Effects of Skin Surface Temperature on Epidermal Permeability Barrier Homeostasis

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1 ORIGINAL ARTICLE Effects of Skin Surface Temperature on Epidermal Permeability Barrier Homeostasis Mitsuhiro Denda 1, Takaaki Sokabe 2, Tomoko Fukumi-Tominaga 2 and Makoto Tominaga 2 Members of the transient receptor potential (TRP) family are temperature sensors, and TRPV1, V3, and V4 are expressed in epidermal keratinocytes. To evaluate the influence of these receptors on epidermal permeability barrier homeostasis, we kept both hairless mouse skin and human skin at various temperatures immediately after tape stripping. At temperatures from 36 to 41C, barrier recovery was accelerated in both cases compared with the area at 341C. At 34 or 421C, barrier recovery was delayed compared with the un-occluded area. 4a-Phorbol 12,13-didecanone, an activator of TRPV4, accelerated barrier recovery, whereas ruthenium red, a blocker of TRPV4, delayed barrier recovery. Capsaicin, an activator of TRPV1, delayed barrier recovery, whereas capsazepin, an antagonist of TRPV1, blocked this delay. 2-Aminoethoxydiphenyl borate and camphor, TRPV3 activators, did not affect the barrier recovery rate. As TRPV4 is activated at about 351C and above, whereas TRPV1 is activated at about 421C and above, these results suggest that both TRPV1 and TRPV4 play important roles in skin permeability barrier homeostasis. Previous reports suggest the existence of a water flux sensor in the epidermis, and as TRPV4 is known to be activated by osmotic pressure, our results indicate that it might be this sensor. Journal of Investigative Dermatology (27) 127, doi:1.138/sj.jid.59; published online 19 October 26 INTRODUCTION One of the most important roles of the skin in terrestrial mammals is to act as a water-impermeable barrier to prevent excessive transcutaneous water loss. A decline in barrier function often parallels increased severity of clinical symptomatology (Elias and Feingold, 21). When the stratum corneum barrier is damaged, a series of barrier-related homeostatic processes is immediately accelerated, and barrier function is restored to its original level (Elias and Feingold, 21). Grubauer et al. (1989) demonstrated that occlusion with a water-impermeable membrane immediately after barrier disruption blocked barrier recovery, whereas occlusion with a water-permeable membrane did not perturb barrier recovery. Moreover, we previously demonstrated that environmental humidity affects barrier function (Denda et al., 1998a). These results suggest that there might be a sensor of water flux from the skin surface and a monitoring system that regulates barrier homeostasis. Recently, members of the transient receptor potential (TRP) receptor family have been reported to act as sensors of temperature or other physical or chemical factors (Tominaga and Caterina, 24), and TRPV1, TRPV3, and TRPV4 were shown to be present in epidermal 1 Shiseido Research Center, Kanazawa-ku, Yokohama, Japan and 2 Okazaki Institute for Integrative Bioscience, Okazaki, Aichi, Japan Correspondence: Dr Mitsuhiro Denda, Shiseido Research Center, , Fukuura, Kanazawa-ku, Yokohama , Japan. mitsuhiro.denda@to.shiseido.co.jp Abbreviations: 2APB, 2-aminoethoxydiphenyl borate; TEWL, transepidermal water loss; TRP, transient receptor potential Received 19 March 26; revised 21 August 26; accepted 26 August 26; published online 19 October 26 keratinocytes (Denda et al., 21; Guler et al., 22; Inoue et al., 22; Chung et al., 23). As TRPV4 is activated by osmotic pressure, it might act as a sensor of water flux from the skin surface (Tominaga and Caterina, 24). Thus, we hypothesized that these receptors contribute to skin permeability barrier homeostasis. In this study, we kept skin from hairless mice and human skin at different temperatures immediately after tape stripping in order to evaluate the effect of temperature on skin permeability barrier homeostasis. We also examined the effects of topical application of activators or inhibitors of TRPV1, TRPV3, and TRPV4 on the recovery of barrier function. RESULTS The barrier recovery of both mouse (Figure 1) and human skin (Figure 2) was accelerated at temperatures between 36 and 41C compared with the area at 341C, whereas at 34 or 421C, it was delayed compared with the un-occluded area (Figures 1 and 2). As the skin surface temperature of the control hairless mice was around 331C, the delays of barrier recovery at 32 and 341C compared with the un-occluded area are considered to have been owing to the occlusion by the temperature control pads. The pads did not cause any apparent irritation. Topical application of capsaicin, a TRPV1 activator, on hairless mouse skin after tape stripping delayed barrier recovery, whereas application of capsazepine, a TRPV1 blocker, accelerated barrier repair. On the other hand, topical application of 4a-phorbol 12,13-didecanone (4a- PDD), a TRPV4 activator, accelerated barrier recovery, whereas application of ruthenium red, a TRPV4 blocker, 654 Journal of Investigative Dermatology (27), Volume 127 & 26 The Society for Investigative Dermatology

2 a hour CNT b hours * CNT P <.1 P <.1 *P <.5 F = , P <.1 n = 8 each c hours 24 hours 1 CNT d 5 CNT Figure 1. Effects of application of different temperatures immediately after tape stripping on hairless mouse skin. Immediately after application of heat at 36 41Cfor(a) 1 hour, barrier recovery was accelerated compared with the area at 341C, whereas application of heat at 32, 34, or 421C delayed barrier recovery compared with un-occluded area. Similar tendencies were observed at (b) 3 and(c) 6 hours after barrier disruption, but not at (d) 24 hours. a hour CNT b hours CNT P <.1 P <.1 *P <.5 F = 3.724, P <.1 n = 4 each c hours 1 24 hours * CNT d 5 CNT * Figure 2. Effects of application of different temperatures immediately after tape stripping of human skin. Similar tendencies to those found for hairless mouse skin (Figure 1) were seen. Immediately after application of heat at 36 41C for(a) 1 hour, barrier recovery was accelerated compared with the area at 341C, whereas application of heat at 34 or 421C delayed barrier recovery compared with un-occluded area. Similar tendencies were observed at (b) 3 and (c) 6 hours after barrier disruption, but not at (d) 24 hours

3 delayed recovery. Application of 2-aminoethoxydiphenyl borate (2APB) and camphor, both TRPV3 activators, did not affect the barrier recovery rate (Figure 3). Topical application of capsazepine blocked the delay of barrier recovery at 421C, whereas application of ruthenium red blocked the acceleration of barrier recovery at 361C (Figure 4). Application of 4a-PDD, a TRPV4 activator, to cultured mouse keratinocytes increased the intracellular calcium level, and the increase was blocked by ruthenium red. These results suggest functional activity of TRPV4 in keratinocytes (Figure 5). DISCUSSION We have recently demonstrated that various neurotransmitter receptors, originally found in the nervous system, also exist in epidermal keratinocytes, and many of them are involved in the mechanisms of epidermal permeability barrier homeostasis (Denda, 26). These receptors influence lamellar body secretion and barrier homeostasis (Denda, 26). The TRP family of polymodal receptors was also discovered mainly in the nervous system, where some of them play a role in thermosensation (Tominaga and Caterina, 24). Among the TRP receptors, TRPV1, TRPV3, and TRPV4 are expressed in epidermal keratinocytes (Denda et al., 21; Inoue et al., 22; Chung et al., 24a, b). We have reported the expression of TRPV1 in human epidermis and keratinocytes (Denda et al., 21). TRPV4 is expressed in rat epidermis and mouse keratinocytes (Guler et al., 22; Chung et al., 24a, b). The TRP receptors also affect ion dynamics in keratinocytes (Inoue et al., 22; Chung et al., 24a, b). TRPV1 is activated by heat (4431C), acidic conditions (pho6.6), and capsaicin. TRPV3 is activated by heat (4351C), mechanical stress, camphor, and 2APB. TRPV4 is activated by heat (4351C), osmotic pressure, and 4a-PDD (Hu et al., 24; Tominaga and Caterina, 24; Xu et al., ). In this study, application of heat between 36 and 41C accelerated the barrier recovery of both hairless mouse and human skin after barrier disruption by tape stripping. TRPV3 and TRPV4 are both activated at temperatures of C and above (Peier et al., 22; Chung et al., 23). However, in this study, topical application of TRPV3 activators did not affect the barrier recovery rate. On the other hand, application of a TRPV4 activator, 4a-PDD, accelerated barrier recovery, whereas application of a TRPV4 blocker, ruthenium red, delayed it. Moreover, the acceleration of barrier repair at 361C was blocked by ruthenium red. These results suggest that TRPV4, but not TRPV3, is associated with epidermal permeability barrier homeostasis. At 421C, the barrier recovery of both hairless mouse and human skin was delayed. TRPV1 is activated at approximately 421C, and topical application of the TRPV1 activator capsaicin delayed barrier recovery, whereas application of P <.1 NS P <.1 P <.1 Control 36 C 36 C + 42 C Ru-Red NS P <.1 42 C + Capsazepine Figure 4. Ruthenium red and capsazepine blocked the effects of heat on the barrier recovery rate. Topical application of capsazepine blocked the delay of barrier recovery by heat at 421C and application of ruthenium red blocked the acceleration of barrier recovery by heat at 361C. a b c Control Control 2APB Capsazepine Campher Capsaicin Hours Hours Control 4α-PDD Ru-RED Hours F = 113.6, P <.1, n = 8 each F = 137.2, P <.1, n = 8 each F = 53.4, P <.1, n = 8 each P <.1 P <.1 Figure 3. Effects of topical application of activators and inhibitors of TRP receptors. (a) Topical application of capsaicin, a TRPV1 activator, to hairless mouse skin after tape stripping delayed barrier recovery, whereas application of capsazepine, a TRPV1 blocker, accelerated barrier repair. (b) Application of 2APB and camphor, both TRPV3 activators, did not affect the barrier recovery rate. (c) On the other hand, topical application of 4a-PDD, a TRPV4 activator, accelerated barrier recovery, whereas application of ruthenium red delayed the recovery. 656 Journal of Investigative Dermatology (27), Volume 127

4 Ratio (34 / 38) μm 4 α-pdd Time (seconds) 1 μm RR Figure 5. TRPV4 is functional in keratinocytes. Mouse primary keratinocytes were treated with the synthetic TRPV4 agonist 4aPDD as indicated, and the changes in intracellular Ca 2 þ were measured in terms of the fluorescence ratio at 34 and 38 nm (see text) every 5 seconds. Ruthenium red (RR) was added to block TRPV4 activation. Data are presented as mean7sem from three independent experiments. Values in each experiment were obtained from 6 to 9 cells. the TRPV1 inhibitor capsazepine accelerated barrier recovery. Moreover, the delay of barrier recovery by 421C temperature conditions was blocked by capsazepine. These results suggest that TRPV1 in epidermal keratinocytes might also be associated with skin permeability barrier homeostasis. Recently, Xu et al. () demonstrated that camphor activates TRPV1 at high concentrations, and Hu et al. (24) reported that 2APB also activates TRPV1 at high concentrations. However, in this study, topical application of camphor and 2APB did not affect the skin barrier recovery rate, whereas both capsaicin and capsazepine significantly affected it. We previously demonstrated that TRPV1 is functional in cultured keratinocytes (Inoue et al., 22). Intracellular calcium was increased by capsaicin and low ph (5.5), and the increase was blocked by capsazepine. However, the responses to capsaicin and low ph in keratinocytes were slower than those in neurons. Thus, TRPV1 in keratinocytes might have different characteristics from those in neurons. An electrophysiological study of TRPV1 in epidermal keratinocytes would be of interest. The difference of the barrier recovery rates of treated skin appeared within 1 hour after barrier disruption. Barrier recovery is thought to occur in two stages. First, immediately after barrier disruption, exocytosis of lamellar bodies, which contain intercellular lipids, is accelerated, and intercellular lipid bilayer structure is formed within 3 minutes after barrier disruption. In the second stage, epidermal lipid synthesis starts 6 12 hours after barrier disruption. Thus, activation or inhibition of TRPV1 and TRPV4 might affect the earlier stage, that is, lamellar body secretion and formation of intercellular lipid bilayer structure between the stratum granulosum and stratum corneum. The mechanisms underlying the present results remain to be clarified. We previously demonstrated that influx of calcium ions into epidermal keratinocytes perturbs lamellar body exocytosis and consequently delays barrier recovery after barrier disruption (Denda et al., 23). TRPV1 is an ionotropic receptor with high Ca permeability. Thus, the delay of barrier recovery by the activation of TRPV1 might be the result of calcium influx into keratinocytes. On the other hand, although TRPV4 is also an ionotropic receptor, with cation permeability, activation of TRPV4 accelerated barrier recovery. In this study, we demonstrated that the activation of TRPV4 caused an increase of intracellular calcium. The activation of TRPV4 might induce some other signaling system that regulates lamellar body secretion or the stability of intercellular lipid bilayer structure after barrier disruption. TRPV4 is activated by an osmotic stimulus (Tominaga and Caterina, 24). Thus, this receptor might play a crucial role as a sensor of humidity or water flux from the skin surface. The existence of a sensory system for water flux or environmental humidity as a part of epidermal permeability barrier homeostasis has been suggested (Grubauer et al., 1989; Denda et al., 1998a), and our results suggest that TRPV4 might be this sensor. Further work is needed to understand the role of TRPV4 in epidermal barrier homeostasis. Although barrier function is repairable, repeated damage or damage under conditions of low environmental humidity may cause epidermal hyperplasia or inflammation (Denda et al., 1996, 1998b). Moreover, a variety of dermatoses, such as atopic dermatitis, psoriasis, and contact dermatitis, are characterized by barrier dysfunction (Grice, 198). Interestingly, for normal humans, the temperature range between 36 and 41C is regarded as a pleasant temperature (Mower, 1976). Our emotional and sensory systems seem to know the appropriate temperature range for skin barrier homeostasis. Our results suggest that it may be possible to treat the above skin problems by appropriately regulating TRP receptors to normalize skin barrier homeostasis, either by adjusting the skin surface temperature or by topical administration of suitable receptor modulators. MATERIALS AND METHODS Materials All experiments were performed on 7- to 1-week-old male hairless mice (HR-1, Hoshino, Japan). All procedures for measuring skin barrier function, disrupting the barrier, and applying the sample were carried out under anesthesia. All experiments were approved by the Animal Research Committee of the Shiseido Research Center in accordance with the National Research Council Guide (National Research Council, 1996). Human skin experiments were carried out on the inner forearm of healthy males, who had given informed consent. The human experiments adhered to the Declaration of Helsinki Principles. 4a-PDD and ruthenium red were purchased 657

5 from Sigma-Aldrich (St Louis, MO). Capsaicin, capsazepine, and 2APB were purchased from Tocris (Bristol, UK). Camphor was purchased from Wako (Osaka, Japan). Capsaicin is a specific agonist of TRPV1 and capsazepine is a specific antagonist of TRPV1 (Caterina et al., 1997). 2APB and camphor are activators of TRPV3 (Chung et al., 23; Moquich et al., ). 4a-PDD is a specific activator of TRPV4 and ruthenium red is a non-specific blocker of TRPV4 (Watanabe et al., 22). Cutaneous barrier function Permeability barrier function was evaluated by measurement of transepidermal water loss (TEWL) with an electric water analyzer, as described previously (Denda et al., 1998a). For barrier recovery experiments, both sides of the flank skin were subjected to repeated tape stripping until the TEWL reached 7 1 mg per cm 2 per hour, as described previously (Denda et al., 1998a). Immediately after barrier disruption, 1 ml of an aqueous solution containing 1 mm reagent or water alone (control) was applied to the treated area. We did not apply the same reagent to both flanks. The treated areas were covered with plastic membrane for 15 minutes and then the membrane was removed. Two points on each side of the flank were measured and 4 8 mice were used to evaluate the effects of each treatment. We always disrupted the barrier between 7 and 8 hours, followed by measurements of barrier repair, to avoid the influence of circadian rhythm on repair rate (Denda and Tsuchiya, 2). TEWL was measured over the same sites at 1, 3, and 6 hours after barrier disruption. The barrier recovery results are expressed as percent recovery, because of day-to-day variations in the extent of barrier disruption. In each animal, the percent recovery was calculated by use of the following formula: (TEWL immediately after barrier disruption TEWL at indicated time point)/(tewl immediately after barrier disruption baseline TEWL) 1%. All experiments were performed on 7- to 1-week-old male hairless mice (HR-1, Hoshino, Japan). All procedures for measurement of skin barrier function, disruption of the barrier, and application of test samples were carried out under anesthesia. Control of skin temperature To achieve a constant required skin temperature, we used a siliconerubber-coated heater (Sakaguchi Dennetsu, Tokyo, Japan) with a Watlow temperature control system (Sakaguchi Dennetsu, Tokyo, Japan). A small cooling pad was used when the required temperature was 321C. The size of the heater was 5 1 cm 2, and it was attached directly to the treated skin surface immediately after tape stripping. After 1 hour, we removed the heater and started to measure the TEWL. Thus, heat was applied for only 1 hour at the beginning of experiments. Un-occluded skin was not heated; during the experimental period, the control skin surface temperature was between 32 and 341C. Preparation of primary keratinocytes from newborn mice Primary keratinocytes were obtained from newborn mice (postnatal day ) using a modification of a reported method (Chung et al., 24b). Pups were decapitated and soaked in 3% iodine tincture for 3 minutes. The trunks were washed with dh 2 O and 7% ethanol, the limbs were removed, and the trunk skin was peeled off. The skin was stripped of fat, and floated (epidermis upward) for 16 hours at 41C in a culture dish containing.% trypsin. The next day, the epidermis was peeled from the underlying tissue. Keratinocytes were harvested from the surface of a dissection plane by gentle scraping and suspended in mouse keratinocyte medium (Chung et al., 23). Cells were pelleted (4 minutes, 1, r.p.m., 41C), resuspended in mouse keratinocyte medium, layered on a density gradient (Lymphoprep, Axis-shield, Oslo, Norway), and centrifuged (15 minutes, 5 r.p.m., 41C). Keratinocytes, located at the interface of the resulting two layers, were collected, pelleted (5 minutes, 1, r.p.m., room temperature), and resuspended in modified chemically defined medium 153 containing 1% heat-inactivated fetal bovine serum. Cells were counted and 5 ml of suspension ( cell/ml) was dropped on 12 mm glass coverslips in a culture dish, and incubated at 371C under 5% CO 2 in air. After 3 hours, modified chemically defined medium 153 containing 1% fetal bovine serum was added to the culture dish, and incubation was continued overnight under the same conditions. The next day, the medium was changed to modified chemically defined medium, and culture was continued until the cells became subconfluent. The medium was replaced with modified chemically defined medium 153 containing 1.2 mm CaCl 2 and assays were conducted after 24 hours. Calcium-imaging experiment Intracellular Ca 2 þ changes in individual cells were measured with a fluorescent Ca 2 þ indicator dye, fura-2, according to Grynkiewicz et al. (1985), by means of dual excitation using a fluorescence imaging system illumination controller (MAC 5, High Speed Filter Wheel Module, Ludl Electronics Products Ltd, Mid Hudson, NY) controlled with appropriate software (IP Lab, Scanalytics Inc., Rockville, MD). To load the dye, cells were incubated in the culture media containing 5 mm fura-2, AM (F121, Molecular Probes, Eugene, OR) at 331C for 1 hour. The glass coverslip was washed, and the fura-2 fluorescence of cells was measured in standard bath solution containing (in mm) 14 NaCl, 5 KCl, 2 MgCl 2, 1 CaCl 2,1 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid, and 1 glucose (ph 7.4) (adjusted with NaOH). A coverslip with fura-2- loaded cells was mounted in a chamber (RC-26G with mount SA-NIK, Warner Instruments Inc., Hamden, CT) on the stage of a microscope (IX7, Olympus with Uplan FLN 1/.3 lenses, Olympus) and a filter set (Chroma, Rockingham, VT). The ratio of fluorescence at 34:38 nm was measured. Briefly, cells were illuminated every 5 seconds at 34 and 38 nm. The emitted fluorescence was projected onto a CCD camera (CoolSNAP ES, Roper Scientific Photometrics, Tucson, AZ) and data were stored on the hard drive of a computer. All experiments were performed at room temperature (24 1C). Chamber temperature was monitored with a thermocouple (TA-3, Warner Instruments, Hamden, CT) and sampled with an A/D converter (Digidata 1322A with pclamp 8.2 software, Axon Instruments, Sunnyvale, CA). Statistics The results are expressed as mean values7sd. The statistical significance of differences between two groups was determined with the two-tailed Student s t-test. For more than two groups, significance was determined by means of analysis of variance with Fisher s protected least significant difference. CONFLICT OF INTEREST The authors state no conflict of interest. 658 Journal of Investigative Dermatology (27), Volume 127

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