Acta Dermato-Venereologica 99-10CompleteContent | Page 16

SHORT COMMUNICATION 901 Signal Transducer and Activator of Transcription 3 in Keratinocytes Regulates Histaminergic Itch but Not Nonhistaminergic Itch Takashi HASHIMOTO 1,2 , Kent SAKAI 1 , Gil YOSIPOVITCH 1 and Tasuku AKIYAMA 1 Dr. Phillip Frost Department of Dermatology and Cutaneous Surgery, Miami Itch Center, University of Miami Miller School of Medicine, Miami, FL 33136, USA, and 2 Department of Dermatology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. E-mail: [email protected] 1 Accepted May 29, 2019; E-published May 29, 2019 Itch, an unpleasant sensation that evokes scratching beha- vior, is divided into two forms: histaminergic and nonhis- taminergic itch. Histaminergic itch is caused by histamine that is secreted principally by mast cells and basophils, while nonhistaminergic itch is evoked by various prurito- gens other than histamine from multiple origins. Recent findings suggest that not only the interaction between pruritogens and peripheral pruriceptor terminals, but also the interplay among peripheral pruriceptor terminals, im- mune cells, and epidermal keratinocytes play roles in itch induction (1, 2). However, the molecular mechanisms of this interplay are not fully understood. Signal transducer and activator of transcription (STAT)3 is a transcription factor (3) that has recently been high- lighted as a key player in itch sensation. In the spinal cord, STAT3 amplifies itch sensation through promoting astrogliosis (4). While STAT3 plays critical roles in the regulation of keratinocyte function (3), whether STAT3 in the skin is also important in itch sensation is still unclear. Since keratinocytes are capable of producing itch medi- ators (2), keratinocyte STAT3 may also modulate itch. The aim of this study was to elucidate the involvement of keratinocyte STAT3 in itch sensation. MATERIALS AND METHODS AND RESULTS To examine the role of STAT3 in keratinocytes in vivo, we cros- sed Stat3 flox mice with K5CreERT2 mice, and tamoxifen was injected to delete STAT3 in keratinocytes. Keratinocyte-specific STAT3-depletion was confirmed through immunohistochemistry (Fig. S1 1 ). Vehicle-treated K5CreERT2:Stat3 flox/+ mice were used as controls. For further information on materials and methods, see Appendix S1 1 . All animal experiments were approved by the Institutional Committee of the University of Miami. We initially assessed the in vivo effect of keratinocyte-specific STAT3 depletion on acute itch in mice. The acute itch was https://www.medicaljournals.se/acta/content/abstract/10.2340/00015555-3229 1 evoked by intradermally injecting either histaminergic or non- histaminergic pruritogen (histamine, serotonin, or chloroquine). All 3 pruritogens evoked scratching responses in control mice. Histamine-evoked scratching behavior was significantly attenuated in STAT3 conditional knockout (cKO) mice while serotonin- and chloroquine-induced scratching responses did not change (Fig. 1). We also examined the impact of keratinocyte STAT3 on chronic itch using ovalbumin (OVA)-induced atopic dermatitis (AD) murine model, which has been shown to involve nonhistaminergic itch (5). We did not find any significant reduction of OVA-induced scratching responses in STAT3 cKO mice (Fig. S2 1 ). These results demonstrate that keratinocyte STAT3 contributes to histaminergic itch, but not nonhistaminergic itch. To determine whether pruritogens elevate intracellular calcium in keratinocytes, we performed calcium imaging experiments on keratinocytes. In keratinocytes from control mice, intracellular calcium gradually increased and reached a maximum level 90- 150 s after application of histamine and serotonin (Fig. S3a–c 1 ). However, chloroquine did not increase intracellular calcium (Fig. S3d 1 ), which is consistent with a previous report (1). The chloroquine receptor, Mas-related G protein coupled-receptor member A3, is expressed exclusively by sensory neurons, but not keratinocytes, suggesting that chloroquine evokes itch without affecting keratinocytes. We next examined whether STAT3 is involved in intracellular calcium elevations by pruritogens in keratinocytes. The increased responses of keratinocytes to histamine and serotonin were sig- nificantly reduced in STAT3 cKO mice (Fig. S3a–c 1 ). This result was confirmed using a STAT3 inhibitor, STA-21. Keratinocytes treated with STA-21 for one hour showed a significant reduction of intracellular calcium responses compared to vehicle-treated keratinocytes (Fig. S3e, f 1 ). A recent report has shown that histamine-induced Ca 2+ influx in keratinocytes is dependent on transient receptor potential cation channel subfamily V member 4 (TRPV4), a highly calcium selec- tive ion channel, and TRPV4-mediated Ca 2+ influx is required for histaminergic itch (2). Thus, we examined the effects of a selective TRPV4 inhibitor, GSK205, on the intracellular calcium responses to histamine and serotonin in keratinocytes. The magnitude of in- tracellular calcium responses of keratinocytes from naive mice was significantly reduced by the application of GSK205 (Fig. S3e, f 1 ). To investigate whether STAT3 is involved in TRPV4 expression, we performed immunostaining experiments on epidermal sheets. In epidermal sheets from control mice, TRPV4 was detected Fig. 1. Effects of in vivo depletion of keratinocyte STAT3 on scratching behaviors. (a) K5CreERT2:Stat3 flox/+ mice were injected with either tamoxifen (cKO) or vehicle (Ctrl). cKO mice showed attenuated histamine-induced scratching behavior in comparison with Ctrl mice. (b,c) Scratching bouts in response to serotonin (b) and chloroquine (c) did not change in cKO mice compared with Ctrl mice. Vertical bars indicate mean + SEM (n = 6–7 mice/group). * p < 0.05 versus Ctrl mice, two-tailed unpaired t-test. This is an open access article under the CC BY-NC license. www.medicaljournals.se/acta Journal Compilation © 2019 Acta Dermato-Venereologica. doi: 10.2340/00015555-3229 Acta Derm Venereol 2019; 99: 901–902