Yes-associated protein promotes the abnormal proliferation of psoriatic keratinocytes via an amphiregulin dependent pathway

Psoriasis is a chronic inflammatory skin disease with high morbidity, poor treatment methods and high rates of relapse. Keratinocyte hyperproliferation and shortened cell cycles are important pathophysiological features of psoriasis. As a known oncogene, Yes-associated protein (YAP) plays a role in promoting cell proliferation and inhibiting cell apoptosis; however, whether YAP is involved in the pathogenesis of psoriasis remains to be determined. Amphiregulin (AREG), a transcriptional target of YAP, was found to be upregulated in psoriasis, and overexpression of AREG promoted keratinocyte proliferation. In the present study, immunohistochemistry showed that YAP expression was elevated in the skin of psoriasis patients and in the Imiquimod (IMQ) mouse model of psoriasis. Knockdown of YAP in HaCaT cells inhibited cell proliferation, caused cell cycle arrest in G0/G1 phase and promoted apoptosis. These changes in YAP-knockdown HaCaT cells were related to changes in AREG expression. We concluded that YAP may play an important role in the regulation of abnormal keratinocyte proliferation via an AREG-dependent pathway and that YAP could be a new target in the treatment of psoriasis.


YAP expression is higher in HaCaT cells than in human primary keratinocytes. HaCaT cells are
immortalized human epidermal keratinocytes, which are a commonly used cell model in psoriasis research [15][16][17][18] . HEK-a cells are primary human epidermal keratinocytes. A431 cells are cSCC cells, which are known to express high levels of YAP 12 . We used qPCR and Western blotting to compare YAP expression at the mRNA and protein levels among HaCaT, A431 and HEK-a cells. The level of YAP expression in HaCaT cells was similar to that in A431 cells, but was much higher than that in HEK-a cells (Fig. 1e,f). These results showed a direct correlation between increased keratinocyte proliferation and increased expression of YAP.

YAP expression is upregulated in the psoriatic skin of mice with IMQ-induced psoriasis.
Knowing that psoriasis patients' skin and HaCaT cells express increased levels of YAP, we then determined whether mice with the psoriatic skin of IMQ-induced psoriasis (a widely used animal model of psoriasis) also exhibits increased YAP expression. After daily application of topical IMQ for 7 days, the IMQ-treated skin became thickened, with erythematous and scaled, in contrast to the skin treated with control cream (Fig. 2a). Immunohistochemistry showed negative or weak YAP staining in the stratum basale of the control group, with a positive rate of 16.667% (1/6), while the IMQ-induced psoriatic skin showed strong YAP staining, with a positive rate of 83.333% (5/6) ( Table 2 and Fig. 2d-g). The difference between the two groups was statistically significant (χ2 = 5.333, P = 0.021). No staining was evident in when the isotype control, matched rabbit IgG isotype control antibody was substituted for the primary antibody ( Supplementary Fig. S2c,d). By qPCR and Western blot analysis, we determined that levels of YAP mRNA and protein were also up-regulated in the IMQ group compared with the control group (Fig. 2b,c). These results demonstrated that YAP expression is also upregulated in the psoriatic skin of the IMQ-induced mouse model of psoriasis, further suggesting that increased levels of YAP in the skin cause increased proliferation of keratinocytes.

YAP knockdown inhibits the proliferation of HaCaT cells. If the hyperproliferation of keratinocytes
is due to the increased YAP level, then blockade of YAP expression should lead to reduced keratinocyte proliferation. To test this hypothesis, HaCaT cells were transfected with YAP-specific siRNAs (si-YAP-1 and si-YAP-2) or with control siRNA and analysed by qPCR 24 h after transfection to determine YAP mRNA levels, and analysed by Western blot 48 h after transfection to determine YAP protein levels. Both the mRNA and protein levels of YAP in HaCaT cells transfected with YAP-specific siRNAs were significantly lower than those in the non-transfected (BLK) and negative siRNA-transfected (si-Ctrl) groups (Fig. 3a,b).
More importantly, knockdown of YAP significantly inhibited the proliferation of HaCaT cells, as determined by the MTT assay (Fig. 3c).

YAP knockdown causes G0/G1 phase arrest in HaCaT cells.
To investigate the role of YAP in cell cycle regulation, a propidium iodide (PI) cell cycle analysis was performed in HaCaT cells 48 h after YAP knockdown. As shown in Fig. 3d, the proportion of cells in G0/G1 phase was significantly higher in the si-YAP groups than in the BLK and si-Ctrl groups, indicating that YAP knockdown led to growth arrest in the G0/G1 phase of the cell cycle. Further qPCR and Western blot analyses showed that the protein levels of cyclin A, cyclin B1, cyclin D1 and cyclin E, and the mRNA levels of CDK2 and CDC25A decreased by different degrees after YAP knockdown, but

YAP knockdown promotes apoptosis of HaCaT cells.
To test whether YAP plays any role in regulating cell apoptosis, we quantified apoptosis in HaCaT cells with vs. without YAP knockdown using 7-AAD/ PE Annexin V staining. Cells in the BLK and si-Ctrl groups showed a low basal apoptosis rate (1.800 ± 0.208% and 1.633 ± 0.418%, respectively). YAP knockdown, however, increased the apoptosis rate to 5.267 ± 1.241% and 5.333 ± 1.477% in the si-YAP-1 and si-YAP-2 groups, respectively (Fig. 4a,b). Further Western blot analysis showed that the protein level of cleaved caspase-3 (C-caspase-3), a marker of apoptosis, was elevated, while the levels of Bcl-2, BAX, and p53 and its down stream targets p21, Puma and Noxa were not affected (Fig. 4c).
AREG and other signalling pathways are involved in the YAP-mediated effects on cell proliferation in HaCaT cells. AREG, a ligand for EGFR, plays an important role in cell growth 21 . AREG promoted the proliferation of HaCaT cells in a dose-dependent manner in culture medium with or without serum ( Supplementary  Fig. S3). Our results showed that in the human samples, AREG could be found in 51.515% (17/33) of the psoriatic skin samples, while and in 20.000% (6/30) of the normal skin samples (Table 3 and Fig. 5a,b). The difference in the positive staining rate of AREG between the psoriatic and normal control skin was statistically significant (χ2 = 6.733, P = 0.009). Furthermore, the relationship between YAP and AREG histoscores showed a significant positive correlation (r = 0.958, P < 0.001). In the mouse samples, AREG could be found in 66.667% (4/6) of the samples from the IMQ-induced psoriasis group and in 16.667% (1/6) of the control samples (Table 4 and  Although the difference in the positive staining rate of AREG between the IMQ-induced psoriatic skin and control skin was obvious, and the relationship between YAP and AREG expression showed a strong positive correlation, neither of these differences reached statistical significance (χ2 = 3.086, P = 0.079; r = 0.894, P = 0.106), probably due to the limited animal sample size. These results indicated that AREG may be also be involved in the pathogenesis of psoriasis, and that AREG was positively related to YAP. In the in vitro studies, the levels of AREG mRNA (Fig. 5e), intracellular AREG protein ( Fig. 5f) and secreted AREG protein (Fig. 5g) in HaCaT cells were reduced after YAP knockdown. In addition, AREG depletion by specific siRNAs resulted in the inhibition of HaCaT cell proliferation. However, the addition of 100 nmol/L AREG to the si-YAP-transfected HaCaT cells partially restored the cell growth ( Fig. 5h), suggesting that YAP regulates cell proliferation through the regulation of AREG expression. Using Western blot analysis, we examined the impact of YAP knockdown on other key signalling pathway molecules. YAP knockdown inhibited STAT3, JAK2 and NF-κB p65 to different degrees but had no effect on p-AKT and p-ERK in HaCaT cells (Fig. 5i).

Discussion
Recently, increasing evidence has implicated the oncogene YAP in the pathogenesis of many human cancers [22][23][24] , since it promotes cell proliferation, regulates the cell cycle, inhibits apoptosis and promotes the migration and invasion of tumour cells. Keratinocyte hyperproliferation and shortened cell cycles are important pathophysiological features of psoriasis, and psoriatic keratinocytes are resistant to apoptosis. These features of psoriatic keratinocytes are similar to those of tumour cells. Therefore, tumour-related regulatory factors and signalling pathways have attracted widespread attention in the study of psoriasis in recent years [25][26][27][28] . Complicated immune networks may be involved in the mechanisms of keratinocyte hyperproliferation, but the details remain unclear. Studies have shown that, immune cells such as CD4+ T cells, CD8+ T cells and Th17 cells, cytokines such as IL-1, IL-6, TNF-α, IL-17, IL-22 and IL-23, and antimicrobial peptides such as LL-37 and psoriasin could lead to   Table 3. Expression of AREG in normal skin and psoriasis tissues. −Negative, score 0; +weakly positive, score 1-4; ++moderately positive, score 5-8; +++strongly positive, score 9-12. **P < 0.01 compared with the control group. normal human primary keratinocytes interfered with their normal differentiation process and led to immortalized proliferation, upregulation of the epithelial proliferation markers p63 and PCNA, and downregulation of the differentiation markers 14-3-3σ and LEKTI. In HaCaT cells, an immortalized epidermal keratinocyte cell line widely used as a cell model of psoriasis 15-18 , we found a high level of YAP that almost reached the level of YAP in the cSCC cell line A431 and was significantly higher than that in primary keratinocytes. Further functional analyses of HaCaT cells showed that, similar to its effects in cSCC, YAP promotes the proliferation, regulates the cell cycle and inhibits the apoptosis of HaCaT cells through modulating cell cycle regulators and apoptosis-related proteins. These results confirm that psoriasis and cSCC share some common pathological mechanisms. However, unlike in cSCC, in which YAP functions through the AKT and ERK pathways, YAP may function through the STAT3, JAK2 and NF-κB pathways in psoriasis. Further studies are needed to identify the specific regulatory sites for YAP activity in psoriasis. AREG is overexpressed in a variety of human cancers, such as colon, breast, lung, liver, prostate, gastric, cancer and pancreatic 36 . In some abnormal epidermal proliferative skin diseases such as psoriasis, AK, warts, keratoacanthoma and cSCC, the expression of AREG is also increased. Overexpression of AREG in HaCaT cells enhances cell proliferation 13,14 . These findings indicate that the proliferative ability of cells is correlated with the expression level of AREG. It is generally believed that AREG is a downstream transcriptional target of YAP 37 . Zhang et al. 38 found that YAP played a role in the phosphorylation of the AREG downstream molecules AKT and ERK, and that knockdown of AREG blocked this effect, suggesting that YAP promotes the expression of AREG. AREG then binds to and activates EGFR, leading to subsequent activation of its downstream signalling pathways. Our study showed that AREG expression was positively correlated with that of YAP in both human and mouse samples and could promote the proliferation of HaCaT cells in a dose-dependent manner. The secretion of AREG was reduced in YAP-depleted HaCaT cells. Adding 100 nmol/L AREG back to the YAP knockdown cells partially restored the growth rate. Therefore, AREG is involved in the YAP-mediated regulation of cell proliferation, cell cycle and apoptosis in psoriatic keratinocytes.
In conclusion, the findings from our study suggest that YAP plays an important role through AREG in the abnormal proliferation and differentiation of keratinocytes in psoriasis. Targeting the YAP-mediated pathways may be have therapeutic efficacy in the treatment of psoriasis.

Materials and Methods
Patient samples. Patient samples were obtained between Jul 2015 and Sep 2016 from the tissue bank of the Department of Dermatology at the Second Affiliated Hospital of Xi'an Jiaotong University. A total of 63 samples were collected, including 33 psoriatic tissues (from 17 males and 16 females, age range = 10-77 years) and 30 normal skin tissues (from 19 males and 11 females, age range = 11-63 years); the samples were obtained from cosmetic surgery. Written informed consent for tissue collection and use was obtained from all adult patients or the parents/legal guardians for patients under 18 years before the study, and an ethics approval was obtained from the Institutional Ethics Committee of Xi'an Jiaotong University.
Immunohistochemistry. Immunohistochemical staining was performed by a standard immunoperoxidase staining procedure 39 . Two pathologists independently observed the results under a microscope. The results were quantified based on the following scoring system: the first score was obtained based on the percentage of positive cells (≤5% = 0, 6-25% = 1, 26-50% = 2, 51-75% = 3, and >75% = 4). The second score was obtained based on the staining intensity (colourless = 0, light yellow = 1, yellowish-brown = 2, and chocolate brown = 3). The overall score of each microscopic field was calculated as the product of the two scores, and the final score was the average of the score in five fields.
Cell culture. The human immortalized epidermal keratinocyte cell line HaCaT and the cSCC cell line A431 were obtained from ATCC (USA) through an authorized local ATCC dealer (Xiangf Biotechnology, Shanghai, China) and were maintained in Dulbecco's modified Eagle's medium (Gibco/Thermo Fisher Scientific, CA, USA). The human epidermal keratinocyte cell line HEK-a was from ScienCell Research Laboratories (CA, USA) and was maintained in keratinocyte medium (ScienCell Research Laboratories). The culture media were supplemented with 10% foetal bovine serum (FBS), penicillin (100 units/ml), and streptomycin (100 mg/ml). Cells were routinely cultured in a humidified incubator at 37 °C and 5% CO 2 .
Transient transfection of siRNA. The siRNA oligonucleotide sequence (shown in Supplementary Table S1) was synthesized by Shanghai GenePharma (Shanghai, China). Twenty-four hours before transient transfection, the cells were seeded at a density of 2 × 10 5 cells/well in 6-well plates. When the confluence reached 70-80%, the cells were transfected with siRNA according to the recommended procedures for LipofectamineTM2000 Transfection Reagent (Invitrogen, Carlsbad, CA).   AREG ELISA. AREG protein levels were calculated by a Human Amphiregulin Quantikine ELISA Kit from R&D Systems following the manufacturer's instructions. Three independent experiments were performed, and three technical replicates were included for each data point.

IMQ mouse models.
Female BALB/c mice (6-8 weeks old) were purchased from Beijing Vital River Company (Beijing, China). The mice were shaved approximately 2 * 2 cm on the back and were then randomly divided into a normal control group and an IMQ model group (6 mice in each group). The IMQ group received a daily topical application of 62.5 mg of 5% IMQ cream (Sichuan Med-Shine Pharmaceutical Co., Ltd; China), and the control group received the same dosage of Vaseline. After 7 days, the mice were sacrificed, and the skin lesions were removed. A portion of the tissues was fixed with 4% paraformaldehyde and embedded in paraffin for further immunohistological studies, and other portions were stored in liquid nitrogen for quantitative real-time PCR and Western blot analysis. All animal protocols were approved by the Institutional Animal Care and Use Committee of Xi'an Jiaotong University.