E3 ubiquitin ligase NEDD4L inhibits epithelial-mesenchymal transition by suppressing the β-catenin/HIF-1α positive feedback loop in chronic rhinosinusitis with nasal polyps

Abstract

Chronic rhinosinusitis with nasal polyp (CRSwNP) is a refractory inflammatory disease with epithelial-mesenchymal transition (EMT) as one of the key features. Since ubiquitin modification has been shown to regulate the EMT process in other diseases, targeting ubiquitin ligases may be a potential strategy for the treatment of CRSwNP. In this study we investigated whether certain E3 ubiquitin ligases could regulate the EMT process in CRSwNP, and whether these regulations could be the potential drug targets as well as the underlying mechanisms. After screening the potential drug target by bioinformatic analyses, the expression levels of three potential E3 ubiquitin ligases were compared among the control, eosinophilic nasal polyp (ENP) and non-eosinophilic nasal polyp (NENP) group in clinical samples, and the significant decrement of the expression level of NEDD4L was found. Then, IP-MS, bioinformatics and immunohistochemistry studies suggested that low NEDD4L expression may be associated with the EMT process. In human nasal epithelial cells (hNECs) and human nasal epithelial cell line RPMI 2650, knockdown of NEDD4L promoted EMT, while upregulating NEDD4L reversed this effect, suggesting that NEDD4L inhibited EMT in nasal epithelial cells. IP-MS and Co-IP studies revealed that NEDD4L mediated the degradation of DDR1. We demonstrated that NEDD4L inhibited the β-catenin/HIF-1α positive feedback loop either directly (degrading β-catenin and HIF-1α) or indirectly (mediating DDR1 degradation). These results were confirmed in a murine NP model in vivo. This study for the first time reveals the regulatory role of ubiquitin in the EMT process of nasal epithelial cells, and identifies a novel drug target NEDD4L, which has promising efficacy against both ENP and NENP by suppressing β-catenin/HIF-1α positive feedback loop.

Introduction

Chronic rhinosinusitis (CRS), a heterogeneous inflammatory disease of the nasal cavities and paranasal sinuses, can be divided into chronic rhinosinusitis with nasal polyps (CRSwNP) and chronic rhinosinusitis without nasal polyps (CRSsNP). Caused by persistent inflammation and tissue remodeling, nasal polyps are upper airway lesions characterized by high mucosal edema, increased vascular exudation, and collagen fiber deposition [1]. As the most common co-morbidity of CRS, NPs afflicts as many as 30% of CRS patients [2] while imposing heavy healthy, psychological, and financial burden worldwide. Based on the presence or absence of eosinophilia, CRSwNP can be subdivided into eosinophilic CRSwNP (ENP) and non-eosinophilic CRSwNP (NENP) [3–5]. Currently, the drugs commonly used to treat CRSwNP, such as corticosteroids and biologics, are significantly more effective in patients with ENP than in patients with NENP [5]. However, NENP patients represent a higher proportion of CRSwNP patients in East Asia [6–8]. Therefore, the search for novel drug targets with good efficacy for all types of CRSwNP has become a hot topic.

Epithelial-mesenchymal transition (EMT), a cellular process in which epithelial cell junctions and polarities are deconstructed, accompanied by the acquisition of mesenchymal properties [9], has been reported to play a key role in the polypogenesis of CRSwNP. During EMT, adhesion junctions are destabilized due to the cleavage and degradation of epithelial cell adhesion protein (E-cadherin, E-cad) at the plasma membrane [10], which is a typical phenomenon in CRSwNP. Since EMT occurs in both ENP patients and NENP patients [11, 12], exploring novel drug targets based on blocking the EMT process is expected to improve the condition of all CRSwNP patients.

Epigenetic modifications regulate most life functions in the human body. Several studies have investigated the mechanisms of epigenetic regulation of EMT in CRSwNP, such as hydroxymethylation and deacetylation [13, 14]. However, as one of the most common post-translational protein modifications, the effect of ubiquitination on EMT in CRSwNP remains a research gap [15]. Taking into account the numerous links between E3 ubiquitin ligases and EMT-related signaling pathways found in other diseases [16, 17], it was hypothesized that certain E3 ubiquitin ligases are involved in the EMT process of CRSwNP.

Materials and methods

Bioinformatics analysis

Chronic rhinosinusitis related GSE36830 expression microarrays were downloaded from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/gds). After quality control and annotation, the differentially expressed gene list of 6 uncinate tissue samples from the control group and 6 NP samples from the CRSwNP group was identified using the Limma R package [18] (|log2 fold change (FC)|>0.2, adjusted P-value < 0.05) (https://www.bioconductor.org/packages/release/bioc/html/limma.html). Heatmap, volcano plot, and Venn diagram were constructed using the R packages ‘Pheatmap’, ‘ggplot2’ and ‘ggVennDiagram’, respectively.

GO and KEGG annotation files were downloaded from the Gene Ontology Consortium website (https://geneontology.org/), and KEGG FTP server (ftp://ftp.bioinformatics.jp/), respectively. The two-tailed hypergeometric test was adopted for the enrichment analysis of the interactors. P < 0.05 and q < 0.05 were considered statistically significant. The E3 ubiquitin ligase of DDR1 was predicted by the UbiBrowser2.0 database (http://ubibrowser.bio-it.cn/ubibrowser_v3/home/index).

To determine the correlation between interacting proteins identified by IP-MS and EMT, a difference analysis of these proteins was performed using edgeR, and P < 0.05 was considered statistically significant. We then performed hypergeometric distribution tests, background genes using all genes in the whole human genome (62,696), 15 of the 1624 interacting proteins obtained by IP-MS were in the EMT gene set (identified by the gene set HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, https://www.gsea-msigdb.org/gsea/msigdb/cards/HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION), the P-values and OR values were calculated using performing fisher’s exact test.

Sample collection

All participants in this study were recruited from the Department of Otolaryngology-Head and Neck Surgery, Renmin Hospital of Wuhan University (Wuhan, China) between January 2021 and May 2021. All enrolled CRSwNP patients were diagnosed according to EPOS 2020 [19]. Patients with any of the following criteria were excluded, 1) age less than 18 years, 2) diagnosis of primary ciliary dyskinesia, posterior nasal polyp, fungal rhinitis, cystic fibrosis, or systemic coagulopathy, 3) aspirin sensitivity, 4) treatment with antibiotics or immunomodulatory drugs within 4 weeks prior to surgery weeks. As for the control group, patients with diseases other than deviated nasal septum will be excluded. During endoscopic sinus surgery (ESS), nasal polyp (NP) tissue was obtained from the middle nasal meatus of CRSwNP patients (n = 30), while the normal nasal mucosa of the control group was obtained from the middle turbinates (MT) of patients with deviated nasal septum (n = 20). Part of the tissue was sectioned in paraffin for morphological experiments, and the other samples were frozen in liquid nitrogen and stored at −80 °C after being washed with normal saline. The demographic and clinical information for all subjects is summarized in Table 1.

Table 1.

Demographics of subjects and methodologies used.

	Control	ENP	NENP
Total No. of subjects	20	14	16
Tissue used	MT	NP	NP
Age (year), mean (SD)	31 (11)	42 (15)	37 (12)
Asthma (No.)	0	0	0
Aspirin sensitivity (No.)	0	0	0
Nasal steroid (No.)	0	0	0
VAS score, mean (SD)	–	13 (9.7)	16 (7.5)
Lund–Mackay CT score, mean (SD)	–	8.9 (3.7)	7.3 (3.4)
Lund–Kennedy score, mean (SD)	–	3.4 (1.3)	3.1 (1.1)
Methodologies used
RNA sequencing (No.)	3	5	5
Morphology (No.)	14	12	12
Tissue extracts (No.)	14	10	9

Immunohistochemistry (IHC)

After deparaffinisation, hydration and washing, tissue sections were neutralized with endogenous peroxidase after antigen retrieval was performed. Tissue sections were then blocked with goat serum for 1 h at 37 °C and incubated with anti-NEDD4L (1:250, ProteinTech Group Inc., Wuhan, China), anti-E-cad antibodies (1:2000, ProteinTech Group Inc., Wuhan, China & 1:600, Fine Test, Wuhan, China), anti-α-SMA (1:100, Boster Bio, Wuhan, China), anti-HIF-1α (1:200, Bioss, Beijing, China), anti-β-catenin (1:100, Bioss, Beijing, China), anti-SMAD3 (1:50, Affinity, Nanjing, China), anti-p-SMAD3 (1:400, Abcam, Cambridge, UK) for 12 h. On the second day, after incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature, the sections were stained with DAB and haematoxylin, the sections were differentiated with hydrochloric acid alcohol, reverse-blue, dehydrated, and sealed after drying. Finally, the sections were observed under a light microscope (BX53, Olympus, Tokyo, Japan). All results were observed and photographed under the microscope. Further statistical analyses were performed using ImageJ software.

Immunofluorescence

After treatments, the hNECs were fixed with 4% paraformaldehyde at 37 °C for 15 min, followed by permeabilizing with triton and blocking with goat serum. Then sections were incubated with anti-HIF-1α (1:200, Bioss, Beijing, China), anti-β-catenin (1:200, Bioss, Beijing, China), and anti-CTHRC1 (1:200, Bioss, Beijing, China) at 4 °C overnight. Next day, after incubating with FITC-labeled goat anti-rabbit IgG (Abcam, Cambridge, UK) at 37 °C for 30 min, the nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, Roche Molecular Biochemicals, Basel, Switzerland) for 5 min. Finally, the sections were observed under a light microscope (BX53, Olympus, Tokyo, Japan). All results were observed and photographed under the microscope. Further statistical analyses were performed using ImageJ software.

Establishment of an NP mouse model

Forty-nine C57BL/6J wild-type mice (4–5 weeks old) were provided by the Animal Experiment Center, Renmin Hospital of Wuhan University and housed in a pathogen-free facility in the Laboratory Animal Center, Renmin Hospital of Wuhan University. The mice were randomly divided into 5 groups, the control group (treated with saline, n = 10), the NP group (treated with OVA + SEB, n = 10), the NP + oe-NC group (treated with OVA + SEB + oe-NC lentiviral particles, n = 9), NP + oe-NEDD4L group (treated with OVA + SEB + oe-NEDD4L lentiviral particles, n = 10) and the dexamethasone group (treated with OVA + SEB+ dexamethasone, n = 10). The murine NP model was established as previously described [14, 20]. On day 0 and day 5, 200 μL phosphate-buffered saline (PBS) containing 25 μg ovalbumin (OVA, Sigma-Aldrich, Germany) and 2 mg of aluminum hydroxide was administered by intraperitoneal injection (i.p.) for sensitization. Subsequently, mice were challenged with 20 μL of 6% OVA by intranasal administration (i.n.) for 13 consecutive weeks. The experimental mice were then challenged with staphylococcal enterotoxin B (10 ng SEB, Toxin Technology, USA) and OVA (i.n.). oe-NC lentiviral particles and oe-NEDD4L lentiviral particles were administered (i.n.) to the respective treatment groups, while the dexamethasone (1 mg/kg) was administered i.p. to the dexamethasone group. Twenty-four hours after the last challenge, the mice were euthanized and dissected. Half of the mouse heads were completely excised, fixed, decalcified, and embedded in paraffin. The nasal mucosa in the heads of the other mice was carefully collected using small curettes.

Hematoxylin and eosin (H&E) staining

First, the paraffin sections were sectioned, deparaffinized, and rehydrated, hematoxylin was used to stain the nucleus, while eosin was used to stain the cytoplasm. The nasal mucosa was then observed and photographed under a light microscope (BX53, Olympus, Tokyo, Japan). Finally, the images were analyzed by ImageJ software.

Culture of the human nasal epithelial cell (hNEC)

Normal human nasal epithelial cells and nasal polyp epithelial cells were obtained from patients who signed informed consent forms prior to surgery. After isolation, the cells were centrifuged and resuspended in PneumaCult™-Explus medium (Stemcell™ Technologies Inc., Vancouver, Canada). Finally, the cells were cultured at 37 °C and 5% CO₂ for further administration.

Culture of cell lines

The human nasal epithelial cell line RPMI-2650 was cultured in RPMI-1640 medium (Servicebio Technology Co., Ltd., Wuhan, China). The cells were cultured at 37 °C and 5% CO₂ for further administration.

Cell grouping and treatment

The RPMI-2650 and hNECs were digested with 0.25% trypsin during the logarithmic growth period and were seeded in a 6-well plate at 1 × 10⁵ cells/well. Cells were cultured at 37 °C, 5% CO₂ until the cell confluence reached 70%–90%, after being washed with PBS, transient cell transfection was performed according to the instructions of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). Cells were divided into several groups and treated with plasmids of oe-NEDD4L, sh-NEDD4L, oe-DDR1, sh-DDR1, deubiquitinase inhibitor (2, 6-diamino-5-thiocyanatopyridin-3-yl) thiocyanate (PR-619) (MCE, NJ, USA), Z-Leu-Leu-Leu-al (MG-132) (MCE, NJ, USA), β-catenin inhibitor indocyanine green-001 (ICG-001) (MCE, NJ, USA), or HIF-1α inhibitor (S)-4-(2-amino-2-carboxyethyl)-N,N-bis(2-chloroethyl)aniline oxide dihydrochloride (PX-478) (MCE, NJ, USA), respectively. The oe-NEDD4L plasmids were synthesized by and purchased from BioRun Inc (Wuhan, China), the sh-NEDD4L plasmids were synthesized by and purchased from Genechem (Shanghai, China), the oe-DDR1 and the sh-DDR1 plasmids were synthesized by and purchased from MiaoLing Plasmid (Wuhan, China).

Immunoprecipitation

hNECs were lysed with NP40 (Beyotime, Shanghai, China) containing protease inhibitors. The lysate was centrifuged to collect the supernatant. Meanwhile, 25 μL Protein A/G magnetic beads were added into 400 μL diluted antibody of NEDD4L (4 μg, ProteinTech Group Inc., Wuhan, China), and the mixture was rotated at 4 °C for 120 min. After magnetic separation and discarding of supernatant, the lysate was added to magnetic beads. After two hours of incubation and rotation, the beads were finally heated in SDS-PAGE loading buffer at 100 °C for 5 min. The final products were stored at −80 °C for further analysis.

IP-MS

After the bite protein was immunoprecipitated, beads were incubated in the reaction buffer (1% SDC/100 mM Tris-HCl, pH 8.5/10 mM TCEP/40 mM CAA) at 95 °C for 10 min. The eluates were diluted and subjected to trypsin digestion (1:50 (enzyme:protein, w/w)) at 37 °C overnight. The next day, the digestion was terminated by the addition of trifluoroacetic acid (TFA). After centrifugation (12,000 × g, 15 min), the peptide was purified, vacuum dried, and stored at −20 °C.

LC-MS/MS data acquisition was performed on a Q Exactive HF mass spectrometer coupled to the UltiMate 3000 RSLCnano system. Peptides were loaded using an auto-sampler and separated on a C18 analytical column (75 μm × 25 cm, C18, 1.9 μm, 100 Å). Raw MS data were analyzed with MaxQuant (V1.6.6) using the Andromeda database search algorithm. Spectra files were searched against the Human database (2022-03-29, 20,377 entries) in Uniprot and the results were filtered with 1% FDR at both protein and peptide levels. Proteins denoted as decoy hits, contaminants, or only identified by sites were removed, and the remaining identifications were used for further analysis.

Homology modeling and molecular docking

The full-length AlphaFold predicted structures of NED4L_HUMAN (Uniprot ID: Q96PU5) and DDR1_HUMAN (Uniprot ID: Q08345) were selected as the receptor protein and ligand protein. The structures of the two biopolymers were analyzed and prepared for the docking experiment. HDOCK server was used for the docking study of the binding mode between the NEDD4L and the DDR1. After sampling all binding modes of two proteins, a scoring function was then used to rank the sampling process and the sampled binding modes.

Protein-Ligand Interaction Profiler (PLIP) was used to provide a comprehensive description and systematic analysis of the binding interface. The interaction details were complemented by pyMOL.

Immunoblotting

By using RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors, the total protein was extracted and temporarily stored at 4 °C for 30 min. After centrifugation at 12,000 r/min for 15 min at 4 °C, the BCA assay was used to measure the concentration of extracted protein before the addition of loading buffer. Samples (40 μg) were loaded onto a 10% sodium dodecyl sulfate-polyacrylamide gel. After electrophoresis, proteins were electrotransferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% skim milk for 1 h, the proteins were incubated at 4 °C overnight in a shaker with various antibodies, which including anti-NEDD4L (1:2500, ProteinTech Group Inc., Wuhan, China), anti-NEDD4L (1:1000, Abclonal, Wuhan, China), anti-E-cad antibodies (1:5000, ProteinTech Group Inc., Wuhan, China), anti-Vimentin (1:5000, ProteinTech Group Inc., Wuhan, China), anti-HIF-1α (1:2000, ProteinTech Group Inc., Wuhan, China), anti-DDR1 (1:1000, GeneTex, TX, USA), anti-α-SMA (1:1000, Cell signaling Technology, MA, USA), anti-β-catenin (1:1000, Cell signaling Technology, MA, USA), anti-CTHRC1 (1:1000, ProteinTech Group Inc., Wuhan, China) and anti-GAPDH (1:5000, ProteinTech Group Inc., Wuhan, China). On the next day, the membranes were incubated with the secondary antibody (1:10,000, ProteinTech Group Inc., Wuhan, China) at room temperature for 1 h on a shaker. Finally, the bands were visualized with enhanced chemiluminescence (Millipore, Burlington, MA, USA) using a gel imaging system (Bio-Rad, Hercules, CA, USA). ImageJ software was used to determine the gray value.

RT-qPCR

TRIzol reagent was used to extract RNA from the tissues. After reverse transcription according to the obtained RNA, cDNA was amplified using the SYBR Green PCR Master Mix (Servicebio Technology Co., Ltd., Wuhan, China). The primer list can be found in supplementary materials (Supplementary Table S1). The Real-Time PCR system, Bio-Rad CFX Connect (Bio-Rad, Hercules, CA, USA) was used to determine the RT-qPCR process.

Statistical analysis

The data and graphs were analyzed by GraphPad Prism 9.3.1 (GraphPad Software, La Jolla, CA, USA) and R 4.2.2. Statistical analyses used in this study include the unpaired t-test, Welch’s t-test, and Mann–Whitney U test for comparison between data from two groups, ordinary one-way ordinary analysis of variance (ANOVA), Kruskal–Wallis test for comparison among data from multiple groups, Pearson correlation test and linear regression for the correlation between two indicators. The specific tests were determined based on the normality and chi-square of the data. The P-value was considered significant if it was less than 0.05.

Results

Forty-seven differentially expressed E3 ubiquitin ligases were identified by bioinformatic analysis

After performing background correction and standardization to reduce variability of GSE36830, principal component analysis (PCA) was used to reduce the dimension and to present the visual coordinates of similarity or difference between the CRSwNP and healthy controls. The results showed that the samples clustered into two distinct groups (Fig. 1a). Then the significantly differentially expressed genes in NPs compared to control tissues were identified (Supplementary Figs. S1, S2). The role of E3 ubiquitin ligases in CRSwNP was explored by taking the intersection of the DEGs of GSE36830 and the gene set of E3 ubiquitin ligases [21]. Forty-seven intersection genes were identified, including 23 upregulated genes and 24 downregulated genes (Fig. 1b). A heat map was used to visualize the differential expression of up- and down-regulated genes among 47 DEGs (Fig. 1c), details of which can be found in supplementary material (Supplementary Table S2).

Fig. 1. Identification of differentially expressed E3 ubiquitin ligases.

a PCA of gene expression in CRSwNP and healthy controls in GSE36830. b Venn diagram illustrating the number of overlapping genes of the DEGs of GSE36830 and the gene set of E3 ubiquitin ligases. c Gene expression heat map of the 47 overlapping genes. d–f The comparisons of CBL, NEDD4L, and NOSIP expression in CRSwNP patients (n = 6) and healthy controls (n = 6) in GSE36830. Data are presented as means ± SD. Welch’s t-test was used except in f, where unpaired t-test was used. * for P < 0.05, ** for P < 0.01.

NEDD4L is downregulated in clinical samples of nasal polyps

The intersecting genes we obtained belong to the E3 ubiquitin ligase subfamily HECT family, RING family, and U-box family, respectively. For efficient exploration of potential drug targets, Neural Precursor Cell Expressed Developmentally Downregulated 4-Like (NEDD4L) of the HECT family, Cbl Proto-Oncogene (CBL) of the RING family, and Nitric Oxide Synthase Interacting Protein (NOSIP) of the U-box family were selected, each of them has the most significant difference in its family (Fig. 1d–f). However, the RT-qPCR results of the clinical samples showed that there was no significant difference in mRNA expression levels of CBL and NOSIP among the control group, the ENP group, and the NENP group (Fig. 2a, c). By contrast, the mRNA and protein expression level of NEDD4L showed a significant decrease in the ENP and NENP groups (Fig. 2b). Furthermore, the difference in the mRNA and protein expression level of NEDD4L between the ENP group and the NENP group was not significant (Fig. 2b, d, e). In addition, NEDD4L expression was correlated with the visual analog scale (VAS) of clinical symptoms (Fig. 2f, g).

Fig. 2. Expression of CBL, NEDD4L, and NOSIP in clinical samples from CRSwNP patients and controls.

a Comparison of the relative mRNA expression level of CBL in controls (n = 3), ENP (n = 5), and NENP (n = 5). b Comparison of the relative mRNA expression level of NEDD4L in controls (n = 3), ENP (n = 5), and NENP (n = 4). c Comparison of the relative mRNA expression level of NOSIP in controls (n = 3), ENP (n = 5), and NENP (n = 5). d, e The relative protein expression level of NEDD4L expression in controls (n = 14), ENP (n = 10), and NENP (n = 9). f, g The correlation between NEDD4L and the clinical parameters of VAS in ENP and NENP. Data are presented as means ± SD. Ordinary one-way ANOVA, Pearson correlation test and linear regression were used. * for P < 0.05, ** for P < 0.01.

Function of NEDD4L interacting proteins correlates with EMT

To comprehensively explore the role of NEDD4L in the epithelium of NPs, potential proteins interacting with NEDD4L were screened using immunoprecipitation-mass spectrometry (IP-MS) and bioinformatic analyses were performed. The results of GO functional enrichment illustrated the biological process (BP), cellular component (CC), and molecular function (MF) of the identified genes (Fig. 3a), while that of KEGG pathway enrichment analyses showed the related signaling pathway (Fig. 3b). A PPI network was constructed based on the top 24 interacting proteins that were identified by IP-MS and the top 25 interacting proteins that were shown in the STRING database (Fig. 3c). The complete results of the GO and KEGG pathway enrichment analyses are available in supplementary materials (Supplementary Tables S3, S4). Notably, “focal adhesion” and “cell-substrate junction” of the CC terms, “cadherin binding” and “tubulin binding” of the MF terms are all related to EMT. Moreover, 15 EMT-associated genes were identified among the interacting proteins (Fig. 3d). To establish the relationship between the interacting proteins and EMT, a hypergeometric distribution test was performed to assess the enrichment of the interacting genes in the EMT gene set, revealing an OR value of 2.895331 and a P-value of 0.0004407 (Fig. 3e).

Fig. 3. Bioinformatic analyses of the identified interacting genes of NEDD4L.

a A Bar chart showing the top 15 enriched GO terms of biological process (BP), cellular component (CC), and molecular function (MF), respectively. b Bubble chart showing all enriched KEGG pathways. c PPI networks of the top 24 interacting proteins identified by IP-MS and the top 25 interacting proteins from the STRING database. The shapes of the nodes represent different gene sources, the circles represent deriving from the String database, the squares represent deriving from the current IP-MS results, and the diamonds represent this gene exists in both the database and the IP-MS results. d Bubble map showing the distribution of EMT-related genes among all interacting genes. e Relative enrichment of the interacting genes in EMT.

NEDD4L expression levels correlate with expression levels of EMT-related biomarkers in the epithelium

The correlation between the expression levels of NEDD4L and the expression levels of EMT-related indicators was verified, given the correlation between NEDD4L and EMT suggested by the IP-MS results. The results derived from GSE36830 showed that NEDD4L expression levels were negatively correlated with the expression levels of two EMT biomarkers, VIM and TWIST1, respectively (Fig. 4a, b). Since the interacting proteins were enriched in the GO term “cadherin binding” (Fig. 3a), E-cad was used as a biomarker of EMT to verify the correlation between NEDD4L and EMT in immunohistochemistry (Fig. 4c). The results showed that NEDD4L and E-cad were significantly reduced in the group of ENP and NENP, and there was no significant difference in NEDD4L and E-cad expression between ENP and NENP (Fig. 4d, e). Furthermore, correlation analysis results indicated that the expression level of NEDD4L was positively correlated with the expression level of E-cad in nasal polyps (Fig. 4f–h).

Fig. 4. Reciprocal expression of NEDD4L and EMT-related indicators.

The results of the two samples in controls were so close that their points coincide in f–h. a The correlation of NEDD4L expression levels and VIM expression levels in NPs (n = 6) and healthy controls (n = 6) in GSE36830. b The correlation of NEDD4L expression levels and TWIST1 expression levels in NPs (n = 6) and healthy controls (n = 6) in GSE36830. c Immunohistochemical stains of E-cad and NEDD4L in controls (n = 14), ENP (n = 12), and NENP (n = 12) (400×). d, e The comparisons of NEDD4L and E-cad expression levels in each group. f The correlation of NEDD4L and E-cad expression in controls and ENP samples. g The correlation of NEDD4L and E-cad expression in controls and NENP samples. h The correlation of NEDD4L and E-cad expression in controls and all NP samples. Data are presented as means ± SD. Ordinary one-way ANOVA, Pearson correlation test and linear regression were used. ** for P < 0.01, **** for P < 0.0001.

Downregulation of NEDD4L exacerbates nasal polyp formation by promoting epithelial-mesenchymal transition

The trends of EMT-related biomarker expression changes were analyzed after the gain and loss function of NEDD4L in RPMI-2650 (Fig. 5a–e) and hNECs (Fig. 5f–j), respectively, to determine the role of NEDD4L in the EMT process of nasal epithelial cells. It was found that decreased expression of the epithelial biomarker E-cad and increased expression of the mesenchymal biomarker α-SMA and vimentin resulted from NEDD4L knockdown, and vice versa. In addition, changes in the expression levels of EMT biomarkers induced by deubiquitinase inhibitor PR-619 could be reversed by the sh-NEDD4L plasmids. These findings suggest that the E3 ubiquitin ligase NEDD4L has a suppressive effect on EMT in nasal epithelial cells.

Fig. 5. Downregulation of NEDD4L exacerbates nasal polyp formation by promoting EMT.

a Relative protein expression level of NEDD4L, E-cad, vimentin, and α-SMA in the groups of negative control (NC), vector, sh-NEDD4L, PR-619, sh-NEDD4L + PR-619 (RPMI-2650). b–e The comparisons of the relative expression levels of NEDD4L, E-cad, vimentin, and α-SMA in each group of RPMI-2650. f Relative protein expression level of NEDD4L, E-cad, vimentin, and α-SMA in the groups of negative control (NC), vector, sh-NEDD4L, PR-619, sh-NEDD4L + PR-619 (hNECs). g–j The comparisons of the relative expression levels of NEDD4L, E-cad, vimentin, and α-SMA in each group of hNECs. * for P < 0.05, ** for P < 0.01. Data are presented as means ± SD. Ordinary one-way ANOVA was used. *** for P < 0.001, **** for P < 0.0001.

NEDD4L mediates β-catenin degradation in nasal epithelial cells

To gain insight into the mechanism by which NEDD4L suppresses EMT, previous studies on the correlation between the EMT process and the top 10 interacting proteins identified by IP-MS were searched. The results showed that correlations between the EMT process and DDR1, MAP9, DCTPP1, and VCP, respectively. Besides, among these interacting proteins, research on the correlation between DDR1 and the EMT process (32 studies) [22, 23] was significantly more prevalent than that of the other interacting proteins (1–2 studies). Furthermore, Ubibrowser2.0 database was used to predict the top 20 potential E3 ligases of DDR1, VCP, MAP9, and DCTPP1. The results showed that NEDD4L was ranked 8th in DDR1’s predicted E3 ligases, while NEDD4L was ranked 41th in the predicted E3 ligases for VCP (Fig. 6b, Supplementary Tables S5, S6). There were no corresponding predicted targets for MAP9 and DCTPP1. Thus, DDR1, a member of the receptor tyrosine kinases which is associated with EMT in tumors, was considered as a potential interacting partner (Fig. 6a). Co-IP assays confirmed that the bite protein NEDD4L interacted with DDR1 in hNECs (Fig. 6c), and further analysis was conducted by docking NEDD4L with DDR1 to explore their interaction region (Fig. 6d). The highest-ranked model revealed a docking score of −334.21 and a confidence score of 0.9755 (Fig. 6e, Supplementary Fig. S3), with 18 pairs of hydrogen bonds (within 4.1 Å) and 3 pairs of salt bridges between the two proteins (Fig. 6f). The hot spot residues involved in the interaction were primarily SER-264, ALA-508, HIS-492, ASN-876, TRP-576, HIS-559, ASP-561, ARG-563, ASN-616, TYR-660, ARG-945, LYS-870 of NEDD4L. LYS-133, GLU-139, LYS-82, VAL-198, THR-201, TYR-203, SER-262, SER-259, ASP-328, ASN-325, SER-264, PHE-263 of DDR1.

Fig. 6. Interaction between NEDD4L and DDR1.

a MS/MS spectra of the peptide “LLLATYAR”. Each peak represents a fragment ion. The colored peaks are the detected b ions (green) and y ions (red). The y1, b2, y5, y6 and y7 ions matched the secondary ion distribution generated after the fragmentation of the theoretical peptide sequence. b E3 ubiquitin ligase of DDR1 predicted in the UbiBrowser2.0 database. c The comparisons of NEDD4L and DDR1 expression in each group after Co-IP. d, e Overall binding structure of DDR1 on the NEDD4L predicted by docking. Gold represents DDR1, blue represents NEDD4L. f Detailed interaction network between NEDD4L and DDR1. Hot spot residues of NEDD4L (gold) and NEDD4L (blue) are displayed as sticks. H-bonds are shown as blue lines, and salt bridges are shown as golden dashed lines. g Immunofluorescent stains of E-cad (green) and vimentin (pink) in each group of hNECs (400×). h, i The comparisons of E-cad and vimentin expression levels in each group. Data are presented as means ± SD. Ordinary one-way ANOVA was used. * for P < 0.05, ** for P < 0.01, *** for P < 0.001.

Subsequently, after the loss function of NEDD4L and DDR1, the expression levels of EMT biomarker E-cad and vimentin were determined by conducting immunofluorescence. The results indicate that the downregulation of NEDD4L leads to a decrease in the expression of E-cad and an increase in the expression of vimentin (Fig. 6g–i). Moreover, these effects were reversed when DDR1 had also been downregulated, suggesting that NEDD4L suppressed the EMT process via DDR1 in NPs.

NEDD4L/DDR1 suppresses EMT through β-catenin/HIF-1α positive feedback loop

The underlying molecular mechanism was determined to further evaluate the effect of NEDD4L. As adhesion junctions consist of E-cad and β-catenin, the disruption of adhesion junctions induced by the downregulation of NEDD4L indicated the potential effect of β-catenin in this process [24, 25]. Therefore, the expression of β-catenin in hNECs was determined. It was found that the protein expression of β-catenin was downregulated by overexpression of NEDD4L, which could be reversed by DDR1 overexpression (Fig. 7a, b). By contrast, the mRNA expression of β-catenin showed an insignificant decreasing trend after overexpression of NEDD4L (Fig. 7c). Co-treatment with MG-132 increased the protein expression of β-catenin (Fig. 7d), indicating the degrading effect of NEDD4L on β-catenin. Further, Chen et al. [26] found that NEDD4L could regulate β-catenin’s function of activating HIF-1α through CTHRC1 in interstitial pulmonary fibrosis, whilst numerous studies have reported the mutual regulatory effect between β-catenin and HIF-1α [27–30], the critical role of inducing EMT in NPs patients necessitates the investigation of the mutual regulatory effect of β-catenin and HIF-1α under the regulation of NEDD4L/DDR1.

Fig. 7. The role of β-catenin/ HIF-1α positive feedback loop regulation under NEDD4L/DDR1 regulation.

a The relative protein expression level of β-catenin in each group of hNECs. b The comparisons of the relative protein expression levels of β-catenin in each group of hNECs. c The comparisons of the relative mRNA expression levels of β-catenin in each group of hNECs. d The comparisons of the relative protein expression levels of β-catenin, CTHRC1 and HIF-1α in each group of hNECs. e Immunofluorescent stains of β-catenin (red), CTHRC1, and HIF-1α in each group of hNECs (400×). f Immunofluorescent stains of β-catenin (red), CTHRC1, and HIF-1α in each group of hNECs (630×). g Mechanism diagram of NEDD4L regulation of the β-catenin/HIF-1α positive feedback loop. h–j The relative protein expression levels and their comparisons of HIF-1α and CTHRC1 in each group. k, l The relative mRNA expression levels of HIF-1α and CTHRC1 in each group. m–o The relative protein expression levels and their comparisons of β-catenin and CTHRC1 in each group. p, q The relative mRNA expression levels of β-catenin and CTHRC1 in each group. Data are presented as means ± SD. Ordinary one-way ANOVA was used. * for P < 0.05, ** for P < 0.01, *** for P < 0.001.

The immunofluorescence results showed that the increased expression of β-catenin, CTHRC1 and HIF-1α under the downregulation of NEDD4L could be reversed by downregulation of DDR1 (Fig. 7e). Besides, the immunofluorescence showed the enhancement of nuclear β-catenin translocation (Fig. 7f), suggesting transcriptional activity of β-catenin was enhanced in hNECs. By co-traiting with the specific inhibitor of β-catenin, ICG-001, as well as the specific inhibitor of HIF-1α, PX-478, it was found that the upregulation of HIF-1α was reversed after adding ICG-001, as well as finding the upregulation of β-catenin/CTHRC1 was reversed after adding PX-478, indicating the existence of positive feedback loop between β-catenin and HIF-1α in NPs (Fig. 7g–q). Furthermore, since the expression trends in protein expressions and mRNA expressions of HIF-1α and CTHRC1 were not consistent (Fig. 7i, j), we further co-treated the oe-NEDD4L group with MG-132, and found that MG-132 could eliminate NEDD4L-mediated downregulation of HIF expression, which indicating the potential degradation effect of NEDD4L on HIF-1α in NPs (Fig. 7d).

Effect of NEDD4L on polyp formation in murine NP model

To validate our findings in vivo, the murine NP model was established (Fig. 8a). In polyp-bearing mice, more NPs, more epithelial disruptions, and lower mRNA expression levels of NEDD4L were found. Upregulation of NEDD4L expression level significantly attenuated nasal polyp formation and epithelial disruption in mice, its effect has no significant difference compared to the application of dexamethasone (Fig. 8b, g–i). Interestingly, an increasing trend in the expression level of NEDD4L was observed in dexamethasone-treated mice (Fig. 8i).

Fig. 8. Effect of NEDD4L on CRSwNP mouse model.

a Schematic illustration of the murine NP model. C57BL/6J mice were treated with saline, ovalbumin (OVA), Staphylococcus enterotoxin B (SEB), lentiviral oe-NC particles, lentiviral oe-NEDD4L particles, and dexamethasone, respectively. i.p. for intraperitoneal, i.n. for intranasal. b Representative images of sinonasal spaces stained with H&E in each group (40×). Nasal polyps are marked with squares and epithelial disruptions are marked with circles. c–f Immunohistochemical stains of E-cad, α-SMA, β-catenin, and HIF-1α in group 1 (n = 5), group 2 (n = 5), group 3 (n = 4), group 4 (n = 5), and group 5 (n = 5) (200×). g, h The comparisons of the number of polypoid lesions and epithelial disruptions in mice of group 1 (n = 5), group 2 (n = 5), group 3 (n = 5), group 4 (n = 5), and group 5 (n = 5). i, j The comparisons of mRNA expression level of NEDD4L and CDH1 in nasal mucosa of mice of group 1 (n = 5), group 2 (n = 5), group 3 (n = 4), group 4 (n = 5), and group 5 (n = 5). k–n The comparisons of E-cad, α-SMA, β-catenin, and HIF-1α expression levels in mice of group 1 (n = 5), group 2 (n = 5), group 3 (n = 5), group 4 (n = 5), and group 5 (n = 5). o, p The comparisons of mRNA expression level of CXCL1 and CCL11 in nasal mucosa of mice of group 1 (n = 5), group 2 (n = 5), group 3 (n = 4), group 4 (n = 5), and group 5 (n = 5). Data are presented as means ± SD; Ordinary one-way ANOVA was used except in c and d, where the Kruskal–Wallis test and Mann–Whitney U test were used. * for P < 0.05, ** for P < 0.01, *** for P < 0.001, **** for P < 0.0001.

Immunohistochemistry and PCR were conducted to confirm the effect of NEDD4L in suppressing EMT in vivo. The mRNA expression of EMT-related biomarker CDH1 was downregulated in polyp-bearing mice, which was reversed by treatment with lv-oe-NEDD4L particles or dexamethasone (Fig. 8j). Consistently, the expression of E-cad in immunohistochemistry showed the same trend with CDH1 (Fig. 8k). By contrast, though the expression differences were not significant, the expression of EMT biomarker α-SMA showed an increased trend in polyp-bearing mice, which was reversed by treatment with lv-oe-NEDD4L particles or dexamethasone (Fig. 8l). The expression difference between the oe-NEDD4L group and the dexamethasone group was insignificant. These results indicated that upregulating NEDD4L could suppress the EMT process of polyp-bearing mice.

Subsequently, the expression levels of β-catenin and HIF-1α were determined to verify the effect mechanism of NEDD4L in vivo. The expression of HIF-1α was upregulated in polyp-bearing mice, which could be reversed by co-treating with oe-NEDD4L or dexamethasone. Correspondingly, though the expression differences were not significant, the expression of β-catenin showed an increased trend in polyp-bearing mice, which was reversed by treatment with lv-oe-NEDD4L particles or dexamethasone. Moreover, the percentage of nuclear β-catenin⁺ cells was significantly increased in polyp-bearing mice, while being downregulated in the oe-NEDD4L group or the dexamethasone group (Fig. 8m). The expression difference between the oe-NEDD4L group and the dexamethasone group was insignificant. These results were largely consistent with what we found in vitro.

Furthermore, the difference in the therapeutic efficacy of targeting NEDD4L for ENP and NENP was compared. CXCL1 plays a crucial role in recruiting neutrophils by activating CXCL2 and CXCR1 [31], whilst CCL11 (eotaxin) plays a crucial role in recruiting and activating eosinophils [32]. Therefore, neutrophil chemotactic cytokines CXCL1 and eosinophil chemotactic cytokines CCL11 were used as indicators. In polyp-bearing mice, mRNA expressions of CXCL1 and CCL11 were upregulated, which were reversed after treatment with lv-oe-NEDD4L particles. By contrast, the reversal of CCL11 by dexamethasone treatment was significantly stronger than that of CXCL1 (Fig. 8o, p).

Discussion

This study initially investigated three ubiquitin ligases that may affect CRSwNP and it was found that significant downregulation of NEDD4L exacerbated EMT in nasal epithelial cells. Additionally, after the gain and loss function of NEDD4L, it was found that upregulation of NEDD4L expression could reverse the EMT process in nasal epithelial cells, thereby inhibiting the development of nasal polyps. To the best of our knowledge, this study established the first link between ubiquitin modifications and the EMT process of CRSwNP, which provided mechanistic insights into the role of NEDD4L in CRSwNP, not only by identifying interacting proteins of NEDD4L but also by enriching the downstream signaling pathways.

NEDD4L, a HECT E3 ubiquitin ligase of the NEDD4 family, plays a role with various proteins in different signaling pathways, including the epithelial sodium channel (ENaC) and Wnt/β-catenin signaling pathway. Our finding reflects the protective role of NEDD4L through reversing the EMT process in CRSwNP, which is similar to previous studies in renal fibrosis [33, 34], pulmonary fibrosis [35], and lung cancer [36]. In addition, several studies have reported that the downregulation of NEDD4L is correlated with poor prognoses in certain tumors, such as gastric cancer [37], prostate cancer [38], and malignant glioma [39]. Given the key role of EMT in tumor invasion and metastasis [24], these correlations could be explained, at least in part, by the inhibitory effect of NEDD4L on the EMT process.

A novel NEDD4L-interacting protein, DDR1, which is a receptor tyrosine kinase mainly expressed in epithelial cells, was identified by IP-MS. Upregulation of DDR1 has been reported in a variety of cancers and fibrotic diseases, including cutaneous proliferative scarring, idiopathic pulmonary fibrosis, and renal fibrosis [40]. Results of Co-IP experiments showed that NEDD4L mediated the degradation of DDR1, thereby inhibiting its downstream signaling pathway. Furthermore, the way NEDD4L binds to DDR1 was simulated by molecular docking, and hot spot amino acid residues were predicted, laying the foundation for future drug research and development. By conducting immunofluorescence, it was found that NEDD4L suppressed the EMT process via DDR1. Besides, it is interesting to note that a variety of collagens can activate DDR1. Since type I, III, and IV collagens are deposited in nasal polyps, especially in the submucosal connective tissue [41], DDR1 may play a role in the pathogenesis of CRSwNP directly or indirectly.

During EMT, the disruption of cell-cell adhesion junction was accompanied by the downregulation of E-cad, which results in some β-catenin no longer binding to E-cad, but activating and playing a transcription factor role in nucleus [24]. Since our study found a correlation between the downregulation of NEDD4L and that of E-cad, the effect of NEDD4L/DDR1 on β-catenin was investigated and a regulatory role of NEDD4L/DDR1 on the downstream β-catenin/ HIF-1α positive feedback loop was found. Borza et al. [42] found that DDR1 could increase the number of activated β-catenin in nucleus by inducing breakpoint cluster region (BCR) phosphorylation, thereby blocking the interaction sites between BCR and β-catenin, which supported our finding that knock-down of DDR1 could reverse the activation of nuclear β-catenin. Moreover, Chen et al. [26] found that β-catenin can bind to the promoter region of CTHRC1 and increase its expression in IPF, their study partially supported our finding of the existence of β-catenin/ HIF-1α positive feedback loop. Upregulation of NEDD4L inhibited two major signaling pathways that mediate EMT in nasal mucosal epithelial cells, namely the Wnt/β-catenin signaling pathway and the HIF-1α signaling pathway. Previous studies have shown that direct silencing of HIF-1α does not reverse the progression of EMT [43], this issue can be addressed by targeting NEDD4L. By comparing the mRNA expression levels and the protein expression levels of β-catenin, CTHRC1, and HIF-1α, whilst co-treating with MG-132, the ubiquitination and degradation roles of NEDD4L on β-catenin, and HIF-1α in NPs were initially found in NPs (Fig. 9). Our results suggested that the regulatory effect of NEDD4L on the EMT process was mainly based on its degradation effects.

Fig. 9. The mechanism of NEDD4L inhibiting EMT progression in nasal polyp patients.

NEDD4L negatively regulates DDR1, which in turn inhibits the phosphorylation of the breakpoint cluster region (BCR), increases the interaction site between BCR and β-catenin, and reduces the amount of activated β-catenin in the nucleus, thereby, NEDD4L could inhibit the Wnt/β-catenin signalling pathway and HIF-1α signalling pathway. Not only that, NEDD4L could also directly inhibit these pathways by degrading β-catenin and HIF-1α. Via the mechanisms mentioned above, NEDD4L could finally suppress the EMT process in nasal mucosa.

To initially determine the efficacy of drugs targeting NEDD4L in patients with different types of CRSwNP, as well as to verify the in vitro results with in vivo experiments, the CRSwNP mice model was established, which could reflect the pathogenesis of different types of CRSwNP and is widely used in studies of ENP and NENP [25, 44]. After OVA + SEB stimulation, the elevation of both non-eosinophilic characteristic CXCL1 and eosinophilic characteristic CCL11 in our mouse model is consistent with the results of previous studies. Besides, the decreased expression of E-cad, the increased expression of α-SMA, and the decreased mRNA expression of CDH1 in polyp-bearing mice could be reversed by upregulating NEDD4L, confirming the regulatory role of NEDD4L in EMT. Furthermore, the increased expressions of β-catenin and HIF-1α in polyp-bearing mice could be reversed by upregulating NEDD4L, confirming the regulatory effects of NEDD4L on the Wnt/β-catenin signaling pathway and the HIF-1α signaling pathway in vivo. In addition, the expression of CCL11 was significantly decreased in the dexamethasone-treated group, while the decrease in CXCL1 expression was not significant, reconfirming the poor effect of traditional corticosteroids on NENP. By contrast, the upregulation of NEDD4L expression significantly reversed the trend of the above indicators, suggesting that targeting NEDD4L is effective for both ENP and NENP.

It should be noted that the present study has several limitations. First, if the differential expression of all 47 differential genes in clinical samples had been examined, the role of ubiquitination modifications in the pathogenesis of CRSwNP could have been more fully elucidated. Secondly, the specific interacting site of NEDD4L with DDR1 could be identified if the mechanisms by which NEDD4L mediates DDR1 degradation were elaborated on. Thirdly, previous studies have shown the ubiquitination and degradation role of NEDD4L on TGF-β signaling pathway. Considering the induction role of TGF-β signaling pathway on EMT process in NPs, the regulatory role of NEDD4L on TGF-β signaling pathway should be further determined. However, though our immunofluorescence experiments of the regulatory role of NEDD4L/DDR1 on TGF-β signaling pathway showed the positive results, our immunoblotting experiment showed the conflicting results. Therefore, the intricate role of TGF-β1 under NEDD4L regulation should be further investigated in the future.

In conclusion, for the first time, the E3 ubiquitin ligase NEDD4L was identified as a novel target for CRSwNP therapy, the interaction of NEDD4L with DDR1 was discovered, and the direct (degradation of β-catenin and HIF-1α) or indirect (mediated degradation of DDR1) inhibition of the β-catenin/HIF-1α positive feedback loop by NEDD4L was demonstrated. Our study opens a new avenue to inhibit the EMT process through ubiquitination modifications in CRSwNP. Since NEDD4L has good efficacy against both ENP and NENP, drug research and development against this target is expected to comprehensively improve the refractory character of CRSwNP.

Author contributions

SYC conceived and designed the study, conducted the experiments and analyzed the data, prepared the figures and drafted the paper. PQL, DXQ, HL, and HQZ provided crucial assistance during the animal experiments. YX edited and revised the manuscript.

Competing interests

The authors declare no competing interests.

Ethical approval

This work was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Renmin Hospital, Wuhan University (No. WDRY2021-K084). The detailed aims of the study and the planned procedures were explained to all patients, who then signed the informed consent documentation. The Institutional Animal Care and Use Committee of the Renmin Hospital of Wuhan University approved our animal experiments which were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (License No. WDRM 20211005).

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-023-01190-8.