SERPINB4 Promotes Keratinocyte Inflammation via p38MAPK Signaling Pathway

Psoriasis is a chronic inflammatory skin disease characterized by infiltration of inflammatory cells and excessive proliferation of epidermal keratinocytes. SERPINB4, as a serine protease inhibitor, has been clearly expressed in the skin lesions and serum of patients with psoriasis, but the specific mechanism of action is not yet clear. Here, we showed that SERPINB4 expression was increased in skin lesions from the imiquimod (IMQ)-treated mice and M5-(a mixture of five proinflammatory cytokines: IL-17A, IL-22, IL-1α, oncostatin M, and TNF-α) treated human immortalized keratinocyte (HaCaT). Knockdown of SERPINB4 by short hairpin RNA attenuated the M5-induced keratinocyte inflammation. Conversely, lentiviral expression of SERPINB4 promoted keratinocyte inflammation. Finally, we observed that SERPINB4 stimulation activated the p38MAPK signaling pathway. Taken together, these results suggest that SERPINB4 has a critical role in psoriasis pathogenesis.


Introduction
Psoriasis, an immune-mediated chronic inflammatory skin disease, is characterized by epidermal hyperproliferation and infiltration of inflammatory cells [1]. As a chronic relapsing and remitting inflammatory skin disease, it affects 2%-3% of the world's population [2]. As the prolonged erythematous scaly patches or plaques in any skin site, it seriously affects the quality of life of the patient [3]. Although the pathogenesis is unclear, hyperactive keratinocytes and immune cells are a key pathological process during psoriasis development, and the crosstalk between these cells contributes to the pathological phenotype.
Squamous cell carcinoma antigen 2 (SCCA2, SERPINB4), as one of the squamous cell carcinoma antigens, has been used as a tumor marker of some squamous cell carcinomas to predict the pathological grade, stage, recurrence, and response to radiotherapy and chemotherapy [4]. SERPINB4 can promote the migration [5] and invasion [6] of cancer cells, and participate in the process of cell senescence [7] and inflammatory reaction [8].
Further, accumulating research has indicated that SER-PINB4 is upregulated in many inflammatory skin diseases including psoriasis [9,10] and atopic dermatitis [11,12], and serum SERPINB4 level is positively correlated with clinical severity of patients. However, the specific mechanism of action in these diseases is still unknown.
In the data obtained in our previous study, we found that SERPINB4 expression was associated with increased psoriasis severity [13]. In this study, we first discovered that the expression of SERPINB4 was upregulated in skin lesions of IMQ-induced psoriasis-like mice and M5-induced psoriasiform cell model, and then examined the biological function of SERPINB4 by knockdown and overexpression approaches in vitro. Our results showed that SERPINB4 may promote keratinocyte inflammation via activation of p38MAPK in psoriasis. Thus targeting SERPINB4 may represent a potent strategy in psoriasis treatment. 2.5. Plasmids and Reagents. Flag-human SERPINB4 was cloned into the pcDNA3.1 plasmid. Short hairpin RNA of (SERPINB4) and short hairpin RNA of GFP (shControl) constructs described as previously were cloned into the pLKO.1 vector (Addgene). shRNA targeting sequences used are sh-GFP: 5′-tacaacagccacaacgtctat-3′; shS-ERPINB4#1 : 5gcagaagcttgaagagaaact-3′; sh-SERPIN4#2 : 5′-ggacaagtttgcagaatatga-3′.

Materials and Methods
2.6. Short Hairpin RNA or Lentiviral Expression. Constructs were transfected along with pMD2.G and psPAX2 into 293T cells. Forty-eight and seventy-two hours after initial transfection, viral supernatant was collected, filtered, supplemented with polybrene (10 µg/ml), and used to infect HaCaT cells. Forty-eight hours after the last infection, cells were selected with appropriate antibiotics. Transduced cells were selected with puromycin or G418.

Enzyme-Linked Immunosorbent Assay (ELISA).
To detect the levels of CXCL8 in the cell culture supernatant, the cell culture supernatants were collected, and the CXCL8 level was measured using CXCU8 ELISA kit (Thermo Fisher Scientific, 88-8086), according to the manufacturer's instructions.
2.10. Statistical Analysis. All statistical analyses were performed with the GraphPad Prism 8.0 software (GraphPad Software). Data from at least three independent experiments are presented as the mean AE standard deviation (SD). Student t-test (comparisons between two groups) or one-way ANOVA followed by Kruskal-Wallis test (more than two groups) were used to analyze the statistical significance. p<0:05 was considered to be statistically significant.

Serpinb3a (Mouse Homolog of SERPINB4) Was Induced and Activated in IMQ-Induced Psoriasiform Lesions in Mice.
Given that increased SERPINB4 expression was associated with psoriasis severity, we are committed to discussing its role in the pathogenesis of psoriasis. First, we established a psoriasis-like skin disorder in BALB/c mice using IMQ treatment, according to previous reports [14]. We applied IMQ cream on the shaved back skin and right ear of BALB/c mice for 6 consecutive days. As shown in Figure 1(a), mice treated daily with control cream did not show any sign of inflammation, but in IMQ-treated mice, hematoxylin-eosin staining of sections of the back skin showed that the thickness of the epidermis increased and inflammatory cells infiltrated obviously. Daily IMQ treatment of the right ear of the mice led to a significant increase in ear thickness, the ear changes were statistically significant on the 5, 6, and 7 days (the fifth day: IMQ: mean AE SD: 40.13 AE 13.36, Control: mean AE SD: 0 AE 0, p<0:01; the sixth day: IMQ: mean AE SD: 69.68 AE 9.499, Control: mean AE SD: 0 AE 0, p<0:01; the seventh day: IMQ: mean AE SD: 79.24 AE 30.72, Control: mean AE SD: 0 AE 0, p<0:01) (Figure 1(b)). Using real-time PCR, we found that serpinb3a expression in the back skin of IMQtreated mice (mean AE SD: 15.62 AE 11.89) was significantly upregulated compared with that of control mice (mean AE SD: 4.367 AE 3.559, p<0:05) (Figure 1(c)). The IMQ-treated mice showed raised expression of T17 cell chemotactic factor (Ccl20), neutrophil chemotactic factors (Cxcl1, Cxcl2), and antimicrobial peptide S100a7a in skin lesions compared with the control mice (Ccl20: IMQ: mean AE SD:13.89 AE 11.86,   (Figure 1(d)). Taken together, these results suggested that ser-pinb3a may be related to the pathogenesis of psoriasis.

SERPINB4 Overexpression Promoted Keratinocyte
Inflammation. Next, we constructed SERPINB4 lentiviral overexpression cell line (Figures 4(a) and 4(b)). 3.5. SERPINB4 Activated p38 MAPK Pathway. To elucidate the molecular mechanism of the effect of SERPINB4 on keratinocyte inflammation, we first analyzed the effect of SERPINB4 on the p38 MAPK signaling pathway. Because previous studies have shown that M5 activated p38MAPK signaling pathway in psoriasis and activation of p38MAPK pathway contributed to the regulation of the production of inflammation mediators [20][21][22]. As shown in Figure 5 (Figure 5(b)). Next, we pretreated HaCaT cell with SB203580, an inhibitor of p38MAPK, before transfected with SERPINB4. As shown in Figure 5

Discussion
Squamous cell carcinoma antigens (SCCAs) are members of the serpin family of endogenous protease inhibitors [23]. SCCA is encoded by two highly homologous genes (SCCA1 and SCCA2), which are arranged in tandem at chromosome 18q21.3 [24]. SCCA1 and SCCA2 belong to class B serine family protease inhibitors (SERPINs), which were later named SERPINB3 and SERPINB4 [25]. Tissue distribution studies have shown that SERPINB3 and SERPINB4 are coexpressed in the suprabasal layers of the stratified squamous epithelium of the tongue, esophagus, uterine cervix, vagina, tonsil, Hassall's corpuscles of thethymus, and some areas of the skin [26]. SERPINB3/SERPINB4 has been found to be upregulated in a variety of cancer tissues, including esophageal cancer [27], cervical cancer [28], head and neck cancer [29], liver cancer [30], breast cancer [31], and so on. Studies have found that SERPINB3/SERPINB4 can promote the migration and invasion ability of cancer cells, and participate in the process of cell aging and inflammatory response.
In recent years, increasing evidence has implicated that serine proteases are critical for epidermal barrier homeostasis and their aberrant expression or activity is associated with chronic skin diseases. Elevated levels of the serine protease inhibitors SERPINB3 and SERPINB4 are seen in patients with atopic dermatitis and psoriasis, further clinical studies found that SERPINB4 is closely associated with the clinical severity of these two diseases. However, their mechanistic role in these skin diseases is unknown. In this study, we found that SERPINB4 expression was upregulated in skin from IMQ-induced psoriasis-like mice as well as in M5-induced psoriasiform cell model. Silencing SERPINB4 expression using shRNA-expressing lentivirus vector in vitro reduced inflammation in M5-induced psoriasiform cell model. Next, we further proved that p38 MAPK is an important signaling pathway mediating the effects of SERPINB4 on keratinocyte inflammation. Taken together, these in vitro and in vivo results pointed toward a potential role of SERPINB4 in the pathogenesis of psoriasis. Guilloteau et al. [19] found stimulation keratinocytes with a combination of five cytokines (M5) induced inflammation that recapitulates some features of psoriasis including increased cell inflammation and cell proliferation in vitro and in vivo. In this study, we also found that treatment with M5 induced mRNA levels of chemokines (CXCL1, CXCL2, and CCL20) and antimicrobial peptide (S100A7) and the secretion of CXCL8 in HaCaT cell. Meanwhile, we further found that M5 treatment can led to upregulation of SERPINB4 mRNA and protein expression. To further confirm whether some connection between SERPINB4 and inflammation or not in psoriatic keratinocytes, we constructed SERPINB4 silence and overexpression cell lines. We found that knockdown of SERPINB4 inhibited cell inflammation. Conversely, overexpression of SER-PINB4 promoted keratinocytes inflammation. These results indicated that SERPINB4 promoted keratinocyte inflammation in M5-treated HaCaT cell.
In the process of Ras-associated cytokine production and tumorigenesis, Catanzaro et al. [32] showed that endogenous SERPINB4 can promote the secretion of cytokine IL-6. Overexpression of SERPINB4 in human keratinocytes promoted the secretion of IL-1α and IL-6 cytokines [33]. In this study, we demonstrated that SERPINB4 stimulated expression of chemokines CXCL1, CXCL2, CXCL8, and antimicrobial peptides (S100A7) in M5-induced HaCaT cell. How SERPINB4 regulates inflammation in M5-induced psoriasis-like keratinocyte is still unknown. Previous studies have reported that MAPK was an important inflammation signal pathway in psoriasis. Excessive activation of the MAPK (p38, JNK, and ERK1/2) signaling pathway has a key role in regulating the production of inflammatory mediators in psoriasis [20][21][22][23]. And p38 MAPK signaling pathway was activated in M5-induced karatinocytes [34,35]. We speculated that SER-PINB4 may promote inflammation through p38MAPK pathway in the process of psoriasis-like inflammation. Indeed, silencing SERPINB4 reduced the phosphorylation level of p38MAPK in keratinocyte. In summary, SERPINB4 expression was upregulated in patients with psoriasis as well as in vivo and in vitro models of psoriasis. SERPINB4 promoted keratinocyte inflammation, which was associated with activation of p38MAPK signaling pathway.

Conclusion
This study suggested that SERPINB4 promoted keratinocyte inflammation through p38MAPK signal pathway ( Figure 6). SERPINB4 expression was upregulated in skin lesions of IMQ-induced psoriasis-like mice and M5 induced psoriasiform cell model. We confirmed SRPINB4 promoted cell inflammation through SERPINB4 knockdown and overexpression mehtods. Furthermore, SERPINB4 promoted cell inflammation via the p38MAPK pathway. Taken together, our results suggested that SERPINB4 may be implicated in the pathogenesis of psoriasis.

Data Availability
The data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that they have no conflicts of interest.