WDPCP modulates cilia beating through MAPK/ERK pathway in chronic rhinosinusitis


 Background: Cilia loss and dysfunction is one of the typical pathological features of chronic rhinosinusitis. Tryptophan-aspartic acid (W-D) repeat containing planar cell polarity effector (WDPCP) has been proven to be an essential element for ciliogenesis in human nasal epithelium, but its role in the beating of cilia remains unclear. Cilia beating requires energy from the mitochondria, which is regulated by the MAPK/ERK pathway. In this study, we sought to investigate the role of WDPCP and its underlying mechanism behind the dysfunction in the beating of cilia in chronic rhinosinusitis.Methods: We demonstrated WDPCP expression in the epithelium of nasal polyps. We also investigated the MAPK/ERK pathway in primary human sinonasal epithelial cells to explore the function of WDPCP. The air-liquid interface culture system was used as a model to verify the role of WDPCP and the MAPK/ERK pathway in the beating of cilia.Results: With the dysfunction of cilia beating, we observed a low expression of WDPCP in the epithelium of nasal polyps. Within the in vitro study, we found that WDPCP was critical for mitochondrial biogenesis and mitochondrial function in human sinonasal epithelial cells, possibly due to the activation of the MAPK/ERK pathway. The mitochondrial dysfunction caused by U0126 or lacking WDPCP could be partially recovered by dexamethasone.Conclusion: The low expression of WDPCP in nasal epithelium could affect mitochondria via the MAPK/ERK pathway, which may contribute to the dysfunction in the beating of cilia in chronic rhinosinusitis.

Mitochondrial structure was detected via fluorescent MitoTracker (Invitrogen), which was cultured with HSECs at 1:500 dilution at 37 °C for 1 hour. Then the cells were fixed and stained with DAPI for

Western blot analysis
Total protein from nasal mucosa and HSECs were extracted in RIPA lysis buffer. Protein concentrations were determined by bicinchoninic acid assay (BCA). Samples which contained 30 µg of protein were resolved via SDS-PAGE in 10%~12% Tris-glycine gels and transferred onto a polyvinylidene fluoride membrane (Milipore) and blocked with 5% bovine serum albumin (BSA). The membranes were incubated overnight with monoclonal mouse anti-NRF1 (Abcam, ab175932), monoclonal mouse anti-NRF2 (Abcam, ab62352), monoclonal mouse anti-TFAM (Abcam, ab131607), polyclonal rabbit anti-COX4 (Cell signaling, 4850S) at 1:1000, then incubated with an appropriate secondary antibody (1: 10,000) for 1 hour. The relative protein levels were quantified by densitometric image analysis of bands using Fiji (National Institutes of Health) and were normalized against β-actin.

Scanning electron microscopy
The membranes which contained HSECs from the Transwell inserts were fixed with 2.5% glutaraldehyde for 4 hours at room temperature. Subsequently, the cells were progressively dehydrated in 30%, 50%, 70% and 90% ethanol for one time per concentration at 15-minutes intervals, 3 times in 100% ethanol for 15 minutes, and 3 times in 100% tert-butyl alcohol for 15 minutes. The filters were then glued onto scanning electron microscopic (SEM) stubs and sputter coated to a thickness of 12 nm by gold palladium. The surface of the Transwell membrane was examined at an accelerating voltage of 10 kV using a Quanta-400 scanning electron microscope (FEI).

Cell stimulation and transfection
Human sinonasal epithelial cells were transfected with siRNA targeted to WDPCP using Lipofectamine 3000 (Invitrogen) following the manufacturer's instructions. HSECs were stimulated by U0126 (10 µM) or dexamethasone (0.01 mg/mL). After 24 hours of incubation, the protein was collected for further experiments.

Mitochondrial DNA copy number analysis
Total DNA was extracted from HSECs using the QIAamp DNA Mini Kit (Qiagen). The mitochondrial DNA (mtDNA) copy number was determined by amplifying genes encoding genomic DNA and mitochondrial DNA. The mtDNA levels were quantified by quantitative real-time polymerase chain reaction on a Roche Light Cycler 96 (Roche) using HV1 primers (forward: 5'-TTGCACGGTACCATAAATACTTGAC-3', reverse: 5'-GAGTTGCAGTTGATGTGTGATAGTTG-3'). Nuclear gene β-globin primers (forward: 5'-ACACAACTGTGTTCACTAGC-3', reverse: 5'-CAACTTCATCCACGTTCACC-3') were used as a nuclear control. Relative quantification of mitochondrial DNA copy number was calculated after using the 2 −ΔΔCT method to obtain the expression fold change.

Mitochondrial membrane potential measurement
The mitochondrial membrane potential was evaluated with JC-1 fluorescent dye (Beyotime, China). JC-1 displayed red or green fluorescence depending on the mitochondrial potential. Normal mitochondrial membrane potential exhibited red fluorescence, whereas damaged mitochondria exhibited green fluorescence. The HSECs were stained with JC-1 in culture media for 20 minutes at 37°C and were then washed for 3 times with washing buffer. The mitochondrial membrane potential was measured by flow cytometry (BD Biosciences).

Measurement of ATP levels
An ATP Assay Kit (Beyotime, China) was used to conduct the ATP assay. Briefly, HSECs were collected in lysis buffer and centrifuged at 12,000 × g for 5 minutes at 4 °C. ATP detection reagent was added into 96-wells, then the standards and samples were added into the wells and the detection solution was mixed. Chemiluminescence was detected by a Synergy H1 Hybrid Multi-Mode Reader (BioTek, USA). The levels of ATP were calculated based on the standard curve and were normalized to the protein content.

Cilia beating frequency
Both ciliated HSECs in ALI culture and clinical samples collected within 30 minutes were used for cilia beating frequency (CBF) analysis. The room temperature was controlled at 25 ℃, and the humidity was maintained at 70-80%, which would not significantly affect the CBF [25]. A high-speed digital video camera (Basler AG) captured images at 100 frames per second and Sisson-Ammons Video Analysis (SAVA) software (National Instruments) was used for video analysis. Each measurement was obtained 3 times and recorded 15 seconds each time.

Statistical analysis
Data were presented as mean ± standard error of the mean (SEM), and analyzed using GraphPad PRISM 7 (GraphPad Software). One-way ANOVA, Student's t-test, one sample t-tests and Fisher's exact tests were performed for statistical analysis. A value of p <0.05 was considered statistically significant.

Expression of WDPCP and the cilia beating function were decreased in nasal polyps
In our previous study, we have previously discovered that the expression of WDPCP was reduced in the epithelial layer of mucosa from patients with CRS compared with control subjects, accompanied with cilia loss [18]. According to immunohistochemical staining, we discovered that WDPCP was mainly expressed in the cytoplasm of nasal mucosa epithelium and the staining (brown color) was reduced in nasal polyps ( Figure 1A). Western blot also showed decreased protein expression of WDPCP in nasal polyp tissue compared with ethmoid sinus tissue from control subjects ( Figure 1B-C). Consistent with the protein levels, RT-PCR demonstrated a decrease of WDPCP mRNA levels in nasal polyps ( Figure   1D). Moreover, to explore the function of cilia, we measured the cilia beating frequency (CBF), and found that the cilia from nasal polys beat at a slower rate compared with control mucosa ( Figure 1E).
Additional movie files show the cilia beating in more detail (see Additional file 1-2).

WDPCP potentially acted through the MAPK/ERK pathway to regulate ciliogenesis and cilia function
Evidence showed that WDPCP participated in ciliogenesis and could possibly affect the beating of cilia [16]. To investigate the potential mechanism, we detected the changes of expression in the MAPK/ERK pathway. We silenced the expression of WDPCP in HSECs by transferring small interfering RNAs (Si-WDPCP). Twenty-four hours post-transfection, the expression of phosphorylated ERK1/2 (p-ERK1/2) was significantly decreased in HSECs compared to the control. However, the expression of MEK1/2, p-MEK1/2 and ERK1/2 remained constant after WDPCP silencing (Figure 2A-B).
HSECs could differentiate into respiratory epithelium when they were grown in an air-liquid interface (ALI). It took approximately 14 days to obtain fully mature cilia. This ALI culture was also used to explore the role of WDPCP and the MAPK/ERK pathway in ciliogenesis and cilia beating. The cilium in HSEC ALI cultures was detected on Day-14 by scanning electron microscopy and immunofluorescence staining. HSECs which lacked WDPCP at the beginning of ciliogenesis, differentiated into fewer and shorter cilia compared with the controls (Figure 2C-D). Similarly, HSECs cultured with MAPK/ERK inhibitor U0126 also developed fewer and shorter cilia ( Figure 2C-D). We also examined the cilia beating frequency (CBF) on Day-14. HSECs treated with Si-WDPCP and U0126 displayed slower cilia beating frequencies compared with controls ( Figure 2E). Additional movie files show the cilia beating of HSECs in more detail (see Additional file [3][4][5].

WDPCP regulated mitochondrial biogenesis through the MAPK/ERK pathway
Substantial evidence suggested that ATP from mitochondria is essential for the beating of cilia [26][27][28].
Thus, we hypothesized that WDPCP acts on mitochondria through the MAPK/ERK pathway to affect cilia beating in CRS. First, we measured the mitochondrial DNA (mtDNA) copy number to observe the number of mitochondria in HSECs. We found that the mtDNA copy numbers were decreased in HSECs after WDPCP silencing or after being cultured with MAPK/ERK pathway inhibitor U0126.
Glucocorticoids are known to be the mainstay of medical treatment for CRS. Based on previous research, we explored the effects of glucocorticoids on the mitochondria. mtDNA copy numbers were not significantly decreased after treatment with dexamethasone in Si-WDPCP HSECs and HSECs inhibited by U0126 ( Figure 3A).
Decreased mitochondrial biogenesis or increased apoptosis can lead to the reduction in the number of mitochondria. To understand the specific reasons of mitochondrial reduction, we examined the expression of biomarkers for both mitochondrial biogenesis and apoptosis. Western blot results revealed that the apoptosis markers, Cleaved Caspase 3 and Cleaved Caspase 7 were not activated in HSECs which lacked WDPCP nor HSECs inhibited by U0126 ( Figure 3B). However, we found the low expression of NRF1, NRF2, TFAM and COX4 in Si-WDPCP HSECs compared with controls ( Figure 3C-D).
Interestingly, NRF1 protein levels were increased after dexamethasone treatment in Si-WDPCP HSECs ( Figure 3C-D). Additionally, the protein expression of NRF1, NRF2 and TFAM could be inhibited by U0126 ( Figure 3E-F). Negative control siRNAs (Si-NC) were used to rule out the effect of transfection on mitochondria. Moreover, the decreased expression of NRF1, NRF2, TFAM and COX4 was also observed in the nasal epithelium and nasal polyps by immunohistochemical staining ( Figure 3G).

WDPCP stabilized mitochondrial function through the MAPK/ERK pathway
In addition to the number of mitochondria, the function of mitochondria is also critical for the beating of cilia [29]. Flow cytometry analysis revealed a depolarization of the mitochondrial membrane in Si-WDPCP HSECs and U0126-treated HSECs ( Figure 5A-B). We also measured mitochondrial function by analyzing ATP production, and we found that ATP levels were reduced in Si-WDPCP HSECs and U0126treated HSECs ( Figure 5C). Interestingly, dexamethasone could repair the damage of mitochondrial function caused by the lack of WDPCP as well as inhibition by U0126 ( Figure 5A-C). To monitor mitophagy in the HSECs, mitochondria were visualized with fluorescent MitoTracker. From the MitoTracker staining, rod-shaped mitochondria can be detected in the control and Si-NC group, but not in the Si-WDPCP or U0126 group. Despite the use of dexamethasone, the mitochondrial structure was difficult to recover ( Figure 5D).

Discussion
Patients with CRS were subjected to repeated cycles of inflammation and infection, which resulted in severe cilia loss and increased mucus secretion. In addition to direct ciliary loss, the surviving cilia that experienced inflammatory and/or microbial damage appeared to be dysfunctional. However, the reason behind poor ciliation and cilia dysfunction is still unclear. PCP pathway proteins, such as Intu, Vangl2 and WDPCP were important units which controlled ciliogenesis and cilia function. Studies showed that WDPCP governs ciliogenesis in Xenopus embryos [14,17] and human nasal epithelium [18]. Although short cilia might contribute to cilia beating dysfunction [30], it is still unclear whether WDPCP directly regulated the beating of cilia. To our knowledge, this is the first study of WDPCP on cilia function in human sinonasal epithelium. Our data showed that the low expression of WDPCP in nasal polyps was accompanied with a decreased frequency of cilia beating. Following WDPCP silencing in ALI cultures of HSECs, there was a decrease in cilia beating frequency. These data suggest that in addition to its role in the ciliogenesis of HSECs, WDPCP is also critical to cilia beating function.
Mitochondria are highly dynamic organelles that are essential for energy production and cell homeostasis. Mitochondrial function modulates cell survival, metabolism and health status. The impairment of mitochondrial function has been involved in a variety of pathological conditions and diseases. For example, mitochondrial dysfunction could provoke heterotaxy via aberrant ciliogenesis and reduced the beating of cilia [29]. Our finding showed that the expression of mitochondrial biogenesis markers such as NRF1, NRF2, TFAM and COX4 were decreased in the epithelium of nasal polyp. HSECs which lacked the expression of WDPCP had decreased mtDNA copy number, low expression of NRF1, NRF2, TFAM and COX4, and poor mitochondrial function and structure. However, the mitochondrial apoptosis markers were not activated. Overall, these findings support the notion that WDPCP regulated cilia function from its effects on the mitochondria.
The MAPK/ERK pathway regulates a series of cellular biological behaviors. Although the importance of MAPK/ERK activity in morphogenesis and metabolism is emerging [31], the role of the MAPK/ERK pathway in ciliogenesis and the function of human airway epithelium is still unknown. Our results showed that after WDPCP silencing, the expression of p-ERK in HSECs was decreased. Also, when MAPK/ERK activity was inhibited by U0126, HSECs developed fewer and shorter cilia in ALI cultures, a phenotype reminiscent of WDPCP deficiency. Moreover, in the absence of MAPK/ERK activity, the mtDNA copy number and the expression of NRF1, NRF2, and TFAM were decreased in HSECs, and the structure of mitochondria was altered. Collectively, these results demonstrate that WDPCP acts through the MAPK/ERK pathway to regulate ciliogenesis and mitochondrial biogenesis. Consistent with our findings, the role of MAPK/ERK activity in cellular differentiation has already been investigated.
For example, MAPK/ERK activation was involved in the differentiation of nephron progenitors by the Wnt and Notch pathways [32], in addition to the crucial role of the MAPK/ERK pathway in airway development [31]. Our findings highlight the importance of MAPK/ERK activity in airway ciliogenesis and cilia function.
Glucocorticoids and their receptors could modulate mitochondrial function. Glucocorticoids activated nuclear-encoded genes, which resulted in enhanced mitochondrial biogenesis [33,34]. Additionally, glucocorticoids could directly regulate mitochondrial function by interpolating into the mitochondrial membrane [35,36]. Hence, we speculated that glucocorticoids could also repair mitochondrial damage in airway diseases. In order to address this hypothesis, we examined the expression of mitochondrial biogenesis markers and mitochondrial function after treatment with dexamethasone in Si-WDPCP HSECs and U0126-treated HSECs. Interestingly, we found that the decreased NRF1 in

Ethics approval and consent to participate
This study was approved by the Sun Yat-sen Memorial Hospital Institutional Review Board. Written consent has been obtained from all participants.

Consent for publication
All participants signed their written consent for publication.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that there is no potential conflict of interest.      Schematic diagram of WDPCP regulating ciliogenesis and cilia function in nasal epithelium.
Our results showed that WDPCP could work through MAPK/ERK pathway to regulate mitochondrial biogenesis and function. In the case of CRS, HSECs low expressed WDPCP, and phosphorylation of ERK1/2 was partially blocked, further affecting the number and function of mitochondria. This mechanism might lead to the poor ciliation and cilia dysfunction of nasal epithelium in CRS.