Yap Promotes Epithelial Cell Proliferation and Epithelium-derived Innate Cytokine Expression via the NF-κb Pathway in Chronic Rhinosinusitis With Nasal Polyps


 Objectives: The hippo-yes-associated protein (YAP) pathway plays an important role in epithelial cell proliferation and inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP). However, the underlying mechanisms remain unclear. This study intends to investigate the role of YAP and the nuclear factor kappa-B (NF-κB) signalling pathway in nasal epithelial cell proliferation and the expression of epithelium-derived cytokines in CRSwNP.Methods: The expression levels of YAP, TEAD1, Ki-67, and NF-κB in sinonasal mucosa, primary nasal epithelial cells (NPECs), and human nasal epithelial RPMI 2650 cells were detected by RT-qPCR and immunoblotting. NPECs were cultured and treated with verteporfin (VP), a selective YAP inhibitor, YAP shRNA or BAY 11-7082, a small molecule inhibitor of NF-κB. The relationship between cell proliferation and hippo pathway activity was explored using a cell counting kit-8 (CCK-8) assay, 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) labelling and colony formation assay. The cell cycle and apoptosis were examined through flow cytometry (FCM) assay. The epithelium-derived cytokines including interleukin (IL-) 33, IL-25 and thymic stromal lymphopoietin(TSLP) were detected by RT-qPCR.Results: The hippo pathway effector YAP, Ki-67, p65 NF-κB, and cyclin D1were significantly increased in CRSwNP compared with control mucosa; which was accompanied by overexpression of interleukin (IL)-33, IL-25, and thymic stromal lymphopoieth (TSLP). Pharmaceutical inhibition of YAP by VP suppressed cell proliferation of RPMI 2650 cells by blocking cell cycle progression at G0/G1 without inducing obvious cell apoptosis. Furthermore, lentiviral transfection-mediated knockdown of hippo pathway activity reduced the expression of IL-33,,IL-25, TSLP as well as p65 NF-κB in RPMI 2650 cells. Downregulation of NF-κB pathway with BAY 11-7082 in NPECs could decrease the mRNA level of TSLP, IL-33 and IL-25 accordingly.Conclusions: Inhibition of hippo pathway suppressednasal epithelial cell proliferation and declined the expression of epithelium-derived cytokines IL-33 and IL-25 and TSLP expression via the NF-κB signalling pathway in NPECs.

proliferation, and a thickened sub-epithelial basement membrane with a reduced number of vessels and glands but virtually no neuronal structure, goblet cell hyperplasia, or increased extracellular matrix deposition (Watelet et al., 2006;Van Bruaene et al., 2012). Physiologically, the nasal epithelium not only serves as a mechanical barrier through the formation of strong mechanical cohesion by apical (tight and adherens) junctions to protect against environmental factors, microorganisms, and toxins, but also participates in both innate and adaptive immune responses by secreting a large array of antimicrobial host defence molecules .
The hippo-yes-associated protein (YAP) signalling pathway has been indicated to play a critical role in the self-renewal and expansion of stem cells and tissue-speci c progenitor cells, and in tissue regeneration and organ size (Johnson, 2019). In mammals, activation of MST1/2 leads to phosphorylation and activation of LATS1/2. YAP and transcriptional coactivator with PDZ-binding motif (TAZ) are phosphorylated by LATS1/2 on multiple sites, resulting in interaction with 14-3-3 and cytoplasmic retention; YAP/TAZ phosphorylation also leads to YAP/TAZ polyubiquitination and degradation. However, when hippo signalling is inactivated, YAP/TAZ enter the nucleus, competes with VGLL4 for the TEAD family of proteins, and recruits other factors to induce gene transcription associated with cell proliferation and differentiation (Halder and Johnson, 2011;Zhao et al., 2011;Yu et al., 2015). YAP has been implicated in airway epithelial cell proliferation and differentiation. . Recently, we showed that abnormal expression of the hippo-YAP pathway contributed to epithelial proliferation and remodelling in CRSwNP. YAP activity is highly related to epithelia cell proliferation during the pathogenesis of CRSwNP and was positively correlated with the VAS score, Lund-Mackay CT score, and Lund-Kennedy endoscopic score in patients with CRSwNP clinically (Deng et al., 2019). However, the precise mechanisms of cell proliferation, tissue remodelling, and epithelial in ammation are still unclear.
The nuclear factor-kappa B (NF-κB) pathway is thought to play a fundamental role in the regulation and production of pro-in ammatory cytokines and tissue remodelling in nasal polyps NF-κB is a key proin ammatory nuclear transcriptional heterodimer consisting of p50 and p65 subunits. CRSwNP and is related to IL-6 and IL-8 cytokine expression (Xu et al., 2007). Activation of NF-κB contributed to the pathophysiology of CRSwNP by increasing the expression of various cytokines, chemokines, and adhesion molecules associated with p65 (Jung et al., 2019). In addition, Young Hyo Kim suggested that a therapeutic strategy that targets NF-κB in Asian patients with nasal polyps could be established (Kim, 2019).
In this study, we found that abnormal expression of YAP activity related to epithelial proliferation in nasal polyp tissue during the pathogenesis of CRSwNP. In addition, the NF-κB pathway components were increased in CRSwNP tissue and promoted epithelium-derived thymic stromal lymphopoietin (TSLP), IL-25, and IL-33 expression in patients with CRSwNP. The inhibition of YAP-NF-κB may be a therapeutic strategy for reducing epithelial proliferation and epithelium-derived cytokine production in CRSwNP.  Asthma 0 0 Allergic rhinitis 0 0 Parts of the fresh nasal tissues were used for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and histological analysis, and some of the tissues were isolated for cell culture.

Immunohistochemistry in Solid Tissues
Tissue samples were xed in 10% neutral buffered formalin and embedded in para n. Para n sections (4 µm) were prepared for each block. Sections were subjected to heat-induced antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. Nonspeci c staining was blocked with 5% bovine serum albumin (Boster Biotechnology). Sections were conducted with speci c primary antibodies YAP (1:100, CST, #14074), NF-κB (1:100, Servicebio, GB11142 ) and cyclin D1 ( terminal Antigen, 1:1,000, CST, #9247) and rabbit monoclonal anti-GAPDH antibody (1:3,000, Bioworld, AP0063) at 4 ℃ overnight. After washing for three times, the membrane was incubated with HRPconjugatedsecondary antibodies for 1 h at room temperature. Finally, enhanced chemiluminescence (Advansta, K-12045-D50) was used to visualize the immunoblot. The intensity of the bands was determined by Image J software. The ratio of each band/GAPDH was considered as the expression level of the target protein.

RNA Extraction and Real-Time Polymerase Chain Reaction Analysis
The quantitative real-time PCR was performed as previously reported (Deng et al., 2019). All primers used are listed in Table 2. The total RNA (1 µg) of each tissue sample was extracted using RNAiso Plus (TaKaRa, Japan) and reverse-transcribed to cDNA with random hexamer primers and RT-PCR kits (TaKaRa RR047A, Japan). The YAP, IL-25, IL-33 and TSLP were detected. PCR was performed with an ABI 7500 FAST instrument (Foster City, CA, USA) using an SYBR Premix Ex Taq kit (TaKaRa DDR420A, Japan). The relative fold increase of gene expression was calculated using the comparative 2-ΔΔCt method. β2 microglobulin (β2M) was used as the housekeeping gene for normalization.

Construction of The Lentiviral Interference Vectors and Transfection.
Sequence of the shRNA targeting YAP was designed as follows: 5'-GCCACCAAGCTAGATAAAGAA-3' as For lentivirus transfection, the RPMI 2650 cells were grown to 50-70% con uency and infected with YAP-shRNA and the control shRNA with polybrene at a concentration of 8 µg/ml. Cells were washed and switched into the complete medium after transfection after 8 h. The uorescence was used to con rm the successful transfection of YAP-shRNA lentiviral vectors 2 d after transfected. The YAP-shRNA infected cells were detected as GFP-positive cells. Then cells were treated with 10 volumes of ice-cold culture medium (10% FBS) and were washed with cold PBS buffer twice to remove excess dyes and seeded in 6-well plates for 24 h. Next, the cells were treated with various concentrations of VP (0, 10, 15, 20 µM or 0, 2.5, 5, 10 µM) for 24 h. After the treatment, the percentage of cell proliferation was analyzed by using ow cytometry (BD, FACSCalibur, USA) to evaluate the CFSE uorescence intensity. The experiment was done for three times.

Flow Cytometry Assay of Cell Cycle and Apoptosis
24 h after VP treatment at different concentration (0, 10, 15, 20 µM), RPMI 2650 cells were trypsinized and then centrifuged at 1,500 rpm for 5 minutes, followed by wash in cold PBS twice. Next, 70% cold ethanol was added and cells were xed for more than 2 h. A total of 250 µl PI/RNase buffer was added and incubated for 30 minutes at RT. Finally, cells analyzed on a ow cytometer (BD Biosciences).
For apoptosis analysis, cells were stained with the Annexin V-FITC/PI apoptosis detection kit (Bestbio, China). The rate of apoptotic cells was analyzed using a dual laser ow cytometer and estimated using the ModFit software (BD Biosciences).

Statistical Analysis
All data are presented as means and standard error (mean ± SD) from the indicated number of samples.
If not normally distributed, data were expressed as median. In continuous variables, for non-normally distributed data, the Kruskal-Wallis H-test was used to assess signi cant intergroup variability. Normal data were assessed by one-way ANOVA for multiple comparisons. The Mann-Whitney U 2-test was applied for non-normally distributed data or Student's t test was used for unpaired data between two groups analysis. Statistical analysis was performed using IBM SPSS software (SPSS, Inc, Chicago, IL, USA), and GraphPad Prism 6 (GraphPad Software, San Diego, CA, USA). Two-tailed P value less than 0.05 indicates as statistical signi cance.

Aberrant expression of YAP, epithelial cell-derived cytokines, and cyclin D1 in CRSwNP
Firstly, we explored the expression of YAP, the cell proliferation marker Ki-67, and NF-κB in polyp tissues by western blotting analysis. We found that the protein levels of YAP, Ki-67, and NF-κB were signi cantly increased in nasal polyp tissues compared with healthy nasal tissues ( Fig. 1A and B). Since cyclin D1 is related to cell proliferation (Zhang et al., 2020), we detected the expression of YAP, NF-κB, and cyclin D1 by immunohistochemistry in nasal polyps and normal control tissues. We found that the expression levels of YAP, NF-κB, and cyclin D1 were signi cantly increased in nasal polyps. Consistently, YAP, NF-κB, and cyclin D1 were mainly upregulated in the epithelium, and low levels were found in the lamina propria in nasal polyps (Fig. 1C). Moreover, to explore the levels of epithelium-derived innate cytokines, we determined the mRNA levels of TSLP, IL-33, and IL-25 by RT-qPCR. The results showed that the mRNA levels of TSLP, IL-33, and IL-25 were signi cantly upregulated in nasal polyps compared with those of normal control tissues (Fig. 1D). Together, these results indicate that the hippo pathway effector YAP might be involved in epithelium-derived cytokine expression and in ammation via the NF-κB pathway in nasal polyps.

Inhibition of YAP suppresses the proliferation of RPMI 2650 cells
To investigate the impact of YAP on cell proliferation, different density gradients of vertepor n (VP) were used to treat RPMI 2650 cells. We assessed cell viability at 24 h using a CCK-8 assay. Compared with the control group, a dose-dependent decrease in cell viability was observed in the VP group in (Fig. 2A). As shown in Fig. 2B, the cell morphology changed after VP treatment. We further examined the proliferation of the three VP-treated cell groups (10, 15, and 30 µM) by cell counting. After inhibiting YAP expression in RPMI 2650 cells for 24 and 48 h, we found that there was a signi cant reduction in cell proliferation (Fig. 2C). The results demonstrated that downregulation of YAP led to suppression of epithelial cell proliferation. Further testing using 5(6)-carboxy uorescein diacetate succinimidyl ester (CFSE) labelling was performed to con rm the ability of the proliferation inhibition of VP. The results revealed that the proliferation of cells treated with 15 µM and 20 µM VP was greatly reduced (Fig. 2D and E). Colony formation assays also showed much less colony formation in the group treated with 10 µM VP compared with the control group ( Fig. 2F and G). Moreover, the western blotting results revealed that VP considerably decreased the expression levels of YAP and TEAD1 in a dose-dependent manner, together with Ki-67 (Fig. 2H). These results indicate that pharmaceutical inhibition of YAP suppressed the proliferation and colony formation ability of RPMI 2650 cells.

Inhibition of YAP arrests the cell cycle
After RPMI 2650 cells were treated with different concentrations of VP, the effect of YAP on cell cycle progression was analysed by FCM In the 20 µM VP group, we found that 83.81% of cells were in G1 phase and 7.93% were in S phase. In comparison, 86.12% of cells were in G1 phase and 8.58% were in S phase in the 15-µM group, 77.92% of cells were in G1 phase and 11.67% of cells were in S phase in the 5 µM VP group, and 58.87% of cells were in G1 phase and 31.52% of cells were in S phase in the control group ( Fig. 3A and B). In addition, we further explored the protein levels of cyclin D1 and cyclin-dependent kinase (CDK) 4, which are involved in cell cycle regulation. Western blot analysis indicated that both cyclin D1 and CDK4 levels were decreased after VP treatment compared with the untreated group (Fig. 3C).
Furthermore, the apoptosis rate of cells treated with VP was analysed by FACS. The results showed that in RPMI 2650 cells with pharmaceutical inhibition of hippo pathway activity, the apoptosis rate was slightly increased compared with the group without VP treatment. However, the difference between the blank control and the 20-µM VP group was not signi cant (P > 0.05, Fig. 3D). These ndings suggest that downregulation of YAP induced G1 phase arrest, resulting in suppression of cell proliferation.

Inhibition of YAP suppresses NF-κB pathway and epithelium-derived factor expression in RPMI 2650 cells
To further explore whether pharmacological inhibition of hippo pathway activity affects the NF-κB pathway and epithelium-derived factor expression in epithelial cells, we treated RPMI 2650 cells with VP, and knockdown YAP using lentiviral transfection. Western blotting showed that the expression of NF-κB decreased in RPMI 2650 cells treated with VP in a dose-dependent manner (Fig. 4A). Immuno uorescence analysis con rmed the expression of NF-κB in the epithelial cells of nasal polyps treated with 20 µM VP (Fig. 4B). Previous study shows that epithelial cells produces epithelium-derived factors (Mjosberg et al., 2011), and we found that VP signi cantly decreased the mRNA levels of IL-33, TSLP, and IL-25 in a dosedependent fashion (Fig. 4D-F). As expected, both Ki-67 and TEAD1 were signi cantly decreased after YAP knockdown. Cell proliferation, colony formation, and IL-33, TSLP, and IL-25 mRNA expression were also decreased ( Fig. 5A-C). The data demonstrated that inhibition of the hippo pathway by VP or lentiviral transfection could suppress the NF-κB pathway and epithelium-derived factor expression in RPMI 2650 cells.

YAP induced epithelium-derived cytokine expression via NF-κB signalling in epithelial cells derived from nasal polyp tissues
We used epithelial cells derived from nasal polyp tissues to con rm our results. We found that 2.5 µM VP effectively hampered hippo-YAP signalling (Fig. 6A). VP inhibited the expression of NF-κB and the proliferation of epithelial cells derived from nasal polyp tissues (Fig. 6B-F). Moreover, the mRNA levels of TSLP, IL-33, and IL-25 were signi cantly downregulated compared with those of normal control tissues ( Fig. 6H-J). Furthermore, BAY 11-7082, a small molecule inhibitor of the NF-κB pathway, was employed to con rm the expression of VP-induced epithelium-derived factors. After treating NPECs with BAY 11-7082 for 24 h, the protein levels of pIκBα and NF-κB were attenuated signi cantly (Fig. 6K-L). Following treatment with 5 µM BAY 11-7082 for 24 h, the mRNA levels of TSLP, IL-33, and IL-25 were decreased accordingly (Fig. 6M-O), suggesting that the decreased levels of TSLP, IL-33, and IL-25 with inhibition of YAP were mediated by NF-κB suppression.

Discussion
Thus far, the molecular mechanisms contributing to the pathogenesis of nasal polyps, is still unclear. In our previously published study, we provided evidence that the hippo pathway, especially its core effector YAP, played a key role in upregulating nasal epithelial proliferation and remodelling in nasal polyps, which suggested the hippo pathway could be a valuable therapeutic target in nasal polyps.. However, how the hippo pathway regulates the pro-in ammatory effect of epithelial cells is still unknown. Thus, to uncover key insights of the control of hyper-in ammatory responses elicited from epithelial cells in CRSwNP, we identi ed possible pathway in epithelial proliferation and epithelium-derived innate cytokines production. Previously, it was shown that YAP protein level was positively correlated with epithelial basement membrane thickness and the Ki-67 mRNA level, and negatively correlated with the E-cadherin mRNA level in both eosinophilic and non-eosinophilic nasal polyps (Deng et al., 2019). Here, we found that YAP overexpression induced the upregulation of the epithelium-derived innate cytokines TSLP, IL-33, and IL-25 via NF-κB signalling, consistent with other studies that showed that NF-κB plays an important role in CRS (Kim, 2019). These novel ndings provide new insights into the pathophysiology of in ammation in nasal polyps.
CRSwNP was heterogeneous and marked by niches of denuded respiratory epithelium with associated proliferation, basal membrane thickening, and goblet cell hyperplasia (Li et al., 2011;Li et al., 2014). In this study, it showed that aberrant expression of YAP induced epithelial cell proliferation during nasal polyp development. Moreover, when the hippo pathway in RPMI 2650 cells was downregulated, we found that the cells were arrested in G1 phase resulting in repressed cell growth. The cell cycle progression proteins cyclin D1 and CDK4 were also decreased when the hippo pathway was downregulated. Consistent with our ndings, previous study found that YAP-de cient retinal progenitors displayed decreased S-phase cells and altered cell cycle progression (Kim et al., 2016). Furthermore, another study demonstrated that when YAP was decreased in cells from clear cell renal cell carcinoma, cell proliferation was inhibited (G1 phase arrest) and apoptosis was increased (Cao et al., 2014). In our study, however, VP regulation of the hippo pathway showed no effect on cell apoptosis. Future studies are still needed to further address its possible mechanism.
As previously reported, the nasal epithelium contributes to host defence and forms the physical barrier In addition, the NF-κB pathway plays a critical role in host defence against pathogens, and protects cells from apoptosis. When inactive, NF-κB exists as a heterodimer in the cytoplasm and binds inhibitor-kappa B (IκB). When the cells are subjected to various stimuli, such as in ammation, IκB is dephosphorylated and dissociates from NF-κB, of which the most important are IκBα, IκBβ, and IκBε. Similar to another study (Jung et  In summary, we found that hippo/YAP promoted nasal epithelial cell proliferation and inhibition of YAP could downregulate the expression of TSLP, IL-33, and IL-25 via the NF-κB signaling pathway in nasal epithelial cells. Inhibition of the YAP pathway reduced cell proliferation and epithelial in ammation in nasal polyps, which serves as a potential therapeutic approach for treating nasal in ammation.

Con ict of interest
The authors declare that the research was conducted in the absence of any commercial or nancial relationships that could be construed as a potential con ict of interest.   . Data were presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.  Data are presented as mean ± SD. * P < 0.05, *** P < 0.001.