Deficiency of integrin β4 contributes bronchopulmonary dysplasia by compromising cellular stability through the activation of RhoA-(ZO- 1) signaling pathways

Deficiency of integrin β4 contributes bronchopulmonary dysplasia by compromising cellular stability through the activation of RhoA-(ZO- 1) signaling pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Deficiency of integrin β4 contributes bronchopulmonary dysplasia by compromising cellular stability through the activation of RhoA-(ZO- 1) signaling pathways Dongliang Zhang, Xianhui Wang, Jingjing Han, Xiaoyun Shao, Yang Xiang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5893262/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 01 Jul, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Alterations in the composition and remodeling of the lung extracellular matrix (ECM) are critical for lung development. Our research identified that mice with a conditional knockout of integrin β4 (ITGB4) exhibit lung dysplasia. In this study, we investigated the expression of collagen IV (IVcol) and matrix metalloproteinase 9 (MMP9) in both normal and ITGB4-deficient mice using Western blot and immunohistochemistry techniques. Our findings indicate that ITGB4 deficiency results in bronchopulmonary dysplasia, which is characterized by increased deposition of IVcol and reduced expression of MMP9. The zonula occludens-1 (ZO-1), on both normal and IV collagen-coated substrates was assessed using laser confocal microscopy. Concurrently, RhoA activities were quantified via fluorescence resonance energy transfer (FRET) microscopy. The findings indicated a significant disruption of ZO-1 in ITGB4-deficient cells, accompanied by an dcrease in RhoA activity.However, RhoA activity was enhanced in ITGB4 −/− cells on the type IVcollagen-coated substrate. Furthermore, the application of rhosin resulted in an enhanced expression of ZO-1 in ITGB4 −/− cells. These findings indicate that reduced expression of ITGB4 leads to elevated levels of IV collagen and hinders the adaptation of bronchial epithelial cells. Integrin β4(ITGB4) collagen IV( IV col) MMP9 ZO-1 RhoA Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bronchopulmonary dysplasia (BPD) is a prevalent chronic pulmonary disorder predominantly affecting preterm infants(Dani et al. 2023 ). The etiology of BPD is primarily attributed to pulmonary injury and complications arising from mechanical ventilation(Thébaud et al. 2019 ). Numerous studies have substantiated that children with BPD exhibit impaired lung architecture, restricted pulmonary vascular development, and compromised lung parenchyma(Ramos-Navarro et al. 2022 , Wang and Tsao 2020 ). Nevertheless, the precise pathophysiological mechanisms underlying BPD remain elusive. A notable clinical characteristic of BPD is the aberrant remodeling of the extracellular matrix (ECM), which hinders the development of lung parenchyma (Busch, Lorenzana and Ryan 2021 , Hu, Ling and Ren 2022 ). ECM is a three-dimensional spatial structure ubiquitous in all tissues, characterized by its dynamic nature and its critical role in the generation, expansion, and formation of organ branches(Ko et al. 2023 , Hahn and Sundar 2023 ). Conversely, aberrant ECM remodeling results in uncontrolled hyperplasia and the disruption of intercellular connective structures, thereby facilitating pathological progression (Lu et al. 2011 ). Prior research has demonstrated that infants with BPD exhibit elevated levels of collagen in the lungs, along with abnormal collagen scaffolds and fibers(Chi et al. 2022 ). In the lungs, type IV collagen predominantly constitutes the basement membrane (BM) (Lu, Weaver and Werb 2012 , Hall et al. 2022 ). Research indicates that the mechanical force generated by cells, regulated through the BM structure, stimulates epithelial branching morphogenesis during lung development via Rho GTPase(Sherwood 2021 ). Additionally, studies have demonstrated that type IV collagen (IV col) is a crucial component that facilitates cell migration by providing the initial anchorage for cells to exert traction(Chen, Lin and Yang 2014 , Jeong et al. 2019 ). Collagenases within the matrix metalloproteinase (MMP) family possess the capability to specifically degrade native fibrous collagen(Lv et al. 2018 ). Under physiological conditions, the equilibrium between MMPs and collagen is crucial for preserving the normal architecture and functionality of tissues(Hwang and Jeong 2010 ). Conversely, in pathological states, the overexpression or heightened activity of MMPs can result in the excessive degradation of collagen, thereby compromising the normal structural integrity and function of tissues(Mummidi et al. 2019 ). Integrins, which are transmembrane receptors, mediate the connection between cells and their extracellular environment. It is known to play critical roles in virtually every side of the behavior of epithelial cells, including cell proliferation, polarity, differentiation, and migration(Gahmberg, Grönholm and Madhavan 2022 ). Therefore, integrin in airway epithelial cells may play a crucial role in lung development and serve as a key factor in cellular sensing of the extracellular matrix and environmental signaling. Our research team discovered that integrin β4 (ITGB4) expression was reduced in the bronchial mucosa of both animal models and asthmatic patients(Liu et al. 2010 ). In individuals with asthma, a high frequency of base variation was observed at specific sites within the regulatory region of the ITGB4 gene, correlating with increased asthma susceptibility(Xiang et al. 2014 ). Furthermore, our prior research has demonstrated that a deficiency in integrin β4 leads to increased stiffness in lung tissue(Chi et al. 2022 ). In the present study, we further investigate the underlying mechanisms of lung dysplasia associated with ITGB4 defects. RESULTS ITGB4 Deficiency Resulted in Abnormal Lung Structure To avoid the lethal effect of ITGB4 null on mice, we constructed a conditional ITGB4 deficiency model known as CCSP–rtTA tg/− /TetOCre tg/− /ITGB4 fl/fl , in which ITGB4 was deleted only in airway epithelial cells, following the protocols elaborated in previous studies (Chi et al. 2022 ). We defined the normal group as ITGB4 +/+ , and the ITGB4 conditional knockout group as ITGB4 −/− . To examine the expression of ITGB4 in airway epithelial cells of mice, we performed RT-PCR and Immunohistochemical staining. As illustrated in Figs. 1A and 1B, ITGB4 was selectively deleted in the airway epithelium. Histological examination using hematoxylin and eosin (H&E) staining was performed to assess the airway and alveolar structures at postnatal days 2 (P2) and 28 (P28), in order to evaluate the impact of ITGB4 deficiency. The results indicated that the epithelial surface of the airway was disrupted, integrity was compromised, and cellular arrangement was disorganized (Fig. 1C). In the ITGB4 group, there were observations of dilated airspaces surrounded by thickened alveolar walls, a lack of alveolar septation, and increased alveolar size (Fig. 1D). IVcol expression increased at all stages of lung development Collagen deposition occurs during the development of bronchial dysplasia(McGowan and McCoy 2014 ). Subsequently, we assessed the expression levels of major lung collagen at postnatal day 28 (P28) using quantitative reverse transcription PCR (qRT-PCR). Our findings indicated an upregulation of type IV collagen (IV col) expression (Fig. 2 A). Notably, the expression of IV col, a principal component of the basal membrane, was elevated in the lung tissue of integrin β4-deficient mice at various stages of lung development. To corroborate these findings, we further analyzed the expression of IV col at different developmental stages using western blotting. The results demonstrated a significant increase in IV col expression in the ITGB4 −/− groups at both P2 and P28 (Fig. 2 B). Next, we assessed the expression of IVcol at P2 and P28 days using immunohistochemistry. The results indicated a substantial deposition of IVcol in the airway epithelium of ITGB4 −/− group mice (Fig. 2 .C) Increased IVcol expression was associated with decreased MMP9 expression. Collagen deposition in the lung is generally thought to be associated with a decrease in the MMP family (Fig. 3A, B). Therefore, we employed immunohistochemistry to detect the expression of MMP9, MMP2, and MMP14 in the lung tissues of mice at postnatal days 2 (P2) and 28 (P28). The results indicated that MMP9 expression in the lung tissue of both P2 and P28 mice in the ITGB4-/- group was significantly reduced (Fig. 3C). In conclusion, the increase in IVcol expression in ITGB4-/- group mice at P2 and P28 is closely related to the decrease in MMP9 expression. ITGB4 deficiency disrupted the tight junctions between cells, particularly on the type IV collagen-coated substrate To investigate whether ITGB4 influences intercellular junctions in a collagen-dependent manner, we prepared two distinct culture substrates: a standard substrate and a type IV collagen-coated substrate. We cultured 16HBE14o-cells on both the standard and the collagen-coated substrates, subsequently dividing the cells into the following subgroups: normal control groups (ITGB4 +/+ ), ITGB4-knockdown groups (ITGB4 −/− ), and nonsense-RNA groups (NC). Upon staining cells with the tight junction protein ZO-1, we observed that normal cells exhibited well-developed cell–cell adhesions. Conversely, in cells subjected to ITGB4 knockdown, the cell–cell junctions were disrupted, indicating that ITGB4 is essential for maintaining optimal cell–cell junctional integrity. Notably, the expression of ZO-1 in cells cultured on a substrate coated with type IV collagen (IVcol) was more significantly affected (Fig. 4) RhoA activity was enhanced in ITGB4−/−cells on the type IVcollagen-coated substrate The activation levels of RhoA in cells cultured on both normal and IVcol-coated substrates were assessed using a unimolecular effector-based RhoA FRET biosensor(Chi et al. 2022 ). The findings indicated significantly higher levels of RhoA activity in normal cells. Conversely, RhoA activity was diminished in ITGB4 −/− cells cultured on normal substrates.Notably, ITGB4 −/− cells cultured on the IVcol-coated substrate exhibited elevated RhoA activity (Fig. 5A, B). Rhosin, a specific inhibitor of RhoA activation, was employed in this study. The findings indicated that the expression of ZO-1 in ITGB4 −/− cells was significantly enhanced following Rhosin treatment, particularly on the IVcol-coated substrates (Fig. 5C). Discussion Cells continuously communicate with their surrounding extracellular matrix (ECM) through both mechanical and chemical signals to maintain homeostasis. The ECM undergoes controlled remodeling to sustain normal tissue homeostasis and function. In this study, we developed a CCSP/TetO-Cre mouse model to conditionally knockdown ITGB4 in the airway epithelium. Our findings indicate defective lung alveolar development and increased tissue stiffness in the animal model (Chi et al. 2022 ). Abnormal stiffening of the extracellular matrix (ECM) during pathogenesis is partially attributable to the excessive deposition of ECM proteins, which arises from a disruption in the equilibrium between matrix production and degradation(Cox and Erler 2011 ). Consequently, we initially investigated the collagen composition within the pulmonary ECM and observed an upregulation in the expression of type IV collagen. A plausible explanation for the observed discrepancy between the expression levels of type IV collagen and type I collagen mRNA and their corresponding proteins is the accelerated degradation of extracellular type IV collagen and type I collagen by matrix metalloproteinase-9 (MMP-9). Additionally, the intracellular uptake of degraded type IV collagen molecules was observed. Type IV collagen constitutes the primary collagen of the interstitial lung architecture and serves as a substrate for MMP2 and MMP9(Sun et al. 2023 ). Previous studies have confirmed that MMP9 specifically degrades basement membrane type IV collagen (Hu et al. 2020 ). In our study, we examined the expression levels of MMP9, MMP2, and MMP14, and found that only MMP9 expression was significantly reduced. This reduction in MMP9 expression suggests a correlation with the deposition of type IV collagen. Matrix metalloproteinase 9 (MMP9) plays a crucial role in modulating barrier function and maintaining the integrity of tight junction proteins, including zonula occludens-1 (ZO-1) (Nighot et al. 2015 ). In our subsequent in vitro analysis, we investigated the expression levels of ZO-1 in 16HBE14o- cells. To this end, we prepared two distinct culture substrates: a standard substrate and an IVcol-coated substrate. Our findings indicated that the expression of ZO-1 was significantly more compromised in cells cultured on the IVcol-coated substrate. Consequently, it can be concluded that deficiency in ITGB4, reduced expression of MMP9, deposition of type IV collagen, and compromised integrity of ZO-1 collectively contribute to the development of bronchopulmonary dysplasia in vivo. It has been documented that elevated RhoA activity leads to a reduction in ZO-1 expression, thereby disrupting tight junctions between cells and ultimately compromising cellular stability(Li et al. 2018 ). In our investigation, we observed that deficiencies in ITGB4 resulted in heightened RhoA activity on an IVcol-coated substrate. Conversely, the application of RhoA inhibitors led to an increase in ZO-1 expression. These findings collectively suggest that the cellular responses elicited by ITGB4 defects are intricately linked to the effects of RhoA activation on ZO-1 expression. Materials and methods Mice All the animal experiments in this study are conducted in accordance with ARRIVE (Animal Research: Reporting of in vivo) guidelines. Drug injection euthanasia Pentobarbital sodium injection: 150mg/kg intraperitoneal injection of the drug. Mouse models: Generation of transgenic mice All animal studies were approved by the No.201803079 Central South University at XiangYa Animal Care and Use Committee. All the methods were carried out in accordance with the relevant guidelines and regulations.The mice were housed under barrier conditions in air-filtered, temperature-controlled units under a 12-hour light-dark cycle and with free access to food and water. The generation of ITGB4fl/flmice was described previously. CCSP–rtTAtg/−/TetO-Cretg/tg mice expressing Cre recombinase under the control of the clara-cell secretory protein (CCSP) promoter on a C57BL/6 background were described elsewhere(Ceteci et al. 2012). To generate mice that had ITGB4 conditionally knocked out in their airway epithelial cells, ITGB4fl/fl mice were bred with CCSP–rtTAtg/−/TetO-Cretg/tg mice to generate the CCSP–rtTAtg/−/TetO-Cretg/−/ITGB4fl/fl triple transgenic mice. Only male mice were used for the studies. To induce Cre expression in the respiratory epithelium and produce ITGB4 −/− mice, doxycycline (Dox; 1% in drinking water) was administered to 8-week-old mice. The Dox treatment was continued throughout the experiment. The Dox-treated ITGB4fl/fl male littermates lacking either CCSP–rtTA, TetO-Cre or both were used as ITGB4 +/+ mice. Triple transgenic mice not treated with Dox were used as the negative control. 4.2 Small interfering RNA synthesis and transfection ITGB4 mRNA silencing was achieved using the siRNA technique, as previously described(Liu et al. 2010). The effective ITGB4 siRNA(Zhang, Rozengurt and Reed 2010) (5‘-CAGAAGAUGUGGAUGAGUU-3’) and nonsense siRNA (5‘-UUCUCCGAACGUGUCACGU-3’) were designed and synthesized by Guangzhou RiboBio (RiboBio Inc., Guangzhou, China). The transfections of 16HBE14o- cells with negative control siRNA and effective silencing siRNA were performed by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer’s instructions. The efficiency of siRNA gene silencing was measured using RT-PCR and western blotting assay. 4.3 Lung Histology and Immunohistochemical Staining Paraffin-embedded lung sections were stained with hematoxylin and eosin (H&E). Immunohistochemical staining was performed to detect the expression of IV col and MMP9 in lung paraffin sections by using the following antibodies: anti-collagen IV antibody (1:200, Abcam, ab6586), and anti-MMP9 antibody (1;200, Affinity, AF5228). Zeiss Discovery.V8 Stereo microscopes (Carl Zeiss MicroImaging GmbH) and Axio-Cam ICc3 camera (Spectra Service) were used to take photos. Zeiss AxioVision Rel. 4.7 software was used for image analysis. 4.4 Western blot analysis The total protein of the cells was gathered with RIPA Lysis Buffer (Thermo Scientific, USA) including 1% phenylmethanesulfonyl fluoride on ice. Detection of protein concentration with a BCA Kit (Takara, Tokyo, Japan, T9300A). Lysates (50 μg) were separated on10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane that was blocked with 5% bovine serum albumin (BSA). The membranes were incubated with antibodies: β-actin (Sigma-Aldrich, St Louis, MO, USA, A5441); Anti-Collagen IV antibody (1:1000, abcam, ab6586). And then The membranes were washed three times with TBST (TBS 0.1% Tween-20) and then incubated with the secondary antibody for 1 h at room temperature: Goat Anti-Rabbit IgG H&L (HRP,ab205718). The GADPH was used as the loading control. 4.5 Cell culture Cell culture as previously described(Huang et al. 2020).16HBE14o- cells (an in vitro cultured engineered human bronchial epithelial cell line) were obtained from Professor Dieter Gruenert, University of California San Francisco.The cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml streptomycin, and 100 U/ml penicillin in a humidified air atmosphere containing 5% CO 2 at 37 °C. Cell culture reagents were purchased from Gibco (Invitrogen, Grand Island, NY, USA). 4.6 Immunofluorescence Immunofluorescent staining was performed in airway epithelial cells which were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were washed twice, and permeabilized with 0.3% Triton X-100 in PBS for 5 min, cells were incubated with ZO-1 antibody (1:200, Abcam, ab221547) overnight. The nuclei were stained with 4’,6-diamidino-2-phenylindole (Sigma-Aldrich) for 2 min. The images were acquired using a Zeiss LSM710confocal microscope (Carl Zeiss). 4.7 FRET RhoA FRET biosensor is a gift from Professor Klaus Hahn at the University of North Carolina, which was previously described(Pertz et al. 2006). Briefly in work mechanism, the biosensor consists of a Rho-binding domain of the effector rhotekin (RBD), which specifically binds to GTP-RhoA, cyan fluorescent protein (CFP), an unstructured linker of optimized length, yellow fluorescent protein (YFP) and full-length RhoA. The detailed principles and procedures can be found in our published article(Chi et al. 2022). Simply, after the biosensor was activated by GTP-loading, and the RBD binds to Rho, the relative orientation of the two fluorophores was modified to increase FRET efficiency. After transfection with RhoA biosensor for 36-48h, the cells were detached from the normal and IV-coated substrates with gentle accutase digestion solution, and then seeded on fibronectin-coated 15-mm glass-bottom cell culture dish (801002, NEST, China) for 3-6 h. During the imaging process, the cells were maintained in 5% CO2 without serum at 37 ℃. The emission ratio was generated and computed by using the FluoCell software package in MatLab to represent the FRET efficiency before being quantified by using Prism. 4.8 Statistical Analysis Statistical analysis was performed by using Prism 5.01 (Graph-Pad Software, San Diego California, USA). Data were presented as mean ± SEM. The student’s t-test (two -tailed) was used to evaluate the differences between two groups. For three or more groups, data were evaluated with one-way analysis-of-variance followed by Bonferroni for multiple comparisons. A significant difference was considered by p value (< 0.05). Error bars (SD) and P values were determined from the results of three or more independent experiments. The ANOVA with post hoc Student’s t test was used for analysis of statistical significance. Declarations Funding This work was supported by Basic Research of Yancheng City (Natural Science Foundation) (YCBK2024009); Changsha Natural Science Foundation, (kq24022310). Acknowledgements The RhoA FRET Biosensor is a gift from Professor Klaus Hahn at University of North Carolina. Data availability The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. References Busch, S. M., Z. Lorenzana & A. L. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5893262","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":411681598,"identity":"b5da33c5-767f-45f9-954a-2938e675b99b","order_by":0,"name":"Dongliang Zhang","email":"","orcid":"","institution":"Jiangsu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Dongliang","middleName":"","lastName":"Zhang","suffix":""},{"id":411681599,"identity":"0b700eb5-ff3c-4171-bb91-6a691f8db144","order_by":1,"name":"Xianhui Wang","email":"","orcid":"","institution":"Jiangsu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xianhui","middleName":"","lastName":"Wang","suffix":""},{"id":411681600,"identity":"8cb5e3dd-f8d6-4247-8105-4141e61987a2","order_by":2,"name":"Jingjing Han","email":"","orcid":"","institution":"Jiangsu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Jingjing","middleName":"","lastName":"Han","suffix":""},{"id":411681601,"identity":"e47b00a7-fd68-4424-8abb-2d3c09acaf8d","order_by":3,"name":"Xiaoyun Shao","email":"","orcid":"","institution":"Jiangsu Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyun","middleName":"","lastName":"Shao","suffix":""},{"id":411681602,"identity":"b823c0e9-65a5-4eb7-8328-99bc17933360","order_by":4,"name":"Yang Xiang","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xiang","suffix":""},{"id":411681603,"identity":"c197f45f-31d4-4443-be7b-12480cb334ec","order_by":5,"name":"Yinxiu Chi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIie2RvWrDMBCAJQSdrqijDf2hb3DBQyiY5FUUDJ485BFkAskrqC+S+YSgk5usgQzN1PmyZ2jUtUReO+gbpfs4fUiITOafQoz184vYn8hgDVrbUUN6t2yrST9I4mX7WDoaVVQADgsbBuUdhxqtSc9r16F32C769ScFwD2gIMnn7rZSHFoTW6oN7MxVOcJUWVW+b28rWDQUtzz1jvBXebN0p+6TyrUCMEj7dYrKDpDMmNLEhPBqaYhRNK4Uw7eJD6sm9iNGNVA6v0q26E1XMV/iV4bA5jKba73yfE4o4sH8OZI2MR/XUPo+k8lkMuIHWB9j67V2tOYAAAAASUVORK5CYII=","orcid":"","institution":"Jiangsu Medical College","correspondingAuthor":true,"prefix":"","firstName":"Yinxiu","middleName":"","lastName":"Chi","suffix":""}],"badges":[],"createdAt":"2025-01-24 07:08:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5893262/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5893262/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-05983-1","type":"published","date":"2025-07-01T15:58:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":75941084,"identity":"29c3ea7d-03e5-43d7-98a5-c2102ef4a8f6","added_by":"auto","created_at":"2025-02-10 18:37:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":539003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSilencing efficiency of ITGB4 and lung structure of ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+/+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) ITGB4 expression was detected by Immunohistochemical staining. (B) RT-PCR analysis of ITGB4 mRNA expression levels in ITGB4\u003csup\u003e+/+\u003c/sup\u003e and ITGB4\u003csup\u003e-/-\u003c/sup\u003e mice. (C) The epithelial surface of the airway is broken, integrity is impaired, and cellular arrangement is disrupted in H\u0026amp;E-stained paraffin sections of P2 and P28 lungs in ITGB4\u003csup\u003e-/-\u003c/sup\u003e mice. (D) Dilated airspaces with thickened alveolar septa were seen in H\u0026amp;E-stained paraffin sections of P2 and P28 lungs in ITGB4\u003csup\u003e-/-\u003c/sup\u003e mice. (**\u003cem\u003eP\u0026lt;0.01\u003c/em\u003e). Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/01a5b0b00207da5ca117800b.png"},{"id":75941489,"identity":"c321b685-0981-405b-8ce0-e60cc30f5ce8","added_by":"auto","created_at":"2025-02-10 18:45:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":323796,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of collagens between ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+/+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eand ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The mRNA expression levels of collagens were detected by RT-PCR. (B) The expression levels of IVcol were detected by western blot in different stages. (C) Immunostaining for IVcol in airway epithelium in ITGB4\u003csup\u003e+/+\u003c/sup\u003eand ITGB4\u003csup\u003e-/-\u003c/sup\u003elungs. Scale bars: 20μm. (D) Immunostaining for IVcol in alveolar septa in ITGB4\u003csup\u003e+/+\u003c/sup\u003eand ITGB4\u003csup\u003e-/-\u003c/sup\u003elungs. Scale bars: 20μm. (n=3 independent experiments, **\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/30f8940bc9da3af6c4f09ecc.png"},{"id":75941040,"identity":"2304ad6a-6ce0-4ffd-9c19-ff293fc35177","added_by":"auto","created_at":"2025-02-10 18:29:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":481842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of MMP family between ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+/+ \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eand ITGB4\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003emice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,B) The mRNA expression levels of MMP9, MMP2 and MMP14 were detected by RT-PCR at P2 and P28. (C) Immunostaining for MMP9 in ITGB4\u003csup\u003e+/+\u003c/sup\u003eand ITGB4\u003csup\u003e-/-\u003c/sup\u003elungs. Scale bars: 20μm. (n=3 independent experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05,**\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01). Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/c7ec062a0cd7d74f1cf65151.png"},{"id":75941038,"identity":"c105cc9b-6a66-4037-a8cf-9a2c131dfe32","added_by":"auto","created_at":"2025-02-10 18:29:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":798992,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of Zo-1 in 16HBE14o-cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescence images of Zo-1 (green) and DAPI (blue) in the different groups of cells Scale bars: 20μm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/8693cea7245b427e4b832a69.png"},{"id":75941045,"identity":"430527ca-292c-4e8d-a598-06ce218b9837","added_by":"auto","created_at":"2025-02-10 18:29:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":352270,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRhoA activity and the expression of zo-1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;(A, B) Images of the emission ratios of YFP/CFP-based RhoA biosensor in cells on normal substrate and an IVcol-coated substrate. Cells are shown on the left, the right panels show the emission ratios of the YFP/CFP-based RhoA biosensors (C) Representative images of the expression for NC and ITGB4\u003csup\u003e-/-\u003c/sup\u003e cells before and after Rhosin treatment. (n=3 independent experiments, *\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05). Data are presented as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/783530f837c0c7429290d297.png"},{"id":86179906,"identity":"3214898b-b7b1-4207-91b3-75b5fa850f32","added_by":"auto","created_at":"2025-07-07 16:20:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3434156,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5893262/v1/c5683897-7157-4760-a3a7-98d9d2636375.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Deficiency of integrin β4 contributes bronchopulmonary dysplasia by compromising cellular stability through the activation of RhoA-(ZO- 1) signaling pathways","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBronchopulmonary dysplasia (BPD) is a prevalent chronic pulmonary disorder predominantly affecting preterm infants(Dani et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The etiology of BPD is primarily attributed to pulmonary injury and complications arising from mechanical ventilation(Th\u0026eacute;baud et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Numerous studies have substantiated that children with BPD exhibit impaired lung architecture, restricted pulmonary vascular development, and compromised lung parenchyma(Ramos-Navarro et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e, Wang and Tsao \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Nevertheless, the precise pathophysiological mechanisms underlying BPD remain elusive. A notable clinical characteristic of BPD is the aberrant remodeling of the extracellular matrix (ECM), which hinders the development of lung parenchyma (Busch, Lorenzana and Ryan \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2021\u003c/span\u003e, Hu, Ling and Ren \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). ECM is a three-dimensional spatial structure ubiquitous in all tissues, characterized by its dynamic nature and its critical role in the generation, expansion, and formation of organ branches(Ko et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2023\u003c/span\u003e, Hahn and Sundar \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Conversely, aberrant ECM remodeling results in uncontrolled hyperplasia and the disruption of intercellular connective structures, thereby facilitating pathological progression (Lu et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Prior research has demonstrated that infants with BPD exhibit elevated levels of collagen in the lungs, along with abnormal collagen scaffolds and fibers(Chi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the lungs, type IV collagen predominantly constitutes the basement membrane (BM) (Lu, Weaver and Werb \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2012\u003c/span\u003e, Hall et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Research indicates that the mechanical force generated by cells, regulated through the BM structure, stimulates epithelial branching morphogenesis during lung development via Rho GTPase(Sherwood \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Additionally, studies have demonstrated that type IV collagen (IV col) is a crucial component that facilitates cell migration by providing the initial anchorage for cells to exert traction(Chen, Lin and Yang \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Jeong et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Collagenases within the matrix metalloproteinase (MMP) family possess the capability to specifically degrade native fibrous collagen(Lv et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Under physiological conditions, the equilibrium between MMPs and collagen is crucial for preserving the normal architecture and functionality of tissues(Hwang and Jeong \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Conversely, in pathological states, the overexpression or heightened activity of MMPs can result in the excessive degradation of collagen, thereby compromising the normal structural integrity and function of tissues(Mummidi et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIntegrins, which are transmembrane receptors, mediate the connection between cells and their extracellular environment. It is known to play critical roles in virtually every side of the behavior of epithelial cells, including cell proliferation, polarity, differentiation, and migration(Gahmberg, Gr\u0026ouml;nholm and Madhavan \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Therefore, integrin in airway epithelial cells may play a crucial role in lung development and serve as a key factor in cellular sensing of the extracellular matrix and environmental signaling. Our research team discovered that integrin β4 (ITGB4) expression was reduced in the bronchial mucosa of both animal models and asthmatic patients(Liu et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In individuals with asthma, a high frequency of base variation was observed at specific sites within the regulatory region of the ITGB4 gene, correlating with increased asthma susceptibility(Xiang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Furthermore, our prior research has demonstrated that a deficiency in integrin β4 leads to increased stiffness in lung tissue(Chi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the present study, we further investigate the underlying mechanisms of lung dysplasia associated with ITGB4 defects.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eITGB4 Deficiency Resulted in Abnormal Lung Structure\u003c/h2\u003e\n \u003cp\u003eTo avoid the lethal effect of ITGB4 null on mice, we constructed a conditional ITGB4 deficiency model known as CCSP\u0026ndash;rtTA\u003csup\u003etg/\u0026minus;\u003c/sup\u003e/TetOCre\u003csup\u003etg/\u0026minus;\u003c/sup\u003e/ITGB4\u003csup\u003efl/fl\u003c/sup\u003e, in which ITGB4 was deleted only in airway epithelial cells, following the protocols elaborated in previous studies (Chi et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). We defined the normal group as ITGB4\u003csup\u003e+/+\u003c/sup\u003e, and the ITGB4 conditional knockout group as ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e. To examine the expression of ITGB4 in airway epithelial cells of mice, we performed RT-PCR and Immunohistochemical staining. As illustrated in Figs.\u0026nbsp;1A and 1B, ITGB4 was selectively deleted in the airway epithelium. Histological examination using hematoxylin and eosin (H\u0026amp;E) staining was performed to assess the airway and alveolar structures at postnatal days 2 (P2) and 28 (P28), in order to evaluate the impact of ITGB4 deficiency. The results indicated that the epithelial surface of the airway was disrupted, integrity was compromised, and cellular arrangement was disorganized (Fig.\u0026nbsp;1C). In the ITGB4 group, there were observations of dilated airspaces surrounded by thickened alveolar walls, a lack of alveolar septation, and increased alveolar size (Fig.\u0026nbsp;1D).\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIVcol expression increased at all stages of lung development\u003c/h3\u003e\n\u003cp\u003eCollagen deposition occurs during the development of bronchial dysplasia(McGowan and McCoy \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Subsequently, we assessed the expression levels of major lung collagen at postnatal day 28 (P28) using quantitative reverse transcription PCR (qRT-PCR). Our findings indicated an upregulation of type IV collagen (IV col) expression (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA). Notably, the expression of IV col, a principal component of the basal membrane, was elevated in the lung tissue of integrin \u0026beta;4-deficient mice at various stages of lung development. To corroborate these findings, we further analyzed the expression of IV col at different developmental stages using western blotting. The results demonstrated a significant increase in IV col expression in the ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e groups at both P2 and P28 (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, we assessed the expression of IVcol at P2 and P28 days using immunohistochemistry. The results indicated a substantial deposition of IVcol in the airway epithelium of ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e group mice (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e.C)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIncreased IVcol expression was associated with decreased MMP9 expression.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollagen deposition in the lung is generally thought to be associated with a decrease in the MMP family (Fig.\u0026nbsp;3A, B). Therefore, we employed immunohistochemistry to detect the expression of MMP9, MMP2, and MMP14 in the lung tissues of mice at postnatal days 2 (P2) and 28 (P28). The results indicated that MMP9 expression in the lung tissue of both P2 and P28 mice in the ITGB4-/- group was significantly reduced (Fig.\u0026nbsp;3C). In conclusion, the increase in IVcol expression in ITGB4-/- group mice at P2 and P28 is closely related to the decrease in MMP9 expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eITGB4 deficiency disrupted the tight junctions between cells, particularly on the type IV collagen-coated substrate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate whether ITGB4 influences intercellular junctions in a collagen-dependent manner, we prepared two distinct culture substrates: a standard substrate and a type IV collagen-coated substrate. We cultured 16HBE14o-cells on both the standard and the collagen-coated substrates, subsequently dividing the cells into the following subgroups: normal control groups (ITGB4\u003csup\u003e+/+\u003c/sup\u003e), ITGB4-knockdown groups (ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e), and nonsense-RNA groups (NC). Upon staining cells with the tight junction protein ZO-1, we observed that normal cells exhibited well-developed cell\u0026ndash;cell adhesions. Conversely, in cells subjected to ITGB4 knockdown, the cell\u0026ndash;cell junctions were disrupted, indicating that ITGB4 is essential for maintaining optimal cell\u0026ndash;cell junctional integrity. Notably, the expression of ZO-1 in cells cultured on a substrate coated with type IV collagen (IVcol) was more significantly affected (Fig. 4)\u003c/p\u003e\n\u003ch3\u003eRhoA activity was enhanced in ITGB4\u0026minus;/\u0026minus;cells on the type IVcollagen-coated substrate\u003c/h3\u003e\n\u003cp\u003eThe activation levels of RhoA in cells cultured on both normal and IVcol-coated substrates were assessed using a unimolecular effector-based RhoA FRET biosensor(Chi et al. \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e). The findings indicated significantly higher levels of RhoA activity in normal cells. Conversely, RhoA activity was diminished in ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells cultured on normal substrates.Notably, ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells cultured on the IVcol-coated substrate exhibited elevated RhoA activity (Fig. 5A, B). Rhosin, a specific inhibitor of RhoA activation, was employed in this study. The findings indicated that the expression of ZO-1 in ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells was significantly enhanced following Rhosin treatment, particularly on the IVcol-coated substrates (Fig. 5C).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCells continuously communicate with their surrounding extracellular matrix (ECM) through both mechanical and chemical signals to maintain homeostasis. The ECM undergoes controlled remodeling to sustain normal tissue homeostasis and function. In this study, we developed a CCSP/TetO-Cre mouse model to conditionally knockdown ITGB4 in the airway epithelium. Our findings indicate defective lung alveolar development and increased tissue stiffness in the animal model (Chi et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Abnormal stiffening of the extracellular matrix (ECM) during pathogenesis is partially attributable to the excessive deposition of ECM proteins, which arises from a disruption in the equilibrium between matrix production and degradation(Cox and Erler \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Consequently, we initially investigated the collagen composition within the pulmonary ECM and observed an upregulation in the expression of type IV collagen. A plausible explanation for the observed discrepancy between the expression levels of type IV collagen and type I collagen mRNA and their corresponding proteins is the accelerated degradation of extracellular type IV collagen and type I collagen by matrix metalloproteinase-9 (MMP-9).\u003c/p\u003e \u003cp\u003eAdditionally, the intracellular uptake of degraded type IV collagen molecules was observed. Type IV collagen constitutes the primary collagen of the interstitial lung architecture and serves as a substrate for MMP2 and MMP9(Sun et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Previous studies have confirmed that MMP9 specifically degrades basement membrane type IV collagen (Hu et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In our study, we examined the expression levels of MMP9, MMP2, and MMP14, and found that only MMP9 expression was significantly reduced. This reduction in MMP9 expression suggests a correlation with the deposition of type IV collagen.\u003c/p\u003e \u003cp\u003eMatrix metalloproteinase 9 (MMP9) plays a crucial role in modulating barrier function and maintaining the integrity of tight junction proteins, including zonula occludens-1 (ZO-1) (Nighot et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In our subsequent in vitro analysis, we investigated the expression levels of ZO-1 in 16HBE14o- cells. To this end, we prepared two distinct culture substrates: a standard substrate and an IVcol-coated substrate. Our findings indicated that the expression of ZO-1 was significantly more compromised in cells cultured on the IVcol-coated substrate. Consequently, it can be concluded that deficiency in ITGB4, reduced expression of MMP9, deposition of type IV collagen, and compromised integrity of ZO-1 collectively contribute to the development of bronchopulmonary dysplasia in vivo.\u003c/p\u003e \u003cp\u003eIt has been documented that elevated RhoA activity leads to a reduction in ZO-1 expression, thereby disrupting tight junctions between cells and ultimately compromising cellular stability(Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In our investigation, we observed that deficiencies in ITGB4 resulted in heightened RhoA activity on an IVcol-coated substrate. Conversely, the application of RhoA inhibitors led to an increase in ZO-1 expression. These findings collectively suggest that the cellular responses elicited by ITGB4 defects are intricately linked to the effects of RhoA activation on ZO-1 expression.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eMice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the animal experiments in this study are conducted in accordance with ARRIVE (Animal Research: Reporting of in vivo) guidelines. Drug injection euthanasia Pentobarbital sodium injection: 150mg/kg intraperitoneal injection of the drug.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMouse models:\u003c/strong\u003e Generation of transgenic mice All animal studies were approved by the No.201803079 Central South University at XiangYa Animal Care and Use Committee. All the methods were carried out in accordance with the relevant guidelines and regulations.The mice were housed under barrier conditions in air-filtered, temperature-controlled units under a 12-hour light-dark cycle and with free access to food and water. The generation of ITGB4fl/flmice was described previously. CCSP\u0026ndash;rtTAtg/\u0026minus;/TetO-Cretg/tg mice expressing Cre recombinase under the control of the clara-cell secretory protein (CCSP) promoter on a C57BL/6 background were described elsewhere(Ceteci et al. 2012). To generate mice that had ITGB4 conditionally knocked out in their airway epithelial cells, ITGB4fl/fl mice were bred with CCSP\u0026ndash;rtTAtg/\u0026minus;/TetO-Cretg/tg mice to generate the CCSP\u0026ndash;rtTAtg/\u0026minus;/TetO-Cretg/\u0026minus;/ITGB4fl/fl triple transgenic mice. Only male mice were used for the studies. To induce Cre expression in the respiratory epithelium and produce ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u0026nbsp;\u003c/sup\u003emice, doxycycline (Dox; 1% in drinking water) was administered to 8-week-old mice. The Dox treatment was continued throughout the experiment. The Dox-treated ITGB4fl/fl male littermates lacking either CCSP\u0026ndash;rtTA, TetO-Cre or both were used as ITGB4\u003csup\u003e+/+\u003c/sup\u003e mice. Triple transgenic mice not treated with Dox were used as the negative control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eSmall interfering RNA synthesis and transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eITGB4 mRNA silencing was achieved using the siRNA technique, as previously described(Liu et al. 2010).\u0026nbsp;The effective ITGB4 siRNA(Zhang, Rozengurt and Reed 2010)\u0026nbsp;(5\u0026lsquo;-CAGAAGAUGUGGAUGAGUU-3\u0026rsquo;) and nonsense siRNA (5\u0026lsquo;-UUCUCCGAACGUGUCACGU-3\u0026rsquo;) were designed and synthesized by Guangzhou RiboBio (RiboBio Inc., Guangzhou, China). The transfections of 16HBE14o- cells with negative control siRNA and effective silencing siRNA were performed by using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer\u0026rsquo;s instructions. The efficiency of siRNA gene silencing was measured using RT-PCR and western blotting assay.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3 Lung Histology and Immunohistochemical Staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Paraffin-embedded lung sections were stained with hematoxylin and eosin (H\u0026amp;E). Immunohistochemical staining was performed to detect the expression of IV col and MMP9 in lung paraffin sections by using the following antibodies: anti-collagen IV antibody (1:200, Abcam, ab6586), and anti-MMP9 antibody (1;200, Affinity, AF5228). Zeiss Discovery.V8 Stereo microscopes (Carl Zeiss MicroImaging GmbH) and Axio-Cam ICc3 camera (Spectra Service) were used to take photos. Zeiss AxioVision Rel. 4.7 software was used for image analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.4 Western blot analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe total protein of the cells was gathered with RIPA Lysis Buffer (Thermo Scientific, USA) including 1% phenylmethanesulfonyl fluoride on ice. Detection of protein concentration with a BCA Kit (Takara, Tokyo, Japan, T9300A). Lysates (50 \u0026mu;g) were separated on10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene fluoride membrane that was blocked with 5% bovine serum albumin (BSA). The membranes were incubated with antibodies: \u0026beta;-actin (Sigma-Aldrich, St Louis, MO, USA, A5441); Anti-Collagen IV antibody (1:1000, abcam, ab6586). And then The membranes were washed three times with TBST (TBS 0.1% Tween-20) and then incubated with the secondary antibody for 1 h at room temperature: Goat Anti-Rabbit IgG H\u0026amp;L (HRP,ab205718). The GADPH was used as the loading control.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.5\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCell culture\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell culture as previously described(Huang et al. 2020).16HBE14o- cells (an in vitro cultured engineered human bronchial epithelial cell line) were obtained from Professor Dieter Gruenert, University of California San Francisco.The cells were cultured in high-glucose Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/ml streptomycin, and 100 U/ml penicillin in a humidified air atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026nbsp;\u0026deg;C. Cell culture reagents were purchased from Gibco (Invitrogen, Grand Island, NY, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.6 Immunofluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunofluorescent staining was performed in airway epithelial cells which were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were washed twice, and permeabilized with 0.3% Triton X-100 in PBS for 5 min, cells were incubated with ZO-1 antibody (1:200, Abcam, ab221547) overnight. The nuclei were stained with 4\u0026rsquo;,6-diamidino-2-phenylindole (Sigma-Aldrich) for 2 min. The images were acquired using a Zeiss LSM710confocal microscope (Carl Zeiss).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.7 FRET\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRhoA FRET biosensor is a gift from Professor Klaus Hahn at the University of North Carolina, which was previously described(Pertz et al. 2006). Briefly in work mechanism, the biosensor consists of a Rho-binding domain of the effector rhotekin (RBD), which specifically binds to GTP-RhoA, cyan fluorescent protein (CFP), an unstructured linker of optimized length, yellow fluorescent protein (YFP) and full-length RhoA. The detailed principles and procedures can be found in our published article(Chi et al. 2022). Simply, after the biosensor was activated by GTP-loading, and the RBD binds to Rho, the relative orientation of the two fluorophores was modified to increase FRET efficiency. After transfection with RhoA biosensor for 36-48h, the cells were detached from the normal and IV-coated substrates with gentle accutase digestion solution, and then seeded on fibronectin-coated 15-mm glass-bottom cell culture dish (801002, NEST, China) for 3-6 h. During the imaging process, the cells were maintained in 5% CO2 without serum at 37\u0026nbsp;℃. The emission ratio was generated and computed by using the FluoCell software package in MatLab to represent the FRET efficiency before being quantified by using Prism.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.8 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed by using Prism 5.01 (Graph-Pad Software, San Diego California, USA). Data were presented as mean \u0026plusmn; SEM. The student\u0026rsquo;s t-test (two -tailed) was used to evaluate the differences between two groups. For three or more groups, data were evaluated with one-way analysis-of-variance followed by Bonferroni for multiple comparisons. A significant difference was considered by p value (\u0026lt; 0.05). Error bars (SD) and P values were determined from the results of three or more independent experiments. The ANOVA with post hoc\nStudent\u0026rsquo;s t test was used for analysis of statistical significance.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Basic Research of Yancheng City (Natural Science Foundation) (YCBK2024009); Changsha Natural Science Foundation, (kq24022310).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe RhoA FRET Biosensor is a gift from Professor Klaus Hahn at University of North Carolina.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBusch, S. 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Reed (2010) HLA class I molecules partner with integrin \u0026beta;4 to stimulate endothelial cell proliferation and migration. \u003cem\u003eSci Signal,\u003c/em\u003e 3\u003cstrong\u003e,\u003c/strong\u003e ra85.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Integrin β4(ITGB4), collagen IV( IV col), MMP9, ZO-1, RhoA","lastPublishedDoi":"10.21203/rs.3.rs-5893262/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5893262/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlterations in the composition and remodeling of the lung extracellular matrix (ECM) are critical for lung development. Our research identified that mice with a conditional knockout of integrin β4 (ITGB4) exhibit lung dysplasia. In this study, we investigated the expression of collagen IV (IVcol) and matrix metalloproteinase 9 (MMP9) in both normal and ITGB4-deficient mice using Western blot and immunohistochemistry techniques. Our findings indicate that ITGB4 deficiency results in bronchopulmonary dysplasia, which is characterized by increased deposition of IVcol and reduced expression of MMP9. The zonula occludens-1 (ZO-1), on both normal and IV collagen-coated substrates was assessed using laser confocal microscopy. Concurrently, RhoA activities were quantified via fluorescence resonance energy transfer (FRET) microscopy. The findings indicated a significant disruption of ZO-1 in ITGB4-deficient cells, accompanied by an dcrease in RhoA activity.However, RhoA activity was enhanced in ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003ecells on the type IVcollagen-coated substrate. Furthermore, the application of rhosin resulted in an enhanced expression of ZO-1 in ITGB4\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e cells. These findings indicate that reduced expression of ITGB4 leads to elevated levels of IV collagen and hinders the adaptation of bronchial epithelial cells.\u003c/p\u003e","manuscriptTitle":"Deficiency of integrin β4 contributes bronchopulmonary dysplasia by compromising cellular stability through the activation of RhoA-(ZO- 1) signaling pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-10 18:29:52","doi":"10.21203/rs.3.rs-5893262/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-18T06:02:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-16T21:06:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-16T07:05:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8470889756879929353086402879352918122","date":"2025-02-24T17:44:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"284066237648855483115205698651030743987","date":"2025-02-24T16:05:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-02-24T15:48:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-02-24T15:21:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-02-05T16:53:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-02-04T10:46:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-01-24T07:00:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ca649d6-7cfd-4ef5-8186-2a0245b9dc92","owner":[],"postedDate":"February 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:12:54+00:00","versionOfRecord":{"articleIdentity":"rs-5893262","link":"https://doi.org/10.1038/s41598-025-05983-1","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-07-01 15:58:28","publishedOnDateReadable":"July 1st, 2025"},"versionCreatedAt":"2025-02-10 18:29:52","video":"","vorDoi":"10.1038/s41598-025-05983-1","vorDoiUrl":"https://doi.org/10.1038/s41598-025-05983-1","workflowStages":[]},"version":"v1","identity":"rs-5893262","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5893262","identity":"rs-5893262","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}