Transient Receptor Potential Vanilloid 3 Activation Research Articles

TRPV3-ANO1 interaction positively regulates wound healing in keratinocytes

Transient receptor potential vanilloid 3 (TRPV3) belongs to the TRP ion channel super family and functions as a nonselective cation channel that is highly permeable to calcium. This channel is strongly expressed in skin keratinocytes and is involved in warmth sensation, itch, wound healing and secretion of several cytokines. Previous studies showed that anoctamin1 (ANO1), a calcium-activated chloride channel, was activated by calcium influx through TRPV1, TRPV4 or TRPA1 and that these channel interactions were important for TRP channel-mediated physiological functions. We found that ANO1 was expressed by normal human epidermal keratinocytes (NHEKs). We observed that ANO1 mediated currents upon TRPV3 activation of NHEKs and mouse skin keratinocytes. Using an in vitro wound-healing assay, we observed that either a TRPV3 blocker, an ANO1 blocker or low chloride medium inhibited cell migration and proliferation through p38 phosphorylation, leading to cell cycle arrest. These results indicated that chloride influx through ANO1 activity enhanced wound healing by keratinocytes.

KS0365, a novel activator of the transient receptor potential vanilloid 3 (TRPV3) channel, accelerates keratinocyte migration.

Ca2+ signalling mediated by the thermosensitive, non-selective, Ca2+ -permeable transient receptor potential channel TRPV3 is assumed to play a critical role in regulating several aspects of skin functions, such as keratinocyte proliferation, differentiation, skin barrier formation and wound healing. Studying the function of TRPV3 in skin homeostasis, however, is still constrained by a lack of potent and selective pharmacological modulators of TRPV3. By screening an in-house compound library using fluorometric intracellular Ca2+ assays, we identified two chemically related hits. The more potent and efficient TRPV3 activator 2-(2-chloro-3-isopropylcyclopent-2-en-1-yl)-4-methylphenol (KS0365) was further evaluated in fluo-4-assisted Ca2+ assays, different Ca2+ imaging approaches, electrophysiological studies, cytotoxicity and migration assays. KS0365 activated recombinant and native mouse TRPV3 more potently and with a higher efficacy compared with 2-APB and did not activate TRPV2 or TRPV4 channels. The activation of TRPV3 by KS0365 super-additively accelerated the EGF-induced keratinocyte migration, which was inhibited by the TRP channel blocker ruthenium red or by siRNA-mediated TRPV3 knockdown. Moreover, KS0365 induced strong Ca2+ responses in migrating front cells and in leading edges of keratinocytes. The selective TRPV3 activator KS0365 triggers increases in [Ca2+ ]i with most prominent signals in the leading edge and accelerates migration of keratinocytes. TRPV3 activators may promote re-epithelialization upon skin wounding.

Molecular determinants for the chemical activation of the warmth-sensitive TRPV3 channel by the natural monoterpenoid carvacrol

Transient receptor potential vanilloid 3 (TRPV3), robustly expressed in the skin, is a nonselective calcium-permeable cation channel activated by warm temperature, voltage, and certain chemicals. Natural monoterpenoid carvacrol from plant oregano is a known skin sensitizer or allergen that specifically activates TRPV3 channel. However, how carvacrol activates TRPV3 mechanistically remains to be understood. Here, we describe the molecular determinants for chemical activation of TRPV3 by the agonist carvacrol. Patch clamp recordings reveal that carvacrol activates TRPV3 in a concentration-dependent manner, with an EC50 of 0.2 mM, by increasing the probability of single-channel open conformation. Molecular docking of carvacrol into cryo-EM structure of TRPV3 combined with site-directed mutagenesis further identified a unique binding pocket formed by the channel S2-S3 linker important for mediating this interaction. Within the binding pocket consisting of four residues (Ile505, Leu508, Arg509, and Asp512), we report that Leu508 is the most critical residue for the activation of TRPV3 by carvacrol, but not 2-APB, a widely used nonspecific agonist and TRP channel modulator. Our findings demonstrate a direct binding of carvacrol to TRPV3 by targeting the channel S2-S3 linker that serves as a critical domain for chemical-mediated activation of TRPV3. We also propose that carvacrol can function as a molecular tool in the design of novel specific TRPV3 modulators for the further understanding of TRPV3 channel pharmacology.

TRPV3 enhances skin keratinocyte proliferation through EGFR-dependent signaling pathways.

Transient receptor potential vanilloid 3 (TRPV3) is highly expressed in skin keratinocytes where it forms Ca2+-permeable nonselective cation channels to regulate various cutaneous functions. TRPV3 expression is upregulated in many skin disorders. Here, we examined how TRPV3 affects keratinocyte proliferation and investigated the underlying mechanism. Topical application of TRPV3 agonist, carvacrol, increased skin thickness in wild type (WT) mice but not in TRPV3 knockout (KO) mice. Carvacrol promoted proliferation of human keratinocytes HaCaT cells at concentrations ≤ 100 μM, but at 300 μM, it decreased cell viability, suggesting a nonmonotonic proliferative effect. Suppression of TRPV3 expression abolished carvacrol-induced cell proliferation while overexpression of TRPV3 enhanced HaCaT cell proliferation. Carvacrol also stimulated Ca2+ influx and proliferation of primary keratinocytes prepared from WT but not TRPV3 KO mice, suggesting that carvacrol-stimulated cell proliferation was dependent on TRPV3-mediated Ca2+ influx. Mechanistic investigation demonstrated that carvacrol stimulated TGFα release and increased phosphorylation levels of EGFR, PI3K, and NF-κB, effects abolished by suppression of TRPV3 expression and CaMKII inhibition. Moreover, inhibition of CaMKII, EGFR, PI3K, or NF-κB diminished carvacrol-induced cell proliferation. We conclude that while strong activation of TRPV3 may cause cell death, moderate activation of TRPV3 promotes cell proliferation in keratinocytes through Ca2+/CaMKII→TGFα/EGFR→PI3K→NF-κB signaling. Graphical abstract Headlights 1. Carvacrol induces epidermal hyperplasia and keratinocyte proliferation. 2. TRPV3 mediates carvacrol-induced epidermal hyperplasia and keratinocyte proliferation. 3. TRPV3 acts through Ca2+/CaMKII→TGFα/EGFR→PI3K→NF-κB signaling to promote keratinocyte proliferation.

Pharmacological Activation of Thermo-Transient Receptor Potential Vanilloid 3 Channels Inhibits Hair Growth by Inducing Cell Death of Hair Follicle Outer Root Sheath.

Hair growth starts from hair follicles that reside in dermis, and abnormal hair growth is an early sign of hair follicle disease or systemic illness such as alopecia or hair loss. Therefore, identifying a target critical for dysfunctional hair follicles is fundamental to alleviating dermatologic or systemic diseases with hair abnormalities. The warm temperature-activated Ca2+-permeable transient receptor potential vanilloid 3 (TRPV3) channel protein is abundantly expressed in the skin keratinocytes, and dysfunctional TRPV3 causes human congenital Olmsted syndrome, characterized by skin diseases and alopecia, indicating an important role for TRPV3 in hair follicle development and hair growth. To validate TRPV3 as a therapeutic target, we investigated the impact of pharmacological modulation of TRPV3 on hair growth using a combination of biochemical and cell biology, immunohistochemical, whole-cell patch clamp, RNA interference, and pharmacological approaches. We found that functional TRPV3 channel proteins are highly expressed in hair follicle outer root sheath (ORS) cells as detected by Western blot analysis, immunohistochemical staining, and electrophysiological techniques. Pharmacological activation of TRPV3 by agonist natural carvacrol induces cell death of ORS cells, and topical application of carvacrol to mouse dorsal skin also inhibits hair growth. Conversely, specific inhibition of TRPV3 by inhibitor natural forsythoside B and short-hairpin RNA reverses the cell death induced by carvacrol-mediated TRPV3 activation in human ORS cells. Furthermore, forsythoside B results in a significant reversal of hair growth inhibition induced by agonist carvacrol. Altogether, our findings demonstrate that TRPV3 channel is critical for regulation of hair growth, and inhibition of TRPV3 may represent a promising therapy for hair loss or hair follicle-related skin diseases.

Activation of transient receptor potential vanilloid 3 channel (TRPV3) aggravated pathological cardiac hypertrophy via calcineurin/NFATc3 pathway in rats.

Cardiac hypertrophy is a compensatory response to mechanical stimuli and neurohormonal factors, ultimately progresses to heart failure. The proteins of some transient receptor potential (TRP) channels, Ca2+‐permeable nonselective cation channel, are highly expressed in cardiomyocytes, and associated with the occurrence of cardiac hypertrophy. Transient receptor potential vanilloid 3 (TRPV3) is a member of TRP, however, the functional role of TRPV3 in cardiac hypertrophy remains unclear. TRPV3 was elevated in pathological cardiac hypertrophy, but not in swimming exercise‐induced physiological cardiac hypertrophy in rats. TRPV3 expression was also increased in Ang II–induced cardiomyocyte hypertrophy in vitro, which was remarkably increased by carvacrol (a nonselective TRPV channel agonist), and reduced by ruthenium red (a nonselective TRPV channel antagonist). Interestingly, we found that activated TRPV3 in Ang II–induced cardiomyocyte hypertrophy was accompanied with increasing intracellular calcium concentration, promoting calcineurin, and phosphorylated CaMKII protein expression, and enhancing NFATc3 nuclear translocation. However, blocking or knockdown of TRPV3 could inhibit the expressions of calcineurin, phosphorylated CaMKII and NFATc3 protein by Western blot. In conclusion, the activation of TRPV3 aggravated pathological cardiac hypertrophy through calcineurin/NFATc3 signalling pathway and correlated with the protein expression levels of calcineurin, phosphorylated CaMKII and NFATc3, revealing that TRPV3 might be a potential therapeutic target for cardiac hypertrophy.

Transient receptor potential vanilloid-3 (TRPV3) activation plays a central role in cardiac fibrosis induced by pressure overload in rats via TGF-β1 pathway.

Cardiac fibrosis is a common pathologic change along with pressure overload. Recent studies indicated that transient receptor potential (TRP) channels played multiple roles in heart. However, the functional role of transient receptor potential vanilloid-3 (TRPV3) in cardiac fibrosis remained unclear. The present study was designed to investigate the relationship between TRPV3 activation and pressure overload-induced cardiac fibrosis. Pressure overload rats were successfully established by abdominal aortic constriction (AAC), and cardiac fibrosis was simulated by 100nM angiotensin II (Ang II) in neonatal cardiac fibroblasts. Echocardiographic parameters, cardiac fibroblast proliferation, cell cycle, intracellular calcium concentration ([Ca2+] i ), and the protein expressions of collagen I, collagen III, transforming growth factor beta 1 (TGF-β1), cyclin E, and cyclin-dependent kinase 2 (CDK2) were measured. Echocardiographic and histological measurements suggested that the activation of TRPV3 exacerbated the cardiac dysfunction and increased interstitial fibrosis in pressure overload rats. Further results showed that TRPV3 activation upregulated the expressions of collagen I, collagen III, TGF-β1, cyclin E, and CDK2 in vivo and in vitro. At the same time, blocking TGF-β1 pathway could partially reverse the effect of TRPV3 activation. These results suggested that TRPV3 activation exacerbated cardiac fibrosis by promoting cardiac fibroblast proliferation through TGF-β1/CDK2/cyclin E pathway in the pressure-overloaded rat hearts.

The human transient receptor potential vanilloid 3 channel is sensitized via the ERK pathway

The transient receptor potential vanilloid 3 (TRPV3) channel is a Ca2+-permeable thermosensitive ion channel widely expressed in keratinocytes, where together with epidermal growth factor receptor (EGFR) forms a signaling complex regulating epidermal homeostasis. Proper signaling through this complex is achieved and maintained via several pathways in which TRPV3 activation is absolutely required. Results of recent studies have suggested that low-level constitutive activity of TRPV3 induces EGFR-dependent signaling that, in turn, amplifies TRPV3 via activation of the mitogen-activated protein kinase ERK in a positive feedback loop. Here, we explored the molecular mechanism that increases TRPV3 activity through EGFR activation. We used mutagenesis and whole-cell patch clamp experiments on TRPV3 channels endogenously expressed in an immortalized human keratinocyte cell line (HaCaT) and in transiently transfected HEK293T cells and found that the sensitizing effect of EGFR on TRPV3 is mediated by ERK. We observed that ERK-mediated phosphorylation of TRPV3 alters its responsiveness to repeated chemical stimuli. Among several putative ERK phosphorylation sites, we identified threonine 264 in the N-terminal ankyrin repeat domain as the most critical site for the ERK-dependent modulation of TRPV3 channel activity. Of note, Thr264 is in close vicinity to a structurally and functionally important TRPV3 region comprising an atypical finger 3 and oxygen-dependent hydroxylation site. In summary, our findings indicate that Thr264 in TRPV3 is a key ERK phosphorylation site mediating EGFR-induced sensitization of the channel to stimulate signaling pathways involved in regulating skin homeostasis.

Acceleration of Skin Barrier Restoration via Activation of Transient Receptor Potential Vanilloid 3 (TRPV3) by Agrimonia pilosa Leaf

The formation and maintenance of the skin barrier are controlled by the proliferation and differentiation of epidermal keratinocytes, and impaired skin barrier homeostasis resulting from abnormal keratinocyte function causes many skin disorders, including atopic dermatitis and psoriasis. Keratinocyte differentiation is regulated by intra‐ and extracellular calcium ion concentrations. Recently, it was reported that the transient receptor potential vanilloid 3 (TRPV3) ion channel is functionally expressed in human keratinocytes. Activation of TRPV3 increased intracellular calcium signaling, which in turn increased transglutaminase 1, 3 activities as well as the subsequent formation of cornified cell envelopes in the epidermis. It was also reported that activation of TRPV3 promotes epithelial cell proliferation and wound healing in oral epithelia. Thus, TRPV3 activation may be a potential therapeutic target for skin barrier recovery in various dermatological diseases. The leaves of Agrimonia pilosa Ledeb (Rosaceae, AP), which have traditionally been used in East Asia to treat conditions including sore throat, parasitic infection, and eczema, have been reported to possess anti‐inflammatory and anti‐allergic effects in lipopolysaccharide‐ and ovalbumin‐induced mouse models. We performed whole‐cell patch‐clamp and tape stripping tests to determine whether AP extract enhances barrier recovery via TRPV3 activation. We found that APH2O potently activated human TRPV3 and subsequently activated TGase activity in vitro. We also confirmed that topical application of APH2O improved the recovery of skin barrier disruption induced by tape stripping in mice. APH2O may be useful for preventing and treating skin barrier disorders, including inflammatory skin diseases such as atopic dermatitis and psoriasis.Support or Funding InformationThis research was supported by the Convergence of Conventional Medicine and Traditional Koran Medicine R&D program funded by the Ministry of Health & Welfare through the Korean Health Industry Development Institute (SHIDI) (To. JH Nam, HI15C0256).

Activation of transient receptor potential vanilloid 3 by the methanolic extract of Schisandra chinensis fruit and its chemical constituent γ-schisandrin.

Transient receptor potential vanilloid 3 (TRPV3) is a non-selective cation channel with modest permeability to calcium ions. It is involved in intracellular calcium signaling and is therefore important in processes such as thermal sensation, skin barrier formation, and wound healing. TRPV3 was initially proposed as a warm temperature sensor. It is activated by synthetic small-molecule chemicals and plant-derived natural compounds such as camphor and eugenol. Schisandra chinensis (Turcz.) Baill (SC) has diverse pharmacological properties including antiallergic, anti-inflammatory, and wound healing activities. It is extensively used as an oriental herbal medicine for the treatment of various diseases. In this study, we investigated whether SC fruit extracts and seed oil, as well as four compounds isolated from the fruit can activate the TRPV3 channel. By performing whole-cell patch clamp recording in HEK293T cells overexpressing TRPV3, we found that the methanolic extract of SC fruit has an agonistic effect on the TRPV3 channel. Furthermore, electrophysiological analysis revealed that γ-schisandrin, one of the isolated compounds, activated TRPV3 at a concentration of 30 µM. In addition, γ-schisandrin (~100 µM) increased cytoplasmic Ca2+ concentrations by approximately 20% in response to TRPV3 activation. This is the first report to indicate that SC extract and γ-schisandrin can modulate the TRPV3 channel. This report also suggests a mechanism by which γ-schisandrin acts as a therapeutic agent against TRPV3-related diseases.

Increased activity of TRPV3 in keratinocytes in hypertrophic burn scars with postburn pruritus

Postburn pruritus is a common distressing sequela of burn wounds. Empirical antipruritic treatment often fails to have a satisfactory outcome, as the mechanism of it has not been fully elucidated. The aim of this study was to evaluate the manifestation of transient receptor potential vanilloid 3 (TRPV3), transient receptor potential ankyrin 1 (TRPA1), and other related receptors in postburn pruritus. Sixty-five burn patients with (n = 40) or without (n = 25) pruritus were investigated, including skin biopsies. Keratinocytes and fibroblasts from skin biopsy samples were separated. Real time-PCR showed that mRNA of TRPV3 was significantly increased in keratinocytes from pruritic burn scars than in keratinocytes from nonpruritic burn scars. With TRPV3 activation, intracellular Ca2+ concentrations were more significantly increased in keratinocytes from pruritic burn scars than in those from nonpruritic ones. Additionally, mRNA and protein levels of protease-activated receptor 2 (PAR2) and neurokinin-1 receptor (NK1R) were also significantly increased in pruritic burn scars. In conclusion, it was confirmed that TRPV3, PAR2, and NK1R were highly expressed in pruritic burn scars. These results may help determine a novel mechanism for postburn pruritus.

Modulatory effects of the fruits of Tribulus terrestris L. on the function of atopic dermatitis-related calcium channels, Orai1 and TRPV3

ObjectiveTo examine the effects of Tribulus terrestris L. (T. terrestris) extract on the modulation of calcium channels to evaluate its use in topical agents for treatment of atopic dermatitis. MethodsThe 70% methanol extract of T. terrestris was prepared. Human HEK293T cells with over-expressed calcium release-activated calcium channel protein 1 (Orai1), transient receptor potential vanilloid 1, or transient receptor potential vanilloid 3 (TRPV3) were treated with T. terrestris extract. Modulation of ion channels was measured using a conventional whole-cell patch-clamp technique. ResultsT. terrestris extract (100 μg/mL) significantly inhibited Orai1 activity in Orai1-stromal interaction molecule 1 co-overexpressed HEK293T cells. In addition, T. terrestris extract significantly increased the TRPV3 activity compared with 2-Aminoethyl diphenylborinate (100 μmol/L), which induces the full activation of TRPV3. ConclusionsOur results suggest that T. terrestris extract may have a therapeutic potential for recovery of abnormal skin barrier pathologies in atopic dermatitis through modulating the activities of calcium ion channels, Orai1 and TRPV3. This is the first study to report the modulatory effect of a medicinal plant on the function of ion channels in skin barrier.

Overexpression of TRPV3 Correlates with Tumor Progression in Non-Small Cell Lung Cancer.

(1) Background: Transient receptor potential vanilloid 3 (TRPV3) is a member of the TRP channels family of Ca2+-permeant channels. The proteins of some TRP channels are highly expressed in cancer cells. This study aimed to assess the clinical significance and biological functions of TRPV3 in non-small cell lung cancer (NSCLC); (2) Methods: Immunohistochemistry was used to detect the expression of TRPV3 in NSCLC tissues and adjacent noncancerous lung tissues. Western blot was used to detect the protein expressions of TRPV3, CaMKII, p-CaMKII, CyclinA, CyclinD, CyclinE1, CDK2, CDK4, and P27. Small interfering RNA was used to deplete TRPV3 expression. A laser scanning confocal microscope was used to measure intracellular calcium concentration ([Ca2+]i). Flow cytometry was used to analyze cell cycle; (3) Results: TRPV3 was overexpressed in 65 of 96 (67.7%) human lung cancer cases and correlated with differentiation (p = 0.001) and TNM stage (p = 0.004). Importantly, TRPV3 expression was associated with short overall survival. In addition, blocking or knockdown of TRPV3 could inhibit lung cancer cell proliferation. Moreover, TRPV3 inhibition could decrease [Ca2+]i of lung cancer cells and arrest cell cycle at the G1/S boundary. Further results revealed that TRPV3 inhibition decreased expressions of p-CaMKII, CyclinA, CyclinD1, CyclinE, and increased P27 level; (4) Conclusions: Our findings demonstrate that TRPV3 was overexpressed in NSCLC and correlated with lung cancer progression. TRPV3 activation could promote proliferation of lung cancer cells. TRPV3 might serve as a potential companion drug target in NSCLC.

TRPV3 Channel Negatively Regulates Cell Cycle Progression and Safeguards the Pluripotency of Embryonic Stem Cells.

Embryonic stem cells (ESCs) have tremendous potential for research and future therapeutic purposes. However, the calcium handling mechanism in ESCs is not fully elucidated. Aims of this study are (1) to investigate if transient receptor potential vanilloid-3 (TRPV3) channels are present in mouse ESCs (mESCs) and their subcellular localization; (2) to investigate the role of TRPV3 in maintaining the characteristics of mESCs. Western blot and immunocytochemistry showed that TRPV3 was present at the endoplasmic reticulum (ER) of mESCs. Calcium imaging showed that, in the absence of extracellular calcium, TRPV3 activators camphor and 6-tert-butyl-m-cresol increased the cytosolic calcium. However, depleting the ER store in advance of activator addition abolished the calcium increase, suggesting that TRPV3 released calcium from the ER. To dissect the functional role of TRPV3, TRPV3 was activated and mESC proliferation was measured by trypan blue exclusion and MTT assays. The results showed that TRPV3 activation led to a decrease in mESC proliferation. Cell cycle analysis revealed that TRPV3 activation increased the percentage of cells in G2 /M phase; consistently, Western blot also revealed a concomitant increase in the expression of inactive form of cyclin-dependent kinase 1, suggesting that TRPV3 activation arrested mESCs at G2 /M phase. TRPV3 activation did not alter the expression of pluripotency markers Oct-4, Klf4 and c-Myc, suggesting that the pluripotency was preserved. Our study is the first study to show the presence of TRPV3 at ER. Our study also reveals the novel role of TRPV3 in controlling the cell cycle and preserving the pluripotency of ESCs.

The thermosensitive TRPV3 channel contributes to rapid wound healing in oral epithelia.

The oral cavity provides an entrance to the alimentary tract to serve as a protective barrier against harmful environmental stimuli. The oral mucosa is susceptible to injury because of its location; nonetheless, it has faster wound healing than the skin and less scar formation. However, the molecular pathways regulating this wound healing are unclear. Here, we show that transient receptor potential vanilloid 3 (TRPV3), a thermosensitive Ca(2+)-permeable channel, is more highly expressed in murine oral epithelia than in the skin by quantitative RT-PCR. We found that temperatures above 33°C activated TRPV3 and promoted oral epithelial cell proliferation. The proliferation rate in the oral epithelia of TRPV3 knockout (TRPV3KO) mice was less than that of wild-type (WT) mice. We investigated the contribution of TRPV3 to wound healing using a molar tooth extraction model and found that oral wound closure was delayed in TRPV3KO mice compared with that in WT mice. TRPV3 mRNA was up-regulated in wounded tissues, suggesting that TRPV3 may contribute to oral wound repair. We identified TRPV3 as an essential receptor in heat-induced oral epithelia proliferation and wound healing. Our findings suggest that TRPV3 activation could be a potential therapeutic target for wound healing in skin and oral mucosa.

Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part, due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 overexpressing human embryonic kidney-293 (HEK-293) cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

TRPV3.

Transient receptor potential vanilloid-3 (TRPV3) is a Ca(2+)-permeable nonselective cation channel widely expressed in skin keratinocytes, as well as oral and nasal epithelia. TRPV3 is activated by innocuous warm as well as noxious hot temperatures. Activation of TRPV3 in skin keratinocytes causes release of multiple substances, which in turn regulate diverse functions including skin barrier formation, hair growth, wound healing, temperature sensing, and itch and pain perceptions. While several natural and synthetic ligands have been described for TRPV3, only one of them, farnesyl pyrophosphate, is naturally produced in animal cells. Together with the use of genetic mouse models, applications of these compounds have revealed not only the physiological functions but also regulatory mechanisms of TRPV3 channel by extracellular Ca(2+), Mg(2+), and protons as well as intracellular Ca(2+)-calmodulin, ATP, phosphatidylinositol 4,5-bisphosphate, polyunsaturated fatty acids, protons, and Mg(2+). Gain-of-function genetic mutations of TRPV3 in rodents and humans have been instrumental in unveiling the critical role of this channel in skin health and disease.

Intracellular Proton-mediated Activation of TRPV3 Channels Accounts for the Exfoliation Effect of α-Hydroxyl Acids on Keratinocytes

α-Hydroxyl acids (AHAs) from natural sources act as proton donors and topical compounds that penetrate skin and are well known in the cosmetic industry for their use in chemical peels and improvement of the skin. However, little is known about how AHAs cause exfoliation to expose fresh skin cells. Here we report that the transient receptor potential vanilloid 3 (TRPV3) channel in keratinocytes is potently activated by intracellular acidification induced by glycolic acid. Patch clamp recordings and cell death assay of both human keratinocyte HaCaT cells and TRPV3-expressing HEK-293 cells confirmed that intracellular acidification led to direct activation of TRPV3 and promoted cell death. Site-directed mutagenesis revealed that an N-terminal histidine residue, His-426, known to be involved in 2-aminoethyl diphenylborinate-mediated TRPV3 activation, is critical for sensing intracellular proton levels. Taken together, our findings suggest that intracellular protons can strongly activate TRPV3, and TRPV3-mediated proton sensing and cell death in keratinocytes may serve as a molecular basis for the cosmetic use of AHAs and their therapeutic potential in acidic pH-related skin disorders.

Activation of Transient Receptor Potential Vanilloid-3 Inhibits Human Hair Growth

In the current study, we aimed at identifying the functional role of transient receptor potential vanilloid-3 (TRPV3) ion channel in the regulation of human hair growth. Using human organ-cultured hair follicles (HFs) and cultures of human outer root sheath (ORS) keratinocytes, we provide the first evidence that activation of TRPV3 inhibits human hair growth. TRPV3 immunoreactivity was confined to epithelial compartments of the human HF, mainly to the ORS. In organ culture, TRPV3 activation by plant-derived (e.g., eugenol, 10-1,000 μM) or synthetic (e.g., 2-aminoethoxydiphenyl borate, 1-300 μM) agonists resulted in a dose-dependent inhibition of hair shaft elongation, suppression of proliferation, and induction of apoptosis and premature HF regression (catagen). Human ORS keratinocytes also expressed functional TRPV3, whose stimulation induced membrane currents, elevated intracellular calcium concentration, inhibited proliferation, and induced apoptosis. Of great importance, these effects on ORS keratinocytes were all mediated by TRPV3, as small interfering RNA-mediated silencing of TRPV3 effectively abrogated the cellular actions of the above agonists. These findings collectively support the concept that TRPV3 signaling is a significant player in human hair growth control. Therefore, TRPV3 and the related intracellular signaling mechanism might function as a promising target for pharmacological manipulations of clinically relevant hair growth disorders.