in the pathogenesis of highly prevalent dermatoses such as acne and seborrhea,
and their dysfunction contributes to the development of dryness-accompanied skin diseases, including
atopic dermatitis (AD) [102,103,105,110].
The clinical observation that cannabinoid abuse can be accompanied by acne, already highlights
how cannabinoid signaling may influence human sebocyte biology [
88
]. Indeed, expression of
CB
1
(in the differentiated, central cells) and CB
2
(predominantly in the basal, non-differentiated
sebocytes) receptors in human SGs was first demonstrated by Ständer and her co-workers in 2005 [
111
].
When exploring the functional relevance of these findings, it has been shown that CB
2
is likely to
contribute to the maintenance of homeostatic sebaceous lipogenesis (SLG), since siRNA-mediated
silencing of the receptor significantly decreased lipid production, whereas administration of AEA and
2-AG (30
µ
M) led to excessive lipid synthesis via the activation of a CB
2
ERK1/2 MAPK
PPAR
pathway [
112
]. Later on, the major eCB synthesizing (NAPE-PLD, DAGL
α
and
β
) and degrading
(MAGL and FAAH) enzymes were found to be expressed both in cultured human immortalized
SZ95 sebocytes [
108
,
109
,
113
] and in situ in human SGs, with the sole exception of DAGL
α
[
114
],
the expression of which was observed to be much weaker as compared to the endogenous tissue
positive control [115] sweat glands.
It has also been demonstrated that certain EMT-inhibitors (VDM11 and AM404), but, intriguingly,
not the FAAH-inhibitor URB597, promoted SLG, and VDM11-induced elevation of the eCB-tone
suppressed the pro-inflammatory action of the Toll-like receptor (TLR)-4 activator lipopolysaccharide
(LPS) [
114
]. Considering that, as mentioned above, SG hypoplasia and dysfunction contributes
to the development of dryness-accompanied skin diseases [
102
,
103
], and that such diseases often
have inflammatory components, a moderate (i.e., not excessive, seborrheic/acnegenic) elevation of
physiological SLG together with the suppression of the release of pro-inflammatory cytokines and
chemokines could exert beneficial effects. Thus, the available data [
114
] highlight the possibility that
eCB transport inhibitors might have beneficial effects in diseases with skin dryness such as AD. Future
studies are therefore invited to explore the exact impact of VDM11 treatment on the sebaceous lipidome
to reduce the possibility of potential acnegenic side effects.
Interestingly, that study also demonstrated that human sebocytes were involved in the metabolism
of PEA and OEA [
114
]. Moreover, the expression of an important cellular target of the latter, namely
GPR119, was also identified on human sebocytes. The available scarce evidence suggests that the
OEA
GPR119
ERK1/2 MAPK signaling chain may be a previously unknown promoter of sebocyte
differentiation, and therefore dysregulation of this pathway may contribute to the development of
seborrhea and acne [
116
]. This seems to be particularly interesting, since GPR119 has recently emerged
as a promising therapeutic target in type 2 diabetes mellitus. Although the tested synthetic agonists
have not passed yet phase II clinical trials [
117
], and both endogenous and synthetic agonists of GPR119
may exhibit biologically relevant signaling bias [
73
], these preliminary findings warn of the risk of
unexpected cutaneous side effects upon administration of GPR119 activators exhibiting “OEA-like”
signaling preference [116].
Intriguingly, besides the aforementioned “classical” members of the ECS, functional expression
of several ECS-related TRP channels was also demonstrated. The mostly Ca
2+
-permeable ion
channels TRPV1, TRPV2, TRPV3 and TRPV4 [
118
120
] were shown to be expressed on human
sebocytes. Importantly, in a striking contrast to the “classical” cannabinoid signaling, activation
of the TRPV channels was proven to decrease SLG. Moreover, activation of TRPV3 led to a significant
Molecules 2019,24, 918 6 of 56
pro-inflammatory response in the sebocytes as revealed by the up-regulated expression and increased
release of several pro-inflammatory cytokines [
119
], a phenomenon recently demonstrated on human
epidermal keratinocytes as well [121].
Notably, the best-studied non-psychotropic pCB, i.e., CBD (10
µ
M), was found to exert complex
anti-acne effects by normalizing several pro-acne agents-induced excessive SLG, and by exerting
anti-proliferative and anti-inflammatory actions, without influencing homeostatic SLG or viability
of human sebocytes. Importantly, the lipostatic and anti-proliferative effects were found to be
mediated by the TRPV4
[Ca
2+
]
IC
ERK1/2 MAPK
and nuclear receptor interacting protein 1
(NRIP1, a.k.a. RIP140)
signaling pathway, whereas the anti-inflammatory actions were coupled
to the (most likely indirect) activation of the adenosine A
2A
receptor
cAMP
↑→
tribbles homolog 3
(TRIB3)
↑→
p65-NF-
κ
B
pathway [
120
]. This, together with the fact that CBD was shown to suppress
proliferation [
122
] and differentiation [
59
] of human keratinocytes, and to exert potent anti-bacterial
effects [123], collectively argue that it may be an efficient anti-acne agent in vivo as well.
This concept was further supported by a small, single-blind, split-face study, in which a cream
containing 3% Cannabis seed extract was applied twice daily to the cheeks of patients for 12 weeks.
The treatment was found to be efficient in reducing sebum production and erythema compared to
the vehicle treated side [
124
]. Moreover, a synthetic CBD containing special topical formulation
(“BTX 1503”) exhibited promising anti-acne potential in a small phase Ib clinical trial [
125
], and its
efficacy is now being tested in a randomized, double-blind, vehicle-controlled phase II clinical study
(ClinicalTrials.gov ID: NCT03573518).
Last, but not least, it should also be noted that effects of several other non-psychotropic pCBs,
namely CBC, CBDV, CBG, CBGV and THCV were also assessed in human sebocytes. This latter study
found an intriguing functional heterogeneity between the tested pCBs, with CBC, CBDV and most
especially THCV behaving in a “CBD-like” manner (potent complex anti-acne effects
in vitro
), whereas
CBG and CBGV being more “eCB-like” substances (slight, but significant promotion of SLG together
with potent anti-inflammatory activity) [
126
]. Although the exact impact of CBG and CBGV on the
sebaceous lipidome remains to be tested in future studies, the available evidence suggests that, similar
to the aforementioned EMT-inhibitors VDM11 and AM404, they might have therapeutic value in
dryness- and inflammation-accompanied skin diseases. The putative SG-related translational potential
of cannabinoid signaling is summarized in Table 1.
2.2. Hair Growth Disorders: Alopecia, Effluvium, Hirsutism, Hypertrichosis
Hair follicles (HF) are unique miniorgans of the human body. They exhibit immune privilege
(IP), i.e., they can be characterized by low or absent major histocompatibility complex (MHC) class Ia
and
β
2 microglobulin expression leading to an ineffective self-peptide presentation, and they secrete
several immunosuppressants to create an immunoinhibitory milieu [
127
,
128
]. Besides this, HFs are
characterized by life-long cycles of growing (anagen), regressive (catagen) and “quasi-quiescent”
(telogen) life phases collectively referred to as the “hair cycle” [
129
]. Importantly, dysregulation of this
cycle (e.g., premature termination or abnormal prolongation of the anagen phase) lies at the base of
several clinically important hair growth disorders leading to unwanted hair loss (i.e., various alopecia
forms) or undesired hair growth (hirsutism and hypertrichosis).
Similar to SGs, the biology of HFs is also influenced by cannabinoids. Indeed, as mentioned
above, abuse of certain synthetic cannabinoids was shown to result in hair loss and graying [
88
],
and it is well-proven that CB
1
is expressed in human HFs, whereas regarding the expression of CB
2
contradictory findings have been published so far [
111
,
130
133
]. Of great importance, prototypic eCBs
(i.e., AEA and 2-AG) were shown to be produced in human HFs, among which 30
µ
M AEA (but not
2-AG) was proven to inhibit hair growth by inducing premature catagen entry in a CB
1
-dependent
manner, but, somewhat surprisingly, it did not influence the pigmentation of HFs.
Molecules 2019,24, 918 7 of 56
Table 1.
Overview of the putative sebaceous gland-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Disease Intervention Level of Evidence References
Dry skin
EMT-inhibition (elevation of the
eCB-tone) In vitro (cell culture) data [112,114]
CBG, CBGV In vitro (cell culture) data [126]
Acne & Seborrhea
CBD (via activating TRPV4 and
A2A receptors)
In vitro (cell culture) and ex vivo
(organ culture) data [120]
BTX 1503 (synthetic CBD
containing cream)
Successful phase Ib and ongoing
phase II clinical trials
[125]ClinicalTrials.gov
ID: NCT03573518
THCV, CBC, CBDV In vitro (cell culture) data [126]
3% Cannabis seeds extract cream
single-blind, split-face study [124]
Reduction of the eCB-tone In vitro (cell culture) data [112,114]
GPR119-antagonism 1Hypothesis based on preliminary
in vitro (cell culture) data [114,116]
1
Note that effects of GPR119 antagonism have not been tested yet; however, in light of the available scarce data,
interfering with GPR119 signaling might deserve systematic experimental exploration.
In line with these findings, 2–20
µ
M THC was also shown to inhibit hair shaft elongation,
and to induce catagen entry, but, unlike AEA, it also suppressed melanogenesis in anagen VI HFs,
highlighting an intriguing functional heterogeneity between cannabinoids, which might have reflected
the aforementioned (Section 1.2, Figure 2b) signaling bias of the tested compounds. Importantly,
CB
1
itself was greatly up-regulated in the hair matrix keratinocytes both in AEA- and interferon-
γ
(IFN-
γ
)-induced catagen, supporting the concept that it may play a role in the termination of the HF
growth phase [
130
]. The idea that CB
1
is a negative regulator of HF growth was further supported by
animal data. Indeed, an orally administered rimonabant analogue CB
1
antagonist (“compound 3”)
promoted hair growth (and had antiobesity effects) in C57BL/6J mice, in which high fat diet induced
obesity was accompanied by alopecia. Interestingly, however, the effect of the CB
1
antagonist did not
develop if it was applied topically [134].
Besides CB
1
, several cannabinoid-responsive TRPV channels (namely TRPV1, TRPV3 and TRPV4)
were shown to be functionally expressed in human HFs, and to promote the onset of catagen
phase [135138]
, which, considering that all three channels are heat-sensitive [
45
,
94
,
95
,
139
], may be an
evolutionary relic of warmth-induced shedding. Last, but not least, preliminary evidence suggests
that CBD may concentration-dependently promote (0.1
µ
M) or suppress (10
µ
M) hair shaft elongation,
most likely in adenosine receptor and TRPV4-dependent manners, respectively [140].
Finally, considering the well-known anti-inflammatory and immunosuppressive effects of
cannabinoids [
31
,
33
,
93
,
141
145
], it is not surprising that certain data suggest involvement of
cannabinoid dysregulation in the development of alopecia areata (AA). AA is an autoimmune disease
characterized by localized or global hair loss due to the collapse of the HF IP and the subsequent
autoaggression of cytotoxic T cells leading to premature catagen entry. Importantly, several lines
of evidence suggest that a loss-of-function single-nucleotide polymorphism (C1858T substitution;
“R620W variant”; “rs2476601”) of PTPN22 (a phosphatase involved in synthesizing AEA [
48
], which
normally suppresses T-cell proliferation), which leads to its rapid degradation, is coupled to several
autoimmune diseases (for details, see [
31
]), including alopecia areata (AA) [
146
152
]. Although
PTPN22 has several other functions besides AEA synthesis [
153
], and eCB levels were not measured
yet in lesional skin of AA patients, one might hypothesize based on the above correlation that a
decrease in the anti-inflammatory eCB-tone induced by PTPN22 dysfunction might contribute to the
onset of the disease. Thus, elevation of the eCB-tone as well as direct CB
1
agonism might be promising
tools to prevent the onset/relapse of AA. Finally, albeit only scant evidence is available, it is noteworthy
that some experimental [
140
] and pilot clinical data [
154
] highlight the possibility that carefully selected
doses of topically applied CBD might also exert beneficial effects in AA. Further studies, as well as
well-controlled clinical trials are therefore invited to elucidate the putative therapeutic potential of
Molecules 2019,24, 918 8 of 56
the cutaneous cannabinoid and related signaling systems in AA. Putative hair-related translational
potential of the cannabinoid signaling is summarized in Table 2.
Table 2.
Overview of the putative hair-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Disease Intervention Level of Evidence References
Unwanted hair growth
(hirsutism,
hypertrichosis)
Certain CB1agonists Ex vivo (organ culture) data [130]
TRPV1, TRPV3 and TRPV4
activators Ex vivo (organ culture) data [135138]
Unwanted hair loss
(different non-immune
alopecia forms)
Certain CB1
antagonists/inverse agonists
Ex vivo (organ culture) and
in vivo (mouse) data [130,134]
TRPV1, TRPV3 and TRPV4
antagonists Ex vivo (organ culture) data [135138]
Alopecia areata
Elevation of the eCB-tone;
certain CB1agonists, low
doses of CBD 1
Hypothesis based on the
available data [31,130,140,146152,154]
1
Note that well-controlled studies proving the efficiency of the indicated interventions are missing; however,
in light of the available data, cannabinoid signaling might exert beneficial effects in alopecia areata, therefore it
deserves systematic experimental exploration.
2.3. Melanocytes & Pigmentation Disorders
Primary human melanocytes were shown to produce AEA and 2-AG [
155
], and to express
GPR119 (only mRNA data) [
156
], CB
1
, CB
2
and TRPV1 together with NAPE-PLD, DAGL, FAAH and
MAGL [
155
]. However, expression of MAGL in normal human epidermal melanocytes was questioned
in a recent study stating that this enzyme was only expressed in melanoma cells, where its expression
correlated with the aggressiveness of the tumor [157].
In functional studies, 100–150
µ
M
β
-caryophyllene was found to inhibit spontaneous
melanogenesis of mouse B16 melanoma cells [
158
], whereas 5
µ
M AEA was shown to induce
apoptosis of primary human melanocytes most likely by activating TRPV1. Lower (
3
µ
M) AEA
concentrations however, dose-dependently stimulated melanogenesis and tyrosinase activity in a
CB
1
-dependent manner through the activation of p38 and ERK1/2 MAPK, as well as the cAMP
response element-binding protein (CREB), but without influencing the cAMP level [155].
In line with these observations, CBD was also shown to enhance melanogenesis and tyrosinase
activity of primary human epidermal melanocytes by (most probably indirectly) activating the same
CB
1
-coupled signaling pathway [
60
]. Although these data argue that CB
1
agonism may be a potent tool
to treat hypopigmentation, other findings suggest that the overall effects of the eCB-signaling might be
more complex. Indeed, by using co-cultures of a human melanotic melanoma cell line (SK-mel-1) and
HaCaT keratinocytes (a spontaneously immortalized human epidermal keratinocyte cell line [
159
]),
Magina and her co-workers found that CB
1
agonism reduced both spontaneous and UVB-induced
melanogenesis, highlighting that the local tissue microenvironment may have an important role in
regulating melanocyte functions [
160
]. Finally, in contrast to AEA, OEA (10–50
µ
M) was shown to
markedly inhibit melanin synthesis and tyrosinase activity in
α
-MSH-stimulated B16 mouse melanoma
cells in a PPAR
α
-independent manner. Its effects were found to be coupled to the activation of p38
and ERK1/2 MAPK, as well as of Akt signaling cascades, and inhibition of the CREB pathway
(unfortunately, putative involvement of GPR119 was not assessed) [
161
]. Thus, (endo)cannabinoid
signaling appears to exert a complex regulatory role in melanocytes; however, the results are greatly
model-dependent (mono-cultures vs. co-cultures; human vs. mouse data).
It is also noteworthy that eCB-dysregulation may also contribute to the development of vitiligo,
a chronic skin disease characterized by localized or generalized de-pigmentation, having a rather
complex, but chiefly autoimmune pathogenesis [
162
,
163
]. Indeed, similar to AA, the 1858 C/T missense
single nucleotide polymorphism of PTPN22 (R620W; rs2476601) was shown to be associated with a
higher vitiligo risk [
164
167
]. Interestingly, however, this association seems to be ethnicity-dependent,
Molecules 2019,24, 918 9 of 56
since no such correlation was found in Turkish and Jordanian patients [
168
,
169
]. Although one should
keep in mind that the actual levels of eCBs have never been investigated in lesional skin of vitiligo
patients, and that PTPN22 has other, ECS-independent biological functions [
153
], immunosuppressive
cannabinoid signaling might have therapeutic value in vitiligo. This bold hypothesis has to be tested
in future targeted studies. Putative melanocyte-related translational potential of the cannabinoid
signaling modulation is summarized in Table 3.
Table 3.
Overview of the putative pigmentation-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Disease Intervention Level of Evidence References
Hypopigmentation
Elevation of the
eCB-tone/activation of CB
1
(?)
In vitro (monoculture of primary
human epidermal melanocytes) [155]
Administration of CBD (via
activating CB1)
In vitro (monoculture of primary
human epidermal melanocytes) [60]
Hyperpigmentation
Elevation of the
eCB-tone/activation of CB
1
(?)
In vitro (co-culture of SK-mel-1 and
HaCaT keratinocytes) [160]
β-caryophyllene In vitro (mono-culture of B16
melanoma cells) [158]
Vitiligo Elevation of the eCB-tone 1Hypothesis
based on literature data
[164167]
1
Note that well-controlled studies proving the efficiency of cannabinoids are missing; however, in light of the
available data, elevation of the eCB-tone might exert beneficial effects in vitiligo, therefore it deserves systematic
experimental exploration.
2.4. Epidermal Keratinocytes
2.4.1. Proliferation and Differentiation
Several members of the ECS (AEA, 2-AG, CB1, CB2, NAPE-PLD, FAAH, multiple TRP channels,
etc.) have been shown to be expressed on human epidermal keratinocytes [
45
,
91
,
92
,
95
,
111
,
133
], and the
functional activity of the putative EMT was also demonstrated on these cells [
170
]. What’s more,
one of the first pieces of morphological and biochemical evidence indicating that transport and
hydrolysis of AEA are two spatially and functionally distinct processes was also provided in HaCaT
keratinocytes [171].
Based on the available functional evidence, the homeostatic eCB-tone appears to play a role in
regulating proliferation/differentiation balance, as well as pro-inflammatory mediator production and
release by epidermal keratinocytes. Indeed, activation of CB
1
by 1
µ
M AEA was shown to prevent
differentiation induced by the combination of 12-O-tetradecanoylphorbol 13-acetate (a “general” PKC
activator) and elevated [Ca
2+
]
EC
in confluent 2D keratinocyte cultures, as revealed by abrogated
cornified envelope formation [
170
]. Importantly, AEA was also able to prevent differentiation-induced
up-regulation of several differentiation markers (keratin (K)-1, K10, involucrin and transglutaminase 5)
by increasing DNA methylation, through a p38, and, to a lesser extent, an ERK1/2 MAPK-dependent
pathway, again, in a CB
1
-dependent manner [
172
,
173
]. On the other hand, higher (3–30
µ
M)
concentrations of AEA were found to suppress proliferation and to induce apoptosis of HaCaT
and primary human epidermal keratinocytes
in vitro
, as well as in situ in full-thickness human skin
organ culture (hSOC) via sequentially activating first CB1and then indirectly TRPV1 [174]. Likewise,
24-h treatment of hSOC with 30
µ
M arachidonyl-2
0
-chloro- ethylamide (ACEA; CB
1
-specific agonist)
suppressed proliferation (monitored by the ratio of Ki-67 positive nuclei), and this effect could be
abrogated by the CB
1
-selective antagonist/inverse agonist AM251 (1
µ
M). Intriguingly, although
the above ACEA treatment also decreased staining intensity of two proliferation-associated keratins
(K6 and K16), this effect could not be prevented by the said CB
1
blocker [
175
]. Finally, in a pilot
hSOC experiment, 48-hr treatment with 1
µ
M ACEA down-regulated K1 and up-regulated K10
expression [176].
Molecules 2019,24, 918 10 of 56
In line with the above observations, 0.5–1
µ
M CBD and CBG (but interestingly, not CBDV) also
exerted differentiation-impairing effects in HaCaT keratinocytes (suppression of K1, K10, involucrin
and transglutaminase 5 expression) via increasing DNA methylation by selectively enhancing DNA
(cytosine-5)-methyltransferase 1 (DNMT1) expression. Although CBG was found to act in a CB
1
and
CB
2
independent manner, quite surprisingly, CB
1
antagonism could partially prevent the action of
CBD [
59
]. The role of non-classical cannabinoid targets in mediating pCB actions was further confirmed
in another model system, where 1–10
µ
M of THC, CBD, CBN, and CBG exerted anti-proliferative
actions (72-h treatments) on HPV-16 E6/E7 transformed human keratinocytes (“CRL-2309 KERT”),
in a TRPV1, CB1and CB2independent manner [122].
These observations, albeit being slightly nebulous, collectively support the concept that
slight/moderate CB
1
activation may operate as suppressor of the differentiation, whereas its activation
by high concentrations of AEA or ACEA rather leads to anti-proliferative and pro-apoptotic events.
However, certain pieces of evidence suggest that the role of CB
1
might be even more complex,
and context-dependent.
2.4.2. Barrier Formation
Indeed, by assessing wild-type as well as CB
1/
and CB
2/
global KO mice, another team
showed that absence of CB
1
delayed, whereas lack of CB
2
accelerated permeability barrier recovery
after tape-stripping [
177
]. In line with these observations, lamellar body secretion as well as expression
of certain late differentiation markers (filaggrin, loricrin, involucrin, as well as ratio of apoptotic cells)
were increased in CB
2/
mice and were decreased/abnormal in CB
1/
animals, suggesting that
differentiation of epidermal keratinocytes was indeed less efficient in the latter case [
177
]. In line
with these data, both topically applied AEA and a synthetic CB
1
agonist (
α
-oleoyl oleylamine serinol;
α
-OOS) were found to accelerate barrier recovery following tape-stripping in another study [
178
].
Although the apparent contradiction between these
in vivo
animal data and the aforementioned
findings obtained in cultured keratinocytes as well as in ex vivo hSOCs has not been resolved yet,
one can speculate that the difference most likely lies at the base of the CB
1
expression in other cell
types, and hence in disturbed intercellular communication. Alternatively, delayed barrier repair in
CB
1/
animals may be due to the elevated baseline secretion of thymic stromal lymphopoietin
(TSLP) [
179
], a pro-inflammatory mediator driving T
h
2-type cutaneous inflammation in AD, since T
h
2
cytokines are known to impair the epidermal barrier [
180
,
181
]. Further experiments, ideally using
keratinocyte-specific CB
1
and CB
2
KO mice, are now invited to dissect the exact role of CB
1
/CB
2
and
eCB signaling in keratinocyte differentiation.
2.4.3. Keratin Disorders
Epidermolytic ichthyosis (EI), pachyonychia congenita (PC) and epidermolysis bullosa (EB) are
rare genodermatoses caused by function-impairing mutations in different keratins (EI: K1 or K10; PC:
K6, K16 or K17; EB: K5 or K14) [
182
]. Thus, pharmacologically induced down-regulation of the mutated,
dysfunctional keratins, and ideally, up-regulation of other ones capable of compensating the role of
the mutated molecules, is thought to be an innovative, novel approach in these
diseases [176,182]
.
Since irrespective of the above open questions, it seems to be safe to assume that appropriate
modulation of the eCB signaling and/or administration of various pCBs may be capable of inducing
marked alterations in the keratin expression profile in human epidermis, it is not surprising that such
interventions were already suggested to be exploited in these diseases [176,182].
Along these lines, it is important to note that according to a recent observational study reporting
3 cases of self-initiated topical CBD use in patients with EB, CBD may improve quality of life in such
patients. Indeed, one patient was weaned completely off oral opioid analgesics, and all 3 patients
reported faster wound healing, less blistering, and amelioration of pain. The authors concluded
that the effects might have been due to the anti-inflammatory activity of CBD, but in light of the
above data, one can speculate that CBD might have beneficially modulated the keratin expression
Molecules 2019,24, 918 11 of 56
profile as well [
183
]. Likewise, in another small pilot study, three EB patients, who were prescribed
pharmaceutical-grade sublingually administered cannabinoid-based medicine (CBM) comprising THC
and CBD, reported improved pain scores, reduced pruritus and decreased overall analgesic drug
intake [
184
]. Further studies are therefore invited to exploit putative therapeutic potential of the
(endo)cannabinoid signaling in the clinical management of keratin diseases.
Putative keratinocyte-related translational potential of cannabinoid signaling modulation is
summarized in Table 4.
Table 4.
Overview of the putative keratinocyte-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Disease Intervention Level of Evidence References
Epidermolysis bullosa
Topical CBD Case report of 3 patients [183]
Sublingual THC and
CBD containing CBM oil Case report of 3 patients [184]
Pachyonychia congenita ACEA (and maybe other
CB1agonists) Ex vivo (hSOC) [175]
Epidermolytic ichthyosis
ACEA (and maybe other
CB1agonists) Pilot ex vivo (hSOC) [176]
Barrier disruption CB1activation and/or
CB2blockade In vivo (CB1/and
CB2/mice) [177]
2.5. Cutaneous Inflammation
2.5.1. General Considerations
Another key function of cannabinoid signaling is to control local immune responses in the skin.
Several lines of evidence demonstrate that both eCBs and pCBs can modulate immune functions,
and they are generally considered to be anti-inflammatory agents [
31
,
33
,
141
,
142
]. Of great importance,
immune effects of cannabinoids are not only exerted on “professional” immune cells, but also on
non-immune cells (e.g., keratinocytes, sebocytes).
As mentioned above, many cell types of the skin express pathogen- and danger-associated
molecular pattern recognizing receptors. These cells are also capable of producing anti-microbial
peptides and lipids, and can initiate and coordinate local immune responses as well, by producing
various pro- and anti-inflammatory cytokines and chemokines [
4
,
16
18
,
20
]. These processes are under
the tight control of the cutaneous cannabinoid system [33,9193].
Indeed, as it was elegantly demonstrated in the groundbreaking work of Karsak and her
co-workers, homeostatic eCB-signaling through CB
1
and CB
2
receptors is a key mechanism, which
keeps the production and release of pro-inflammatory cytokines and chemokines under control in
epidermal keratinocytes [
143
]. Dinitrofluorobenzene (DNFB)-induced allergic inflammation was more
severe in CB
1/
/CB
2/
double KO mice as compared to the wild-type, whereas the inflammatory
response was significantly suppressed in FAAH
/
animals, as well as in THC-treated (5 mg/kg
subcutaneously injected or 30
µ
g topically administered) wild-type mice. Intriguingly, however,
the CB
2
-selective agonist HU-308 (5 mg/kg subcutaneously injected or 10
µ
g topically administered)
failed to induce significant alleviation, suggesting that both CB
1
and CB
2
are needed for the effect in
this inflammatory model system [143].
In line with this concept, 24-h treatment with the TLR4 activator LPS (5
µ
g/mL) was found
to up-regulate CB
1
and CB
2
mRNA expression in primary human keratinocytes. Moreover, in the
presence of 10
µ
g/mL LPS, the CB
2
-selective JWH-015 promoted wound closure (scratch assay of
human keratinocyte-fibroblast co-culture), elevated TGF-
β
-release, and exerted anti-inflammatory
effects in a CB
1
and CB
2
-dependent manner. Since JWH-015 could be successfully delivered into
porcine skin, the authors concluded that it may be a powerful future anti-inflammatory agent [
185
].
Molecules 2019,24, 918 12 of 56
Similarly, novel synthetic CB
2
-activators suppressed chemokine (C-C motif) ligand 8 (CCL8; a.k.a.
monocyte chemoattractant protein 2 [MCP-2]) release from poly-(I:C)-stimulated (100
µ
g/mL; 6 h)
HaCaT keratinocytes in a CB
2
-dependent manner, since co-administration of AM630 (100 nM) could
prevent the action [186].
The fundamental role of homeostatic eCB-signaling in controlling epidermal inflammatory
responses was further supported by a recent study demonstrating that activation of TLR2 by
lipoteichoic acid (LTA; 10
µ
g/mL; 24 h) led to the up-regulation of FAAH-activity as well as
expression at the protein (but intriguingly, not at the mRNA) level in human keratinocytes [
144
].
Moreover, FAAH-inhibitors could prevent the LTA-induced pro-inflammatory response in a CB
1
/CB
2
receptor-dependent manner. Co-administration of the CB
1
and CB
2
antagonists/inverse agonists
AM251 and AM630 (both at 1
µ
M) prevented the action; however, the compounds were only tested in
combination, leaving the individual roles of CB
1
and CB
2
unexplored. Moreover, following topical
application, the FAAH-inhibitors alleviated dust mite-induced cutaneous inflammation of NC/Tnd
mice with the same efficiency as the positive control tacrolimus [
144
]. Likewise, topical administration
of sulfur mustard and nitrogen mustard at concentrations that induced tissue injury in mice led to
up-regulation of FAAH (as well as of CB
1
, CB
2
, and PPAR
α
). These alterations persisted throughout
the wound healing process, and FAAH-inhibitors were found to be highly effective in suppressing
vesicant-induced cutaneous inflammation in this study too [
187
]. Collectively, these data highlight the
possibility that by regulating homeostatic eCB signaling, FAAH may be an important regulator of the
initiation and maintenance of cutaneous inflammatory processes. Thus, restoration of the homeostatic
eCB tone by e.g., FAAH-inhibitors may be a promising tool in alleviating skin inflammation [93].
Besides eCBs and THC, other pCBs also deserve attention as potential topical anti-inflammatory
agents. Indeed, in a croton oil-induced murine cutaneous inflammation model [
188
], topical
administration of several pCBs (CBC, CBCV, CBD, CBDV,
8
-THCV,
8
-THC,
9
-THC; 0.1–1
µ
mol/cm
2
) was found to exert significant anti-inflammatory effects as revealed by reduced ear
swelling [
189
]. Moreover, in poly-(I:C)-stimulated HaCaT cells (100
µ
g/mL, 6 h), CBD (5–20
µ
M)
elevated the levels of AEA and concentration-dependently inhibited poly-(I:C)-induced release of
CCL8 (a.k.a. MCP-2), IL-6, IL-8, and TNF-
α
. The effects could be reversed by CB
2
(AM630; 0.1
µ
M) and
TRPV1 (5’-iodo-resiniferatoxin [I-RTX]; 1
µ
M) antagonists, without any cytotoxic effect. Importantly,
low micromolar (1–20
µ
M) concentrations of THCV, CBC and CBG were also efficient, but exhibited
inferior efficacy compared to CBD [
190
]. Finally, as mentioned above, CBD (10
µ
M; A
2A
receptor
dependent action) [
120
], as well as CBG, CBGV, CBC, CBDV, and THCV (all in 0.1
µ
M) [
126
] were
found to exert anti-inflammatory effects in human sebocytes, whereas CBD (0.1
µ
M; adenosine receptor
dependent action) was also shown to be effective in alleviating poly-(I:C)- induced pro-inflammatory
response in cultured human plucked HF-derived outer root sheath (ORS) keratinocytes [140].
Interestingly, other
in vitro
and
in vivo
studies have found that in certain inflammation models
activation of CB
1
alone may also be sufficient to induce potent anti-inflammatory actions. Indeed,
the IFN-
γ
-induced pro-inflammatory response (elevated production of T
h
1- and T
h
17-polarizing
cytokines IL-12 and IL-23) was prevented by 2.5
µ
M AEA pre-treatment in HaCaT keratinocytes
in a CB
1
-dependent manner [
191
]. Moreover, keratinocyte-specific CB
1/
mice exhibited a
stronger pro-inflammatory reaction (higher up-regulation of IL-4, CCL8 [a.k.a. MCP-2], TSLP,
and eosinophilic activity) in fluorescein isothiocyanate (FITC)-induced atopic-like inflammation,
and showed delayed barrier repair following FITC challenge. Furthermore, keratinocytes of
keratinocyte-specific CB
1/
mice secreted more TSLP under un-stimulated conditions [
179
]. By using
the same mice strain, very similar data (increased and prolonged contact hypersensitivity responses
with enhanced reactive epidermal acanthosis and inflammatory keratinocyte hyperproliferation)
were obtained in DNFB-induced cutaneous inflammation. Finally, primary cultures of CB
1
-deficient
keratinocytes released increased amounts of CXCL10 and CCL8 (a.k.a. MCP-2) after stimulation with
IFN-
γ
, highlighting keratinocyte CB
1
-signaling as a master regulator of T cell-dependent cutaneous
inflammation in the effector phase of contact hypersensitivity [192].
Molecules 2019,24, 918 13 of 56
Surprisingly, however, certain experimental data appear to contradict this simplistic picture.
Indeed, 4 kJ/m
2
(400 mJ/cm
2
) UVB-irradiation induced inflammation in wild-type mice; however,
CB
1/
/CB
2/
double KO animals appeared to be protected [
87
]. Moreover, the same UVB
irradiation was shown to induce fast (
30 min) phosphorylation and internalization of CB
1
and
CB
2
in overexpressor HEK293 cells, and it also activated ERK1/2, p38 and JNK MAPK cascades
in wild-type, but not in CB
1/
/CB
2/
double KO, mouse embryonic fibroblasts [
87
]. Finally,
elevation of TNF-
α
level following UVB treatment was higher in the epidermis of wild-type than
in the epidermis of CB
1/
/CB
2/
double KO mice [
87
]. Since the authors found that UVB
(9 kJ/m
2
900 mJ/cm
2
) or UVA (60 or 120 kJ/m
2
6 and 12 J/cm
2
) irradiation induced a substantial
lowering in K
i
values in a competition binding assay using membrane fractions of CB
1
or CB
2
overexpressing cells, they concluded that CB
1
and CB
2
could directly be activated by UV-irradiation.
Thus, the UVB
CB
1
/CB
2
NF-
κ
B activation axis was suggested to play a key role in UV-induced
inflammation [87].
At this point, it is important to note that physiological relevance of such high UV doses
is questionable, since the minimal erythema dose of narrow-band UVB irradiation phototype-
dependently ranges typically between ~300 and 900 mJ/cm
2
. However, the observed phenomena may
contribute to the beneficial therapeutic effects in psoriasis and scleroderma, since the maximal doses
of UVB and UVA for psoriasis or scleroderma treatment may reach 1.5 J/cm
2
(UVB, psoriasis) and
130 J/cm2(UVA, scleroderma) [193195].
Finally, to add a further layer to the complexity of the system, it is noteworthy that another
cannabinoid-responsive receptor (namely TRPV4) was also found to play a role in detecting UVB.
Indeed, UVB-induced sunburn and pain was found to be mediated via direct (i.e., UVB-induced)
activation of TRPV4 ion channels in epidermal keratinocytes, and the subsequent release of
endothelin-1 [86].
2.5.2. Role of “Non-Classical” Cannabinoid Targets
Having discussed the importance of keratinocyte CB
1
(and CB
2
) mediated (mostly)
anti-inflammatory signaling, it should also be noted that several lines of evidence highlight the
existence of additional, so far un-identified, non-classical anti-inflammatory cannabinoid pathways
in the skin. Indeed, topical application of THC (30
µ
g) was found to be efficient in alleviating
DNFB-induced allergic ear swelling and myeloid immune cell infiltration not only in wild-type but also
in CB
1/
/CB
2/
double KO mice. Moreover, THC suppressed the IFN-
γ
production of CD3+ T cells,
decreased the release of CCL2 and of IFN-
γ
-induced CCL8 and CXL10 from epidermal keratinocytes,
and limited the recruitment of myeloid immune cells
in vitro
in a CB
1
/CB
2
receptor-independent
manner [196].
Obviously, in case of pCBs, potential effects (activation, antagonism or desensitization) on various
TRP channels and many other targets (e.g., adenosine receptors or PPARs) have to be taken into
consideration [
31
33
,
35
,
40
,
41
,
45
,
46
,
53
57
] as well; thus, their “net” biological effects will always be
determined by a mixture of multiple molecular actions. With respect to this point, albeit detailed
overview of the roles of TRP channels, adenosine receptors, and PPARs in cutaneous biology lies
far beyond the scope of the current review, we have to emphasize that the activation of the most
skin-relevant TRP channel, i.e., TRPV3 [
197
], results in an elevated production and release of several
pro-inflammatory cytokines from human epidermal keratinocytes [
121
] and human sebocytes [
119
].
Thus, the ability of CBD, THCV and CBGV to activate (and then desensitize) TRPV3 [
55
] may also
contribute to their context-dependent pro- or anti-inflammatory actions. Moreover, considering
the concentrations needed to activate anti-inflammatory adenosine receptors (high nanomolar
range in case of CBD in plucked HF-derived outer root sheath keratinocytes [
140
] or 1
µ
M in
murine brain “b.end5” endothelial cells [
198
]) and the rather pro-inflammatory TRPV channels (low
micromolar range [
55
,
56
]), their efficiency may theoretically exhibit reverse dose-dependence, i.e.,
Molecules 2019,24, 918 14 of 56
superior anti-inflammatory activity at the more adenosine receptor-specific nanomolar than in the
TRPV-activating micromolar concentrations.
2.5.3. Role of “Non-Classical” Cannabinoid Ligands
Besides the “classical” pCBs, plant-derived active substances exhibiting potential cannabimimetic
effects have also been investigated in various model systems. Indeed, CB
2
activating Echinacea
purpurea-derived alkylamides were shown to reduce the TLR3 activator poly-(I:C) (20
µ
g/mL; 3 h)
induced mRNA expression as well as release of pro-inflammatory cytokines (IL-6 and IL-8) in HaCaT
keratinocytes; however, it has not been investigated whether the actual effects were indeed coupled to
the activation of CB
2
. The same Echinacea extract containing Linola
®
Plus Cream was proven to be
well-tolerated, and it reduced local SCORAD not only compared to baseline, but also compared to a
comparator product Imlan
®
Creme Pur. Moreover, it resulted in significantly improved lipid barrier
(with higher levels of overall epidermal lipids, ceramide EOS [
ω
-esterified fatty acid+sphingosine
sphingoid base], and cholesterol at day 15 compared to baseline as well as significantly greater number
of intercellular lipid lamellae) in respective clinical trials [199].
With respect to the non-classical ECS-related endogenous ligands, it is noteworthy that orally
administered (10–30 mg/kg) PEA exerted sustained anti-inflammatory effects in spontaneously Ascaris
hypersensitive Beagle dogs, which were challenged with intradermal injections of Ascaris suum
extract, substance P, and anti-canine IgE [
200
]. Moreover, in HaCaT cells, stimulation with poly-(I:C)
(100
µ
g/mL; 24 h) elevated the levels of PEA, OEA, and AEA (but decreased the level of 2-AG).
Moreover, exogenous PEA (10
µ
M) inhibited poly-(I:C)-induced expression and release of CCL8 (a.k.a.
MCP-2), in a TRPV1- (but not PPARα) dependent manner [200].
Intraperitoneally applied PEA (5–10 mg/kg) was also able to inhibit DNFB-induced ear
inflammation in mice
in vivo
, in a TRPV1-dependent manner. Moreover, DNFB treatment increased
ear skin PEA levels (interestingly, in CB
1
/CB
2
double KO mice, the elevation was higher than in
wild-type), and up-regulated TRPV1, PPAR
α
and NAPE-PLD (PEA and AEA synthesizing enzyme) in
keratinocytes [
201
]. Importantly, the authors reported that PEA (5 mg/kg; i.p.) reduced ear swelling,
the number of mast cells, as well as the expression of VEGF and its receptor FLK-1 in a CB
2
-dependent,
but PPAR
α
-independent manner in the late, allergic stage of the same model system, whereas the
anti-pruritic effect of PEA was mediated in a CB
2
- and PPAR
α
-dependent manner [
202
]. Interestingly,
PEA and OEA, but not AEA or 2-AG, were up-regulated in the epidermis of sodium lauryl sulfate
(SLS)-challenged (2.5%; 24 h) buttock skin of 10 healthy volunteers. Although UVB-irradiation, which
resulted in a similar erythema, had no effect on the above eCB levels [
203
], UVA- and UVB-irradiation of
human CDD 1102 KERTr keratinocytes (UVA: 30 J/cm
2
; UVB: 60 mJ/cm
2
) and CCD 1112Sk fibroblasts
(UVA: 20 J/cm
2
; UVB: 200 mJ/cm
2
) decreased cytosolic, and increased cell membrane CB
1
, CB
2
and
TRPV1 expression (post-irradiation day 1). Intriguingly, both UVA and UVB irradiation were found
to decrease AEA levels, whereas 2-AG was only reduced by UVB [
204
]. Although the authors did
not investigate if the “cytosolic” CB
1
fraction represents mitochondrial [
81
,
82
] or lysosomal [
83
]
CB
1
expression, one might speculate that the elevated production of reactive oxygen species (ROS)
observed upon UV-irradiation might have been (at least in part) due to a reduced mitochondrial CB
1
expression leading to increased mitochondrial activity. However, putative expression and functional
role of intracellular CB
1
sub-populations in epidermal keratinocytes remains to be elucidated in future
targeted studies.
Finally, with respect to PEA it should also be noted that a PEA- and organic osmolyte-containing
topical product (Physiogel
®
A.I. Cream) significantly inhibited the development of UV light (UVB
20%, UVA 80%; produced by a solar UV simulator)-induced erythema and thymine dimer formation
in normal human skin. However, it did not alter the ratio of Ki-67+ proliferating keratinocytes and
the expression of p53 and ICAM-1. Hence, PEA might become a novel tool to alleviate UV-induced
photodamage [205].
Molecules 2019,24, 918 15 of 56
2.5.4. Putative ECS- Endogenous Opioid System (EOS) Interplay
As mentioned above, the ECS may interact with several other signaling pathways, including
the endogenous opioid system (EOS). Indeed, intraplantar administration of the CB
2
-selective
agonist AM1241 (10
µ
M) stimulated
β
-endorphin release from keratinocytes via the activation of
a CB
2
-G
i/o
-G
βγ
-ERK1/2 MAPK-Ca
2+
signaling pathway [
206
]. The released
β
-endorphin was then
found to activate local neuronal
µ
-opioid receptors thereby inhibiting nociception in rats, which was not
the case for CB
2/
animals [
207
]. Similarly, capsaicin-induced pain was dose-dependently alleviated
in mice by intraplantar injection of the highly CB
2
-selective agonist
β
-caryophyllene (18
µ
g) [
208
],
most likely via stimulating β-endorphin release from the keratinocytes.
Intriguingly, further ECS-EOS interplay was evidenced in a few additional studies. Indeed,
electroacupuncture (EA) was found to increase CB
2
expression on keratinocytes and infiltrating
inflammatory cells in inflamed skin tissues of rats [
209
]. EA and CB
2
stimulation reduced inflammatory
pain via activating
µ
-opioid receptors, and EA increased endogenous opioid expression in keratinocytes
as well as in infiltrating immune cells at the inflammatory site through CB
2
activation [
210
].
Furthermore, EA or AM1241 (1 mg/kg; s.c.) treatment significantly decreased the mRNA and protein
levels of IL-1
β
, IL-6 and TNF-
α
in inflamed skin tissues in a CB
2
-dependent manner, since pretreatment
with the CB
2
-selective antagonist/inverse agonist AM630 (150
µ
g/kg; s.c.) abrogated the effect of
EA. Collectively, these data suggest that EA may reduce inflammatory pain and pro-inflammatory
cytokine production by activating CB2[211].
2.5.5. Selected “Skin-Relevant” Professional Immune Cells: Langerhans Cells and Mast Cells (MC)
As we discussed above, several lines of evidence demonstrate that cutaneous cannabinoid
signaling profoundly influences the immunogenic behavior of skin resident non-immune cells.
Unfortunately, albeit effects of cannabinoid signaling on immune cells in general are well
documented [31,33,141,142,212,213], much less data are available about their skin-relevant aspects.
Indeed, according to the sole available paper, murine epidermal Langerhans cells express CB
2
at
the mRNA level. Moreover, the authors showed that 2-AG level was increased in oxazolone-induced
dermatitis, and that treatment with the CB
2
-selective antagonist SR144528 attenuated the inflammatory
response; thus, they concluded that “CB
2
and 2-AG play important stimulative roles in the sensitization,
elicitation, and exacerbation of allergic inflammation” [
214
]. Although it cannot be excluded that
2-AG
CB
2
signaling axis may model- and context-dependently play such roles, the data should be
interpreted carefully, since the authors could not find CB
2
positivity in epidermal keratinocytes. Thus,
targeted studies are urgently invited to explore ECS of Langerhans cells, preferably in human skin,
or in human monocyte-derived model systems.
In contrast to Langerhans cells, several cannabinoid-relevant studies have been conducted on
different mast cell (MC) models, among which, we summarize the most important and skin-relevant
ones below. MCs are important professional immune cells of the cutaneous immune system. They are
able to detect several different potential danger signals, and, by producing and on-demand releasing
a number of different soluble mediators, they can influence a wide-array of biological processes,
including tissue remodeling, wound healing, fibrosis, local immune responses, itch, or even hair
growth [
215
225
]. Although there are a number of cell lines (rat: RBL-2H3; human: HMC-1, LAD1,
LAD2, etc.) generally capable of mimicking several aspects of human MC biology, one should not
forget how important environmental signals are in regulating and fine-tuning MC activity [
215
224
].
Maybe because of this limitation of the
in vitro
systems, partially conflicting results have been obtained
with respect to the effects of the cannabinoid signaling.
First, PEA was identified as an endogenous activator of CB
2
on RBL-2H3 cells as well as Wistar
rat peritoneal MCs, where its administration resulted in an anti-inflammatory phenotype, whereas
AEA was found to be ineffective [
226
]. Later, PEA (1-10
µ
M) was shown to suppress anti-canine
IgE-induced activation of skin MCs ex vivo in freshly isolated dog skin specimens [
227
], while in
another study, enhanced local MC proliferation and (maybe compensatory) elevation of levels of
Molecules 2019,24, 918 16 of 56
PEA and other bioactive lipid mediators were found in canine AD [
228
]. Finally, the NAAA-inhibitor
2-pentadecyl-2-oxazoline-derivative of PEA (“PEA-OXA” 10 mg/kg p.o.) reduced MC activation
in carragenan–induced inflammation in rats in a PPAR
α
-independent manner [
229
]. Last, but not
least, ultramicronized PEA (PEA-um) decreased compound 48/80-induced vasodilation and MC
degranulation in organ-cultured skin of dogs [
230
]. Taken together, the available evidence strongly
suggests that appropriately chosen concentrations of PEA may be efficient in suppressing MC
degranulation in the skin [231].
Next, by using RBL-2H3 cells and bone marrow MCs, another group found that 1–10
µ
M of
metAEA (a FAAH-resistant AEA-analogue) increased the level of cAMP (2 h), and suppressed
anti-DNP IgE-induced degranulation in a CB
1
-dependent manner, whereas the CB
2
-selective agonist
JWH-015 decreased cAMP level in a CB2-dependent manner [232]. Interestingly, CBD (3–10 µM) and
THC (15
µ
M) were found to trigger activation of RBL-2H3 cells via inducing Ca
2+
-influx. Although
the mechanism of action was not uncovered in this study, one might speculate that CBD and THC
might have activated certain TRPV channels, which were already shown to mediate MC activating
signals [233235].
On the other hand, WIN55,212-2 and CP 55,940 (two non-selective synthetic cannabinoids
activating both CB
1
and CB
2
) could prevent IgE-DNP-induced activation of RBL-2H3 cells [
236
].
Finally, semi-synthetic CB
1
activators as well as AEA (10
µ
M) inhibited the release of inflammatory
mediators without causing cytotoxicity in RBL-2H3 cells, and dose-dependently suppressed MC
proliferation. Topical application of the above CB
1
agonists suppressed the recruitment of MCs into
the skin in an oxazolone-induced mouse model of AD, and reduced the blood level of histamine [
237
].
By using the human HMC-1 cell line, another group described functionally active EMT and
inducible FAAH expression in MCs, but they did not find CB
1
or CB
2
expression [
238
], in spite of the
fact that presence of CB
1
and CB
2
was shown in human skin MCs [
111
]. Moreover, in HMC-1 cells
neither AEA nor PEA (10
µ
M both) affected tryptase release triggered by 500 ng/mL A23187 (a Ca
2+
ionophore) [
238
]. Interestingly, unlike CB
1
and CB
2
, GPR55 was found to be expressed on HMC-1
cells. In this study, PEA was found to reduce PMA (a general activator of classical and novel PKC
isoforms) induced nerve growth factor (NGF) release in a GPR55-dependent manner (confirmed by
GPR55 RNA
i
). Thus, by regulating NGF release from activated MCs, PEA was suggested to influence
NGF-induced angiogenesis [239].
In contrast to the above data, 30
µ
M WIN55,212-2 was found to CB
2
-dependently prevent
degranulation of LAD2 cells induced by the supernatant of human HPV18-positive SW756 cervical
carcinoma cells [
240
]. Moreover, AEA inhibited Fc
ε
RI-dependent degranulation and cytokine synthesis
in murine bone marrow-derived MCs via the activation of CB
2
/GPR55 receptor heteromers [
241
],
and VCE-004.3, as well as VCE-004.8, two PPAR
γ
and CB
2
receptor activating derivatives of CBD,
could also reduce MC degranulation in bleomycin-induced murine fibrosis [242,243].
It is also noteworthy that over activation of the aforementioned pro-inflammatory [
119
,
121
],
and skin-wise highly relevant [
197
] TRPV3 ion channel may promote MC proliferation too. Indeed,
DS-Nh mice and WBN/Kob-Ht rats (possessing Gly573 to Ser [“Nh” mutation] or Gly573 to Cys [“Ht
mutation] gain-of-function mutations of TRPV3) exhibiting hairless phenotype and suffering from
pruritic dermatitis, were reported to have increased MC numbers. This supported the concept that
TRPV3 might promote MC proliferation and activity [
244
], inviting the hypothesis that appropriate
doses of TRPV3-desensitizing pCBs might exert MC-suppressive effects too.
As discussed above, the available cellular model systems provided somewhat controversial
data especially with respect to the expression and role of CB
1
and CB
2
, which might have been
the consequence of the lack of appropriate tissue microenvironment. To overcome these issues,
unconventional methods to study human MC biology were also employed. By using human HF,
as well as human nasal polyp organ cultures to study the biology of MCs in situ, a crucial regulatory
role for CB
1
was demonstrated [
245
,
246
]. Although expression of CB
2
was not confirmed [
245
], both HF
connective tissue sheath and mucosal MCs were shown to be tightly controlled by the ECS. Indeed,
Molecules 2019,24, 918 17 of 56
excessive activation and maturation of MCs from resident progenitors was limited via tonic CB
1
stimulation by locally synthesized eCBs [
245
,
246
]. Thus homeostatic eCB signaling, and especially
appropriate function of CB
1
appears to be a key gate-keeper of MC functions in situ, therefore elevation
of the eCB-tone, administration of PEA as well as blockade/desensitization of certain TRP channels
by well-selected doses of certain pCBs hold out the promise of having great translational potential as
potent suppressors of unwanted MC overactivation.
2.5.6. Selected Inflammatory Diseases: Psoriasis (PSO)
Psoriasis (PSO) is a chronic inflammatory skin disorder, often accompanied by additional
non-cutaneous symptoms (e.g., arthritis), and its pathogenesis is still not fully understood. Indeed,
genetic [
247
] and epigenetic [
248
] abnormalities, as well as alterations in the cutaneous microbiota [
249
],
pH [
250
], or, most importantly, IL-17 signaling [
251
,
252
] are known to be involved in its development,
and it is surely accompanied by a disturbance in the dynamic cross-talk between epidermal
keratinocytes and professional cutaneous immune cells. This inappropriate communication then leads
to pathological inflammatory processes and to a disturbance in the proliferation/differentiation balance
of epidermal keratinocytes [
249
,
253
256
]. Since, as discussed above, proliferation/differentiation as
well as immune activity of epidermal keratinocytes are under the tight control of the eCB signaling,
it is not surprising that therapeutic exploitation of various cannabinoids in PSO has already been
suggested by multiple authors [237,257261].
Beyond of the abovementioned theoretical reasons (i.e., dose-dependent differentiation-
modulating, as well as anti-proliferative and anti-inflammatory effects of various cannabinoids in the
skin), there are a few additional pieces of evidence supporting the concept that eCB-dysregulation
may contribute to the development of PSO. Indeed, the promoter of the PTPN22 gene was found to
be hypomethylated resulting in its strong up-regulation in lesional skin of PSO patients as compared
to the adjacent non-lesional skin [
262
]. Intriguingly, however, the C1858T substitution (“R620W
variant”; “rs2476601”; a loss-of-function single-nucleotide polymorphism) in PTPN22 was found to
be positively associated with PSO in Saudi patients [
263
], and other SNPs (“rs3789604”, “rs1217414”,
“rs6679677”) were also found to be related to PSO in other subjects [
264
266
]. Others, however, found
that C1858T substitution is only associated with higher susceptibility of psoriatic arthritis, but not of
PSO itself [
267
269
], whereas again others did not find any significant association between PTPN22
and PSO [270274], leaving the putative role of PTPN22 dysfunction in PSO rather controversial.
A much more important indicator of the potential involvement of eCB dysregulation in the
pathogenesis of PSO is that a recent study found elevated AEA and 2-AG levels in the plasma of
these patients. Moreover, in the granulocytes of the patients, activities of FAAH and MAGL were
increased, and GPR55 expression was also up-regulated. With respect to the “classical” receptors,
the authors found that expression CB
1
was only increased in granulocytes of patients suffering
from psoriatic arthritis, whereas CB
2
was up-regulated in those PSO patients, who had no joint
complications [
275
]. Moreover, RNAseq of skin biopsies obtained from 25 PSO patients revealed that,
compared to region-matched skin of healthy subjects, several important “cannabinoid- relevant” genes
were differentially expressed. Findings in this study include, but are not limited to down-regulation of
adenosine A
1
, A
2A
, A
2B
and A
3
receptors, CB
1
, CB
2
, PPAR
α
and PPAR
γ
, whereas FAAH1 (but not
FAAH2), TRIB3, TRPV1 and TRPV3 were up-regulated at the mRNA level in itchy lesional skin of PSO
patients [
276
]. Thus, alterations in the ECS can indeed be observed in PSO patients, indicating that
certain cannabinoids may possess therapeutic potential.
Along this line, it is important to emphasize that NRIP1, which has previously been shown to be
an important CBD target gene [
120
], was found to be overexpressed both in skin and peripheral blood
monomorphonuclear cells (PBMC) of PSO patients [277]. Importantly, its down-regulation in HaCaT
keratinocytes could significantly suppress proliferation and induce apoptosis, whereas in isolated
CD4+ T cells it reduced RelA/p65 NF-
κ
B expression and IL-17 release [
277
]. Moreover, in NRIP1
/
mice, the PSO-mimicking inflammation induced by imiquimod (a TLR7/8 agonist widely used to
Molecules 2019,24, 918 18 of 56
trigger PSO-like cutaneous symptoms in mice [
278
]) was delayed, and RelA/p65 NF-
κ
B expression was
also reduced in the lesions [
277
]. Collectively, these data suggested that NRIP1 may be a multifaceted
therapeutic target in PSO. Since CBD was found to TRPV4-dependently down-regulate NRIP1 in
human sebocytes [
120
], one might speculate that, by activating the same signaling axis, it could exert
beneficial effects in PSO as well. On the other hand, it is also important to note that another CBD
target gene, namely TRIB3, which was shown to be adenosine A
2A
receptor-dependently up-regulated
in human sebocytes [
120
], was found to be up-regulated in PSO lesions compared to non-lesional
skin, and TRIB3-silencing exerted anti-proliferative effects in HaCaT keratinocytes [
279
]. Further
studies are therefore invited to explore how CBD regulates these PSO-relevant signaling pathways in
actual patients.
2.5.7. Selected Inflammatory Diseases: AD
Although AD and PSO are two markedly different diseases, their pathogeneses still show some
similarities in certain aspects. Indeed, impaired keratinocyte differentiation leading to defects in
the cutaneous barrier functions, as well as disturbed keratinocyte—immune cell communication
and pathological inflammatory processes can be observed in both diseases, but, obviously, the exact
contributors (i.e., involved key cytokines, dysregulated barrier genes, etc.) are different [
280
287
].
Thus, similar to PSO, cannabinoid signaling may theoretically possess therapeutic value in AD as
well [288290].
Indeed, in skin samples of dogs suffering from AD, CB
1
and CB
2
immunoreactivity [
291
], as well
as levels of PEA [
228
] were shown to be higher than in skin samples of healthy animals, and not
less than 18 genetic variants of PTPN22 were shown to be likely to be associated with AD in West
Highland white terriers [
292
]. With respect to the human data, it is noteworthy that RNAseq of skin
biopsies obtained from 25 AD patients revealed that, compared to region-matched skin of healthy
subjects, several important “cannabinoid-relevant” genes were differentially expressed. These included,
but were not limited to the finding that CB
1
, CB
2
and GPR18 were down-regulated, whereas TRPV1
and TRPV2 were up-regulated at the mRNA level in itchy lesional skin of AD patients [
276
]. Thus,
alterations in the ECS can indeed be observed in AD, indicating that certain cannabinoids may possess
therapeutic potential.
Indeed, pharmacological blockade of TRPV1 has recently emerged as potential novel therapeutic
possibility in managing AD [
293
], and according to certain
in vitro
and animal data, TRPV3 antagonists
also seem to be promising anti-AD candidate drugs [
119
,
121
,
244
]. Future studies are therefore urgently
invited to explore if TRPV3 desensitizing pCBs [
55
], most especially CBGV, which has been proven to
exhibit anti-inflammatory and moderate sebostimulatory effects [
126
], indeed exert beneficial effects
in AD.
With respect to the “classical” receptors, it is noteworthy that the orally available CB
2
agonist
S-777469 (1–10 mg/kg) significantly suppressed DNFB-induced ear swelling in BALB/c mice in a
dose-dependent manner, and alleviated mite antigen-induced AD-like skin lesions in NC/Nga mice
(10–30 mg/kg) as revealed by reduced epidermal thickness, as well as MC and eosinophil numbers.
Moreover, dust mite-challenge was found to elevate the 2-AG level in the skin of NC/Nga mice, while
S-777469 could suppress 2-AG (0.5
µ
M)-induced migratory response of differentiated EoL-1 (human
eosinophilic leukemia cell line) and HL-60 (human monocytic cell line) cells
in vitro
. Thus, the authors
concluded that S-777469 may act via inhibiting cutaneous inflammation by blocking the actions of
2-AG [294].
Although the concept that CB
2
activation may be beneficial in AD was further supported by a
recent study demonstrating the efficiency of a CB2-activating Echinacea purpurea extract in alleviating
AD symptoms [
199
], a few additional data argue that the overall picture may be more complex. JTE-907,
a CB
2
antagonist/inverse agonist, was found to exert anti-pruritic activity in NC mice suffering from
chronic AD-like dermatitis [
295
]. In line with these observations, in another study JTE-907 as well as
Molecules 2019,24, 918 19 of 56
SR144528 (another CB
2
blocker) suppressed DNFB-induced ear swelling (0.1–10 mg/kg p.o. in both
cases), probably via inhibiting 2-AGCB2-driven migration of certain immune cells [296].
Intriguingly, unlike CB
2
, CB
1
was found to exert clearly beneficial effects in murine cutaneous
inflammation models. Indeed, as mentioned above, topical application of AEA (0.5%) as well as of
α
-oleoyl oleylamine serinol (
α
-OOS; a newly developed CB
1
agonist; 1%) were shown to accelerate
epidermal permeability barrier recovery following tape-stripping, as revealed by transepidermal water
loss measurement [
178
,
237
], whereas lack of CB
1
was found to delay epidermal barrier recovery in
CB1/mice [177]. Moreover, administration of α-OOS resulted in anti-inflammatory effects in both
acute (12-O-tetradecanoylphorbol-13-acetate-induced) and chronic (oxazolone- induced) inflammation
models [
178
,
237
]. Further details of the potent cutaneous anti-inflammatory effects of CB
1
are reviewed
above (see Section 2.5.1). Finally, highly selective FAAH-inhibitors (WOBE440 and -479) could
efficiently alleviate dust mite-induced “atopic-like” cutaneous inflammation in NC/Tnd mice [144].
With respect to the “non-classical” cannabinoids, it is noteworthy that the NAAA-inhibitor
ARN077 dose-dependently suppressed edema formation and scratching in DNFB-induced dermatitis.
Moreover, it also increased tissue PEA content, and normalized circulating levels of various cytokines
(IL-4, IL-5, IFN-
γ
) and IgE in a PPAR
α
-dependent manner, since the effects did not develop in
PPAR
α/
mice. Thus, NAAA-inhibition and the elevation of PEA level were identified as a promising
tool in AD and maybe in other inflammatory disorders of the skin [297].
In another study, PEA was found to selectively activate PPAR
αin vitro
(EC
50
= 3.1
±
0.4
µ
M),
and it up-regulated mRNA expression of PPAR
α
following topical application to mouse skin.
Moreover, in carrageenan-induced paw edema as well as in phorbol ester-induced ear edema,
PEA was found to attenuate inflammation in wild-type mice, but had no effects in PPAR
α/
animals.
Importantly synthetic PPAR
α
agonists GW7647 (150 nmol/cm
2
topically) and Wy-14643 (20 mg/kg;
i.p.) PPAR
α
-dependently mimicked these effects, and the edema suppressing activity of OEA was
also mediated by PPAR
α
[
298
]. In line with these observations, PEA-um was found to be effective
and safe in reducing pruritus and skin lesions, as well as in improving quality of life in dogs with
moderate AD and moderate pruritus [
299
]. Last, but not least, the “ATOPA” study assessing efficiency
of a special PEA-containing cream (Physiogel
®
A.I. Cream) found a substantial improvement in the
objective and subjective symptoms (decline of pruritus and loss of sleep) of AD after regular skin care
with the cream, and a reduced use of topical corticosteroids was also observed [
300
]. In line with
these observations, PEA and N-acetylethanolamine were found to be effective in asteatotic AD in a
randomized, double-blind, controlled study involving 60 patients [301].
2.5.8. Selected Inflammatory Diseases: Systemic Sclerosis (SSc)
Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by vascular abnormalities,
and fibrosis of the skin and of other organs, including the heart, kidneys, lungs, etc. Its etiology is
still nebulous, but genetic [
302
] and epigenetic [
303
,
304
] factors, as well as abnormalities in the gut
microbiota [
305
], and oxidative stress [
306
] were shown to play a role in its development. The initial
trigger is considered to be an autoimmune reaction against endothelial cells leading to the characteristic
vascular abnormalities, but inappropriate immune cell—fibroblast cross-talk leading to progressive
fibrosis and differentiation of fibroblasts to
α
-smooth muscle actin (
α
-SMA) positive myofibroblasts
are also very important [307].
Similar to many other diseases with an autoimmune component, the association between PTPN22
SNPs and SSc was already suggested by multiple studies [
308
]. Indeed, the aforementioned R620W
polymorphism was found to be a risk factor in French Caucasian [
309
] population, whereas another
study found association with the anti-centromere antibody and anti-topoisomerase I antibody positive
subsets of the disease [
310
313
]. Other groups, however, found no evidence of association between
SSc and R620W polymorphism in Spanish, Columbian and French patients [
314
316
], and other
variants (R263Q and G788A) were not identified as risk factors either [
313
,
317
]. On the other hand,
comparison of plasma samples obtained from 59 Italian SSc patients and 28 age- and sex-matched
Molecules 2019,24, 918 20 of 56
healthy volunteers revealed an elevated 2-AG level in the plasma of SSc patients [
318
]. Although these
data are definitely not more than mere indirect pointers indicating the putative involvement of eCB
dysregulation in SSc, additional evidence suggests that certain cannabinoids may have therapeutic
value in this disease [319,320].
First of all, expression of CB
1
and CB
2
was already demonstrated in human dermal fibroblasts.
Moreover, following a 24-h incubation, both UVA (20 J/cm
2
) and UVB (200 mJ/cm
2
) irradiation
decreased the levels of AEA and 2-AG, but increased the expression of CB
1
, CB
2
, GPR55 and TRPV1
in human “CCD 1112Sk” foreskin fibroblasts [
321
]. Interestingly, both UVA and UVB irradiation
appeared to alter cellular distribution of CB
1
, CB
2
and TRPV1, increasing membrane, and decreasing
cytosolic fractions of the receptors [204].
Up-regulation of CB
1
and CB
2
by pro-inflammatory challenges was further evidenced by
LPS-treatment (10
µ
g/mL; 24 h). Importantly, in this system biological effects of the receptors were also
tested by the co-administration of JWH-015, which was found to partially suppress the LPS-induced
pro-inflammatory response in a CB
1
and CB
2
dependent manner [
185
], inviting the hypothesis that
CB
1
/CB
2
activators may exert beneficial anti-inflammatry effects in SSc. However, several additional
studies have challenged this simplistic theory, arguing that the eCB-signaling may play a more complex
regulatory role in vivo.
Indeed, expression of FAAH (more precisely: FAAH1) was found to be decreased in dermal
cells (morphologically characterized to be fibroblasts) of SSc patients [
322
]. Furthermore, FAAH
/
C57Bl/6 mice with strongly increased levels of eCBs were more sensitive to bleomycin-induced
fibrosis than wild-type animals, as revealed by higher myofibroblast count and hydroxyproline
content, as well as by more pronounced dermal thickening [
322
]. Consistently, pharmacological
inhibition of FAAH-activity by JNJ 1661010 (4 mg/kg four times a day, i.p.) significantly exacerbated
bleomycin-induced fibrosis. Of great importance, CB
1
(AM281; 10 mg/kg four times a day, i.p.), but not
CB
2
(AM630; 2.5 mg/kg four times a day, i.p.), antagonism completely abrogated the pro-fibrotic
effects of FAAH inhibition [322].
At this point, an important controversy has to be mentioned with respect to the expression
of FAAH in human fibroblasts. In contrast to the above findings, a recent study (describing that a
missense polymorphism [A458S] of FAAH2 may contribute to the development of psychiatric disorders
including anxiety and mild learning disability) found that human dermal fibroblasts only express
FAAH2, but not FAAH1 [
64
]. Since FAAH2 is not expressed in mice and rats, but shares the substrate
spectrum of FAAH1 (however, it has less affinity towards AEA and N-acyl taurines), and conventional
FAAH-inhibitors can inhibit its activity [
49
], targeted studies are invited to determine the expression
patterns and putative roles of FAAH1 and FAAH2 in human fibroblasts under physiological as well as
pathological conditions.
Irrespective of the expression pattern of FAAH1 and-2, CB
1
appears to play a rather pro-fibrotic
role
in vivo
, and could theoretically become a promising pharmacological target, especially, since CB
1
(as well as CB
2
[
323
] and TRPV4 [
324
]) were reported to be over-expressed in cultured lesional
fibroblasts of patients suffering from diffuse cutaneous systemic sclerosis (dcSSc) compared with
healthy controls [
323
]. However, since the authors did not provide appropriate densitometry analyses,
the apparent alterations in the level of the loading control
β
-actin question the validity of this
conclusion [323].
In line with the above observations, bleomycin-treatment induced less dermal thickening in
TRPV4
/
[
324
] as well as CB
1/
mice as compared to wild-type animals. Moreover, activation
of CB
1
by the selective agonist ACEA (intraperitoneal injections twice a day at a concentration of
7.5 mg/kg for 4 weeks) further worsened bleomycin-induced dermal thickening. When assessing the
mechanism of action, the authors found that, quite surprisingly, T cell and macrophage infiltration
was significantly reduced in CB
1/
mice following bleomycin challenge; whereas ACEA treatment
could further increase it in wild-type animals. Last, but not least, the phenotype of CB
1/
mice was
mimicked by transplantation of CB
1/
mouse bone marrow into CB
1+/+
mice, demonstrating that
Molecules 2019,24, 918 21 of 56
CB
1
exerted its pro-fibrotic effects indirectly by regulating infiltrating leukocytes. These data suggested
that CB
1
played a key role in positively regulating leukocyte infiltration in bleomycin-induced
fibrosis in C57BL/6 mice [
325
]. This concept was further supported by additional evidence
obtained in the non-inflammatory TSK-1 (“tight-skin”) mouse model of SSc model. TSK-1 mice
carry a dominant mutation in the fibrillin 1 gene leading to accumulation of collagen fibers in the
hypodermis, and thereby to progressive hypodermal thickening. In contrast to the aforementioned
bleomycin-induced fibrosis, TSK-1 lacks inflammatory infiltrates, therefore abnormal fibroblast
activation is not dependent on the release of inflammatory mediators from various immune cells [
326
].
Of great importance, lack of CB
1
did not prevent fibrosis in the inflammation-independent TSK-1 mouse
model, highlighting that CB
1
signaling of the infiltrating immune cells is crucial in the development of
bleomycin-induced fibrosis [325].
On the other hand, another study revealed that the role of CB
1
is very likely to be even more
complex. In fibroblasts isolated form SSc patients, adenosine A
2A
receptors were found to be
overexpressed, and the A
2A
receptor antagonist ZM-241385 (1
µ
M; 24 h) could suppress pathologically
elevated
α
-SMA expression of these cells [
75
]. Moreover the selective A
2A
receptor agonist CGS-21680
(1
µ
M; 24 h) increased collagen production, and myofibroblast trans-differentiation (as monitored
by
α
-SMA expression) both in healthy and in SSc fibroblasts, most likely via activating the ERK1/2
MAPK pathway [
75
]. Collectively, these data strongly argue that abnormally increased activity of
A
2A
may contribute to the pathogenesis of SSc [
75
]. Of great importance, A
2A
receptor was found to
heteromerize with CB
1
(co-immunoprecipitation) in healthy as well as in SSC fibroblasts. Interestingly,
although high (10
µ
M) concentration of the non-selective CB
1
and CB
2
agonist WIN55,212-2 suppressed
collagen synthesis, its lower concentrations (when applied alone) had no effect on it. On the other
hand, the combination of WIN55,212-2 and ZM-241385 (1
µ
M both) suppressed collagen production
of SSc fibroblasts. Since, when applied alone at 1
µ
M, none of the compounds influenced collagen
production, the authors concluded that by blocking A
2A
, ZM-241385 most likely indirectly antagonized
its functional heteromer (i.e., CB
1
) as well, thus the remaining suppressive effect might have been
coupled to the activation of CB
2
. Indeed, the CB
2
antagonist/inverse agonist AM630 could prevent
this effect at an unexpectedly low (1 nM) concentration, whereas its higher concentrations (5–1000
nM) had no effects, or could further enhance (20–80
µ
M) the actions of the WIN55,212-2+ZM-241385
combination [
75
]. Since AM630 was reported to be a “protean” ligand, i.e., under certain conditions
(e.g., following 24-h pre-incubation of the cells with 10
µ
M SR144528, another CB
2
-selective inverse
agonist) it may behave not only as an antagonist/inverse agonist, but also as a low potency (>25
µ
M)
agonist at CB
2
[
327
], the authors speculated that the latter phenomenon was the consequence of a
putative paradoxical CB
2
-activating effect of AM630 [
75
]. Taken together, despite of the lack of certain
key control experiments (e.g., determination of the percentage of co-localization/heteromerization of
CB
1
and A
2A
; assessment of the effects of CB
1
and CB
2
selective agonists; reversal of the effects of the
A
2A
agonist by a selective CB
1
antagonist/inverse agonist), this study added an important new layer
to the complexity of eCB-signaling [
75
], highlighting that, besides the effects on various immune cells,
direct actions on fibroblasts may also be important.
Having dissected CB
1
, it is noteworthy that the role of CB
2
was further investigated in a skin
excisional wound model of BALB/c mice. The animals were treated with either the CB
2
agonist
GP1a, or with the antagonist AM630 (both in 3 mg/kg/day i.p.), where GP1a and AM630 induced
opposing cellular effects. GP1a decreased collagen deposition, reduced the levels of TGF-
β
1, TGF-
β
receptor I and phosphorylated mothers against decapentaplegic homolog 3 (p-Smad3), but elevated the
expression of its inhibitor, Smad7, whereas AM630 increased collagen deposition and the expression
levels of TGF-
β
1, TGF-
β
receptor I and p-Smad3. Although the authors did not assess the effects
of co-treatments, these results indicated that CB
2
can modulate fibrogenesis and the TGF-
β
/Smad
profibrotic signaling pathway during skin wound repair in BALB/c mice [328].
Similarly, in a hypochlorite-induced BALB/c mice fibrosis model, WIN-55,212 (CB
1
and CB
2
agonist) and JWH-133 (a selective CB
2
agonist) prevented the development of skin and lung fibrosis,
Molecules 2019,24, 918 22 of 56
and reduced fibroblast proliferation as well as the development of anti-DNA topoisomerase I
autoantibodies. Experiments performed in CB
2/
mice revealed that hypochlorite administration
in these animals led to earlier and enhanced development of lung fibrosis and higher skin fibroblast
proliferation rate. Moreover, CB
2/
mice exhibited higher anti-DNA topoisomerase I autoantibody
levels, and higher increase in splenic B cell count than wild-type animals [
329
]. Finally, CB
2/
mice were more sensitive to bleomycin-induced dermal fibrosis than wild-type animals. Importantly,
the phenotype of CB
2/
mice was mimicked by transplantation of CB
2/
bone marrow into
wild-type animals, whereas CB
2/
mice transplanted with bone marrow from CB
2+/+
mice did not
exhibit an increased sensitivity to bleomycin-induced fibrosis, indicating that CB
2
expressed by the
leukocytes is crucially important in this model of experimental fibrosis as well [330].
Along these lines, several exocannabinoids were also assessed in SSc. Ajulemic acid (AJA;
a.k.a. CT-3, IP-751, JBT-101, anabasum or lenabasum) is a synthetic, cannabinoid-derived, orally
bioavailable PPAR
γ
and CB
2
receptor activator, which has already been shown to exert remarkable
anti-inflammatory and anti-fibrotic effects in various systems [
319
,
320
,
331
]. The effects of AJA included,
but were not limited to prevention of bleomycin-induced dermal fibrosis, and a modest reduction in its
progression when started 3 weeks after the onset of the symptoms. Moreover, AJA strongly reduced
collagen production by SSc fibroblasts
in vitro
in a PPAR
γ
-dependent manner [
332
]. Importantly,
AJA showed anti-fibrotic efficiency in case of both “preventive” (i.e., administered from Day 0) and
“therapeutic” (i.e., administered from post-bleomycin application Day 8) treatment in a DBA/2 mice
model of lung fibrosis [333].
Encouraged by the above promising preclinical data, clinical investigation of AJA was also
initiated. A multicenter, double-blind, randomized, placebo-controlled phase II trial assessing AJA
efficiency in subjects with dcSSc was recently completed (ClinicalTrials.gov ID: NCT02465437). In this
study, the AJA group showed higher Combined Response Index for Systemic Sclerosis (CRISS)
score (i.e., greater improvement) as compared to the placebo group, suggesting that AJA may
have potential as a novel drug in the management of SSc. Importantly, a phase III multicenter,
double-blind, randomized, placebo-controlled study has already been announced (ClinicalTrials.gov
ID: NCT03398837) to assess the efficacy and safety of AJA (lenabasum) for the treatment of dcSSc.
Approximately 354 subjects are planned to be enrolled in this study at about 60 sites in North America,
Europe, Australia, and Asia; the planned treatment duration is 1 year. Moreover, it is also noteworthy
that efficiency and safety of AJA is currently assessed in some other diseases (namely dermatomyositis
and cystic fibrosis) as well, and certain pre-clinical data suggest that it may exert beneficial effects in
rheumatoid arthritis and multiple sclerosis too (summarized in [319]).
Besides AJA, certain CBD-derivatives also exhibited promising potential in SSc. Indeed, another
PPAR
γ
and CB
2
co-activator (and CB
1
antagonist), namely “VCE-004.3” (a semi-synthetic CBD quinol
derivative) was also found to alleviate bleomycin-induced scleroderma as well as exerting potent
anti-fibrotic effects via activating PPAR
γ
and CB
2
[
242
]. Similarly, another PPAR
γ
- and CB
2
-activating
CBD aminoquinone (VCE-004.8) could inhibit TGF
β
-induced Col1A2 gene transcription and collagen
synthesis, as well as TGF
β
-induced myofibroblast differentiation, and it also impaired wound-healing.
In bleomycin-induced fibrosis, VCE-004.8 reduced dermal thickness and collagen accumulation around
blood vessels, it prevented degranulation of MCs, infiltration and activation of macrophages, as well as
infiltration of T-cells. In addition, VCE-004.8 abrogated the bleomycin-induced up-regulation of several
key genes associated with fibrosis (e.g., Col3A1, Col1A2, IL-1
β
and IL-13) [
243
]. Of great importance,
EHP-101, an oral lipid formulation of VCE-004.8, was found to alleviate bleomycin-induced skin
and lung fibrosis. Indeed, EHP-101 (25 mg/kg p.o.) prevented macrophage infiltration and dermal
thickening, it suppressed vascular cell adhesion molecule 1 (VCAM1), tenascin C as well as
α
-SMA
expression, and it normalized vascular CD31 positivity [
334
]. Moreover, RNAseq analysis of skin
biopsies demonstrated that EHP-101 influenced inflammatory as well as epithelial-mesenchymal
transition transcriptomic signatures. Indeed, bleomycin-induced alterations of several TGF-
β
-regulated
genes (e.g., matrix metalloproteinase-3, cytochrome b-245 heavy chain, lymphocyte antigen 6E,
Molecules 2019,24, 918 23 of 56
VCAM1 and integrin alpha-5) were reversed by EHP-101 treatment. Moreover, EHP-101 could reduce
expression of key SSc biomarker genes e.g., C-C motif chemokine 2 (CCL2) or the interleukin 13
receptor subunit alpha 1 (IL-13R
α
1). Collectively, these data strongly argue that VCE-004.8 containing
formulations deserve further attention as orally active agents to alleviate symptoms of SSc and maybe
other fibrotic diseases as well [334].
With respect to the non-CB
1
/non-CB
2
cannabinoid-activated pathways, it should also be noted
that administration of WIN55,212-2 (1–10
µ
M) reduced expression of TGF-
β
and CTGF, as well as
deposition of the extracellular matrix, and suppressed transdifferentiation of scleroderma fibroblasts
into myofibroblasts and abrogated resistance to apoptosis. The anti-fibrogenic effect of WIN55,212-2
most likely involved inhibition of the ERK1/2 MAPK pathway, but, surprisingly, could not be
prevented by selective CB
1
and CB
2
antagonists [
323
]. Anti-fibrotic effects of WIN55,212-2 were further
dissected in another study. Here, co-treatment with WIN55,212-2 (1 mg/kg/day s.c.) prevented skin
fibrosis in a DBA/2J mouse model of bleomycin-induced scleroderma. Administration of WIN55,212-2
prevented bleomycin-induced fibroblast activation (monitored by
α
-SMA positivity) and subcutaneous
adipose tissue atrophy, suppressed subcutaneous infiltration of various immune cells, and reduced
dermal fibrosis, as well as epidermal hypertrophy. Moreover, it decreased TGF-
β
, CTGF and PDGF-BB
expression, and inhibited phosphorylation of SMAD2/3 [
335
]. Thus, further, targeted studies are
necessary to unveil the exact mechanism of the potential anti-fibrotic effects of WIN55,212-2.
Last, but not least, it should also be noted that TRIB3, a potential cannabinoid target gene,
was recently found to be greatly overexpressed in SSc fibroblasts, as well as in mice fibroblasts
following bleomycin challenge [
336
]. Moreover, it was also demonstrated that breaking the
TRIB3
TGF-
β
/Smad self-activating positive feedback loop by TRIB3 knock-down exerted potent
anti-fibrotic effects [
336
]. Considering that, in human sebocytes CBD up-regulated TRIB3 in an A
2A
receptor-dependent manner [
120
], and that A
2A
receptors were found to be overexpressed in SSc
fibroblasts [
75
], further studies are invited to dissect if dysregulation of the putative A
2A
TRIB3
pro-fibrotic pathway plays a role in the pathogenesis of SSc. Thus, just like in PSO, up-regulation of
TRIB3 appears to be undesirable. Intriguingly, however, down-regulation of another CBD target gene,
namely NRIP1 (deletion of which in mouse embryonic fibroblasts suppressed fibroblast proliferation,
enhanced autophagy, and delayed oxidative and replicative senescence [
337
]) promises to exert
beneficial effects.
Taken together, these findings indicate that activation of CB
2
and/or PPAR
γ
as well as antagonism
of CB
1
and/or A
2A
adenosine receptors may become potent tools in the management of SSc and maybe
in other fibrotic diseases as well. Thus, systematic studies are invited to explore the putative therapeutic
potential of cannabinoids characterized by such “molecular fingerprints”. Such cannabinoids may
include pepcan-12 (a negative allosteric modulator of CB
1
, but a positive allosteric modulator
of CB
2
[
338
]), or THCV, which (albeit the available data about its pharmacology are somewhat
controversial) was reported to be CB
1
antagonist and CB
2
agonist [
57
]). However, use of pCBs, which
have the capability to activate the potentially pro-inflammatory TRPV3 [
119
,
121
] or the pro-fibrotic
TRPV4 [
324
,
339
] ion channels could even be detrimental. On the other hand, since activation of TRPV1
expressed on the sensory nerve fibers was shown to be beneficial in SSc because of the release of certain
sensory nerves-derived neuropeptides, e.g., calcitonin gene-related peptide [
340
], it seems to be almost
unpredictable what the net effect of TRPV-activating pCBs would be in SSc. Systematic studies are
therefore invited to explore putative therapeutic potential of these compounds in SSc and maybe in
other fibrotic diseases too.
Putative inflammation-related translational potential of the cannabinoid signaling modulation is
summarized in Table 5.
Molecules 2019,24, 918 24 of 56
Table 5.
Overview of the putative inflammation-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Disease Intervention Level of Evidence References
Sunburn CB1&CB2antagonism (?) 1Cell culture, as well as KO-validated
animal data [87]
TRPV4 antagonism Cell culture, as well as KO-validated
animal data [86]
Allergic
inflammation,
atopic dermatitis
(AD)
CB1and/or CB2agonism;
FAAH-inhibition
Cell culture, as well as KO-validated
animal data [143,144,186,187,191,192]
Topical CBC, CBCV, CBD, CBDV,
8-THCV, 8-THC, 9-THC In vivo mouse data [189]
TRPV3 blockade or desensitization Cell culture data [119,121]
Echinacea purpurea-derived alkylamides Cell culture data and clinical trials [199]
PEA
Cell culture data, animal data and human
clinical trials [200202,205]
CB2blockade (?) 1Animal data [214]
Excessive MC
activity
PEA Cell culture data [226]
Ex vivo dog skin organ culture data [227]
PEA-OXA (NAAA-inhibititor) Animal data [229]
Activation of CB1Cell culture data [232,236,237]
Ex vivo human HF and nasal polyp organ
culture data [245,246]
Activation of CB2Cell culture data [232,236,242,243]
Activation of PPARγCell culture data [242,243]
TRPV3 blockade or desensitization 2Hypothesis predicted based on animal
data [244]
PSO
CB1activators (e.g., ACEA) via
suppressing hyper-proliferation and K6
& K16 expression
Cell culture as well as ex vivo hSOC data [174,175,182]
NRIP1Cell culture as well as NRIP1/
mice data [277]
TRIB3Cell culture data [279]
AD
TRPV1 antagonism Ongoing phase II and III clinical trials [293]
TRPV3 antagonism or desensitization
(candidate: CBGV) 2Cell culture data [119,121]
FAAH-inhibition Animal data [144]
CB1activators Animal data [178]
CB2activators Clinical study [199]
Animal data [294]
CB2antagonists (?) 1Animal data [295]
NAAA-inhibitors or PPARαagonists Animal data [297299]
PEA Human clinical studies [300,301]
EMT-inhibition 2Hypothesis based on cell culture data [114]
CBG, CBGV 2Hypothesis based on cell culture data [126]
SSc
TRPV4 blockade Animal data [324]
CB1antagonism Animal data [325]
Cell culture data [75]
A2A antagonism Cell culture data [75]
CB2activators
Cell culture data [75]
Animal data [328]
KO-validated animal data [329,330]
AJA (CB2and PPARγactivator)
Cell culture data [332]
Animal data [333]
Completed phase II clinical trial, ongoing
phase III trial
NCT02465437
NCT03398837
[319]
VCE-004.3 (CB2and PPARγactivator;
CB1antagonist) Cell culture and animal data [242]
VCE-004.8/EHP-101 (CB2and PPARγ
activator) Cell culture and animal data [243,334]
TRIB3Animal data [336]
Pepcan-12 or THCV 2Hypothesis based on the available data [57,338]
1
Question marks indicate controversial data, which appear to contradict the majority of findings.
2
Note that
well-controlled studies proving the efficiency of the indicated interventions are missing; however, in light of the
available scarce data, they deserve systematic experimental exploration.
Molecules 2019,24, 918 25 of 56
2.6. Wound Healing
Considering that cannabinoid signaling regulates fibroblast functions, proliferation and
differentiation of epidermal keratinocytes, as well as cutaneous inflammation, it is not surprising that
it influences the complex [341345] process of cutaneous wound healing as well.
Murine data obtained after skin incision suggested that the expression pattern of CB
1
[
346
] and
CB
2
[
131
] can be characterized by dynamic alterations during wound healing in various immune cells
as well as in fibroblasts/myofibroblasts. Besides this, several additional lines of evidence support the
concept that CB1and especially CB2can influence wound healing.
First, as mentioned above, in the presence of LPS, JWH-015 promoted wound closure in a
scratch assay of human keratinocyte-fibroblast co-culture in a CB
1
and CB
2
-dependent manner [
185
].
Moerover, VCE-004.8 (a PPAR
γ
/CB
2
dual agonist) was found to inhibit TGF
β
-mediated myofibroblast
differentiation, and to concentration-dependently (1–10
µ
M) impair human dermal fibroblast migration
in a scratch assay [
243
]. Likewise, in a skin excisional model of BALB/c mice the CB
2
agonist
GP1a markedly attenuated fibrogenesis, whereas the CB
2
blocker AM630 enhanced fibrotic events
during skin wound healing via regulating the TGF-
β
/Smad pro-fibrotic signaling pathway [
328
].
Intriguingly, however, by using the same CB
2
agonist-antagonist pair, others have shown that
CB
2
agonism promoted migration of HaCaT keratinocytes
in vitro
, and enhanced re-epithelization
in vivo
in a BALB/c mice excisional wound model (3 mg/kg daily, i.p.), by inducing partial
epithelial to mesenchymal transition [
347
]. Theoretically, such a dual effect (i.e., promotion of
keratinocyte migration together with suppression of fibroblast activity) could be desirable to achieve
scarless healing.
It should also be noted that abrogation of FAAH activity was found to accelerate skin wound
healing in mice. Moreover, it stimulated migration of human keratinocytes, as well as differentiation
of human fibroblasts to myofibroblasts. Intriguingly, however, these effects were not coupled to the
elevated eCB-tone, but rather to an increase in the level of certain N-acyl taurines, and the subsequent
(most likely indirect) activation of TRPV1 and epidermal growth factor receptor [348].
Topically applied platelet-rich plasma (PRP) [
349
] is widely used in regenerative medicine, since it
improves tissue repair, and exerts potent analgesic effects [
350
]. In a recent study, administration of 5%
(v/v) PRP pooled from
10 donors was shown to induce IL-8 and neutrophil gelatinase-associated
lipocalin (NGAL) release from human NCTC 2544 keratinocytes via the activation of the RelA/p65
NF-
κ
B pathway. Moreover, it has also been shown that PRP contained AEA, 2-AG, PEA and OEA,
and that PRP-treatment induced AEA, 2-AG and OEA release from the keratinocytes. Of great
importance, local administration of PRP before formalin injection into the hind paw of mice reduced
the early response of the formalin-evoked nociceptive behavior by 42%, and completely abolished
the late response. This anti-nociceptive effect was abrogated by local administration of CB1(AM251),
CB
2
(AM630), and TRPV1 (I-RTX) blockers [
350
]. These data suggest that the clinically observed
beneficial effects of PRP might be in part mediated through the ECS.
Interestingly, although potentially cannabinoid-responsive TRP channels are known to be involved
in regulating several aspects of cutaneous (patho)physiology, including keratinocyte and fibroblast
functions, barrier formation and regeneration, inflammation, etc. [
3
,
45
,
94
,
351
353
], only scant data
are available with respect to cutaneous wound healing. Indeed, activation of TRPV3 with the
combination of 1 mM camphor and 100
µ
M 2-APB induced NO production in cultured primary
murine keratinocytes, which facilitated keratinocyte migration, and improved wound healing in
mice [
354
]. On the other hand, TRPV2 antagonists (e.g., tranilast) may be efficient in preventing
hypertrophic scar formation and contractures [355,356].
With respect tothe efficiency of pCBs, only scarce evidence is available. Importantly, as mentioned
above, three patients suffering from epidermolysis bullosa reported faster wound healing following
self-administration of CBD [
183
]. Besides this, it should also be noted that a flax fiber-derived
“CBD-like” compound as well as other bioactive substances in the flax fiber extract may promote
wound healing, as they exerted anti-inflammatory activity, promoted migration of human keratinocytes
Molecules 2019,24, 918 26 of 56
and fibroblasts, and enhanced collagen production [
357
,
358
]. Thus, further studies are needed to assess
putative efficiency of well-selected TRPV-modulating pCBs in cutaneous wound management. Putative
wound healing-related translational potential of the cannabinoid signaling modulation is summarized
in Table 6.
Table 6.
Overview of the putative wound healing-relevant therapeutic potential of cutaneous
cannabinoid signaling.
Condition Intervention Level of Evidence References
Excisional wound FAAH-inhibition and the subsequent
elevation of N-acyl taurines Animal data [348]
Full-thickness wound TRPV3 activation Animal data [354]
In vitro wound models TRPV2 antagonism Cell culture data [355,356]
EB Topical CBD Case report of 3
patients [183]
2.7. Itch
According to the definition of the German physician Samuel Hafenreffer, itch is an “unpleasant
sensation that elicits the desire or reflex to scratch.” Pruritus, especially when it becomes chronic
(>6 weeks), can severely impair quality of life. Although our understanding regarding its mechanism
has grown a lot in the past years, there are still quite a few open questions [
359
]. Obviously,
it would be far beyond the scope of the current paper to overview the pathogenesis of pruritus
in details, especially, since comprehensive overviews have been published recently about itch in
general [
215
,
359
,
360
], as well as about the role of various (mostly cannabinoid-responsive) TRP
channels in its
development [94,95,361363]
. Indeed, among others, all ionotropic cannabinoid
receptors (i.e., TRPV1-4, TRPA1, and TRPM8) have been shown to play a role in the complex cutaneous
intercellular communication network between epidermal keratinocytes, immune cells (e.g., MCs) as
well as sensory nerves leading to itch sensation [
94
,
95
,
361
363
]. Thus, antagonizing or desensitizing
such TRP channels by well-selected topically applied pCBs may hold out the promise of alleviating
pruritus. Clinical trials are therefore invited to exploit putative therapeutic efficiency of topically
applied, carefully selected pCBs in itch.
With respect to the effects of the “classical” ECS and to its related mediators, much less evidence is
available. On one hand, “rs12720071”, “rs806368”, “rs1049353”, “rs806381”, “rs10485170”, “rs6454674”,
and “rs2023239” polymorphisms of CB
1
were not associated with uremic pruritus [
364
], but the
synthetic THC analogue dronabinol (5 mg at bedtime) was reported to decrease pruritus for 4–6 h in 3
patients suffering from intractable cholestatic itch [365].
The latter preliminary data suggested that the ECS and CB
1
may have anti-pruritic activity.
However, especially in case of CB
1
modulation, one has to carefully differentiate between
behavioral effects exerted via activating/antagonizing central nervous system CB
1
, and peripheral,
partially non-neuronal actions. Indeed, i.p. administration of the CB
1
antagonist/inverse agonist
rimonabant (SR141716A) induced head scratching behavior in mice, which could be prevented
by the 5-HT
2A
/5-HT
2C
antagonist ketanserin [
366
]. However, this effect was likely to be rather
a central than a peripheral action of rimonabant, since LH-21 (another CB
1
antagonist with
relatively poor brain-penetration) did not induce head scratching behavior [
367
]. In line with
these observations, intraperitoneally administered WIN55,212-2 (1–10 mg/kg) dose-dependently
suppressed scratching in BALB/c mice, which were intradermally injected with 5
µ
g/50
µ
L serotonin.
Importantly, the intrathecally applied CB1antagonist/inverse agonist AM251 (1 µg), but not the CB2
antagonist/inverse agonist AM630 (4
µ
g), could partially prevent anti-pruritic effects [
368
], indicating
that activation of spinal CB1may possess anti-pruritic activity.
Besides the above data, certain reports argue that not only brain and spinal, but also peripheral CB
1
may be a potent contributor in itch. Indeed, as mentioned above, RNAseq of the skin of AD and PSO
patients suffering from severe itch revealed that CB
1
and CB
2
were significantly down-regulated in both