Role of TRP Channels in Skin Diseases

Chapter 17


Role of TRP Channels in Skin Diseases



Mathias Sulk1,2; Martin Steinhoff1,3,*    1 Department of Dermatology, University of California San Francisco (UCSF), San Francisco, California, USA
2 Department of Dermatology, University Hospital Münster (UKM), Münster, Germany
3 Charles Institute for Translational Dermatology, University College Dublin (UCD), Dublin, Ireland
* Corresponding author: martin.steinhoff@ucd.ie




Introduction


Transient receptor potential (TRP) ion channels are a heterogeneous group of nonselective cation channels. They are expressed on many cells or structures in the skin such as sensory nerves, keratinocytes, melanocytes, immune cells, endothelial cells, and fibroblasts. Therefore, TRP channels play a significant role for normal skin function (e.g. skin barrier, temperature sensation), but moreover, are also crucial under pathologic conditions. An involvement of many TRP channels is suggested for a large variety of skin diseases, like inflammation, skin cancer, and hair disorders.


TRPV Subfamily


The vanilloid (TRPV) subfamily of TRP channels consists of four nonselective cation channels (TRPV1, TRPV2, TRPV3, and TRPV4), which are modestly Ca2 +-permeable and two highly Ca2 +-selective channels (TRPV5 and TRPV6). The nonselective cation channels are expressed by sensory nerves (TRPV1, -2, -3) and skin cells (TRPV1, -2, -3, -4), and are mainly known for their activation by different temperatures, therefore defined as “thermo TRPs.” In addition, the two highly Ca2 +-selective channels (TRPV5 and TRPV6) exist, which are major calcium transporters in epithelial cells, in particular TRPV5 in the kidney and TRPV6 in the intestine. As TRPV1-4 and TRPV6 are expressed in skin cells, they have been extensively studied for their role in skin homeostasis as well as diseases.


TRPV1


Capsaicin, the pungent ingredient in hot chili peppers, activates the founding member among the TRPV-channels, TRPV1. After activation through capsaicin or elevated temperatures (∼ 42 °C), TRPV1 mediates pain [1] and leads to neurogenic inflammation [2]. Moreover, recently, a role in histamine-induced itch was described [3], and therefore, TRPV1 is considered to be a good target to treat pain or itch (see Chapters 5, 6, 8, and 16 within this book for further details).


Classically, TRPV1 is expressed by sensory neurons of the dorsal root and trigeminal ganglia. However, because TRPV1 is also expressed in many nonneuronal tissues, including human skin and immune cells, a role of TRPV1 in skin diseases (in addition to painful and pruritic conditions) can be anticipated.


TRPV1 Mediates Neuronal Sensations in Response to Skin Stimuli


Because TRPV1 plays a pivotal role in pain perception, it was also described to mediate nociceptive side effects associated with different topical skin treatments. A common side effect of tacrolimus, clotrimazole, and retinoids is a burning sensation. Recently, tacrolimus was shown to have direct effects on cultured sensory neurons by regulating the phosphorylation of TRPV1, which might be an explanation for its transient burning sensation [4]. Moreover, using TRPV1-knockout (KO) mice, TRPV1 was shown to induce nocifensive behavior after intraplantar injections of clotrimazole [5] and retinoids [6].


Notably, TRPV1 is also assumed to play a role in sensitive skin, and therefore TRPV1-antagonism might help the stinging, burning, and tightness that patients report in various skin diseases such as eczema, lupus erythematodes, or rosacea, for example [7]. Because patients with rosacea often report neuronal sensations (in particular, burning pain), a role of TRPV1 in rosacea is assumed [8,9], where activation of TRPV1 by rosacea trigger factors (e.g., UV radiation, temperature changes, exercising, alcohol) might lead to neurogenic inflammation associated with flushing, prolonged, erythema, burning pain, and leukocyte recruitment.


Thermosensation and thermal hyperalgesia are major roles of TRPV1 in human and murine skin. Initially, it was shown in vitro that temperatures around 42 °C activate TRPV1 [10]. Using TRPV1-KO mice, a role of TRPV1 in thermosensation and inflammation-induced thermal hyperalgesia was also demonstrated in vivo [1]. Notably, also other thermosensors, other than TRPV1, are considered to be involved in the response to noxious heat [11], such as TRPV2, for example. In line with these data, a study with human healthy volunteers using a TRPV1-antagonist also confirmed the role of TRPV1 in thermosensation and thermal hyperalgesia in human skin [12].


TRPV1 is also considered to play an important role in mediating pruritic stimuli and histamine-induced itch. Furthermore, it was shown to be differentially expressed in human pruritic skin diseases and that TRPV1-modulation might be helpful to improve itch. Indeed, TRPV1-antagonists improve itch in murine atopic dermatitis [13,14]. Accordingly, capsaicin treatment improved itch in patients with prurigo nodularis [15] and notalgia paresthetica [16]. In addition, an increased staining intensity of TRPV1 in keratinocytes and nerve fibers was found in prurigo nodularis [17].


Nonneuronal Expression of TRPV1 in the Skin


As a classical neuronal receptor, TRPV1 is expressed in the peripheral (e.g., trigeminal and dorsal root ganglia; skin C and Aδ fiber afferents; Ref. [18]) as well as the central nervous system (e.g., spinal cord dorsal horn, area postrema; Ref. [10]).


In the skin, nonneuronal cells are also considered to express TRPV1. The most abundant cell type in the epidermis is keratinocytes. An expression of TRPV1 was demonstrated in many studies in murine and human skin on the mRNA and protein level, but a functional role is still discussed because not all groups were able to demonstrate TRPV1 functionality using calcium assays [19]. This is underlined by a recent study demonstrating that not all antibodies against TRPV1 are specific, and that certain anti-TRPV1 antibodies produce unspecific staining in TRPV1-KO mice of mouse urothelium [20]. Within the epidermis, basal keratinocytes show a stronger immunostaining as compared to suprabasal keratinocytes [17,21]. A modulation of TRPV1 expression levels (on the protein and mRNA-level) was observed after UV-radiation of human skin. After using a twofold minimal erythema dose (MED) of ultraviolet A/ultraviolet B (UVA/UVB) radiation, epidermal TRPV1 mRNA and protein-level up-regulation was found [22], whereas down-regulation of TRPV1 mRNA was observed using a fivefold MED after UVC-radiation in whole skin [23]. This indicates a different modulation of TRPV1 mRNA in the epidermal and dermal compartment, dependent on the type and dosage of UV-irritation.


In addition to epidermal keratinocytes, the inner and outer root sheath and infundibulum of hair follicles, differentiated sebocytes, sweat gland ducts, and the secretory portion of eccrine sweat glands express TRPV1 [17,21,24]. However, because of the unclear situation of the specificity of antihuman TRPV1 antibodies and the contradictory calcium assays, the exact role of a functional TRPV1 in those structures awaits further clarification. Notably, capsaicin was described to improve apocrine chromhidrosis [25], which may be at least in part explained via TRPV1 activation in human sweat gland epithelial cells, or the surrounding peripheral nerves, which regulate sweat gland function. Here, further studies are demanded to clarify the role of TRPV1 in the regulation of human skin appendices.


Among other skin cells, melanocytes [26] and fibroblasts [27,28] have also been discussed to express TRPV1, albeit conflicting data exist [17,21]. Thus, further studies are also needed to clarify the role of TRPV1 on human melanocytes and fibroblasts.


A potential role of extraneural TRPV1 on blood vessels is clearer, at least in mice [29]. Within the dermal compartment of the skin, expression of TRPV1 on vascular tissue (endothelium, smooth muscle cells) is assumed [21,30], and also a TRPV1-dependent release of vasoactive mediators by perivascular nerves indicates a role of TRPV1 in vasodilation. Supporting this hypothesis, it has been reported in vitro that TRPV1-activation on endothelial cells could mediate nitric oxide (NO) release [31], and moreover, in humans, capsaicin increased the blood flow and produced erythema [32]. These findings correlate well with the historical studies of Jancso and Szolcsanyi, who had already demonstrated the pivotal role of capsaicin of vasoregulation and neurogenic inflammation 45 years ago [33].


Finally, immune cells (mast cells [17,21], neutrophils [34], primary human CD4-T-cells [35], dendritic cells [3638], and epidermal Langerhans cells [21]) have also been demonstrated to express TRPV1. However, based on the aforementioned problems with specific antibodies and assays, the functional relevance of TRPV1 in immune cells needs further exploration.


Activation of TRPV1 Under Inflammatory Conditions and Its Role in Inflammatory Skin Diseases


The first described activators of TRPV1 were capsaicin (and its ultrapotent analogue resinferatoxin) and temperature changes [33,39]. Later, also other substances or mediators have been demonstrated to activate TRPV1 that are ultimately involved in inflammation. In particular, TRPV1 is activated by low PH [40] and lipoxygenase products such as leukotriene B4 [41] and is sensitized by ATP [42], bradykinin [43], prostaglandins (PGE2, PGI2) [44], histamine [40], reactive oxygen species [45], proteases, and PAR2 [46]. Thus, various components of the “inflammatory soup” can potentially activate or sensitize TRPV1 signaling. This fact not only explains the role of TRPV1 in inflammatory hyperalgesia, but also suggests a critical role in modulating immune responses and therefore the development of inflammatory skin diseases in general.


Neuronal TRPV1 also induces acute inflammatory conditions via a neurogenic mechanism by releasing calcitonin gene related peptide (CGRP) and substance P (SP) from sensory nerve endings, defined as neurogenic inflammation [2]. Less understood is the role of TRPV1 in chronic inflammatory conditions, and this awaits further clarification.


Especially for chronic inflammatory diseases, the role of TRPV1 on nonneuronal skin cells (e.g., endothelial cells, keratinocytes, fibroblasts, immune cells) has to be considered, but awaits further clarification because of conflicting findings regarding a rather pro-inflammatory or anti-inflammatory role of TRPV1 in allergic contact dermatitis [13,47].


However, most studies suggest a rather pro-inflammatory effect of TRPV1 on nonneuronal skin cells. In vitro, it was demonstrated that TRPV1-activation of cultured keratinocytes with capsaicin induced the up-regulation of cyclooxygenase-2 (COX-2) and increased the release of proinflammatory mediators like interleukin 8 (IL-8) and prostaglandin E2 [48,49]. In outer root sheath keratinocytes, interleukin 1 beta was inducible by TRPV1 [24]. Moreover, TRPV1 mediates the UV- and heat-shock-induced up-regulation of matrix metalloproteinase-1 (MMP-1) on the protein and mRNA-level in cultured keratinocytes via a calcium-dependent mechanism [5052]. In line with these data, capsaicin evokes the release of leukotriene B4 (but reduced PGE2 levels) in human keratinocytes and dermal fibroblasts [28]. In mast cells, capsaicin treatment induced the release of IL-4, but failed to mediate mast cell degranulation [53]. So far, conflicting results exist about TRPV1’s role in dendritic cell function where capsaicin was suggested to effect cell maturation (increased antigen-presenting and costimulatory molecules) or cell migration to the lymph nodes [38,54]. Of note, it was elucidated that nickel, which is associated with nickel-induced contact dermatitis, directly activates TRPV1 in vitro using HEK293 and CHO cells [55], which might also suggest a role for TRPV1 in dermatitis.


Indeed, several in vivo studies demonstrated the proinflammatory involvement of TRPV1 in inflammatory skin diseases. For example, capsaicin enhanced allergic contact dermatitis in the guinea pig [56]. In hairless mice, TRPV1-antagonism reduced the UV-induced mRNA and protein expression levels of MMP-2, MMP-3, MMP-9, or MMP-13, and of the proinflammatory cytokines IL-1β, IL-2, IL-4, TNF-α, and COX-2 [57].


Formaldehyde (an inducer of inflammation and pain) provoked skin inflammation (edema and neurotrophin-expression) that was improved in TRPV1-KO mice [58]. Because formalin is an activator of TRPA1, and the receptors for formaldehyde in the setting of human skin are not exactly clear, this observation has to await further clarification in the human system. Of further notice, TRPV1-antagonism in NC/Nga-mice with atopic-like dermatitis diminished the degranulation of mast cells, attenuated the TH2 type-immune response (decreased serum IgG1-levels), and led to decreased scratching behaviors in mice [13].


Based on these findings, it was concluded that repeated capsaicin applications could improve inflammatory responses by depleting the release of pro-inflammatory neuropeptides (e.g., SP, CGRP) from TRPV1-positive sensory nerve endings. Indeed, topical capsaicin (4-6 times daily, for 2 weeks) decreased symptoms and inflammation in patients with prurigo nodularis [15], indicating an anti-inflammatory and antipruritic effect of continued TRPV1 stimulation in humans.


In a mouse model of psoriasis, topical capsaicin application over 5 days reduced the expression levels of TNFα, NF-κB, IL-17, IL-23 and thus improved skin inflammation [59]. Therefore, an anti-inflammatory role of continued TRPV1 application can be assumed in addition to its proinflammatory role in acute (neurogenic) inflammation, which has its maximum 1 h after stimulation and decreases within 6 h.


In vitro, TRPV1-activation appears to regulate cytokine function. In monocyte-derived dendritic cells, TRPV1 inhibited cytokine-induced dendritic cell differentiation and activation and thus suppressed phagocytotic activity, as well as pro-inflammatory cytokine secretion [36]. In sebocytes, capsaicin suppressed lipid synthesis and expression levels of the proinflammatory cytokine IL-1β, suggesting a beneficial effect of TRPV1-agonism in patients with acne vulgaris [60].


In addition, two further in vivo studies support the idea of an anti-inflammatory component of TRPV1 activation in certain skin diseases. Using TRPV1-KO mice, a protective role of TRPV1 in acute oxazolone-induced contact dermatitis was demonstrated where TRPV1-KO mice showed enhanced edema and increased levels of TNF-α, whereas the accumulation of immune cells was not changed. After comparison with NK-1-receptor- and CGRP-receptor KO mice, which showed a pro-inflammatory role for both neuropeptide receptors in acute contact dermatitis, it was concluded that TRPV1 might down-regulate the sensitivity to inflammatory stimuli in the skin under certain conditions [47]. Moreover, TRPV1-KO mice exhibited more severe ulceration after trichloroacetic acid-peeling, which may be due to decreased release of protective cytokines [61].


In summary, the aforementioned results indicate that neural TRPV1 modulates acute neurogenic inflammation (1-6 h). Under other acute inflammatory condition such as acute allergic contact dermatitis (2-3 days), TRPV1 may be pro- as well as anti-inflammatory. One possible explanation is species differences, the timing when the inflammation was measured (first hours vs. 2-3 days), and the models used (different stimuli and triggers). For example, TRPV1-KO mice showed normal edema formation and scratching behavior in the oxazolone-model of acute contact dermatitis [62]. Thus, TRPV1 is able to modulate acute inflammatory conditions, but conflicting data regarding a rather pro-inflammatory or protective role still exist.


Less information exists about a potential role of TRPV1 in chronic inflammatory skin diseases. Notably, very recently a pivotal role of TRPV1 (and the sodium channel Nav1.8) has been demonstrated in a mouse model of psoriasis (through topical imiquimod-application). TRPV1 and Nav1.8 were able to modulate immune reactions by regulating dermal dendritic cell function [63]. Together, these preliminary results underline the importance of TRPV1-nociceptors in chronic inflammation [8]. However, further studies, especially in human disease states and inflammatory models of different acuity, are demanded to finally clarify the involvement of TRPV1 on neuronal and nonneuronal cells in inflammatory skin diseases.


Antiproliferative Effects of TRPV1 in Hair Follicles and Keratinocytes


Because keratinocytes in the root sheath and infundibulum of human hair follicles express TRPV1 [17,21], a role for TRPV1 in hair growth was suggested. Indeed, in human cultured outer root sheath keratinocytes, TRPV1-activation enhanced the expression levels of hair-growth-inhibitory mediators/cytokines IL-1β or TGF-β2 and reduced the expression levels of certain hair growth promoters (hepatocyte growth factor, insulin-like growth factor-I, stem cell factor) [24]. Moreover, in organ-cultured human scalp hair follicles, capsaicin-treatment induced diminished hair shaft elongation and proliferation and induced apoptosis and thus hair follicle regression [24]. These in vitro findings were in line with an in vivo study using TRPV1-KO mice, which demonstrated a delay in the hair follicle cycling [64]. Thus, TRPV1-antagonism may be beneficial for the treatment of alopecia, whereas TRPV1-agonism might improve hirsutism.


In accord with TRPV1’s antiproliferative role in hair follicle function, several studies also provide evidence for antiproliferative effects of TRPV1 activation in keratinocytes. Capsaicin induced apoptosis and suppressed proliferation in cultured human keratinocytes [24,65] and sebocytes [60]. Moreover, TRPV1-KO mice developed higher numbers of skin tumors, which was explained by the ability of TRPV1 to degrade the EGF (epidermal growth factor) receptor, a receptor that is markedly up-regulated in cancer cells [66]. Therefore, an antiproliferative effect of TRPV1 can be concluded. Consequently, TRPV1 agonism might help skin conditions with increased levels of proliferation (e.g., skin cancer, psoriasis), whereas chronic TRPV1 antagonism (e.g., as a treatment for chronic pain or itch) could potentially increase the risk for skin cancer. This, however, has to be thoroughly investigated in humans and mice.


TRPV1 is Important for Epidermal Barrier Formation


The skin serves as a water-impermeable barrier to prevent excessive water loss. As temperature or other physical or chemical factors could alter barrier function, a role of TRPV1 in epidermal barrier homeostasis was considered. Accordingly, it was found in vivo that TRPV1-agonism with capsaicin hampered barrier recovery after tape stripping of hairless mouse skin, whereas TRPV1-antagonism improved barrier recovery [67]. Moreover, TRPV1-antagonism led to improved transepidermal water loss, a reformation of the neutral lipid layer and normalization of loricrin and filaggrin expression levels after tape stripping in mice [14]. Therefore, TRPV1 may play a protective role for epidermal barrier formation.


TRPV2


TRPV2 was the second described member of the TRPV-Ion channel family. It is also modestly Ca2 + permeable and probably involved in certain skin diseases. Recently it was described that TRPV2 plays a role in innate immunity by regulating immune cell function (e.g., macrophages and mast cells), although TRPV2 was initially shown to be expressed on neuronal tissue (e.g., brain, dorsal root ganglia [DRGs]) [6870].


TRPV2 is activated by extreme heat (> 52 °C), and because immunostainings revealed colocalization with TRPV1 in DRGs [71], a role of TRPV2 as a thermosensor for noxious heat was postulated. On the other hand, a recent study denied a relevant functional role of TRPV2 for thermosensation in vivo by demonstrating that TRPV2-KO mice did not show any behavioral differences to noxious heat under normal or inflammatory conditions [11]. Of note, hypo-osmolarity [72] and cell stretching [73] also activated murine TRPV2, indicating a potential role of this channel in mechanotransduction.


Because TRPV2 is colocalized with CGRP and SP on DRGs [18], an indirect role of TRPV2 in promoting neurogenic inflammation was assumed, albeit direct evidence is still lacking both in rodents and humans.


However, TRPV2 has been implicated in inflammatory processes. Many immune cells express TRPV2, among them macrophages (including dendritic cells and epidermal Langerhans cells), mast cells, neutrophil granulocytes [34], and lymphocytes [74], although functional evidence is still lacking in several of those cells. Interestingly, recent studies concluded that TRPV2 is essential for mast cell and macrophage function. Indeed, TRPV2 was essential for phagocytosis [75], migration [76], and cytokine (TNFα, IL-6) production in murine macrophages [77]. In contrast, TRPV2 mediated degranulation and activation in murine [75] and human [76] cultured mast cell lines. Therefore, targeting TRPV2 may play a beneficial role in diseases in which macrophages and mast cells play a pivotal role.


In line with these data, increased expression levels of TRPV2 were observed in dermal immune cells of patients with rosacea, which exhibits increased levels of macrophages and mast cells, beside Th1 lymphocytes [9]. Of note, keratinocytes of human skin were also shown to express TRPV2 [9,18,78], but the exact role of keratinocyte-derived TRPV2 remains unclear, and further studies are needed.


TRPV3


Another important TRPV channel implicated in many skin diseases is TRPV3 [79,80]. It is only modestly Ca2 +-permeable, and high expression levels were found in human [8183] and murine [8486] keratinocytes. In addition to skin, TRPV3 was also found in other organs like tongue [87], mouse testis [88], cornea [89,90], colon [91], larynx [92], and inner ear [93] and neuronal tissue (brain, peripheral nerves), but only low—if any—expression in mouse DRGs [70,82,84,94]. In addition, B- and T-lymphocytes [95], as well as dermal dendritic cells [96], were described to express TRPV3.


The Keratinocyte as a “Forefront” of Neural Signaling: TRPV3’s Role in Thermosensation, Pain and Itch


Keratinocytes build the outer layer of the skin and are able to integrate different stimuli. As TRPV3 is a member of the “thermo-TRPs,” keratinocyte TRPV3 was originally shown to be activated by innocuous temperatures (range from 31-39 °C) [84]. Recent studies [97], however, have questioned its originally believed role [98,99] as an important temperature sensor under physiological temperatures in murine skin.


Furthermore, several chemical skin “sensitizers” activate TRPV3 such as camphor [98], carvacrol, thymol, or eugenol [87]. Keratinocyte TRPV3 is assumed to mediate their neuronal sensations via ATP [99] and PGE2 release that stimulate their corresponding receptors on primary afferent neurons [100]. Hence, TRPV3 was shown to play a role in pain under inflammatory conditions (mechanical hyperalgesia) [100102]. Thus, a TRPV3 antagonist (in particular 17(R)-resolvin D1) may be used as an analgesic [103]. (See Chapter 11 for further details.)


In addition, TRPV3 was reported to be involved in itch, another neuronal skin sensation. Animal studies showed that mice with a TRPV3 gain-of-function mutation (Gly573Ser) developed a pruritic dermatitis [88]. Thus, TRPV3-antagonism may be a future therapy for chronic histamine-independent itch [104,105]. (See Chapter 16 for further details.)


TRPV3 and Skin Barrier Function, Keratinocyte Proliferation, Skin Homeostasis, and Wound Healing


TRPV3 may also play a role in skin barrier function, keratinocyte proliferation, skin homeostasis, and wound healing. Recently, TRPV3 was shown to build a signaling complex with TGF-α/EGFR [106], two growth factors that regulate keratinocyte proliferation and differentiation [107], to modulate the activity of transglutaminases to induce keratinocyte terminal differentiation [108]. Therefore, a reduced activity of the TGF-α/EGFR-complex in TRPV3-KO mice resulted in a reduced transglutaminase-activity, reduced terminal differentiation, and thus impaired barrier integrity [106]. In HaCaT-keratinocytes, TRPV3 was sensitized by cholesterol, which has been suggested to control keratinocyte differentiation and to induce epidermal cornification [109,110]. Thus, TRPV3 may be critically involved in skin barrier function, although more conclusive human data are still lacking.


A recent in vitro study reported that α-hydroxyl acids, which are extensively used as exfoliative substances in skin cosmetic products, mediate their high epidermal cell turnover via TRPV3. Thus, α-hydroxyl acids, in particular the mild glycolic acid, activate TRPV3 by intracellular acidification to induce apoptosis, which helps skin renewal [111]. In line with this data, it was demonstrated in human organ-cultured hair follicles and cultures of human outer root sheath keratinocytes that TRPV3 stimulation suppresses cell proliferation and induces keratinocyte apoptosis [83]. Furthermore, TRPV3 antagonism prolonged cell survival of thermally stressed cells in a primary human keratinocyte culture [78].


Of note, TRPV3 also plays a role in skin homeostasis, NO-metabolism, and wound healing. Activation of TRPV3 induced NO production in cultured keratinocytes, which facilitated keratinocyte migration and—in vivo—improved wound healing [112]. Based on the fact that NO regulates vascular tone and vasodilation, it can be hypothesized that NO released by keratinocytes via TRPV3-activation affects the vascular tone and mediates vasodilation and therefore erythema [113]. In line with this, functional TRPV3 was detected in endothelial cells, and carvacrol (a TRPV3 agonist) was able to induce vasodilation in cerebral arteries [114]. Together, these studies in humans and rodents imply a role for TRPV3 in skin vasodilation and erythema.


TRPV3 and Hair Growth


As TRPV3 is highly expressed in human and rodent skin (hair follicle keratinocytes included), and TRPV3-KO mice and mice with a TRPV3-gain-of-function mutation exhibit an abnormal or defective hair morphology, a role of TRPV3 in hair growth has been implicated. Indeed, it was demonstrated that TRPV3 activation inhibits hair growth in human organ-cultured hair follicles and cultures of human outer root sheath keratinocytes. The stimulation of TRPV3 stopped hair shaft elongation, inhibited proliferation, and induced apoptosis and hair follicle regression [83]. These in vitro findings of an antiproliferative, inhibitory role of TRPV3 on hair growth were confirmed in two different in vivo models:


(1) TRPV3 gain-of-function mutations are associated with spontaneous hairlessness [88,115]. Two rodent strains, in particular DS-Nh mice (bearing a TRPV3 Gly573Ser mutation) and WBN/Kob-Ht rats (bearing a TRPV3 Gly573Cys mutation; Ref. [115]) were described to express a gain-of-function mutation, where the TRPV3-channel is constitutively active [116] and, therefore, have a hairless phenotype. Notably, DS-Nh mice persisted in the anagen phase, whereas no regeneration phase (telogen) was observed [117].


(2) TRPV3-KO mice exhibit a wavy hair coat and curly whiskers, similar to the abnormal hair morphogenesis in mice with loss-of-function mutations in the genes for TGF-α/EGFR [106,107].


Thus, hair-follicle derived TRPV3 might be a future target to treat hair disorders, and TRPV3 antagonism might improve certain forms of alopecia, whereas TRPV3 agonists might help patients with hirsutism.


TRPV3 and Inflammation


Because keratinocytes link the body surface with the environment and, thus, are the first target cells for harmful stimuli from the “outside,” keratinocyte-derived TRPV3 may also be involved in skin barrier function and inflammatory processes. Because keratinocytes have been shown to express functional TRPV3, which can be activated by several external stimuli, a role of keratinocyte-derived TRPV3 in inflammation can be anticipated. However, the precise mechanism of how TRPV3 may regulate epidermal function in human disease is still unexplored.


However, in vitro observations reported that murine keratinocytes release proinflammatory mediators after TRPV3-activation and, moreover, that TRPV3 is sensitized under inflammatory conditions. Indeed, after stimulation with a TRPV3-agonist (eugenol), mouse keratinocytes were shown to release the proinflammatory cytokine IL1-α, and that bradykinin, as well as histamine, sensitize TRPV3 [87]. Interestingly, it was demonstrated that primary mouse keratinocytes from transgenic mice that overexpress TRPV3 release PGE2 in response to TRPV3 stimulation [100]. In addition, in mouse keratinocyte and DRG coculture systems, keratinocyte-derived TRPV3 was involved in the release of ATP after heating [99], and ATP was demonstrated to reduce the sensitivity of TRPV3 to its agonists [118]. Furthermore, arachidonic acids, which regulate important inflammatory processes, were shown to potentiate TRPV3 channel function in mouse keratinocytes [119].


In vivo studies confirmed the proinflammatory role of TRPV3 in mice. As mentioned earlier, mice with a TRPV3-Gly573 mutation (gain-of-function; Ref. [116]) have a hairless phenotype, but also develop spontaneous dermatitis and show elevated mast-cell-numbers [115]. DS-Nh mice (TRPV3-Gly573Ser mutation) and WBN/Kob-Ht rats (TRPV3-Gly573Cys mutation) both show elevated serum IGE-levels [120,121]. In addition, DS-Nh mice show increased serum levels for IL-4 and IL-13, increased nerve growth factor (NGF) serum and tissue levels, and a severe scratching behavior and, therefore, were described as a potential model for atopic dermatitis [122,123]. In line with these data, Gly573Ser-transgenic mice had also significantly elevated levels of IgE, CCL11, CCL17, IL-13, IL-17, MCP-1, thymic stromal lymphopoietin, increased mast cell numbers, and higher NGF-release from keratinocytes [88,124]. Further studies showed that the Gly573Ser mutation might contribute to the development of hapten-induced dermatitis and increased dendritic cell responses [124,125]. Thus, TRPV3 may be also involved in adaptive immunity.


Importantly, TRPV3 also appears to play an important role in human skin. In accord with the earlier described in vitro and in vivo findings, the first TRPV3-related skin “TRP channelopathy” was recently identified in humans. It is caused by mutations in the trpv3-gene in humans, defined as Olmsted syndrome. It is a rare congenital keratinizing disorder characterized by bilateral mutilating excessive epidermal thickening of the palms and soles (palmoplantar keratoderma) and periorificial keratotic plaques. Clinically, it is often heterogenic, and different mutations (not only in the trpv3-gene) underlie this skin disease. In addition to hyperkeratosis, alopecia and severe itching occur in most cases, sometimes infections, and, rarely, squamous cell carcinomas in the keratotic areas. It was recently found that Olmsted syndrome is caused by missense gain-of-function mutations in the trpv3 gene (Gly573Ser, Gly573Cys, Trp692Gly) [126,127], leading to elevated apoptosis of keratinocytes and, thus, skin hyperkeratosis. In addition, other point mutations, particularly Trp521Ser [128], G573Ala [129], and Leu673Phe [130] were detected, of which the G573Ala mutation was associated with dermal infections, eosinophilia, and elevated IgE-levels [129]. Moreover, the Leu673Phe mutation in the trpv3-gene caused acute flares of inflammation and erythromelalgia [130]. Thus, modulation and possibly antagonism of TRPV3 might be a future therapy for Olmsted syndrome or erythromelalgia.


Importantly, TRPV3 is thought to play a role in other inflammatory skin diseases. In line with the assumption that DS-Nh-TRPV3 gain-of-function mice could be considered as a model for human atopic dermatitis, higher expression levels of TRPV3 were found in the skin of patients with atopic dermatitis [124]. In addition, TRPV3 was implicated in rosacea, a common chronic inflammatory skin disease in which increased gene expression levels for TRPV3 mRNA and enhanced immunoreactivity for TRPV3 were found [9].


In summary, TRPV3 plays an important role for many processes in the skin. As it is expressed by keratinocytes, which subsequently release inflammatory mediators on TRPV3 stimulation, TRPV3 may be an important epidermal receptor to “sense” “outside danger” during inflammation and to regulate epidermal barrier function. TRPV3 may also regulate neuronal stimuli such as pain or itch, although this is not fully understood in humans. Thus, TRPV3 may regulate skin homeostasis, barrier integrity, and hair growth, and TRPV3-stimulation results in the release of different factors from keratinocytes, which are able to initiate and maintain inflammation.


TRPV4


Like TRPV3, TRPV4 is also a nonselective cation channel, which is modestly permeable to Ca2 +, activated by moderate heat (25-34 °C), endogenous inflammatory metabolites (e.g., arachidonic acid metabolites), and exogenous compounds (phorbol esters, like 4α-PDD) and is robustly expressed in murine and human skin. Mouse [86,131] and human [132,133] keratinocytes, as well as mast cells, macrophages, fibroblasts, vascular tissue [134,135], and Merkel cells [136] were shown to express TRPV4, albeit a functional role for most cells is unclear. In addition, TRPV4 was found to be expressed in the trachea, kidney, salivary gland [137], liver, heart [138], inner ear hair cells, and neuronal tissue (brain, sympathetic, and parasympathetic nerve fibers and only at low levels in DRGs) [70,137,139,140].


TRPV4 in the Skin—A Sensor and Nociceptor for Outside (and Inside) Stimuli


As a member of the “thermo-TRPs” family, TRPV4 was described to be activated by moderate heat (25-34 °C) [134,140]. Indeed, mice lacking TRPV4 showed an altered temperature behavior under normal [141] and under inflammatory [132,142] conditions. Conflicting with these findings, other studies did not observe a role of TRPV4 as a temperature sensor and regarded the differences in KO mice compared to wild-type mice rather as strain-dependent [97].


Interestingly, as TRPV4 is expressed in vascular endothelium and skin sensory nerve endings, and hypotonicity or osmotic changes activate TRPV4 [136,138,143], this channel may be involved in hypotonic stimulus-induced nociception [139].


In addition, a role for TRPV4 in mechanosensation and mechanical hyperalgesia is assumed. Recent observations reported that TRPV4 is capable of sensing mechanical stimuli [144,145] and that it is involved in mechanical hyperalgesia under inflammatory conditions [132,146] or after DRG-compression [147].


TRPV4 and Inflammation


TRPV4 plays also a role in inflammation, as inflammatory mediators activate TRPV4. It is also found on CGRP-coexpressing sensory nerves [148] and on several immune cells. Indeed, inflammatory mediators (e.g., metabolides of arachidonic acid [135], PGE2 [139], and histamine [149]) and receptors involved in inflammation (e.g., PAR2 [146]) were shown to activate and/or sensitize TRPV4. Moreover, it was shown that mast cells [150,151] and macrophages [152] express functional TRPV4, whereas the expression of TRPV4 in human leukocytes [153] and dendritic cells [37] was demonstrated, but its functionality in these cells remains unclear.


TRPV4 on keratinocytes was also described to be involved in inflammatory conditions. Stimulation of TRPV4 in a keratinocyte cell line caused the release of IL-8 indicating a role of TRPV4 in neutrophil recruitment [154].


An in vivo study performed in keratinocyte-specific TRPV4-KO mice demonstrated that epidermal TRPV4-KO leads to a reduced release of the proinflammatory cytokine IL-6 and consequently, diminished macrophage and neutrophil numbers after generating UVB-induced photodermatitis. Of importance, epidermal TRPV4 increased the concentration of the proalgesic and inflammatory mediator endothelin 1 (ET-1). Thus, TRPV4 and ET-1 may play a role in human acute photodermatitis (“sunburn”), indicating a role of keratinocyte-derived TRPV4 as a potential target for UVB-induced sunburn [132]. In addition to the aforementioned in vitro and in vivo studies, the role of TRPV4 in inflammation was also investigated in human skin. TRPV4-expression levels were increased in dermal cells of rosacea, a chronic inflammatory skin disease [9].


TRPV4 and Skin Barrier


Recent studies report that TRPV4 plays an important role in mediating skin barrier integrity and accelerating barrier recovery. Initially, Denda et al. described a temperature-dependent change in barrier recovery after tape stripping of murine and human skin. Using TRPV4 agonists and antagonists, it was elucidated that TRPV4 elevates Ca2 + levels in mouse primary keratinocytes, and thus activation of TRPV4 may accelerate barrier recovery [67].


In line with this, using TRPV4-deficient mice, it was found that epidermal TRPV4 promotes the development and maturation of intercellular junctions by binding to β-catenin, an adaptor protein linking intercellular adhesion molecules (E-cadherin) and the cytoskeleton (actin fibers) [131,155]. In human cultured keratinocytes it was shown that TRPV4-activation was important for barrier formation by mediating Ca2 +-influx and thus promoting cell-cell junction development to augment intercellular barrier integrity. Supporting this hypothesis in a translational setting, the same authors demonstrated ex vivo in human skin tissue that TRPV4-activation leads to accelerated barrier recovery after the disruption of the stratum corneum [133].


In human epidermal keratinocytes, TRPV4-activation strengthened the tight junction-associated barrier, measured by an increased transepithelial electric resistance, and up-regulated tight junction-structural proteins (occludin and claudin-4) [156].


In summary, keratinocyte TRPV4 appears to be important for skin barrier integrity and contributes to cell-cell junction development, which prevents excess skin dehydration.


TRPV4 and Skin Cancer


Due to its expression on keratinocytes, it was recently found that TRPV4 might also play a role in nonmelanoma skin (NMS) cancer [154]. Immunohistochemical studies with human skin samples of NMS cancer showed that TRPV4 expression was down-regulated in malignant lesions. Therefore, TRPV4 might serve as a “biomarker” for skin certain NMS cancers [154] and, similar to protease-activated receptor 2 (PAR-2), may be a negative regulator during carcinogenesis [157].


TRPV5 and TRPV6


Within the TRPV channel subfamily, TRPV5 and TRPV6 are exemptions regarding their Ca2 + permeability. Whereas TRPV1-V4 are only modestly permeable to calcium, TRPV5 and TRPV6 are highly Ca2 + selective and, therefore, are major calcium transporters in epithelial cells. In particular, TRPV5 appears to play an important role in the kidneys and TRPV6 in the intestine [158]. Moreover, TRPV6 is also expressed by keratinocytes [159,160], whereas a role for TRPV5 in human skin is unknown.


Calcium homeostasis is important for keratinocyte proliferation, differentiation, and barrier function [158]. As TRPV6 is a selective calcium channel, it was shown in vitro that silencing of TRPV6 in human primary keratinocytes decreased the calcium-induced expression of keratinocyte differentiation markers (involucrin, transglutaminase-1, cytokeratin-10). Moreover, TRPV6 was demonstrated to mediate, at least in part, the pro-differentiating effects of vitamin D3. Thus, it is concluded that TRPV6 is essential for calcium- and/or vitamin D3-mediated keratinocyte differentiation and, therefore, also plays a role in promoting skin barrier function [160]. Moreover, TRPV6 may be involved in skin repair and in wound healing [161].


In line with these in vitro data, it was shown in vivo that deletion of the trpv6 gene in mice leads to decreased skin calcium levels, thinner layers of stratum corneum and in 20% of animals to alopecia and dermatitis [159].


In summary, TRPV6 might be important for skin barrier function, keratinocyte proliferation, and differentiation and, therefore, could be a promising target in inflammatory responses, hair growth disorders, and wound healing.


TRPA1


A number of endogenous and exogenous molecules, which act in and on the skin, activate TRPA1 (TRP-ankyrin) to mediate inflammation, pain, and itch.


Role of TRPA1 in Thermosensation, Mechanosensation, Skin Sensitization, Pain, and Itch


Similar to TRPV1 to V4, TRPA1 was believed to be activated by temperature changes and thus to mediate thermosensation. Initially, it was described that TRPA1 is activated by temperatures < 17 °C, which resembles the threshold for noxious cold in humans [162]. This is supported by findings that TRPA1 is colocalized with TRPV1 on DRGs [163]. Thus, TRPA1 was linked to the detection and sensation of noxious cold [164]. On the other hand, TRPA1 was not required for the initial detection of noxious cold in TRPA1-KO mice [165]. Therefore, it was speculated that TRPA1 mediates cold hypersensitivity (and is activated by cold temperatures) only under pathological conditions, for example, when endogenous activators [sensitizing inflammatory mediators] are present [166]. However, highly conflicting results exist with regard to the heat activation of TRPA1 [164,167], which could in part be explained by species differences [168]. For example, it was shown that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1 [168]. Consequently, the exact role of TRPA1 in the detection of noxious cold in various species remains controversial.


In addition to temperature, mechanical stimuli might also be able to activate TRPA1 in the skin [169]. This is underlined by the finding that TRPA1 was expressed in structures of the skin that mediate mechanosensation: for example, it is present in the mechanoperceptive nerve endings in epidermis and around Meissner corpuscles [170]. Therefore, a role for TRPA1 in mechanosensation [171174] and mechanical hyperalgesia was postulated [175]. Conflicting results obtained with TRPA1-KO mice, however, did not confirm the activation of TRPA1 by mechanical stimuli and questioned the postulated role for TRPA1 in mechanosensation and mechanical hyperalgesia [165]. Thus, TRPA1’s role in mechanosensation and mechanical hyperalgesia needs further clarification in various species.


Many skin sensitizers, pungent natural products, and irritant substances activate TRPA1 and are able to produce inflammation and pain. In particular, cinnamaldehyde (active ingredient in cinnamon), isothiocyanates (pungent substances in mustard oil, horseradish, and wasabi), allicin (garlic), and formalin (skin irritant) have been shown to activate TRPA1 [165,176]. Moreover, TRPA1 is activated by environmental chemicals such as formalin [177] and cigarette smoke [178180], which contain abundant aldehydes. Thus, TRPA1 might also be implicated in the skin ageing effects of cigarette smoke.


Skin sensitizers are capable of producing pain, which may also be mediated by TRPA1. Indeed, TRPA1 plays an important role in nociception [162]: Activation of TRPA1 produces pain behavior in mice [165], and recently a gain-of-function mutation in the human TRPA1 gene has been linked familial episodic pain syndrome [181]. (See Chapter 9 for further details.)


In addition to nociception, TRPA1 is also considered to be important in the sensation and perception of itch. In particular, TRPA1 mediates histamine independent itch and is necessary for chronic itch, which might suggest TRPA1 as a good target for novel therapies against pruritic skin diseases [182185]. (See Chapter 16 for further details.)


Nonneuronal Expression of TRPA1 in the Skin


In mice, TRPA1 is predominantly found in TRPV1-expressing (CGRP- and SP-positive) nociceptive (DRG and TRG) neurons [163] that innervate the skin and terminate around Meissner corpuscles [170]. Human peripheral nerves also express TRPA1 [186,187]. Moreover, nonneuronal cells in the skin also express TRPA1, including both murine [170,188] and human [28,154,186,189] epidermal and hair follicle keratinocytes. In addition, up-regulated TRPA1 was found in keratinocytes of lesional skin from patients with atopic dermatitis [187] and solar keratosis [154]. However, the expression of functional TRPA1 in keratinocytes is currently debated because conflicting results exist [62,163].


Immune cells also express TRPA1. In a murine atopic dermatitis model, a colocalization of TRPA1 was found with eosinophils, Langerhans cells, macrophages, T cells, and mast cells [187]. Furthermore, endothelial cells [190], melanocytes [189,191], and fibroblasts [28,189] were found to express TRPA1. Of note, the specificity of the antimurine and antihuman TRPA1 antibodies is still a matter of debate. In addition, clear functional data for TRPA1 in human cells is still lacking. That said, the activation of TRPA1 on murine endothelial cells results in vasodilation [190], which might indicate a role of TRPA1 in erythema [28].


TRPA1 in Keratinocytes: Differentiation and Barrier Function


A role for TRPA1 in keratinocyte proliferation, differentiation, and barrier function has been proposed. TRPA1 activation was demonstrated to evoke increased calcium levels in undifferentiated keratinocytes [192]. After activation of TRPA1 in primary human keratinocytes, genes involved in keratinocyte differentiation and proliferation (e.g., heat shock proteins, cyclins, and cyclin-dependent kinases) showed changes [189]. In line with these data, a recent study found that TRPA1 staining was increased in solar keratosis [154]. Following tape stripping of mice skin, TRPA1 activation was demonstrated to accelerate skin barrier recovery and increase lamellar body secretion [188], implicating a protective role for TRPA1 in skin barrier function.


TRPA1 Plays a Major Role in Skin Inflammation


In addition to its role in “sensing” environmental skin sensitizers and irritants, TRPA1 can be also activated under inflammatory conditions by endogenous trigger factors. For example, endogenous pro-inflammatory and nociceptive mediators (prostaglandins, bradykinin, proteases, oxidative stress; Refs. [162], [179], [193], [194]) were shown to activate and sensitize TRPA1. Moreover, TRPA1 appears to be up-regulated under inflammatory conditions [187,195]. Activation of TRPA1 results in inflammation via the release of SP and CGRP by sensory nerve endings (neurogenic inflammation) or by the activation of TRPA1 on nonneuronal cells. It was shown in vitro that the activation of TRPA1 on keratinocytes results in the release of IL1-alpha, IL1-beta [189], and PGE2 [28].


In vivo, activation of TRPA1 with cinnamaldehyde evokes ear edema (SP-dependent) and leukocyte infiltration, which are prevented by specific TRPA1 antagonists [196]. Thus, chemicals that activate TRPA1 are able to enhance acute contact dermatitis induced by fluorescein isothiocyanate (FITC) and increase ear swelling and the migration of dendritic cells [197]. Also, desensitizing TRPA1 by application of topical allyl isothiocyanate leads to a reduced ear edema and inhibited dendritic cell trafficking and maturation [198]. Moreover, substances, which induce acute contact dermatitis, are also able to directly activate TRPA1. For example, it was shown that oxazolone directly activates TRPA1 and that TRPA1 is a major regulator of inflammatory responses in contact dermatitis [62]. In acute (oxazolone-induced) contact dermatitis, TRPA1-KO mice showed decreased ear edema and leukocyte infiltration, diminished CXCL2, IL-4 and IL-6 cytokine levels, and reduced scratching behavior [62].


In chronic, atopic-like dermatitis induced by repeated oxazolone challenges, TRPA1-KO mice displayed less severe skin dermatitis and reduced levels of NGF, SP, and 5-HT [62]. In line with these findings, a role of TRPA1 in atopic dermatitis (caused by transgenic IL-13-production in mouse skin keratinocytes) was indicated. In these mice, an increased number of TRPA1-positive nerve fibers and mast cells was observed. Moreover, TRPA1 was shown to mediate calcium signals in a cultured mast cell line [187]. Whether the in vitro obtained calcium concentrations are sufficient to play a biological role in humans in vivo is currently under investigation. In addition, in human atopic dermatitis patients, an enhanced expression of TRPA1 was found in keratinocytes and dermal cells (including mast cells) as compared to healthy patients [187], indicating an essential role of TRPA1 in inflammatory and pruritic skin diseases (including atopic dermatitis).


TRPA1 is Functional in Melanocytes


As melanocytes were shown to express TRPA1, a functional role of this ion channel in photo-transduction was assumed. This hypothesis was strengthened by findings that UVA evoked typical TRPA1 currents in electrophysiological recordings in a HEK293-cell line. Consequently, indirect activation of TRPA1 via the generation of oxidative stress was hypothesized [199]. Moreover, TRPA1 activation is able to increase intracellular calcium levels in melanoma cell lines, and TRPA1 was shown to be functional in melanocytes [200]. In accord, it was demonstrated that physiological doses of UVA radiation can induce retinal-dependent G protein-coupled calcium responses, which activate TRPA1 in human epidermal melanocytes. This provides evidence that TRPA1—together with calcium release from intracellular stores [201]—mediates extraocular phototransduction in melanocytes, which results in melanin synthesis [191].


The TRPC (Canonical) Family


The seven members (TRPC1-C7) of the modestly calcium-permeable TRPC family by sequence homology could be subdivided into two groups: (1) TRPC1/C4/C5 and (2) TRPC3/C6/C7. (Of note, TRPC2 is only a pseudogene in humans [202,203]). Moreover, heteromultimeric TRPC-channel formations for TRPC1/TRPC4/TRPC5 and TRPC3/TRPC6/TRPC7 have been described [204]. Because TRPC channels are modest calcium permeable, they are assumed to contribute to receptor-operated Ca2 + entry (activation of TRPC3, TRPC6, TRPC7 in a diacylglycerol (DAG)-dependent mechanism), and also a role in store-operated Ca2 +-entry has been discussed for TRPC1, TRPC4, TRPC5 (activation via PLC) [203,205].


TRPC Channels are Expressed in Skin Cells and in Skin-Innervating Nerves


In the skin, TRPC channels (TRPC1, TRPC3, TRPC4, TRPC5, and TRPC6) are expressed by keratinocytes [206], where they are considered to be involved in keratinocyte differentiation and proliferation. In addition, fibroblasts [207] and immune cells were described to express TRPC channels. In particular, B-cells express TRPC7 [208], whereas primary human CD4+ T-cells express TRPC1 and TRPC3 [35]. Vascular tissue (endothelium and vascular smooth muscle cells) also expresses TRPC: these channels are thought to modulate vascular function, implying a role for TRPC channels in erythema and edema formation (reviewed in [205]).


Some TRPC channels (TRPC1, TRPC3, TRPC4, and TRPC5) are expressed on DRG and trigeminal ganglia (TG) neurons (70). TRPC5 was shown to be expressed on peripheral intraepidermal nerve endings involved in the sensation of cold temperatures [209]. Another TRPC family member, TRPC1, was demonstrated to mediate (directly or indirectly) mechanical stimuli in cultured DRG neurons [210], which was strengthened by similar findings using TRPC1-deficient mice [211].


TRPC Channels are Involved in Keratinocyte Differentiation and Proliferation


All TRPC channels (TRPC1 to TRPC7) are expressed on keratinocytes [206,212], where they are involved in calcium homeostasis. As calcium is a major regulator of the epidermal keratinocyte turnover, several studies indicate a role of TRPC channels in keratinocyte differentiation and proliferation.


TRPC1 expression was described in human keratinocytes [213], and an involvement in store-operated calcium signaling in human cultured primary keratinocytes has been suggested [206]. Moreover, it is expressed in human gingival keratinocytes, and it was shown that TRPC1 mediates calcium-induced keratinocyte differentiation, as indicated by decreased involucrin levels after TRPC1 knockdown using siRNA [214]. Increased TRPC1 signaling was shown in Darier’s disease [215]. In this human skin disease, loss-of-function mutations in the gene (ATP2A2) that encodes SERCA2 lead to impaired keratinocyte differentiation, increased proliferation, and lower rates of apoptosis [216]. By a combination of immunohistochemistry and Western blots, it was demonstrated that TRPC1 protein was overexpressed in keratinocytes of patients with Darier’s disease and also in SERCA2+/− mice. Therefore, Darier’s disease keratinocytes show an increased calcium signaling and, thus, an increased proliferation rate (as compared to normal keratinocytes). Moreover, TRPC1 overexpression in a keratinocyte cell line promotes cell survival by inhibiting apoptosis (higher levels of antiapoptotic proteins) indicating that TRPC1 signaling might be a key factor for increased proliferation in Darier’s disease [215].


TRPC4 shows a high basal expression in differentiated keratinocytes [213,217] and was described to be involved in store-operated calcium signaling in human cultured primary keratinocytes [206,217].


Based on these findings, TRPC1 and TRPC4 have been implicated in the pathomechanism of basal cell carcinoma. Indeed, basal cell carcinoma cells did not express TRPC1/TRPC4, which might explain the lack of differentiation in these tumor cells [218].


In vitro and ex vivo, TRPC6 mediates calcium signals and promotes keratinocyte differentiation (keratin 10 expression). In addition, in human skin, triterpenes induced the up-regulation of TRPC6 in keratinocytes. Moreover, triterpenes improve actinic keratosis as indicated by up-regulation of the differentiation marker keratin 10. Thus, TRPC6 promotes keratinocyte differentiation and modulates keratinocyte proliferation, and therefore, TRPC6-activation might be helpful for actinic keratosis [213]. These findings were confirmed by another study [219], where keratinocyte-TRPC6 was studied in vitro and ex vivo. It was shown that a specific TRPC6-activator (hyperforin) evoked TRPC6-mediated calcium influx, and TRPC6 activation subsequently induced keratinocyte differentiation and inhibited proliferation. Therefore, a role of TRPC6 in chronic skin diseases with impaired keratinocyte differentiation and proliferation like psoriasis and atopic dermatitis can be assumed [219,220]. Furthermore, TRPC6 was demonstrated to be essential for the transdifferentiation from fibroblasts into myofibroblasts, and therefore, it might play a role in wound healing and fibrotic diseases [207]. In vitro, cultured fibroblasts from TRPC6 KO mice did not transform into myofibroblasts after TGF-β treatment and in vivo, TRPC6 KO mice showed impaired wound healing [207].


TRPC7 was shown to be involved in DAG-evoked calcium signaling in HaCaT human keratinocytes [212]. However, further in vivo and studies in primary cells are demanded to clarify its role in human disease.


In line with the aforementioned studies that strongly suggest involvement of TRPCs in cell differentiation, proliferation, and apoptosis, it was demonstrated that TRPC channel expression is decreased in patients with psoriasis [221]. In accord, psoriatic keratinocytes show impaired calcium homeostasis (reduced calcium influx). Conversely, TRPC6 activation normalizes differentiation and proliferation levels of psoriatic keratinocytes [221].


Clearly, further studies are needed to clarify the specific role of each TRPC channel in keratinocyte proliferation and differentiation. Apparently (and somewhat confusingly), TRPC1 plays different role in Darier’s disease (TRPC1-overexpression might be pro-proliferative; Ref. [215]) and psoriasis (TRPC1-down-regulation might be pro-proliferative; Ref. [221]).


TRPMs


The group of TRPM channels consists of eight members. Based on their amino acid sequence, they could be subdivided into three groups, namely TRPM1/M3, TRPM4/M5, and TRPM6/7. The remaining two family members, TRPM2 and TRPM8, exhibit only low sequence homology and are therefore ungrouped [202]. TRPM channels are nonselective cation channels with specificity for monovalent (e.g., TRPM4, TRPM5) or divalent (TRPM6, TRPM7) cations [203]. Interestingly, TRPM4 is activated by calcium and can modulate cacium entry into the cells by regulating membrane potential [222]. TRPM channels have been implicated in taste sensation, magnesium-homeostasis, and detection of cold.


Neuronal TRPM Expression and Cold Sensation


TRPM channels are expressed on neuronal tissue like DRGs (TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, TRPM7, TRPM8) [70], as well as in epidermal and dermal nerve fibers where TRPM colocalizes with CGRP and SP [18]. Two members of the TRPM family have been implicated in temperature sensation: TRPM8 is activated at temperatures below 25 °C and therefore serves as a “cold sensor” [176] whereas TRPM3 is considered as a sensor for nociceptive heat [223].


Skin-sensitizing and cooling agents like menthol and icilin also activate TRPM8. In line with its role as a “cold sensor,” TRPM8-KO mice show behavioral deficits in response to cold stimuli [176]. Cold is able to alleviate pain and itch, and TRPM8-activation was shown to modulate nociception and to mediate analgesia in mice [224]. The burning sensation caused by clotrimazole [a treatment of yeast infections of the skin] was explained by its antagonism at TRPM8 [5].


Nonneuronal TRPM-Expression: Keratinocytes and Immune Cells


In the skin, TRPM ion channels are expressed on melanocytes and in malignant melanoma (TRPM1, TRPM2, TRPM7, TRPM8) [225]. Keratinocytes and different immune cells (T cells, mast cells, granulocytes, and monocytes) express these ion channels, making them a potential target for therapies in skin diseases [226]. However, their functional role in disease state is still uncertain.


In particular, TRPM8-activation on mouse-keratinocytes was demonstrated to improve skin barrier recovery after tape stripping and to mediate keratinocyte proliferation, suggesting a role of TRPM8 in wound healing [226].


Inflammatory skin diseases may be improved by modulating immune cell function. A functional role for TRPM2, TRPM4, and TRPM7 in regulating immune cells has been described. For example, TRPM2 is expressed in primary human CD4+-T cells and up-regulated after T cell-stimulation [35]. TRPM2 is functional in T cells [203] although its role in T cell regulation is still uncertain. In addition, TRPM2 is relevant for mast cell degranulation after antigen stimulation [227], as well as granulocyte and monocyte chemokine production [228] regulated by ADP-ribose [229].


TRPM4 was described to mediate membrane depolarization in immune cells. Therefore, TRPM4 activation is considered to modulate calcium signals in T cells affecting cytokine production [230] and mast cell function (alleviated mast cell degranulation) [222]. On the other hand, TRPM4 was implicated in dendritic cell migration [231]. Thus, the pro- or anti-inflammatory role of TRPM4 depends on the specific immune cell, and further studies are needed to determine its role in inflammatory skin diseases.


Of further note, TRPM7 is also expressed in immune cells (lymphocytes, mast cells) and was suggested to be in involved in lymphocyte and mast cell proliferation [203].


TRPM Channels are Implicated in Melanocyte Function and Malignant Melanoma


TRPM1, TRPM2, TRPM7 and TRPM8 are expressed by melanocytes and in malignant melanoma cells, and are thus suggested to play an important role in melanocyte function and malignant melanoma pathophysiology. However, direct evidence for a critical role in human melanoma is still poor.


TRPM1 (Melastatin-1/MLSN-1), the founding member of the TRPM-ion channels, is considered the most important TRP channel in melanocyte function and malignant melanoma pathophysiology. Initially, it was found that highly metastatic, undifferentiated malignant melanomas exhibit a decreased expression of TRPM1 as compared to benign, highly differentiated nevi [232]. Also, TRPM1-expression correlates positively with the differentiation status of melanocytes, and, inversely, with the aggressiveness and tumor thickness of malignant melanoma [232]. Therefore, TRPM1 could serve as a prognostic marker in malignant melanoma [225]. Further studies confirmed this hypothesis [233,234] and even suggested that the TRPM1 mRNA expression pattern could be helpful for the differentiation of Spitz nevi compared to nodular malignant melanomas [235]. Of note, the expression levels of TRPM1 can also be altered during normal melanocyte maturation, which correlated also with its coexpression of its transcription factor MITF (microphthalmia transcription factor) [236].


To provide explanations for the postulated role of TRPM1 as a factor for melanocyte differentiation, further in vitro studies were performed [237,238]. These data suggest a role of TRPM1 as a key factor for melanocyte calcium homeostasis, controlling melanocyte proliferation, differentiation, and melanogenesis. Accordingly, involvement of TRPM1 in calcium homeostasis and melanogenesis was demonstrated using TRPM1-knockdown in cultured primary human melanocytes. Interestingly, TRPM1 knockdown resulted in a decrease of intracellular melanin pigment, and TRPM1 expression levels were reduced by UVB that subsequently decreased calcium influx [237]. This was also confirmed in another study in vitro with primary human neonatal epidermal melanocytes and mouse melanoma cells, where TRPM1 expression was found to correlate with melanin content. Thus, TRPM1 could be important for melanocyte calcium homeostasis and melanogenesis and might serve as a new target for pigmentation disorders [238]. This is also underlined by findings of a decreased TRPM1 expression in unpigmented skin of the Appaloosa horse [239].


Recent studies have found a microRNA (miR-211), which is located within the sixth intron of the TRPM1 gene, to be a key factor in malignant melanoma. MiR-211 is also regulated by the TRPM1 promotor with its transcription factor MITF and may thus play an important role in tumor suppression. In line with this, miR-211 expression was down-regulated in melanoma cells. Therefore, miR-211 in conjunction with TRPM1 may be a key factor for tumor invasiveness and aggressiveness in malignant melanoma. In particular, an increased expression of miR-211, but not TRPM1 decreased the aggressiveness (migration and invasion) of highly malignant human melanomas [240]. MicroRNA miR-211 was also shown to regulate many genes involved in melanoma pathophysiology [241] and therefore could serve as a new target to treat metastatic melanoma [242].


Other than TRPM1, two transcripts of TRPM2 (TRPM2-antisense and TRPM2-tumor enriched) were demonstrated to be up-regulated in melanoma cells. Moreover, overexpression of wild-type TRPM2 was shown to mediate melanoma apoptosis and necrosis [243]. Therefore, TRPM2 could serve as a potential new target for melanoma therapy, although this field of research is still at an infant stage.


TRPM7 was shown to be functional in melanocytes and, different from TRPM1, increased expression levels were found in metastatic melanoma cells [244]. Using the zebrafish with a TRPM7-homozygous null-allele, it was delineated that TRPM7 protects melanocytes from cell death. Hereby, TRMP7 seems to be necessary to detoxify melanin intermediates, and thus, TRPM7 might serve as an important factor for melanocyte homeostasis [244]. Therefore, it could be hypothesized that a decreased expression of TRPM7 could lead to vitiligo, but further studies are necessary to confirm this hypothesis.


TRPM8 could serve as a target in melanoma therapy. Although it was initially shown (albeit only in a small number of patients) that TRPM8 expression directly correlates with melanoma aggressiveness [245], recently a more protective role of TRPM8 was suggested. It was demonstrated in a human melanoma G-361 cell line that TRPM8-activation with menthol decreased the viability of a cultured melanoma cell line in vitro and therefore is able to inhibit melanoma proliferation [246]. Because conflicting data exist, the exact role of TRPM8 in melanoma proliferation, and its expression in different types of melanoma need further investigation.


TRPML3


Last, a member of the transient receptor potential mucolipin subfamily, TRPML3, has recently been shown to be necessary for melanocyte function. These findings arise from mice with a varitint-waddler phenotype, which are deaf, have vestibular defects, and also exhibit pigmentation abnormalities. Here, TRPML3 was shown to be abundantly expressed in melanocytes and that the pigmentation defects are a result of a TRPML3 gain-of-function mutation that causes high intracellular calcium-levels resulting in melanocyte cell death [247].


Summary


In conclusion, TRP ion channels play an important role in the regulation of various the skin cells and structures during health and disease. In addition to their contribution to the normal skin homeostasis, they could serve as targets for new therapies for many different skin disorders including inflammatory skin diseases, genetic disorders (e.g., Darier’s disease), autoimmunity, fibrotic diseases, and delayed wound healing, as well as pigmentation disorders, malignant melanoma, or nonmelanoma skin cancer. Further translational research in human tissue and cells are denuded to finally clarify their importance in disease state.

Nov 18, 2017 | Posted by in PHARMACY | Comments Off on Role of TRP Channels in Skin Diseases
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