Ghrelin Represses Thymic Stromal Lymphopoietin Gene Expression through Activation of Glucocorticoid Receptor and Protein Kinase C Delta in Inflamed Skin Keratinocytes

Ghrelin, a peptide hormone secreted from enteroendocrine cells of the gastrointestinal tract, has anti-inflammatory activity in skin diseases, including dermatitis and psoriasis. However, the molecular mechanism underlying the beneficial effect of ghrelin on skin inflammation is not clear. In this study, we found that ghrelin alleviates atopic dermatitis (AD)-phenotypes through suppression of thymic stromal lymphopoietin (TSLP) gene activation. Knockdown or antagonist treatment of growth hormone secretagogue receptor 1a (GHSR1a), the receptor for ghrelin, suppressed ghrelin-induced alleviation of AD-like phenotypes and suppression of TSLP gene activation. We further found that ghrelin induces activation of the glucocorticoid receptor (GR), leading to the binding of GR with histone deacetylase 3 (HDAC3) and nuclear receptor corepressor (NCoR) NCoR corepressor to negative glucocorticoid response element (nGRE) on the TSLP gene promoter. In addition, ghrelin-induced protein kinase C δ (PKCδ)-mediated phosphorylation of p300 at serine 89 (S89), which decreased the acetylation and DNA binding activity of nuclear factor- κB (NF-κB) p65 to the TSLP gene promoter. Knockdown of PKCδ abolished ghrelin-induced suppression of TSLP gene activation. Our study suggests that ghrelin may help to reduce skin inflammation through GR and PKCδ-p300-NF-κB-mediated suppression of TSLP gene activation.


Introduction
Atopic dermatitis (AD) is a chronic skin inflammatory disease that is characterized by xerosis, intense pruritus, and eczema [1,2]. The estimated prevalence of AD in the worldwide population is 10~20% of children and 2~10% of adults [3,4]. AD disrupts a patient's quality of life through sleep disturbance, and AD is associated with other atopic disorders such as food allergy, allergic rhinitis, and asthma [5,6]. Although topical medications, including corticosteroids and calcineurin inhibitors, are widely used, they have undesirable side effects, which suggest the need for medications with higher efficacy and safety [7][8][9][10]. Although the etiology of AD is very complicated, currently, it is believed that epidermal barrier abnormalities and immune dysfunctions are the main reasons for the pathogenesis [11][12][13].
Thymic stromal lymphopoietin (TSLP), an IL-7-like cytokine, plays a key role in the development and progression of allergic diseases [14,15]. TSLP is highly expressed in lesional skin of AD patients and induces severe itching by directly activating TRPA1-positive sensory neurons [16][17][18]. Recently, anti-TSLP monoclonal antibody has been developed for

GHSR1a Is Required for Ghrelin-Induced Suppression of TSLP Gene Activation
To determine whether the anti-inflammatory effect of ghrelin is mediated through its receptor, growth hormone secretagogue receptor (GHSR) [27,[42][43][44], we first examined the gene expression of a functional receptor, GHSR1a [45][46][47][48]. Ghrelin did not alter the expression of GHSR1a in mRNA and protein level in HaCaT keratinocytes (Figure 2A,B). GHSR1a was expressed in the cell membrane of HaCaT keratinocytes ( Figure 2C). In mouse skin, we found positive signals for GHSR1a in epidermis and hair follicles ( Figure 2D). In addition, GHSR1a was expressed in DNFB-induced AD-like mouse skin (Supplementary Figure S2). To clarify the epidermal expression of GHSR1a, epidermis and dermis were isolated from mouse skin, and Western blot analysis was performed. Consistent with the results from immunohistochemistry, GHSR1a was expressed in the epidermis, which is positive for loricrin expression ( Figure 2E). We further confirmed the presence of GHSR1a by examination of ghrelin-induced calcium mobilization in HaCaT keratinocytes [49,50]. Although ghrelininduced calcium influx, depletion of GHSR1a by siRNA reduced ghrelin-induced calcium influx ( Supplementary Figures S3 and S4A). Next, we examined the effect of GHSR1a depletion on TSLP gene expression. As expected, depletion of GHSR1a by siRNA abolished ghrelin-induced suppression of TSLP gene activation and the gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin ( Figure 2F,G). the effect of GHSR1a depletion on TSLP gene expression. As expected, depletion of GHSR1a by siRNA abolished ghrelin induced suppression of TSLP gene activation and the gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin ( Figure 2F,G).   (FL). DIW was used as solvent for TNFα, flagellin, and ghrelin. Real-time PCR was performed to quantify TSLP and RPLP0 transcripts (n = 3). (H) Ghrelin suppresses TSLP gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin. After HaCaT keratinocytes were transiently transfected with luciferase reporter vector containing the TSLP gene promoter and control Renilla luciferase expression vector, luciferase activity was measured (n = 3). All data represent mean ± S.E.M. Significance values were ** p ≤ 0.01 and *** p ≤ 0.005.
flagellin. After HaCaT keratinocytes were transiently transfected with luciferase reporter vector containing the TSLP gene promoter and control Renilla luciferase expression vector, luciferase activity was measured (n = 3). All data represent mean ± S.E.M. Significance values were ** p ≤ 0.01 and *** p ≤ 0.005.

GHSR1a Antagonist Reduces Ghrelin-Induced Alleviation of AD-like Phenotypes
We next examined whether inhibition of GHSR1a has negative effects on ghrelininduced alleviation of AD-like phenotypes using a GHSR1 antagonist, [D-Lys3]-growth hormone releasing peptide 6 (GHRP6) [36,51,52]. Consistent with the depletion of GHSR1a, GHRP6 suppressed ghrelin induced suppression of TSLP gene activation and the gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin ( Figure 3A,B). Topical application of GHRP6 abolished ghrelin-induced alleviated ADlike phenotypes such as AD score, skin hyperplasia, and infiltrated mast cells ( Figure   was performed to quantify TSLP and RPLP0 transcripts (n = 3). RPLP0 was used as a control. DIW was used as solvent for TNFα, flagellin, ghrelin, and GHRP6. (B) GHRP6 abolishes ghrelin-induced suppression of TSLP gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin. After HaCaT keratinocytes were transiently transfected with luciferase reporter vector containing the TSLP gene promoter and control Renilla luciferase expression vector, luciferase activity was measured (n = 3). (C) GHRP6 abolishes ghrelin-induced alleviation of AD-like phenotypes. After mice were sensitized with DNFB for 7 days, DNFB was further topically applied to the shaved dorsal skin in combination with ghrelin or GHRP6 for 12 days (n = 6/group). Acetone was used as solvent for DNFB. DIW was used as solvent for ghrelin and GHRP6. Representative images are shown. (D) GHRP6 abolishes ghrelin-induced decrease in the severity of AD-like phenotypes. A scoring index of AD was used to determine the severity. (E) Skin tissue sections were stained with hematoxylin and eosin (H&E) or toluidine blue (TB). Representative images are shown. Scale bar, 100 µm. (F) GHRP6 abolishes ghrelin-induced decrease of epidermis thickness in AD-like skin lesions. Epidermis thickness was measured in H&E stained tissue sections (n = 6/group). (G) GHRP6 abolishes ghrelin-induced decrease in infiltration of mast cells in AD-like skin lesions. The number of mast cells was counted from 5 randomly selected low-power fields in TB stained tissue sections (n = 6/group). (H) GHRP6 abolishes ghrelin-induced suppression of TSLP gene activation in AD-like skin lesions. Real-time PCR was performed to quantify TSLP and RPLP0 transcripts from skin (n = 6/group). All data represent mean ± S.E.M. Significance values were * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.005.

Ghrelin Suppresses TSLP Gene Activation through GR Activation
It has been previously reported that subcutaneous infusion of ghrelin increases GHSR1a-dependent glucocorticoid receptor (GR) expression in peritoneal macrophages from injured rat skin [34]. Thus, we examined the effect of ghrelin on GR activation in HaCaT keratinocytes treated with TNFα or bacterial flagellin. Although we did not observe elevation of GR expression by ghrelin ( Figure 4A), ghrelin-induced phosphorylation of GR at serine 211 (S211) and nuclear localization of GR ( Figure 4A,B). GHRP6 abolished ghrelin-induced phosphorylation of GR (S211) and nuclear localization of GR ( Figure 4C,D). We next tested GR-dependent suppression of TSLP gene activation using siRNA against GR (Supplementary Figure S4B). Depletion of GR abolished ghrelin-induced suppression of TSLP gene activation and the gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin ( Figure 4E,F). Consistently, RU-486 (also known as mifepristone), a GR antagonist, abolished ghrelin-induced suppression of TSLP gene activation and the gene promoter activity in HaCaT keratinocytes treated with TNFα or bacterial flagellin ( Figure 4G,H).

Ghrelin Induces Recruitment of GR to nGRE Site on TSLP Gene Promoter
Previous reports and in silico analysis indicated the presence of negative glucocorticoid response element (nGRE) on the TSLP gene promoter [23,[53][54][55]. Promoter reporter assay implied that ghrelin might suppress TSLP gene activation by binding GR to nGRE site on the TSLP gene promoter ( Figure 5A). We also found that ghrelin induces the interaction of GR with NCoR and HDAC3 corepressor in HaCaT keratinocytes treated with TNFα or flagellin ( Figure 5B). Chromatin immunoprecipitation (ChIP) assay consistently demonstrated that ghrelin induces the recruitment of GR, NCoR, and HDAC3 to the nGRE site on the TSLP gene promoter ( Figure 5C). However, GHRP6 or RU-486 treatment reduced the occupancy of GR, NCoR, and HDAC3 at the nGRE site on the TSLP gene promoter induced by ghrelin ( Figure 5D,E).  site on the TSLP gene promoter ( Figure 5A). We also found that ghrelin induces interaction of GR with NCoR and HDAC3 corepressor in HaCaT keratinocytes treated with TNFα or flagellin ( Figure 5B). Chromatin immunoprecipitation (ChIP) assay consistently demonstrated that ghrelin induces the recruitment of GR, NCoR, and HDAC3 to the nGRE site on the TSLP gene promoter ( Figure 5C). However, GHRP6 or RU-486 treatment reduced the occupancy of GR, NCoR, and HDAC3 at the nGRE site on the TSLP gene promoter induced by ghrelin ( Figure 5D,E).

Discussion
Ghrelin is a peptide hormone secreted from enteroendocrine cells of the gastrointestinal tract that has diverse biological functions, including appetite and obesity regulation, inhibition of insulin secretion, and stimulation of growth hormone release [67][68][69][70][71]. Ghrelin also regulates inflammation in tissue damage of the central nervous system, cardiovascular system, and gastrointestinal system [72][73][74][75][76][77][78]. In skin, it has been reported that ghrelin is expressed in several types of cells, including epidermal cells [36,79]. Qu et al., further showed decreased expression of ghrelin in TNFα-treated skin cells and inflamed mouse skin of contact dermatitis and psoriasiform [36]. They also demonstrated that intraperitoneal injection of ghrelin has therapeutic effects on both contact dermatitis and psoriasis through up-regulation of ghrelin levels in skin and attenuation of NF-κB signaling, which leads to reduced secretion of pro-inflammatory cytokines and skin inflammation [36]. Ghrelin also inhibits infiltration of inflammatory cells in skin. For example, intraperitoneal injection of ghrelin decreased the infiltration of inflammatory cells into the dermal layer in bleomycin-induced mouse scleroderma [80]. In skin burn injury, subcutaneous injection of ghrelin reduced infiltration of inflammatory cells, including leukocytes [81].
In this study, we demonstrated that topically applied ghrelin suppresses TSLP gene activation through GHSR1a in AD-like skin. GHSR, a member of the G protein-coupled receptor family, is a receptor for ghrelin [27,82]. Although two variants of GHSR (GHSR1a and 1b) are expressed in diverse tissues, GHSR1a acts as a functional receptor [45,46]. GHSR1a is highly expressed in the hypothalamus and pituitary, but it is weakly expressed in the pancreas, spleen, kidney, and adrenal gland [83][84][85]. Although it has been previously reported that GHSR1a is not detected in mouse and human skin by quantitative RT-PCR [84,85], immunohistochemical examination demonstrated that GHSR1a was present in skin epidermis and annexes surrounding neurofibromas [86]. In skin wounds, granulation tissues are positive for GHSR1a in the dermis [34]. In addition, epidermal stem cells from mouse skin express GHSR1a at the mRNA and protein level [79]. Our immunohistochemical and Western blot analysis demonstrated the presence of GHSR1a in mouse skin epidermis. We also found that HaCaT keratinocytes, a spontaneously transformed immortal keratinocyte cell line from adult human skin, express GHSR1a at the mRNA and protein level as well as GHSR1a-dependent calcium mobilization. Consistent with our results, functional GHSR1a expression has been reported in HaCaT keratinocytes [87].
We demonstrated that topically applied ghrelin-induced GHSR1a activation results in GR activation. Liu et al. suggested that ghrelin induces up-regulation of GR expression by inactivation of p38 mitogen-activated protein kinase (MAPK) and Jun N-terminal kinase (JNK), resulting in activation of anti-inflammatory gene expression and inhibition of proinflammatory gene expression in macrophages [34]. However, we found that ghrelin induces nuclear trans-localization and phosphorylation of GR (S211), which is required for transactivation of target genes, in HaCaT cells rather than increased GR expression [88,89]. Our results further demonstrated that activated GR by ghrelin induces binding of GR with HDAC3 and NCoR corepressor to nGRE site of the TSLP gene promoter, resulting in suppression of TSLP gene activation. Similarly, glucocorticoids suppress TSLP gene activation induced by a vitamin D3 analog through binding of GR to nGRE site [53]. The exact molecular mechanism underlying ghrelin-mediated phosphorylation and nuclear localization of GR in skin is unclear, although it is known that ghrelin may stimulate the secretion of cortisol [90][91][92][93]. It is interesting to test whether ghrelin induces secretion or synthesis of cortisol in skin which is an extra-adrenal source of cortisol synthesis [94,95]. However, ghrelin-induced increased levels of cortisol may be associated with the side effects of cortisol, such as skin atrophy [96,97]. Thus, the exact molecular mechanism of ghrelin in inflammatory skin diseases should be studied.
NF-κB is a critical transcription factor for TSLP gene activation in response to various stimuli [56,57]. We showed that ghrelin decreases occupancy of NF-κB at the TSLP gene promoter without trans-localization of NF-κB p65 into the cytoplasm or degradation of NF-κB p65 in the nucleus. This is reminiscent of our previous report that activation of aryl hydrocarbon receptor (AhR) inhibits NF-κB-dependent TSLP gene activation through PKCδ/p300-mediated reduced acetylation level of NF-κB p65 [66]. Similarly, we found that ghrelin induces nuclear localization of PKCδ, a putative p300 (S89) kinase, and increased phosphorylation of p300 at serine 89 (S89). Given that p300 acetylates NF-κB p65 (K221) and phosphorylation of p300 (S89) inhibits intrinsic lysine acetyltransferase activity of p300 [62,63], phosphorylation of p300 (S89) may be associated with the decreased DNA binding activity and transcriptional activation of NF-κB p65 [58,59,[98][99][100]. Consistent with our previous study [66], we confirmed the important role of PKCδ in ghrelin-mediated negative regulation of TSLP gene activation. It has also been reported that ghrelin activates PKCδ in colon epithelial cells and dopaminergic neurons [101,102].
Epidermal hyperplasia is a histological hallmark promoted by various growth factors and cytokines such as keratinocyte growth factor (KGF), IL-4, IL-13, IL-17A, IL-22, and IL-24 in AD [103,104]. We found that ghrelin suppresses epidermal hyperplasia probably due to inhibition of expression of IL-4, IL-13, and IL-22 genes by ghrelin in DNFB-induced ADlike mouse skin, implying anti-proliferation activity of ghrelin (Supplementary Figure S1). Infiltration of mast cells, eosinophils, and T helper cells is also a characteristic feature of AD. Mast cells are considered the key effector cells in immediate hypersensitivity, and an increased number of mast cells is found in most AD patients [105,106]. High-affinity IgE receptor-mediated activated mast cells are responsible for secretion of various proinflammatory mediators, sensitization to allergens, and IgE elevation [105]. IL-4 induces production of pro-inflammatory cytokines in Th2 cells and IgE in B cells [107]. IgE then promotes infiltration of mast cells resulting in infiltration of other inflammatory cells in AD [105,106]. We further demonstrated that ghrelin suppresses IL-4 gene activation that may cause decreased infiltration of mast cells in DNFB-induced AD-like mouse skin (Supplementary Figure S1). However, it should be studied whether ghrelin suppresses IL-4, IL-13, and IL-22 gene activation , such as the TSLP gene.
In conclusion, we identified the molecular mechanism underlying ghrelin-induced suppression of TSLP gene activation and alleviation of AD-like phenotypes. Specifically, ghrelin induces GR activation. In turn, activated GR binds to nGRE site in the TSLP gene promoter, repressing its expression. Ghrelin also induces nuclear localization of PKCδ and increases phosphorylation and inactivation of p300. This further leads to decreased acetylation and DNA binding activity of NF-κB p65 to the TSLP gene promoter ( Figure 6). Collectively, our results suggest that ghrelin may alleviate inflammatory skin diseases such as AD through suppression of TSLP gene activation in skin keratinocytes.

Animal Model of AD
Adult female BALB/c mice (6 weeks old) (DBL, Chungcheongbuk-do, Korea) were held in a temperature-controlled room (22 • C) at 55% humidity. The committee for experimental animal research at Sogang University approved the animal experiments [IACUCSGU2017-2 (1 June 2017) and IACUCSGU2019-15 (11 September 2019)]. Dermatitis was induced by DNFB in mice as described previously [108]. The sensitization was performed once by topical application of 100 µL of 0.15% DNFB dissolved in acetone to the shaved abdominal skins of mice. A week later, the shaved dorsal skin of mice was topically applied with 100 µL of 0.15% DNFB every 3 days for 12 days. The same mice were also topically applied with 100 µL DIW, 100 µL of 100 µM acyl-ghrelin, or 1 mM [D-Lys3]-GHRP6 daily for 12 days. In the negative control group, 100 µL of acetone or DIW was topically applied to the shaved dorsal skin. As a positive control, DNFB-treated mice were topically applied with 100 µL of 200 µM dexamethasone. After mice were anesthetized with 2% isoflurane, skin lesions were photographed and harvested. Ghrelin and GHRP6 were dissolved in DIW. Dexamethasone was dissolved in ethanol.

Scoring AD-like Phenotypes
The score of the AD-like phenotype was calculated according to the criteria as described previously [109] with slight modifications. The severity score (0 to 12) was defined as the sum of individual scores graded as 0 (none), 1 (mild), 2 (moderate), and 3 (severe) for each of the four symptoms: (i) erythema/hemorrhage, (ii) edema, (iii) excoriation/erosion, and (iv) scaling/dryness. The maximum score was 12.

Calcium Mobilization Assay
Intracellular calcium levels were determined with the FLUOFORTE calcium assay kit (Enzo Life Sciences, New York, NY, USA) according to the manufacturer's protocol.

Quantitative PCR
Total RNA was extracted from HaCaT keratinocytes or mouse skin using Tri-RNA Reagent (Favorgen, Ping-Tung, Taiwan). First-strand cDNA synthesis was performed with PrimeScript RT master mix (Takara, Shiga, Japan). The resulting cDNAs were subjected to real-time PCR using qPCR 2x Premix SYBR (Enzynomics, Daejeon, Korea) with a Stratagene Mx3000p qPCR machine (Agilent Technologies, CA, USA). PCR conditions used to amplify all genes were 10 min at 95 • C and 40 cycles of 95 • C for 15 s and 64 • C for 40 s. Expression data were calculated from the cycle threshold (Ct) value using the ∆Ct method for quantification. RPLP0 was used for normalization. Oligonucleotides are listed in Supplementary Table S1.

Isolation of Epidermis and Dermis from Mouse Skin
Epidermis was prepared from mouse dorsal skin as described previously [110] with minor modifications. Briefly, the dorsal skin was scraped off subcutaneous tissue, including the fat, until the skin was semi-translucent. Then the skin was placed dermis side down in a Petri dish filled with 0.25% trypsin solution for 2 h at 32 • C. Next, the epidermis was scraped from the dermis. The epidermis and dermis were washed twice in ice-cold PBS and used for protein extraction.

Immunoprecipitation and Western Blot Analysis
For immunoprecipitation, HaCaT keratinocyte lysates were prepared as described previously [111]. After centrifugation of lysates, the supernatants were incubated overnight at 4 • C with the anti-GR antibody (3660S, Cell Signaling), followed by incubation with protein A agarose beads (Amicogen, Daejeon, Korea). Protein/antibody/protein A agarose beads were washed extensively and then dissolved in an SDS sample buffer. Normal IgG (12-

Promoter Reporter Assay
Human TSLP gene promoter was amplified using primers 5 -CCCGGTACCGGGAAA-CTCCATTATTACACCCTT-3 and 5 -CCGGCTAGCACACTAAACTCTTCCCA CCACGAG-3 . PCR product was cloned into pGL3 vector (Promega, Madison, WI, USA). The negative glucocorticoid response element (nGRE) site of the TSLP gene was mutated (CCTC-CGGGAGA→GAACGGGAGA) [53]. HaCaT keratinocytes were transfected transiently with pGL3 plasmids containing human TSLP gene promoter in conjunction with a control thymidine kinase promoter-driven Renilla luciferase. HaCaT keratinocytes were harvested and luciferase activity was measured using a dual luciferase reporter assay system (Promega, Madison, WI, USA). Reporter activity is represented as fold activation relative to Renilla luciferase activity.

Statistical Analyses
All quantitative data are presented as mean ± S.E.M. for three independent experiments. The differences between the two groups were evaluated by a paired t-test. Significance values were * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.005.

Conflicts of Interest:
The authors declare no conflict of interest.