CTNNAL1 participates in the regulation of mucus overproduction in HDM‐induced asthma mouse model through the YAP‐ROCK2 pathway

Abstract Our previous study indicated that adhesion molecule catenin alpha‐like 1(CTNNAL1) is downregulated in airway epithelial cells of asthma patients and asthma animal model but little is known about how the CTNNAL1 affects asthma pathogenesis. To reveal the direct relationship between asthma and CTNNAL1, CTNNAL1‐deficient mouse model in bronchopulmonary tissue was constructed by introducing CTNNAL1‐siRNA sequence using adeno‐associated virus (AAV) as vector. The mouse model of asthma was established by stimulation of house dust mite (HDM). After HDM‐challenged, there was marked airway inflammation, especially mucus hypersecretion in the CTNNAL1‐deficient mice. In addition, the CTNNAL1‐deficient mice exhibited an increase of lung IL‐4 and IL‐13 levels, as well as a significant increase of goblet cell hyperplasia and MUC5AC after HDM exposure. The expression of Yes‐associated protein (YAP), protein that interacted with α‐catenin, was downregulated after CTNNAL1 silencing and was upregulated due to its overexpression. In addition, the interaction between CTNNAL1 and YAP was confirmed by CO‐IP. Besides, inhibition of YAP could decrease the secretion of MUC5AC, IL‐4 and IL‐13 in CTNNAL1‐deficient 16HBE14o‐cells. Above results indicated us that CTNNAL1 regulated mucus hypersecretion through YAP pathway. In addition, the expression of ROCK2 increased when CTNNAL1 was silenced and decreased after YAP silencing, and inhibition of YAP decreased the expression of ROCK2 in CTNNAL1‐deficient HBE cells. Inhibition of ROCK2 decreased MUC5AC expression and IL‐13 secretion. In all, our study demonstrates that CTNNAL1 plays an important role in HDM‐induced asthma, mediating mucus secretion through the YAP‐ROCK2 pathway.


| INTRODUC TI ON
Asthma is an airway chronic inflammatory disease characterized by airflow obstruction, bronchial hyperresponsiveness, mucus hypersecretion and airway inflammation. [1][2][3] It is reported that the airway epithelium is an essential controller of inflammatory, immune and regenerative responses in asthma. 4,5 In response to allergen stimulation, the airway epithelium secretes fluids, antimicrobial proteins and mucins, which, together with club cells, represent a significant part of the immunomodulatory barrier of the airway epithelium and help orchestrate innate pulmonary immunity. [6][7][8] Bronchial mucous glands produce mucous plugs within the airway lumen and ectasia of the gland ducts. 9,10 Mucous plugs are increased in fatal asthma and may contribute to asphyxia due to an abundance of mucous plugs found during the autopsy. 11,12 Bronchial epithelial cells are the first barrier against environmental pollutants and allergen stimuli. 13,14 Epithelium dysfunction is involved in the pathogenesis of lung inflammation disorders, such as asthma. 15,16 Bronchial epithelial cells of asthma patients often show significant structural damage and loss of functional homeostasis. Our previous study showed that the adhesion molecule catenin alpha-like-1 (CTNNAL1) was downregulated in asthma patients and in ovalbumin-stressed asthma mouse model. 17 Downregulation of CTNNAL1 expression leads to epithelial dysfunction. 17,18 Airway resistance was highly correlated with CTNNAL1 expression levels. 18 In addition, CTNNAL1 promoted melanoma progression, metastasis and chemoresistance. 19 Furthermore, CTNNAL1 correlated with the invasion of breast cancer, prostate cancer and lung cancer. [20][21][22] It has been demonstrated that CTNNAL1 was identified as a part of the Rho signalling pathway, serving as a scaffold protein for Lbc, 23 a member of the dbl family of Rho guanine nucleotide exchange factors (GEFs). 24 Rho GTPases play an important role during the organization of the actin cytoskeleton and the formation of focal adhesion proteins. 25 Rho-associated protein kinase (ROCK) is serine/threonine kinase that is downstream target of the GTPases RhoA. 26,27 There are two isoforms of ROCK-ROCK1 and ROCK2. The two ROCK homologs share 64% identity in their primary amino acid sequences, with the highest homology (92%) within the kinase domains and the coiled-coil domains being the most diverse (55%). There are tissuespecific differences in ROCK1 and ROCK2 expression, as well as differences in the subcellular localization of ROCK1 and ROCK2. 27 It is reported that the ROCK inhibitor, fasudil, decreased the mucus secretion and MUC5AC expression in HDM-induced mice. 28 Yes-associated protein (YAP), a transcription factor, could regulate cell proliferation and differentiation by inducing many genes activation or repression, such as AP-1 29 and NR4A1. 30 In addition,YAP also mediates the integrity of the actomyosin cytoskeleton and the intracellular mechanotransduction pathway. Moreover, it has been reported that YAP interacted with the ROCK2 promoter region in an actomyosin activity-dependent manner but not ROCK1. 31 In this study, we demonstrated that CTNNAL1 deficiency caused severer airway inflammation and mucus hypersecretion in response to HDM exposure. Inhibition of ROCK2 could down-regulate MUC5AC expression when CTNNAL1 was silenced. CTNNAL1 deficiency could promote mucus hypersecretion through YAP-ROCK2 signalling pathway.

| Ethics statement
This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethics Committee Institute of Clinical Pharmacology of the Central South University. All surgeries were performed under sodium pentobarbital anaesthesia, and all efforts were made to minimize suffering.

| Animals and challenge protocols
C57bl/6j mice were obtained from the experimental animal center,

| Trachea sampling and treatment
Mouse tracheae were taken from the AAV5-CON305 and AAV5-CTNNAL1-RNAi mice groups. Mouse tracheae were seeded into sixwell cell culture plates and cultured in serum-free medium. After 24 h of culture, HDM was applied to the culture system for 48 h.

| Lung histology and immunohistochemical staining
Paraffin-embedded lung tissue sections were stained using haematoxylin and eosin (HE) (Sigma) or Masson's trichrome. The inflammation score was measured independently by three pathologists blinded to the experiment. The scores from all three were averaged to give a final score. 34 Morphological changes in fibrotic lungs were quantified according to the criteria proposed by Ashcroft. 35 Grading was scored on a scale from 0 to 8, using the average of microscope field scores.
Immunohistochemical staining was performed on mouse lung paraffin sections using the following antibodies: anti-CTNNAL1 (Ab96184, Abcam) and anti-MUC5AC (MA5-12178, Invitrogen). The mouse lung paraffin sections were incubated with primary antibody and subsequently reacted with relative secondary antibody. The positive cells were brown, and the positive particles were located on the part of antigen. The nuclei were stained blue with haematoxylin. For microscopy, we employed the use of a Moticam Pro Microscope (BA410E, Xiamen, China). Images were obtained via Motic Images Plus3.0 (×64).

| Bronchoalveolar lavage fluid (BALF) collection and cell counting
The lungs were lavaged twice with 1 ml sterile PBS to collect one millilitre of BAL (bronchoalveolar lavage) fluid. The BAL fluid was immediately centrifuged at 1500 g. The total cell count was measured, and a cytospin preparation was performed. The cells were stained with a Diff-Quick reagent (Baxter Dade). A differential count of 300 cells was performed using the standard morphological criteria (accurate quantification of cells recovered by BAL lavage).
Transfections were conducted using Lipofectamine 3000 and p3000 (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer's instructions.

| Rho activity assay
For the Rho activity assay, cells were lysed in RIPA lysis buffer

| Western blot
Proteins from the lung tissues or HBE cells were extracted and analysed using Western blotting as described in our previous study. 36 Western blotting analysis was performed as described previously.

| Immunoprecipitation
Immunoprecipitation was performed according to our previous publications. 37 Briefly, protein from HBE cells was extracted with RIPA lysis buffer (Sigma-Aldrich). An appropriate dilution of anti-CTNNAL1 antibody (Ab96174, Abcam) or anti-YAP antibody (AF6328, Affinity) was added to the centrifuge tube that conjugates with the suspension of Protein G. Mix the antibody-bead mixture at 4°C for 4 h using tube rotator. Then, 50 μg of cell lysates were added into the mixtures and the lysate-bead/antibody conjugate mixtures were incubated overnight at 4°C. Mixtures were further resuspended in 5xSDS loading buffer. Samples were boiled for 5 min and analysed by Western blot. Immunocomplexes were stained with anti-CTNNAL1(ab96174, Abcam) as well as YAP (AF6328, Affinity).

| Quantitative RT-PCR
Total RNA was prepared from whole-lung tissues of mice or human bronchial epithelial cells from different group collected in Trizol

| Statistical analysis
All experiments were run at a minimum of triplicates, and analysis was performed using the PRISM (GraphPad, La Jolla, CA, USA) statistical software. An unpaired t test was used for comparisons between two groups. Differences among multiple groups were determined by ANOVA, followed by Bonferroni correction for multiple comparison testing. All data are presented as the mean values ± standard deviation (SD). p < 0.05 was considered statistically significant.  Figure 1A). The silencing efficiency of CTNNAL1 at 2 and 4 weeks was greater than that of 6 weeks. In the AAV5-CTNNAL1-RNAi mouse group, CTNNAL1 protein expression decreased approximately 65% ( Figure 1B,C), and the mRNA expression decreased 60% compared with control and the AAV5-CON305 mouse group ( Figure 1D). We found that the expression of CTNNAL1 was significantly lower than that found in the AAV5-CON305 group ( Figure 1E,F). Transfection of AAV5-CTNNAL1-RNAi did not influence the expression of CTNNAL1 in the livers or kidneys ( Figure S1B), and did not cause liver or kidney injuries or body weight loss ( Figure S1A,C).

| CTNNAL1 deficiency promoted airway inflammation in HDM-treated mice
We depicted our experimental protocol in Figure 2A.

| CTNNAL1 deficiency decreased the expression of RhoA and ROCK1 but increased the expression of ROCK2
It is reported that Rho/ROCK inhibitor, fasudil, decreased the mucus hypersecretion in HDM-treated mice. 28 In addition, CTNNAL1

| ROCK1 inhibition did not alter MUC5AC expression
To investigate whether ROCK1 contributes to mucus production when CTNNAL1 was silenced, we inhibited ROCK1 by siRNA in airway epithelial cells. As shown in Figure 5, inhibition of ROCK1 did not influence MUC5AC expression in CTNNAL1-deficient HBE cells ( Figure 5A-C).
F I G U R E 2 HDM stress promoted airway inflammation in CTNNAL1-deficient mice. (A) A timeline of AAV injection, allergen sensitization and exposure in this study. (B) Lung histopathology in the AAV5-CON305 or AAV5-CTNNAL1 -RNAi mice exposed to HDM or PBS as a vehicle control. Lung sections were stained with HE (n = 8; ×200, Bars = 100 μm). Lung sections were stained with MASSON (n = 6; ×200, Bars = 100 μm). Giemsa-stained BALF cells shown in the picture (n = 6; ×200, Bars = 100 μm). (C) Inflammation score was measured independently by three pathologists blinded to the experiment (n = 6). (D) Quantification of airway collagen area (n = 6). (E-H) The inflammatory cell profile in the BAL fluid was evaluated. Total cells, neutrophils, lymphocytes and eosinophils were enumerated (n = 6). (I,K) mRNA expression of IL-4 and IL-13 in the lungs was detected by RT-qPCR (n = 5). (J,L) The IL-4 and IL-13 concentration in the BALF was detected using ELISA kits (n = 5). All data are presented as mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01 We also found that IL-13 level was increased in HDM-stressed control group and CTNNAL1-deficient group compared with control group (Figure 6K,L). KD025 eliminated the effects that HDM and CTNNAL1 had on IL-13 expression ( Figure 6K,L).

| CTNNAL1 regulates the expression of YAP
Our results demonstrated that ROCK2 inhibition downregulated mucus secretion when CTNNAL1 was silenced. However, ROCK1 inhibition did not alter MUC5AC expression. It is reported that YAP could interact with the promoter region of ROCK2. 31 Thus, we sought to determine the possible involvement of the YAP-mediated signalling pathways in CTNNAL1-regulated ROCK2 production. The interaction between CTNNAL1 and YAP within the airway epithelial cells was detected via immunoprecipitation ( Figure 7A,B). When CTNNAL1 was silenced, YAP expression was significantly increased ( Figure 7C-E). Meanwhile, Western blot and RT-qPCR analysis showed that CTNNAL1 overexpression-induced YAP expression levels were decreased ( Figure 7F-I).

| CTNNAL1 deficiency increased the expression of ROCK2 via the YAP pathway
To further verify the involvement of the YAP signalling pathway in

| DISCUSS ION
In this study, we found that CTNNAL1 deficiency stimulated airway inflammation and increased inflammatory cells, including eosinophils, neutrophils and lymphocytes in the BALF. Especially, mucus production significantly increased in CTNNAL1-deficient mice. to death. [40][41][42] Under asthmatic conditions, excessive production of MUC5AC contributes to mucous plugs and airflow obstruction. 43,44 In this study, we detected MUC5AC expression, which Then, we found that CTNNAL1 deficiency elevated the levels of IL-13 and IL-4 in the mice exposed to HDM. Moreover, the secretion of IL-4 and IL-13 in CTNNAL1-deficient mice and HDM-exposed control mice was higher than that in control mice. It is reported that IL-13 and IL-4, cytokines released from Th2, are major drivers of asthma and appear to play a prominent role in MUC5AC expression and mucus production. 45,46 Th2 cytokines induce goblet cell hyperplasia, resulting in mucus overproduction and airway inflammation in allergic asthma.
CTNNAL1 was identified as a part of the Rho signalling pathway, serving as a scaffold protein for Lbc. 24 We have found that CTNNAL1  The experiments were performed three times, and the error bars represent means ± SD. *p < 0.05, **p < 0.01, ns:no significance when CTNNAL1 was silenced while decreased after YAP silenced.
When YAP and CTNNAL1 were silenced together, ROCK2 expression returned to normal level. It indicated us that CTNNAL1 deficiency promoted mucus overproduction through YAP-ROCK2 signalling pathway (Figure 9).

| CON CLUS IONS
In summary, we demonstrated that CTNNAL1 deficiency enhanced lung inflammation and mucus hypersecretion through the activation of the YAP-ROCK2 signalling pathway in both lung tissue and in HBE cells, indicating CTNNAL1 could be a therapeutic target of mucus secretion in the future.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in this study are available on request from the corresponding author.

O RCI D
Di Wu https://orcid.org/0000-0003-1249-9286 F I G U R E 9 Schematic model of CTNNAL1 mediating mucus hypersecretion through YAP-ROCK2 signalling pathway. In asthmatic model, CTNNAL1 deficiency stimulated YAP expression, which translocated into nucleus and bound with ROCK2 promoter. ROCK2 led to the expression of MUC5AC, IL-4 and IL-13 increased, which secreted from airway epithelial cells into airway lumen