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-inflammatory effect of epithelial cells is still unknown. Thus, to uncover key insights of the control of hyper-inflammatory responses elicited from epithelial cells in CRSwNP, we identified 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 findings provide new insights into the pathophysiology of inflammation 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 findings, previous study found that YAP-deficient 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 (Shah et al., 2016; Steelant et al., 2016), which served as the first line of defense of nose. In order to maintain the stable balance, airway epithelial cells plays a key role, which are characterised by innate and adaptive immunity, and produce a series of pro-inflammatory cytokines such as TSLP, IL-25, and IL-33 that orchestrate type 2 responses at the mucosa site (Lloyd and Saglani, 2015; Zheng et al., 2018). IL-33 and TSLP mRNA levels were increased in polyp tissues (Hong et al., 2018). In this study, we revealed that nasal polyp tissue had increased mRNA levels of the epithelium-derived innate cytokines TSLP, IL-25, and IL-33 compared with nasal tissue from healthy subjects. Much of the literature suggests that cytokines are involved in the pathogenesis of chronic nasal sinusitis or nasal polyps. However, the mechanism by which cytokine expression is upregulated has not been well documented. Several studies have demonstrated that the NF-κB pathway is highly activated in inflammatory disorders due to its ability to induce transcription of pro-inflammatory genes (Tak and Firestein, 2001; Yamamoto and Gaynor, 2001). 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 inflammation, IκB is dephosphorylated and dissociates from NF-κB, of which the most important are IκBα, IκBβ, and IκBε. Similar to another study (Jung et al., 2019), we found that NF-κB activity involved in the production of epithelium-derived innate cytokines TSLP and IL-33, as well as IL-25 expression in epithelia cells from nasal polyps. Possibly, when an inflammatory process is initiated in nasal polyps, activated NF-κB translocates to the cell nucleus, inducing transcription, particularly of genes encoding inflammatory cytokines, chemokines, and adhesion molecules, such as TSLP, IL-33, and IL-25. This NF-κB-driven effect was further verified in a pharmacological inhibition experiment that showed that nasal polyp epithelial cells treated with BAY 11-7082 (5 µM). The mRNA levels of TSLP, IL-33, and IL-25 were decreased accordingly. Except for epithelial-derived cytokines, multiple inflammatory cytokines were highly expressed during NF-κB activation, such as tumour necrosis factor-α, IL-1β, and IL-6 (Youn et al., 2016; Kim et al., 2018), together indicating that the NF-κB pathway might involved in the pro-inflammation process in epithelial cells.
YAP functions as a transcriptional regulator by acting together with sequence-specific DNA binding factors and transcription cofactors to mediate cell proliferation in developing epithelial tissues and tumours. Our previous data demonstrated that YAP promoted epithelia cell proliferation (Deng et al., 2019). Meanwhile, it has been reported that NF-κB and NF-κB-related inflammatory cytokines are increased in nasal polyps, indicating that NF-κB has a pivotal role in the pathogenesis of CRSwNPs in both Caucasians and Asians (Jung et al., 2019). In the present study, we found that inhibition of YAP could reducethe NF-κB pathway. The interaction was supported by two studies. One study underscores the importance of the YAP1/TAZ–NFκB signaling axis in endodermal organ development and disease, and they found that Hippo suppresses NF-κB signaling in pancreatic progenitors to permit cell differentiation and epithelial morphogenesis (Braitsch et al., 2019). Besides, other researchers found that YAP enhances NF-κB-dependent and independent effects on clock-mediated unfolded protein responses and autophagy in sarcoma (Rivera-Reyes et al., 2018). Similarly, in our study, NPECs were treated with BAY 11-7082 (an inhibitor of IκB phosphorylation) to explore the levels of TSLP, IL-33, and IL-25. It was confirmed that YAP-induced upregulation of TSLP, IL-33, and IL-25 expression could be abrogated by NF-κB inhibition, indicating that YAP could induce TSLP, IL-33, and IL-25 expression in nasal epithelial cells via activation of the NF-κB pathway. Maybe VP is a potential treatment for inhibition of inflammation in nasal polyps by downregulating NF-κB signaling pathway. Further studies are warranted to characterize the roles of YAP and other hippo pathway components in the regulation of NF-κB pathway and cytokine expression in NP. Nevertheless, there are some other interesting foundings for the hippo/YAP-NFκB signaling axis. One study showed that TAK1 mediated YAP phosphorylation, resulting in a reciprocal antagonism between Hippo-YAP/TAZ and NF-κB signalling during osteoarthritis pathogenesis (Deng et al., 2018). The other study showed that TAK1 regulated YAP-derived oncogenicity in pancreatic cancer (Santoro et al., 2020). Further studies are warranted to characterize the exact regulated mechanism of YAP and NF-κB in the regulation of inflammatory cell functions in nasal polyps to find out if there was a bi-directional control mechanism.
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 inflammation in nasal polyps, which serves as a potential therapeutic approach for treating nasal inflammation.