Human S100A7 Induces Mature Interleukin1α Expression by RAGE-p38 MAPK-Calpain1 Pathway in Psoriasis

Psoriatic keratinocytes express exaggerated levels of inflammatory cytokines, and show aberrant hyperproliferation and terminal differentiation in the pathogenesis of psoriasis. The antimicrobial protein hS100A7 (psoriasin) has been found highly expressed in psoriatic skin, but the mechanism and physiological function remain largely unknown. We observed that hS100A7 induces mature interleukin 1α (17kDa) expression in normal human epidermal keratinocytes, which is dependent on RAGE-p38 MAPK and calpain-1 as the inhibitors or knockdown of them completely decreased the expression of mature interleukin1α. Then, we proved mS100a7a15, mature IL-1α and calpain-1 were highly expressed in imquimod-induced psoriasis model and mouse IL-17a-neutralizing antibody treatment attenuated mS100a7a15 expression. At last, PD 151746 (calpain-1 inhibitor) treatment decreased epidermal thickness in imquimod-induced psoriasis model. Taken together, our results suggest that mature IL-1α induced by hS100A7 is via RAGE-p38 MAPK and calpain-1 pathway in keratinocyte and this mechanism may play an important role during psoriasis.


Introduction
Psoriatic skin lesions major feature increased keratinocyte proliferation and abnormal differentiation. [1] The immunopathogenesis involves a dysregulated interaction between epidermal keratinocytes and infiltrating inflammatory cells. [2] The pro-inflammatory cytokine interleukin-1α is constitutively expressed by keratinocytes in vivo and has been shown to be expressed in psoriatic lesional skin. [3] Treatment of wild-type organotypic cultures with interleukin-1α was sufficient to induce hyperkeratosis in an in vitro model of lamellar ichthyosis. [4] IL-1 is likely to be an important mediator in the initiation and maintenance of psoriatic plaques and may represent an attractive therapeutic target. [5][6][7] It has been reported that proteolysis of IL-1α by calpain-1 results in a several-fold increase in bioactivity, which has nearly 50-fold higher affinity for IL-1R than full-length IL-1α. [8] Increased IL-1 activity is a hallmark of many chronic inflammatory conditions, including rheumatoid arthritis, diabetes, atherosclerosis, and psoriasis. [9,10] a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 hS100A7 (psoriasin) belongs to the S100A family of Ca 2+ -binding proteins, it has been reported with many functions, such as antimicrobial, [11] chemotactic activity, [12,13] and associated with some diseases, such as psoriasis, [14] skin tumors, [15,16] atopic dermatitis, [17] and chronic rhinosinusitis. [18] These conditions are characterized by an inflammatory reaction, suggesting the role of hS100A7 in the regulation of inflammation. Our study for the first time reveals that hS100A7 induces mature IL-1α expression and other downstream signaling molecules in vitro and in vivo. It explains the mechanism of hS100A7 function in psoriasis, and provides a new target for the treatment of the disease.

Mice
All mice were housed and bred according to the Shanghai Medical Experimental Animal Care guidelines. Animal protocols were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao-Tong University School of Medicine (SJTUSM IACUC).

Western blot
2mm mouse skin taken from mouse back or cells were lysed by using RIPA buffer (pH 7.4) containing protease inhibitor cocktail (Roche, 04693116001) and then were sonicated on icecold water for 15min. Protein concentrations of the extracts were measured by BCA™ Protein Assay Kit (Pierce, 23225) and equal amount of total protein from each sample was separated with SDS-PAGE and then transferred to nitrocellulose membrane followed by probing with the indicated antibodies.

Cytokine release assay
NHEKs were plated, adhered overnight. Different concentrations of hS100A7 were added and incubated for 5 hours. Supernatants were clarified and cytokines assayed by ELISA (Pepro-Tech, 900-T11).

Imiquimod model of skin inflammation
10-week-old BALB/C mice were given 25 mg of imiquimod (InvivoGen, tlrl-imqs) in the shaved back as previous described [19]. 10 mg of PD151146 was i.p. injected 1 day before 25 mg of imiquimod was used to induce psoriatic skin. On day 5, mice were euthanized and back tissues were collected for immunohistochemical evaluation or mS100a7a15 and IL-1α mRNA or protein analysis.

Immunohistochemistry and H&E staining
Skin samples were fixed with 4% paraformaldehyde for 2 days, then dehydrated through a graded series of ethanol and embedded in paraffin. Sections were cut and stained with haematoxylin-eosin (H&E). Immunohistochemistry was done as previously reported. [20] In Vivo IL-17a neutralization 100 μg of monoclonal mouse IL-17a antibody (R&D, MAB421) was intradermally injected into mouse back skin 24 hrs before experiment. Then imiquimod was injected, mouse skin was taken for analysis of mS100a7a15 expression 3 days later.

Statistical analysis
Two-tailed t-test was used to determine significances between two groups. The significances among multiple groups were determined by One-way ANOVA with GraphPad 5 (San Diego, CA). For all statistical tests, we considered P values <0.05 to be statistically significant.

Results
hS100A7 induces mature IL-1α expression in normal human epidermal keratinocytes were measured by real time PCR. The results demonstrated that hS100A7 treatment in keratinocyte induced IL-1α mRNA expression, but it can't induce IL-1β mRNA expression ( Fig 1A). IL-1α (17 kDa), not IL-1β (17 kDa), is induced by the treatment of hS100A7 in normal human keratinocytes (Fig 1B and 1C). The concentration of IL-1α in cell supernatant is also increased after hS100A7 treatment ( Fig 1D). We also show that mature IL-1a is increased in psoriatic epidermis ( Fig 1E). These data demonstrate that hS100A7 induce mature IL-1α (17 kDa) production in keratinocytes.
P38 MAPK and RAGE are critical to mature IL-1α production induced by hS100A7 The MAPK kinases, involved in the pathogenesis of psoriasis, control several important functions within the cell, such as cell proliferation, differentiation, gene expression, and apoptosis in keratinocytes. [22,23] AKT-mediated signaling is functionally involved in keratinocyte transformation, differentiation and proliferation. [19,24,25] hS100A7 treatment activates p38 MAPK, JNK and AKT, but not ERK kinase (Fig 2A). We thereby used inhibitors of those pathways to treat undifferentiated NHEKs in the presence or absence of hS100A7. Among these inhibitors, p38 MAPK kinases inhibitor (SB202190) significantly blocked the effect of hS100A7 on mature IL-1α production, but this was not blocked by caspase-8 inhibitor, JNK inhibitor SP600125 and AKT inhibitor LY294002 (Fig 2B). P38 MAPK function was identified by specific siRNA, as knockdown of p38 MAPK expression blocked mature IL-1α production induced by hS100A7 (Fig 2C). It has been reported that extracellular hS100A7 binds to the transmembrane receptor RAGE, and RAGE can activate MAPK signaling pathway. [26,27] After knockdown RAGE by siRNA, keratinocytes were treated with hS100A7 (50 ng/ml) for 24 hours. The results showed that hS100A7-induced p38 activation and mature IL-1α expression were depending on RAGE (Fig 2D). All these data suggest that hS100A7 binds to RAGE and actives p38 MAPK, which is involved in mature IL-1α production.

mS100a7a15, calpain-1 and mature IL-1α are induced in IMQ-induced psoriasis model
Application of imiquimod on mouse skin results in the influx of various cells of the immune system, as well as hyperplasia of the epidermis, and IMQ-induced psoriasis-like skin inflammation in mice is mediated via the IL-23/IL-17 axis and IL-1R1/MyD88 signaling. [30][31][32] The psoriasis model was made as previously reported, [19] mS100a7a15 mRNA level was highly expressed in IMQ-induced skin tissue (Fig 4A). Also, mS100a7a15, calpain-1 and mature IL- 1α protein levels were induced in psoriasis-like skin compared to normal skin (Fig 4B). Then mS100a7a15 and calpain-1 positive cells in IMQ-induced psoriasis skin are higher than that in normal skin by immunohistochemistry (Fig 4C). All the data mean that mature IL-1α, mS100a7a15 and calpain-1 are positive expressed in IMQ-induced psoriasis mouse model.

PD151746 ameliorates epidermal hyperplasia in IMQ-treated mice
PD151746 is an inhibitor of calpain protein, the IC50 value of calpain-1 is 260 nM, with a 20-fold higher selectivity of calpain-2. Notably, PD151746 ameliorated psoriatic lesions and reversed epidermal hyperplasia (Fig 5A). Moreover, the PD151746 treatment caused a decrease of epidermal thickness on back (Fig 5B). Importantly, mature IL-1α and calpain-1 expression were decreased in the psoriasis-like lesions in the treatment of PD151746 (Fig 5C). However, IL-1α mRNA expression was not significantly changed (Fig 5D). Taken together, these data reveal that calpain-1 inhibition ameliorates psoriatic lesions in vivo.

IL-17a induces hS100A7 or mS100a7a15 expression in vitro and in vivo
There are many inflammatory cytokines associated with psoriasis, it has been reported IL-17a plays a central role in the expression of psoriasis signature genes in keratinocytes. [33] In order to identify which cytokine is relevant to hS100A7 expression, we treated with NHEKs by different inflammatory cytokines. As shown in Fig 6A, hS100A7 is strongly induced by IL-17a, and IL-22 and IL-36γ also up-regulate the expression of hS100A7. Notably, IL-17a neutralizing antibody treatment in IMQ-induced psoriasis model decreased mS100a7a15 expression at mRNA and protein level (Fig 6B and 6C). All the data demonstrate that hS100A7 or mS100a7a15 expression is associated with IL-17a in vitro and in vivo.

Discussion
This study demonstrated that hS100A7 treatment lead to mature IL-1α production in keratinocytes via RAGE-p38 MAPK-calpain-1 signaling. Several psoriasis-related cytokines, including IL-17a, IL-22 [34] and IL-36γ [35], could up-regulate hS100A7 expression in keratinocytes. IL-17a neutralizing antibody blocked mS100a7a15 expression in IMQ-induced psoriasis skin in vivo. Importantly, in a mouse model of psoriasis, calpain-1 inhibition improved epidermal hyperplasia, alleviated skin inflammation and reduced the expression levels of mature IL-1α and calpain-1. These results indicate that hS100A7 contributes to the pathogenesis of psoriasis by modulating keratinocyte activation. Recent studies have reported that inflammatory cytokines can activate keratinocytes to produce cytokines and chemokines. [36] However, the mechanism of keratinocyte activation in psoriasis is not well understood. Inflammation is a driving force in psoriasis. It has been showed that IL-1α, IL-1β, IL-17A and IL-23 are important to psoriasis progress. However, it is interesting that hS100A7 induces mature IL-1α expression, not IL-1β. Pro-IL-1α can be cleaved to mature IL-1α by neutrophil elastase, granzyme B, chymase, and calpain-1, but neutrophil elastase and chymase also cleave pro-IL-1β. Granzyme B is commonly found in the granules of cytotoxic lymphocytes (CTLs), natural killer cells (NK cells) and cytotoxic T cells. [37] So we hypothesized that calpain-1 is essential to mature IL-1α production induced by hS100A7. Calpain-1 inhibition delays the wound healing, for reducing inflammatory cell recruitment and angiogenesis in the early stages of wound healing. [38] IL-1α plays an important role in chronic inflammation and it plays a pivotal role in triggering and sustaining the inflammatory process in a variety of disease indications. It is thought that targeting IL-1α holds significant promise as a unique therapeutic option for psoriasis and a variety of other diseases. IL-1R signaling is critical in skin inflammation. IL-23, IL-17 and IL-22 are markedly decreased in IL-1RI KO mice compared with WT mice which are treated by imiquimod. [39] Mostly, IL-1α deficiency results in impaired skin inflammation and leukocyte infiltration after UV exposure. Recombinant IL-1α enhances epidermal wound healing by stimulating fibroblast and keratinocyte growth and to induce collagen synthesis by fibroblasts in animal models, and IL-1α knockout mice also affect tissue repair and wound healing in vivo. [40,41] Human S100A7 is up-regulated in psoriatic skin lesions and is localized in epithelial cells and forms homodimers by non-covalent bindings. Increasing evidence suggests that hS100A7 plays critical roles in amplifying the inflammatory process in psoriatic skin, perpetuating the disease phenotype. [42,43] In human cultured keratinocytes, hS100A7 also induce the expression of several differentiation markers, which are overexpressed in psoriatic skin. [44,45] In our study, we confirmed that mS100a7a15 was overexpressed in psoriasis model skin. Mature IL-1α, not IL-1β, is induced by hS100A7 stimulation in keratinocytes, and mature IL-1α is also detected in in vivo. Human S100A7 expression is associated with epidermal thickness in psoriasis. Delphinidin was found to suppress hS100A7 expression in a 3D psoriatic skin equivalent model, and treatment with narrow-band UVB phototherapy induces a reduced production of hS100A7 in peripheral blood mononuclear cells in psoriatic patients. [43,46] So, hS100A7 may serve as a potential therapeutic target for psoriasis with relatively few side effects.

Conclusion
In conclusion, we demonstrate that hS100A7 induces mature IL-1α expression through RAGE-p38 MAPK-calpain-1 pathway and block mature IL-1α by calpain-1 inhibitor PD151746 can ameliorate epidermal hyperplasia in IMQ-treated mice. Human S100A7 is a critical player in the psoriatic maintenance phase, which may be considered as an additional and potential target molecule for psoriasis treatment.