Sorcin regulates alveolarization and airway tissue remodeling during lung morphogenesis

Sorcin regulates alveolarization and airway tissue remodeling during lung morphogenesis | 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 Research Article Sorcin regulates alveolarization and airway tissue remodeling during lung morphogenesis Claudia Tito, Luciana De Angelis, Alessia Iaiza, Annalisa Pia Abbinantefina, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6297074/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Oct, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Sorcin, a key calcium-sensing protein, regulates calcium concentration within the endoplasmic reticulum (ER), promoting apoptosis resistance and ER stress. It also modulates downstream signaling pathways of the epidermal growth factor receptor (EGFR), influencing cellular migration and invasion in non-small-cell lung carcinoma (NSCLC) cell lines. For this purpose, this study investigates the relationship between Sorcin and EGFR expression during lung development at the physiological level. Our study was conducted on WT and Sorcin Knock-out ( SRI −/− ) mice, where we performed various analyses, including histological examination, gene and protein expression analysis, and confocal microscopy. Our findings reveal that SRI −/− mice, compared to wild-type controls, exhibit: 1) impaired alveolarization and abnormal development of bronchi and bronchioles, as observed in histological sections; 2) decreased expression of genes encoding branching morphogenesis markers (e.g., FGF10) and surfactant proteins (e.g., SP-B, SP-C and ABCA3), as shown by real-time PCR; 3) increased glycogen content decreased lipid droplets, indicative of type II pneumocyte immaturity and impaired surfactant lipid production; 4) reduced levels of EGFR, RAS and RAB5C proteins, consistent with defects in lung maturation and surfactant protein recycling, as demonstrated by Western blot analysis; and 5) increased expression of phalloidin, α-smooth muscle actin and vimentin, suggesting increased bronchial thickening associated with airway tissue remodeling. Collectively, these data reveal a novel role for Sorcin in lung alveolarization, pulmonary surfactant production, and airway remodeling associated with bronchial contractility, supporting its involvement in respiratory diseases such as respiratory distress syndrome (RDS), asthma and chronic obstructive pulmonary disease (COPD). lung development Sorcin KO alveolarization branching morphogenesis airway remodeling Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Fetal lung development is a complex process crucial for postnatal respiratory health. Disruptions in this delicate process can lead to fetal lung development disorders, impacting neonatal outcomes and potentially influencing long-term health. Several key factors influence fetal lung maturation, including surfactant proteins (SP-A, SP-B, SP-C, and SP-D)[ 1 ][ 2 ][ 3 ][ 4 ], the ATP-binding cassette sub-family A member 3(ABCA3)[ 5 ], growth factor (FGF-7, FGF-10, EGF and their receptors FGFR and EGFR) [ 6 ][ 7 ][ 8 ][ 9 ][ 10 ], thyroid transcription factor-1 (TTF-1)[ 11 ], transcription factor Sox9[ 12 ], Sonic Hedgehog[ 13 ] and ion transport (calcium and chloride, in particular)[ 14 ][ 15 ][ 16 ][ 17 ]. Lung development progresses through five highly coordinated and regulated stages: the embryonic stage (weeks 4 to 7 of gestation), the pseudoglandular stage (weeks 7–16), the canalicular stage (weeks 16–28), the saccular stage (weeks 28–36), and the alveolar stage (week 36 of gestation to early childhood) [ 18 ] [ 19 ][ 20 ]. Between the pseudoglandular and saccular stages, the number of peripheral lung tubules increases dramatically, followed by sacculation, forming the gas exchange region of the lung. This process involves the development of an extensive pulmonary microvascular capillary bed in close proximity to the epithelial cells. Epithelial cells lining the peripheral lung saccules differentiate into large, squamous alveolar type I (ATI) cells, which comprise the majority of the alveolar surface and facilitate gas exchange. Smaller, cuboidal alveolar type II (ATII) cells, which constitute approximately two-thirds of the alveolar epithelium, produce pulmonary surfactant. Pulmonary surfactant, a mixture of lipids (primarily phospholipids like phosphatidylcholine and phosphatidylglycerol) and proteins (SP-B, SP-C, SP-A and SP-D), is essential for reducing surface tension at the air-liquid interface following the onset of ventilation at birth, enabling efficient gas exchange, maintaining alveolar stability, and preventing end-expiratory atelectasis (lung or lobe collapse). Pulmonary surfactant deficiency due to immaturity is the primary cause of respiratory distress syndrome (RDS) in premature infants, leading to breathing difficulties and poor oxygenation shortly after birth [ 21 ][ 19 ]. Surfactant production and release by ATII cells is a complex process. Phospholipids and surfactant proteins are packaged into cellular structures known as lamellar bodies (LBs), a process involving early endosomes and the formation of small vesicles. Fusion of LBs with the ATII plasma membrane, a Ca 2+ -dependent mechanism, triggers the exocytosis of pulmonary surfactant into the alveolar airspace. Secreted surfactant can be recycled (25–95%) by ATII cells and re-secreted into the alveolar lumen [ 22 ] or degraded by alveolar macrophages[ 23 ][ 24 ]. ABCA3, an ATP-binding cassette (ABC) transporter highly expressed in ATII cells, is crucial for the transport of lipid surfactant into LBs. ABCA3 inactivation in mice results in respiratory failure, surfactant loss, depletion of lung phosphatidylglycerol and impaired LB formation [ 25 ][ 26 ]. Mutations in the ABCA3, SP-B, and SP-C genes are associated with congenital respiratory disorders in infants, children, and adults, leading to respiratory failure. Histological features of affected lung tissue of these patients include reduced or absent LBs, accumulation of eosinophilic material and alveolar macrophages, increased periodic acid-Schiff (PAS) staining (indicating pneumocyte immaturity), thickened alveolar septa, and increased cellularity [ 4 ][ 27 ][ 28 ]. EGFR is essential for lung development. EGFR −/− mice (as those of the ABCA3 −/− mice) exhibit an RDS-like phenotype, with immature lungs, collapsed alveoli, thickened alveolar septa, and insufficient surfactant production. EGFR inactivation also leads to ATII cell immaturity, characterized by increased glycogen content and reduced LB numbers. These alveolarization defects result from impaired branching morphogenesis; EGFR −/− mice show dilated bronchi with fewer tubules and increased mesenchyme [ 8 ][ 9 ][ 10 ][ 20 ]. Sorcin is a highly expressed calcium-binding protein, significantly expressed (top 10% proteins) in the lung. Ca 2+ signaling triggers LB exocytosis and surfactant secretion [ 23 ]. Increased cytoplasmic calcium concentration ([Ca 2+ ] c ), due to Ca 2+ release from intracellular stores and Ca 2+ influx from extracellular space, drives LB fusion with the plasma membrane, triggering their exocytosis and surfactant secretion [ 29 ][ 30 ][ 31 ][ 32 ]. Sorcin regulates calcium homeostasis by modulating several calcium channels/pumps/exchangers, including the sarco/endoplasmic reticulum Ca 2+ pump (SERCA), the sarcolemmal Na + -Ca 2+ exchanger (NCX), the plasma membrane Ca 2+ pump (PMCA), the ryanodine receptors (RyRs) and the L-type voltage-gated calcium channel (LVCC), which are involved in branching morphogenesis and surfactant production. Sorcin also interacts with annexin-7 (a Ca 2+ -dependent membrane binding proteins important in membrane fusion during exocytosis), protein kinase A and Ca 2+ /calmodulin-dependent kinase, all of which are involved in surfactant secretion [ 29 ][ 32 ][ 33 ][ 34 ][ 35 ][ 36 ][ 37 ][ 38 ][ 39 ][ 40 ][ 41 ][ 42 ][ 43 ] [ 44 ]. Furthermore, Sorcin regulates the expression of ATP-dependent ABC efflux pumps like ABCB1 and ABCB4, influencing small molecule export and multidrug resistance in cancers [ 45 ][ 44 ]. We already demonstrated that Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium- dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies [ 46 ]. Of note, Sorcin knockout ( SRI −/− ) reduces EGFR levels in the bronchiolar region of mice lungs [ 46 ]. These results prompted us to analyze the relationship between EGFR and Sorcin at the physiological levels, investigating lung development and surfactants homeostasis during lung morphogenesis using SRI − /− mouse model compared to wild-type controls. Finally, since intracellular calcium regulates airway smooth muscle contraction, we explored Sorcin’s role in airway tissue remodeling associated with altered respiratory function. Materials and methods: Mouse models SRI - /- mice were provided by Hèctor H. Valdivia and Carmen R. Valdivia (Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA). These mice were generated as described in the work of Chen et al. [47]. C57BL/6 wild-type mice were purchased from Jackson laboratory (Bar Harbor, ME, USA) and were housed in the Histology Department-accredited animal facility. All the procedures were approved by the Italian Ministry for Health and were conducted according to the US National Institutes of Health (NIH) guidelines (Approval number: 605/2023-PR). Tissue mouse collection and hematoxylin/eosin staining E13.5 embryo, 3-week-old, 6-week-old and 3-month-old mice were sacrificed by cervical dislocation and lung tissues were collected for histological analysis. Tissues were fixed in 4% paraformaldehyde overnight, dehydrated in ethanol, cleared in xylene at room temperature and then, embedded in paraffin. Formalin-fixed paraffin-embedded (FFPE) lung tissue samples were cut (10μm thick) into SuperFrosts Plus-slides by Leica RM2255 microtome, deparaffinized in xylene and rehydrated gradually up to 100% ethanol. Then, sections were analyzed by routine hematoxylin and eosin staining or periodic acid Schiff (Sigma-Aldrich, #395B) for morphology analysis. The ratio of air-filled space on total lung area (3-weeks old, n=5 mice per condition) and the bronchiolar thickness average (6-week-old and 3-month-old, n=4 mice per condition) were quantified by ImageJ software. Oil red assay Cryosection OCT-frozen lung tissues (3-weeks old) were cut into SuperFrosts Plus-slides by using a Leica cryostat. They were fixed in formalin buffered solution 10% (Sigma‐Aldrich) for 10 min at room temperature (RT), then washed in tap water and stained with oil red solution (Sigma-Aldrich, #O-0625). Images were acquired under the microscope for analysis and quantification of lipid droplets was performed by ImageJ software. n = 4 mice per condition were analysed. Total RNA extraction from tissues, cDNA reverse transcriptase and RT‐qPCR Total lung OCT-frozen tissues (3-week-old) were homogenized in 500 ml of TRIzol RNA Isolation System (Invitrogen) and RNA was extracted using the according to manufacturer instructions. Reverse transcription to cDNA was performed with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), and cDNA was amplified using the SYBR TM Green PCR Master Mix (Thermo Fisher Scientific) on QuantStudioTM 7 Flex Real-Time PCR System, 384 well (Applied Biosystems). The relative expression values were normalized using housekeeping H3 gene. The following oligo sequences were used: H3 FW: 5 ́-GTGAAGAAACCTCATCGTTACAGGCCTGGT-3 ́; H3 RW: 5’- CTGCAAAGCACCAATAGCTGCACTCTGGAA-3’; FGF10 FW: 5’-GCTGTTCTCCTTCACCAAGT-3’ FGF10 RW: 5’-GCCATTGTGCTGCCAGTTAA-3’; SP-C FW: 5’- CCTCAAACG CCTTCTCATCG-3’; SP-C RW: 5’- CAGTGGAGCCGATGGAAAAG-3’; SP-B FW:5’-CCAGAGCCAGATTAACCCCA-3’; SP-B RW:5’- AGAAGTCCTGAGTGTGAGGC-3’; SOX9 FW: 5’-TATCTTCAAGGCGCTGCAAG-3’; SOX9 RW: 5’-GATCAACTTTGCCAGCTTGC-3’; ABCA3 FW: 5’-GACCCTCCTGTTCTGTGTCA-3’; ABCA3 RW: 5’-AGAAGTACA GGAAGCCACCC-3’; Lysate preparation and immunoblotting analysis Total flung OCT-frozen tissues (3-weeks old) were lysed in RIPA buffer and fresh protease inhibitors (PMSF 1 mM, NaF 1 mM, NaVO3 1 mM, Na4P207 5 Mm, Apoprotein 2 μg/ml, Leupeptin 5 μg/ml). After placed 30’ in cold ice, lysates were centrifuged for 10 min at 12,000 × rpm and quantified using Bradford Assay Reagent (Thermo Fisher, #1863028). Protein extracts were separated by SDS-PAGE and transferred into a nitrocellulose membrane. The membrane was incubated overnight with the following primary antibodies: rabbit monoclonal EGFR (1:1000, Cell Signaling Technology, #71655), rabbit polyclonal RAB5C (1:1000, Thermo Fisher Scientific, #PA5101828), mouse monoclonal PANRAS (Ab3) (1:1000, Sigma-Aldrich, #OP40) mouse monoclonal α-Smooth Muscle Actin (1:1000, Sigma-Aldrich, #A5228), mouse monoclonal vimentin (9E7E7) (1:1000, Santa Cruz #66001). As secondary antibodies were used goat anti-mouse (1:10,000 Bethyl, #A90-516P) and anti-rabbit (1:5000, Bethyl, #A120-201P) conjugated to horseradish peroxidase (Bethyl). Protein signals were developed by ECL detection using a ChemiDoc-It Imaging System (UVP, Upland, CA) instrument. Immunofluorescence For immunofluorescence in mice, lung tissues were covered in OCT mounting medium and frozen in liquid nitrogen precooled isopentane. Ten-micrometer cryosections were fixed in 4% paraformaldehyde for 5’, washed in PSB and permeabilized in 0.1% Triton X-100 in PBS. Then, sections were blocked in 5% goat serum for 1 h and incubated overnight at 4 °C with primary antibody SFTPB (1:100 in 1%BSA-PBS solution) (Thermo Fisher Scientific, #PA542000) followed by incubation with Alexa fluor 488 (rabbit)-conjugated secondary antibodies (1:500 in 1%BSA-PBS solution) (Thermo Fisher Scientific). For phalloidin immunofluorescence, sections were incubated with Rhodamine phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. Then, they were counterstained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA), mounted with Vectashield (DBA) and visualized under fluorescence confocal microscopy (Zeiss, Wetzlar, Germany). n = 4 mice per condition were analysed. Results Sorcin deficiency disrupts lung development To investigate Sorcin’s role in lung development, we analyzed lung tissue from wild-type WT and SRI - /- mice at embryonic (E13.5 and E16.5) and adult (3 weeks, 6 weeks, and 3 months) stages. Morphological analysis was performed using hematoxylin and eosin (H&E) staining. While E13.5 embryos showed no significant alterations (data not shown), changes appeared in E16.5 embryos. Lung sections from SRI - /- mice displayed increased cellular density and reduced tubule formation compared to WT mice, suggesting impaired branching morphogenesis (Figure 1A). At 3 weeks, SRI -/- mice maintained high cellular density showing irregular, thinner alveolar walls with fewer septations compared to WT mice (Figure 1B). Quantification of air-filled space relative to total lung area highlighted differences in alveolarization. Similar defects were observed in 6-week and 3-month-old SRI - /- mice. Additionally, hypercellularity around bronchioles, potentially indicative of cellular hyperplasia associated with pathological conditions and inflammation, was observed in SRI -/- mice (Figures 1C-D), a finding corroborated by bronchial wall thickness quantification . Sorcin regulates genes involved in the lung branching morphogenesis and alveolarization process. Given Sorcin’s role in lung epithelium formation, we investigated the molecular mechanisms underlying lung development. Since branching morphogenesis occurs during the pseudoglandular stage of lung development (E10.5-E16.5), we performed gene expression analysis at embryonic day E13.5. RT-qPCR revealed significant downregulation of the transcription factors FGF10 and SOX9 , essential for initial bud formation, in SRI - /- mice compared to WT. As alveolarization occurs in later stages of lung development, surfactant protein genes SP-B and SP-C showed no significant expression differences between the two groups at the embryonic stage (Figure 2A). However, at 3 weeks, SRI -/- mice showed significant downregulation of ABCA3 , SP-B , and SP-C expression, suggesting deficiencies in alveolar surfactant transport and secretion (Figure 2B). These findings may also indicate impaired maturation of lung epithelial ATII pneumocytes, which produce and secrete pulmonary surfactant lipids and proteins. To further investigate Sorcin's role in alveolarization, we analyzed lung tissue lipid composition. Oil Red O staining revealed reduced size and number of lipid droplets, indicating impaired total lipid surfactant content (Figure 2C). Quantification of lipid droplet accumulation is shown in Figure 2D. PAS staining, used to detect glycogen stores, highlighted pneumocyte immaturity and abnormal distribution of glycogen, with SRI -/- sections showing intense magenta staining throughout the cytoplasm of the cells, unlike the WT pattern (Figure 2E). These data suggest impaired alveolarization due to decreased surfactant production, potentially leading to respiratory disease. SRI - /- mice displayed impaired surfactant production and EGFR signaling Surfactant secretion involves intracellular processing of SP-B and SP-C proteins from the ER and Golgi to multivesicular bodies (MVBs) and LBs. This trafficking is mediated by early endosomes, which contribute to LB formation and surfactant secretion. To further investigate Sorcin’s role in surfactant secretion, we analyzed the expression of mature SP-B and Rab5C, an isoform of the Rab5 subfamily which play a critical role in regulating early endosomes. Confocal microscopy showed reduced SP-B expression in SRI -/- mice compared to WT (Figure 3A). Western blot analysis showed no significant differences of Rab5C expression at the E13.5 embryonic stage (Figure 3B). However, at 3 weeks, Rab5C expression was significantly reduced in SRI -/- mice compared to WT (Figure 3C). These results provide insights into of Sorcin's potential role in regulating ATII cell function, surfactant homeostasis and trafficking. These molecular and histological findings are consistent with those observed in EGFR -/- mice, which exhibit reduced tubule formation, abundant mesenchyme during branching morphogenesis, irregular alveoli with thick walls, undifferentiated type II pneumocytes with high glycogen content, and decreased surfactant protein production during alveolarization. To further explore the Sorcin (SRI)-EGFR relationship, we analyzed the expression of EGFR and of its downstream effector, RAS, both critical for lung development. Western blot analysis revealed significant reductions in EGFR and RAS protein levels in SRI -/- mice compared to WT mice, at both E13.5 embryonic stage (Figure 3D) and 3 weeks (Figure 3E). These findings support Sorcin’s involvement in EGFR regulation, influencing lung function both at the physiological level (through impaired lung development) and at the pathological level[46]. Sorcin regulates cytoskeletal airway remodeling and adipose tissue infiltration Sorcin is highly expressed in vascular smooth muscle, where it modulates Ca²⁺ sparks (decreasing their frequency, amplitude, duration and width) by regulating calcium concentration in the ER, mainly through SERCA activation and RYR inhibition, and therefore modulating intracellular Ca²⁺ levels, which are essential for muscle airway contraction [38]. Given the observed increase in bronchiole thickness in SRI -/- mice, we analyzed the cytoskeletal remodeling using phalloidin immunofluorescence. Confocal analysis revealed a significant increase in f-actin expression in SRI -/- mice, suggesting an increased cellular airway smooth muscle (ASM) volume compared to WT mice. Notably, these morphological changes were evident as early as three weeks of age and persisted at three months (Figure 4A-B). Accordingly, we showed a significant increase of α-SMA (alpha-smooth muscle actin) and vimentin expression in SRI -/- mice compared to those WT, supporting ASM remodeling and hypercontractility (Figure 4C). Taken together, these data suggest that calcium depletion in the ER may affect bronchial contraction, ultimately leading to lung structural and airway tissue remodeling, including an increase of ASM. Moreover, we observed early and significant fat accumulation in SRI -/- mice at three months of age, compared to WT (Figure 4D). Adipose tissue infiltration within the airway epithelium has been reported in obesity-related clinical cases and asthma models, both of which are associated with airway wall thickening and inflammation. These findings suggested that Sorcin deficiency contributes to both cytoskeletal and airway tissue remodeling due to mechanostress events, leading to significant lung structural alterations. Discussion Our previous work demonstrated that Sorcin regulates EGFR signaling in non-small cell lung adenocarcinoma, promoting cellular invasion and migration. We discovered important relationships between calcium homeostasis and EGFR signaling pathways and identified Sorcin as a key player of EGFR physiological and pathological roles, linked to Ca2 + dysregulation [ 46 ]. Beyond its role in tumorigenesis, EGFR is crucial for lung development, particularly branching morphogenesis and alveolarization. Branching morphogenesis occurs during the pseudoglandular stage (E9.5-16.6 in mice, approximately 16 weeks of gestation in humans), and promotes the formation of a tree-like bronchial system through interactions between epithelial and mesenchymal cells [ 48 ][ 49 ][ 20 ]. Alveolarization occurs during the last stages of lung development, specifically during the alveolar stage (P5–P30 in mice, around 20 week of gestation in humans, continuing postnatally), involving alveoli formation and maturation, as well as ATI and ATII epithelial cell differentiation. ATII cells play a critical role in producing, secreting, and recycling pulmonary surfactant, which is essential for normal lung function, reducing alveolar surface tension and preventing alveolar collapse. Histological analysis of embryo and newborn EGFR −/− mice lungs revealed reduced tubules and increased interstitial mesenchyme during embryonic development, as well as poorly formed, collapsed alveoli characterized by reduced airspace and thickened septa, compared to control mice[ 8 ][ 9 ]. In vitro (e.g., in lung explant cultures), EGF treatment enhances branching complexity and number, indicating EGFR’s positive regulatory role in epithelial proliferation and migration [ 50 ][ 51 ]. Similarly, our histological analysis of lung tissues from WT and SRI⁻ / ⁻ mice revealed reduced bronchiole formation and increased cellular density during the embryonic stage. Consequently, the SRI⁻ / ⁻ adult mice displayed abnormal bronchiole maturation with increased epithelial cell layers. This atypical bronchial hyperplasia is a hallmark of inflammation and pulmonary fibrosis, contributing to respiratory distress conditions like asthma and chronic obstructive pulmonary disease (COPD)[ 52 ]. Furthermore, persistent hypercellularity and altered alveolar structure with thickened walls indicated impaired alveolarization in SRI⁻ / ⁻ mice. To characterize Sorcin’s role in lung development, we investigated the molecular pathway and markers involved in branching morphogenesis and alveolarization. The highly coordinated interplay of growth factors and transcription factors, such as FGF10[ 53 ][ 54 ][ 55 ] and SOX9 [ 12 ], regulates cellular proliferation within tubules, new bud formation, and branching morphogenesis. The surfactant genes SP-B and SP-C encode components of pulmonary surfactant, produced by ATII cells, essential for postnatal alveolar function. Consistently, SRI⁻ / ⁻ mice showed significantly reduced FGF10 and SOX9 levels during embryonic development (E13.5) compared to controls, suggesting impaired branching morphogenesis. As expected, SP-B and SP-C expression was not altered during the embryonic period, given that ATII differentiation occurs later, during the last step of lung development. Since FGF10 also drives normal and physiological formation of alveoli [ 56 ] and the pool of SOX9 + progenitors represents the precursor of ATI and ATII cells [ 57 ], their reduction suggested alterations in mature lung structures as well. At 3 weeks, SRI⁻ / ⁻ mice exhibited significant decreases in SP-B, SP-C, and ABCA3 (the lipid surfactant transporter in LBs), indicating defective alveolarization, including surfactant trafficking and secretion. These molecular results were consistent with the observed lower lipid surfactant accumulation in SRI⁻ / ⁻ mice, demonstrating impaired secretory pathways and ATII function. Increased glycogen deposits within ATII cells indicated cell immaturity or damage. ATII cells play a critical role in producing and secreting pulmonary surfactant via LB fusion. The surfactant secretion pathway in ATII cells involves the translocation of the precursor proteins pro-SP-B and pro-SP-C from the ER to the Golgi apparatus, followed by their trafficking to small vesicles and MVBs. Within these compartments, SP-B and SP-C undergo maturation before fusing with LBs. The early endosome pathway, regulated by RAB proteins (including the RAB5 family isoforms RAB5a, RAB5b, and RAB5c), sorts these precursors, directing them to the appropriate intracellular compartments for further processing, including small vesicles and MVBs. Huang et al. showed that a negative variant of RAB5c led to altered early endosome that failed the fusion with proSP-B- or proSP-C-containing nascent sorting vesicles, impairing surfactant protein processing and trafficking, and causing interstitial lung disease [ 58 ]. Consistent with this, we observed reduced SP-B and RAB5c protein levels in 3-week-old SRI⁻ / ⁻ mice. Disruption in early endosomal trafficking can impair proper SP-B delivery and processing, potentially leading to surfactant deficiencies and respiratory dysfunction, highlighting the critical role of Sorcin in surfactant secretion. As noted, impaired EGFR signaling is associated with branching morphogenesis and alveolarization defects. Accordingly, both E13.5 and 3-week-old SRI⁻ / ⁻ mice showed decreased EGFR and impairment of its downstream pathway, including RAS protein, compared to WT. These data reinforce the relationship between Sorcin and the EGFR signaling pathway previously demonstrated in our lung adenocarcinoma in vitro study [ 46 ]. Calcium oscillations between ER and cytosol are essential for muscle contraction [ 59 ]. In airway smooth muscle (ASM), force generation begins with the myosin light chain kinase (MLCK) phosphorylation and spreads via actin filament polymerization. Alterations in the contractile apparatus and mechanotransduction pathways can lead to asthma, a chronic inflammatory disease of the airways [ 60 ]. Increased cytosolic Ca²⁺ activates MLCK, leading to ASM contraction and airway narrowing [ 61 ]. Many experimental asthma mouse models show airway remodeling and hyperresponsiveness, including increased ASM contractility due to actin cytoskeletal remodeling and cell proliferation [ 62 ]. Mahan et al. demonstrated that SERCA2 downregulation leads to airway remodeling, highlighting the importance of calcium homeostasis - including the calcium release from ER- for normal lung function. ASM of asthmatics patients showed reduced SERCA2 expression [ 63 ]. Accordingly, 3-week and 3-month-old SRI⁻ / ⁻ mice showed increased f-actin filaments and α-smooth muscle actin expression around the peribronchial region, suggesting cytoskeletal airway remodeling and excessive bronchial contraction, likely due to altered intracellular calcium homeostasis. Asthma symptoms worsen in obese patients, which often exhibit increased airway-associated adipose tissue [ 64 ][ 65 ]. This fat accumulation may contribute to increased bronchial wall thickness and inflammation [ 66 ], potentially explaining the high adipose tissue infiltration observed during lung development in our SRI −/− mice. Conclusions This study provides novel insights into Sorcin’s role in fetal lung development, demonstrating its impact on branching morphogenesis and alveolarization, including surfactant production and secretion. Additionally, altered intracellular calcium levels may directly affect EGFR signaling and bronchial contractility, leading to airway cytoskeletal remodeling and adipose tissue accumulation, which can exacerbate airway obstruction. These combined changes may contribute to respiratory disorder development. This study also reveals novel key cell signaling mechanisms regulating fetal lung development, providing a basis for innovative strategies to enhance lung maturation in clinical conditions where development is compromised. Additionally, it addresses critical challenges by identifying approaches to prevent surfactant-related disorders, such as respiratory distress syndrome, ultimately contributing to improved therapeutic outcomes. Abbreviations ABCA3: ATP-binding cassette sub-family A member 3 α-SMA: alpha-smooth muscle actin ASM: airway smooth muscle ATI: alveolar type I ATII: alveolar type II COPD: chronic obstructive pulmonary disease. ER: Endoplasmic Reticulum EGFR: Epidermal growth factor receptor FFPE: Formalin-fixed paraffin-embedded KO: Knockout LBs: Lamellar bodies LVCC: L-type voltage-gated calcium channel MLCK: Myosin light chain kinase MVBs: Multivesicular bodies NCX: Na+-Ca2+ exchanger NSCLC: Non-small-cell lung carcinoma PAS: Periodic acid-Schiff PMCA: Plasma membrane Ca2+ pump RDS: Respiratory distress syndrome RYRs: Ryanodine receptors SERCA: Sarco/endoplasmic reticulum Ca2+ pump SP-B: Surfactant protein B SP-C: Surfactant protein C SRI: Sorcin TTF-1: Thyroid transcription factor-1 Declarations Author contributions CT, LDA, AI, APA, AB, GM, performed the experiments and contributed to data analysis. CT, SM, GC and FF contributed to the experiment designs and wrote the manuscript. CT, GC, FF, and VP conceived or designed the experiments. LT, ML and AI contributed to the discussion and data analysis. All authors read and approved the final manuscript. Funding The research leading to these results received funding from: PRIN_2022 Prot. 2022WB59LB - PRIN_2022PNRR Prot. P2022C948R - European Union - Next Generation EU, Mission 4, Component 2, CUP B93D21010860004 - CN 00000041 to FF; CNCCS s.c.a.r.l. (National Collection of Chemical Compounds and Screening Center, www.cnccs.it); “Potentiating the Italian Capacity for Structural Biology Services in Instruct-ERIC”, acronym “ITACA.SB” (Project No. IR0000009, CUP B53C22001790006), funded by the European Union’s NextGenerationEU under the MUR call 3264/2021 PNRR M4/C2/L3.1.1; PNRR PE8 Age-IT., co-funding from Next Generation EU [DM 1557 11.10.2022], in the context of the National Recovery and Resilience Plan, Investment PE8 – Project Age-It: “Ageing Well in an Ageing Society”; Project PRIN 2022 MUR 2022HYF8KS to GC. Availability of data and materials The data that support the findings of this study are available from the corresponding author upon reason- able request. Competing interests The authors declare that they have no competing interests Ethics approval and consent to participate All procedures were approved by the Italian Ministry for Health and were conducted in accordance with the US National Institutes of Health (NIH) guidelines (Approval number: 605/2023-PR). Written informed consent was not applicable because this study was conducted on mice. Consent for publication I, the undersigned, give my consent for the publication. Acknowledgements We acknowledge Hèctor H. Valdivia and Carmen R. Valdivia (Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA) for providing SRI⁻ / ⁻ mice. 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Eur Respir J 54:1900857. https://doi.org/10.1183/13993003.00857-2019 Cite Share Download PDF Status: Published Journal Publication published 28 Oct, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Editorial decision: Major Revision 29 Apr, 2025 Reviewers agreed at journal 05 Apr, 2025 Reviewers invited by journal 27 Mar, 2025 Editor assigned by journal 26 Mar, 2025 First submitted to journal 24 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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16:06:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6297074/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6297074/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05870-y","type":"published","date":"2025-10-28T15:57:49+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80616266,"identity":"7554bad3-9c3f-4e39-8670-dd115a70f377","added_by":"auto","created_at":"2025-04-15 08:51:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4185527,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHematoxylin and Eosin staining in lung section of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSRI\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eE16.5 lung sections: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice exhibited increased cellularity and reduced bronchiole formation compared to WT mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB)\u003c/strong\u003e 3-week-old (newborn) lung sections: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice showed impaired bronchiole development, thickened alveolar walls, and reduced alveoli formation compared to WT mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC-D)\u003c/strong\u003e 6-week and 3-month-old lung sections: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice displayed persistent alveolarization defects and bronchiolar epithelial hyperplasia.\u003c/p\u003e\n\u003cp\u003eScale bars, 50 μm.\u003c/p\u003e\n\u003cp\u003eQuantitative analysis of air space and bronchiole thickness was performed using ImageJ software. Data are presented as mean ± SEM. Statistical significance: *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 (Student’s t-test). n = 5 mice per group, 10 lung sections per mouse.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6297074/v1/a2cfb52d6d7ce5ea3863b7c7.png"},{"id":80616263,"identity":"38f9a6c4-9906-48d5-8bb8-ce4ea5de91f7","added_by":"auto","created_at":"2025-04-15 08:51:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2948909,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImpaired branching morphogenesis and lung surfactant production in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSRI\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u0026nbsp; \u003c/strong\u003eE13.5 lung tissue: Embryonic \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice showed significant decreased expression of branching morphogenesis genes (\u003cem\u003eFGF10\u003c/em\u003e, \u003cem\u003eSOX9\u003c/em\u003e), compared to WT mice. Surfactant genes (\u003cem\u003eSP-B\u003c/em\u003e and \u003cem\u003eSP-C\u003c/em\u003e) exhibit no significant difference between the two groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB)\u003c/strong\u003e 3-week-old lung tissue: significantly reduced expression of surfactant genes (\u003cem\u003eSP-B\u003c/em\u003e, \u003cem\u003eSP-C\u003c/em\u003e), and of the phospholipid transporter \u003cem\u003eABCA3\u003c/em\u003e was observed in \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice compared to WT controls. Data are presented as mean ± SEM. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 (determined by Student’s t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC)\u003c/strong\u003e Oil Red O staining: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice exhibited decreased lipid droplet accumulation compared to WT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD)\u003c/strong\u003e Quantification of lipid droplets. Data are presented as mean ± SEM. ***p \u0026lt; 0.001 (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE)\u003c/strong\u003e Periodic acid-Schiff (PAS) staining: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice showed increased glycogen staining compared to WT, indicative of pneumocyte immaturity.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6297074/v1/866f457ea6bf705bf2b61023.png"},{"id":80616267,"identity":"566c93fe-386d-42a5-98f2-1da50be7c158","added_by":"auto","created_at":"2025-04-15 08:51:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1424662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered protein expression in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSRI\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003e mice in lung development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA) \u003c/strong\u003eConfocal microscopy (3 weeks): \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e lung tissue showed reduced SP-B (red) expression compared to WT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eB-C) \u003c/strong\u003eWestern blot analysis: \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice exhibited decreased RAB5C protein levels compared to WT at E13.5 (\u003cstrong\u003eB\u003c/strong\u003e) and 3 weeks (\u003cstrong\u003eC\u003c/strong\u003e). Densitometry quantification shown (mean ± SEM). **p \u0026lt; 0.01 (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD-E) \u003c/strong\u003eWestern Blot analysis: \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice showed significantly decreased EGFR and RAS protein levels compared to WT mice at E13.5 \u003cstrong\u003e(D)\u003c/strong\u003e and 3 weeks \u003cstrong\u003e(E)\u003c/strong\u003e. Densitometry quantification shown (mean ± SEM). *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001 (determined by Student’s t-test).\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6297074/v1/f559eb484a4c7e7892c9fc3c.png"},{"id":80616260,"identity":"bb0f57bd-cd4e-46ef-9056-feb68f9792a0","added_by":"auto","created_at":"2025-04-15 08:51:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3086888,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAirway remodeling and adipose tissue accumulation in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSRI\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003e-/-\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003e \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003elungs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA-B) \u003c/strong\u003eConfocal microscopy at 3 weeks \u003cstrong\u003e(A)\u003c/strong\u003e and 3 months \u003cstrong\u003e(B)\u003c/strong\u003e: \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e lung tissue showed increased phalloidin expression and thickened airway walls compared to WT.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eC)\u003c/strong\u003e Western Blot analysis (3 months): \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice exhibited increased α-SMA and vimentin protein levels compared to WT. Densitometry quantification shown (mean ± SEM). *p \u0026lt; 0.05, **p \u0026lt; 0.01 (Student’s t-test).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD) \u003c/strong\u003eH\u0026amp;E staining: \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e-/-\u003c/em\u003e\u003c/sup\u003e mice showed increased airway adipose tissue accumulation compared to WT.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6297074/v1/45ecf5e5ce54e9e6b07484ac.png"},{"id":95041234,"identity":"158b7109-f8d9-4360-9e5c-b27f558eea38","added_by":"auto","created_at":"2025-11-03 16:11:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12578349,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6297074/v1/38035b6f-461a-4232-b400-401373451e5b.pdf"}],"financialInterests":"","formattedTitle":"Sorcin regulates alveolarization and airway tissue remodeling during lung morphogenesis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFetal lung development is a complex process crucial for postnatal respiratory health. Disruptions in this delicate process can lead to fetal lung development disorders, impacting neonatal outcomes and potentially influencing long-term health.\u003c/p\u003e \u003cp\u003eSeveral key factors influence fetal lung maturation, including surfactant proteins (SP-A, SP-B, SP-C, and SP-D)[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e][\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e][\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the ATP-binding cassette sub-family A member 3(ABCA3)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], growth factor (FGF-7, FGF-10, EGF and their receptors FGFR and EGFR) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], thyroid transcription factor-1 (TTF-1)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], transcription factor Sox9[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Sonic Hedgehog[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] and ion transport (calcium and chloride, in particular)[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e][\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e][\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eLung development progresses through five highly coordinated and regulated stages: the embryonic stage (weeks 4 to 7 of gestation), the pseudoglandular stage (weeks 7\u0026ndash;16), the canalicular stage (weeks 16\u0026ndash;28), the saccular stage (weeks 28\u0026ndash;36), and the alveolar stage (week 36 of gestation to early childhood) [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Between the pseudoglandular and saccular stages, the number of peripheral lung tubules increases dramatically, followed by sacculation, forming the gas exchange region of the lung. This process involves the development of an extensive pulmonary microvascular capillary bed in close proximity to the epithelial cells. Epithelial cells lining the peripheral lung saccules differentiate into large, squamous alveolar type I (ATI) cells, which comprise the majority of the alveolar surface and facilitate gas exchange. Smaller, cuboidal alveolar type II (ATII) cells, which constitute approximately two-thirds of the alveolar epithelium, produce pulmonary surfactant. Pulmonary surfactant, a mixture of lipids (primarily phospholipids like phosphatidylcholine and phosphatidylglycerol) and proteins (SP-B, SP-C, SP-A and SP-D), is essential for reducing surface tension at the air-liquid interface following the onset of ventilation at birth, enabling efficient gas exchange, maintaining alveolar stability, and preventing end-expiratory atelectasis (lung or lobe collapse). Pulmonary surfactant deficiency due to immaturity is the primary cause of respiratory distress syndrome (RDS) in premature infants, leading to breathing difficulties and poor oxygenation shortly after birth [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Surfactant production and release by ATII cells is a complex process. Phospholipids and surfactant proteins are packaged into cellular structures known as lamellar bodies (LBs), a process involving early endosomes and the formation of small vesicles. Fusion of LBs with the ATII plasma membrane, a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent mechanism, triggers the exocytosis of pulmonary surfactant into the alveolar airspace. Secreted surfactant can be recycled (25\u0026ndash;95%) by ATII cells and re-secreted into the alveolar lumen [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] or degraded by alveolar macrophages[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e][\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. ABCA3, an ATP-binding cassette (ABC) transporter highly expressed in ATII cells, is crucial for the transport of lipid surfactant into LBs. ABCA3 inactivation in mice results in respiratory failure, surfactant loss, depletion of lung phosphatidylglycerol and impaired LB formation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e][\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Mutations in the ABCA3, SP-B, and SP-C genes are associated with congenital respiratory disorders in infants, children, and adults, leading to respiratory failure. Histological features of affected lung tissue of these patients include reduced or absent LBs, accumulation of eosinophilic material and alveolar macrophages, increased periodic acid-Schiff (PAS) staining (indicating pneumocyte immaturity), thickened alveolar septa, and increased cellularity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e][\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e][\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. EGFR is essential for lung development. \u003cem\u003eEGFR\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice (as those of the \u003cem\u003eABCA3\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice) exhibit an RDS-like phenotype, with immature lungs, collapsed alveoli, thickened alveolar septa, and insufficient surfactant production. EGFR inactivation also leads to ATII cell immaturity, characterized by increased glycogen content and reduced LB numbers. These alveolarization defects result from impaired branching morphogenesis; \u003cem\u003eEGFR\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice show dilated bronchi with fewer tubules and increased mesenchyme [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e][\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Sorcin is a highly expressed calcium-binding protein, significantly expressed (top 10% proteins) in the lung. Ca\u003csup\u003e2+\u003c/sup\u003e signaling triggers LB exocytosis and surfactant secretion [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Increased cytoplasmic calcium concentration ([Ca\u003csup\u003e2+\u003c/sup\u003e]\u003csub\u003ec\u003c/sub\u003e), due to Ca\u003csup\u003e2+\u003c/sup\u003e release from intracellular stores and Ca\u003csup\u003e2+\u003c/sup\u003e influx from extracellular space, drives LB fusion with the plasma membrane, triggering their exocytosis and surfactant secretion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e][\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e][\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Sorcin regulates calcium homeostasis by modulating several calcium channels/pumps/exchangers, including the sarco/endoplasmic reticulum Ca\u003csup\u003e2+\u003c/sup\u003e pump (SERCA), the sarcolemmal Na\u003csup\u003e+\u003c/sup\u003e-Ca\u003csup\u003e2+\u003c/sup\u003e exchanger (NCX), the plasma membrane Ca\u003csup\u003e2+\u003c/sup\u003e pump (PMCA), the ryanodine receptors (RyRs) and the L-type voltage-gated calcium channel (LVCC), which are involved in branching morphogenesis and surfactant production. Sorcin also interacts with annexin-7 (a Ca\u003csup\u003e2+\u003c/sup\u003e-dependent membrane binding proteins important in membrane fusion during exocytosis), protein kinase A and Ca\u003csup\u003e2+\u003c/sup\u003e/calmodulin-dependent kinase, all of which are involved in surfactant secretion [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e][\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e][\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e][\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e][\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e][\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e][\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e][\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e][\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e][\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e][\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e][\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e][\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Furthermore, Sorcin regulates the expression of ATP-dependent ABC efflux pumps like ABCB1 and ABCB4, influencing small molecule export and multidrug resistance in cancers [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e][\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe already demonstrated that Sorcin and EGFR expression are significantly correlated and associated with reduced overall survival in cancer patients. Mechanistically, Sorcin directly binds EGFR protein in a calcium- dependent fashion and regulates calcium (dys)homeostasis linked to EGF-dependent EGFR signaling. Moreover, Sorcin controls EGFR proteostasis and signaling and increases its phosphorylation, leading to increased EGF-dependent migration and invasion. Silencing of Sorcin cooperates with EGFR inhibitors in the regulation of migration, highlighting calcium signaling pathway as an exploitable target to enhance the effectiveness of EGFR-targeting therapies [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Of note, Sorcin knockout (\u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) reduces EGFR levels in the bronchiolar region of mice lungs [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese results prompted us to analyze the relationship between EGFR and Sorcin at the physiological levels, investigating lung development and surfactants homeostasis during lung morphogenesis using \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u0026minus;\u003cem\u003e/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mouse model compared to wild-type controls. Finally, since intracellular calcium regulates airway smooth muscle contraction, we explored Sorcin\u0026rsquo;s role in airway tissue remodeling associated with altered respiratory function.\u003c/p\u003e"},{"header":"Materials and methods:","content":"\u003cp\u003e\u003cstrong\u003eMouse models\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e-\u003cem\u003e/-\u003c/em\u003e\u003c/sup\u003e mice were provided by H\u0026egrave;ctor H. Valdivia and Carmen R. Valdivia (Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA). These mice were generated as described in the work of Chen et al. [47].\u003c/p\u003e\n\u003cp\u003eC57BL/6 wild-type mice were purchased from Jackson laboratory (Bar Harbor, ME, USA) and were housed in the Histology Department-accredited animal facility. All the procedures were approved by the Italian Ministry for Health and were conducted according to the US National Institutes of Health (NIH) guidelines (Approval number: 605/2023-PR).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue mouse collection and hematoxylin/eosin staining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE13.5 embryo, 3-week-old, 6-week-old and 3-month-old mice were sacrificed by cervical dislocation and lung tissues were collected for histological analysis. Tissues were fixed in 4% paraformaldehyde overnight, dehydrated in ethanol, cleared in xylene at room temperature and then, embedded in paraffin. Formalin-fixed paraffin-embedded (FFPE) lung tissue samples were cut (10\u0026mu;m thick) into SuperFrosts Plus-slides by Leica RM2255 microtome, deparaffinized in xylene and rehydrated gradually up to 100% ethanol. Then, sections were analyzed by routine hematoxylin and eosin staining or periodic acid Schiff (Sigma-Aldrich, #395B) for morphology analysis. The ratio of air-filled space on total lung area (3-weeks old, n=5 mice per condition) and the bronchiolar thickness average (6-week-old and 3-month-old, n=4 mice per condition) were quantified by ImageJ software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOil red assay\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCryosection OCT-frozen lung tissues (3-weeks old) were cut into SuperFrosts Plus-slides by using a Leica cryostat. They were fixed in formalin buffered solution 10% (Sigma‐Aldrich) for 10 min at room temperature (RT), then washed in tap water and stained with oil red solution (Sigma-Aldrich, #O-0625). Images were acquired under the microscope for analysis and quantification of lipid droplets was performed by ImageJ software. n = 4 mice per condition were analysed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTotal RNA extraction from tissues, cDNA reverse transcriptase and RT‐qPCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal lung OCT-frozen tissues (3-week-old) were homogenized in 500 ml of TRIzol RNA Isolation System (Invitrogen) and RNA was extracted using the according to manufacturer instructions. Reverse transcription to cDNA was performed with the High-Capacity RNA-to-cDNA Kit (Applied Biosystems), and cDNA was amplified using the SYBR TM Green PCR Master Mix (Thermo Fisher Scientific) on QuantStudioTM 7 Flex Real-Time PCR System, 384 well (Applied Biosystems). The relative expression values were normalized using housekeeping H3 gene. \u0026nbsp;The following oligo sequences were used: H3 FW: 5 ́-GTGAAGAAACCTCATCGTTACAGGCCTGGT-3 ́; H3 RW: 5\u0026rsquo;- CTGCAAAGCACCAATAGCTGCACTCTGGAA-3\u0026rsquo;; FGF10 FW: 5\u0026rsquo;-GCTGTTCTCCTTCACCAAGT-3\u0026rsquo; FGF10 RW: 5\u0026rsquo;-GCCATTGTGCTGCCAGTTAA-3\u0026rsquo;; SP-C FW: 5\u0026rsquo;- CCTCAAACG CCTTCTCATCG-3\u0026rsquo;; SP-C RW: 5\u0026rsquo;- CAGTGGAGCCGATGGAAAAG-3\u0026rsquo;; SP-B FW:5\u0026rsquo;-CCAGAGCCAGATTAACCCCA-3\u0026rsquo;; SP-B RW:5\u0026rsquo;- AGAAGTCCTGAGTGTGAGGC-3\u0026rsquo;; SOX9 FW: 5\u0026rsquo;-TATCTTCAAGGCGCTGCAAG-3\u0026rsquo;; SOX9 RW: 5\u0026rsquo;-GATCAACTTTGCCAGCTTGC-3\u0026rsquo;; ABCA3 FW: 5\u0026rsquo;-GACCCTCCTGTTCTGTGTCA-3\u0026rsquo;; ABCA3 RW: 5\u0026rsquo;-AGAAGTACA GGAAGCCACCC-3\u0026rsquo;;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLysate preparation and immunoblotting analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal flung OCT-frozen tissues (3-weeks old) were lysed in RIPA buffer and fresh protease inhibitors (PMSF 1 mM, NaF 1 mM, NaVO3 1 mM, Na4P207 5 Mm, Apoprotein 2 \u0026mu;g/ml, Leupeptin 5 \u0026mu;g/ml). After placed 30\u0026rsquo; in cold ice, lysates were centrifuged for 10 min at 12,000 \u0026times; rpm and quantified using Bradford Assay Reagent (Thermo Fisher, #1863028). Protein extracts were separated by SDS-PAGE and transferred into a nitrocellulose membrane. The membrane was incubated overnight with the following primary antibodies: rabbit monoclonal EGFR (1:1000, Cell Signaling Technology, #71655), rabbit polyclonal RAB5C (1:1000, Thermo Fisher Scientific, #PA5101828), mouse monoclonal PANRAS (Ab3) (1:1000, Sigma-Aldrich, #OP40) mouse monoclonal \u0026alpha;-Smooth Muscle Actin (1:1000, Sigma-Aldrich, #A5228), mouse monoclonal vimentin (9E7E7) (1:1000, Santa Cruz #66001). As secondary antibodies were used goat anti-mouse (1:10,000 Bethyl, #A90-516P) and anti-rabbit (1:5000, Bethyl, #A120-201P) conjugated to horseradish peroxidase (Bethyl). Protein signals were developed by ECL detection using a ChemiDoc-It Imaging System (UVP, Upland, CA) instrument. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence in mice, lung tissues were covered in OCT mounting medium and frozen in liquid nitrogen precooled isopentane. Ten-micrometer cryosections were fixed in 4% paraformaldehyde for 5\u0026rsquo;, washed in PSB and permeabilized in 0.1% Triton X-100 in PBS. Then, sections were blocked in 5% goat serum for 1 h and incubated overnight at 4 \u0026deg;C with primary antibody SFTPB (1:100 in 1%BSA-PBS solution) (Thermo Fisher Scientific, #PA542000) followed by incubation with Alexa fluor 488 (rabbit)-conjugated secondary antibodies (1:500 in 1%BSA-PBS solution) (Thermo Fisher Scientific). For phalloidin immunofluorescence, sections were incubated with Rhodamine phalloidin (Thermo Fisher Scientific) diluted in PBS for 30 min. Then, they were counterstained with Hoechst 33342 (Thermo Fisher Scientific, Waltham, MA, USA), mounted with Vectashield (DBA) and visualized under fluorescence confocal microscopy (Zeiss, Wetzlar, Germany).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003en = 4 mice per condition were analysed.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSorcin deficiency disrupts lung development\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate Sorcin\u0026rsquo;s role in lung development, we analyzed lung tissue from wild-type WT and \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e-\u003cem\u003e/-\u003c/em\u003e\u003c/sup\u003e mice at embryonic (E13.5 and E16.5) and adult (3 weeks, 6 weeks, and 3 months) stages. Morphological analysis was performed using hematoxylin and eosin (H\u0026amp;E) staining. While E13.5 embryos showed no significant alterations (data not shown), changes appeared in E16.5 embryos. Lung sections from \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e-\u003cem\u003e/-\u003c/em\u003e\u003c/sup\u003e mice displayed increased cellular density and reduced tubule formation compared to WT mice, suggesting impaired branching morphogenesis (Figure 1A).\u003c/p\u003e\n\u003cp\u003eAt 3 weeks, \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003emice maintained high cellular density showing irregular, thinner alveolar walls with fewer septations compared to WT mice (Figure 1B). Quantification of air-filled space relative to total lung area highlighted differences in alveolarization. Similar defects were observed in 6-week and 3-month-old \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e-\u003cem\u003e/-\u003c/em\u003e\u003c/sup\u003e mice. Additionally, hypercellularity around bronchioles, potentially indicative of cellular hyperplasia associated with pathological conditions and inflammation, was observed in \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice (Figures 1C-D), a finding corroborated by bronchial wall thickness quantification\u003cstrong\u003e.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSorcin regulates genes involved in the lung branching morphogenesis and alveolarization process.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGiven Sorcin\u0026rsquo;s role in lung epithelium formation, we investigated the molecular mechanisms underlying lung development. Since branching morphogenesis occurs during the pseudoglandular stage of lung development (E10.5-E16.5), we performed gene expression analysis at embryonic day E13.5. RT-qPCR revealed significant downregulation of the transcription factors \u003cem\u003eFGF10\u003c/em\u003e and \u003cem\u003eSOX9\u003c/em\u003e, essential for initial bud formation, in \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e-\u003cem\u003e/-\u003c/em\u003e\u003c/sup\u003e mice compared to WT. As alveolarization occurs in later stages of lung development, surfactant protein genes \u003cem\u003eSP-B\u003c/em\u003e and \u003cem\u003eSP-C\u003c/em\u003e showed no significant expression differences between the two groups at the embryonic stage (Figure 2A). However, at 3 weeks, \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice showed significant downregulation of \u003cem\u003eABCA3\u003c/em\u003e, \u003cem\u003eSP-B\u003c/em\u003e, and \u003cem\u003eSP-C\u003c/em\u003e expression, suggesting deficiencies in alveolar surfactant transport and secretion (Figure 2B). These findings may also indicate impaired maturation of lung epithelial ATII pneumocytes, which produce and secrete pulmonary surfactant lipids and proteins.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate Sorcin\u0026apos;s role in alveolarization, we analyzed lung tissue lipid composition. Oil Red O staining revealed reduced size and number of lipid droplets, indicating impaired total lipid surfactant content (Figure 2C). Quantification of lipid droplet accumulation is shown in Figure 2D. PAS staining, used to detect glycogen stores, highlighted pneumocyte immaturity and abnormal distribution of glycogen, with \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e sections showing intense magenta staining throughout the cytoplasm of the cells, unlike the WT pattern (Figure 2E). These data suggest impaired alveolarization due to decreased surfactant production, potentially leading to respiratory disease.\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eSRI\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003csup\u003e-\u003cem\u003e/-\u0026nbsp;\u003c/em\u003e\u003c/sup\u003e\u003c/strong\u003e\u003cstrong\u003emice displayed impaired surfactant production and EGFR signaling\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSurfactant secretion involves intracellular processing of SP-B and SP-C proteins from the ER and Golgi to multivesicular bodies (MVBs) and LBs. This trafficking is mediated by early endosomes, which contribute to LB formation and surfactant secretion. To further investigate Sorcin\u0026rsquo;s role in surfactant secretion, we analyzed the expression of mature SP-B and Rab5C, an isoform of the Rab5 subfamily which play a critical role in regulating early endosomes. Confocal microscopy showed reduced SP-B expression in \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e\u003cem\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice compared to WT (Figure 3A). Western blot analysis showed no significant differences of Rab5C expression at the E13.5 embryonic stage (Figure 3B). However, at 3 weeks, Rab5C expression was significantly reduced in \u003cem\u003eSRI\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice compared to WT (Figure 3C). These results provide insights into of Sorcin\u0026apos;s potential role in regulating ATII cell function, surfactant homeostasis and trafficking.\u003c/p\u003e\n\u003cp\u003eThese molecular and histological findings are consistent with those observed in \u003cem\u003eEGFR\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, which exhibit reduced tubule formation, abundant mesenchyme during branching morphogenesis, irregular alveoli with thick walls, undifferentiated type II pneumocytes with high glycogen content, and decreased surfactant protein production during alveolarization. To further explore the Sorcin (SRI)-EGFR relationship, we analyzed the expression of EGFR and of its downstream effector, RAS, both critical for lung development. Western blot analysis revealed significant reductions in EGFR and RAS protein levels in \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice compared to WT mice, at both E13.5 embryonic stage (Figure 3D) and 3 weeks (Figure 3E). These findings support Sorcin\u0026rsquo;s involvement in EGFR regulation, influencing lung function both at the physiological level (through impaired lung development) and at the pathological level[46]. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSorcin regulates cytoskeletal airway remodeling and adipose tissue infiltration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSorcin is highly expressed in vascular smooth muscle, where it modulates Ca\u0026sup2;⁺ sparks (decreasing their frequency, amplitude, duration and width) by regulating calcium concentration in the ER, mainly through SERCA activation and RYR inhibition, and therefore modulating intracellular Ca\u0026sup2;⁺ levels, which are essential for muscle airway contraction [38]. Given the observed increase in bronchiole thickness in \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u0026nbsp;\u003c/em\u003emice, we analyzed the cytoskeletal remodeling using phalloidin immunofluorescence. Confocal analysis revealed a significant increase in f-actin expression in \u003cem\u003eSRI\u003csup\u003e-/-\u003c/sup\u003e\u003c/em\u003e mice, suggesting an increased cellular airway smooth muscle (ASM) volume compared to WT mice. Notably, these morphological changes were evident as early as three weeks of age and persisted at three months (Figure 4A-B). Accordingly, we showed a significant increase of \u0026alpha;-SMA (alpha-smooth muscle actin) and vimentin expression in \u003cem\u003eSRI\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice compared to those WT, supporting ASM remodeling and hypercontractility (Figure 4C).\u003c/p\u003e\n\u003cp\u003eTaken together, these data suggest that calcium depletion in the ER may affect bronchial contraction, ultimately leading to lung structural and airway tissue remodeling, including an increase of ASM.\u003c/p\u003e\n\u003cp\u003eMoreover, we observed early and significant fat accumulation \u003cem\u003ein SRI\u003csup\u003e-/-\u0026nbsp;\u003c/sup\u003e\u003c/em\u003emice at three months of age, compared to WT (Figure 4D). Adipose tissue infiltration within the airway epithelium has been reported in obesity-related clinical cases and asthma models, both of which are associated with airway wall thickening and inflammation. These findings suggested that Sorcin deficiency contributes to both cytoskeletal and airway tissue remodeling due to mechanostress events, leading to significant lung structural alterations.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur previous work demonstrated that Sorcin regulates EGFR signaling in non-small cell lung adenocarcinoma, promoting cellular invasion and migration. We discovered important relationships between calcium homeostasis and EGFR signaling pathways and identified Sorcin as a key player of EGFR physiological and pathological roles, linked to Ca2\u0026thinsp;+\u0026thinsp;dysregulation [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond its role in tumorigenesis, EGFR is crucial for lung development, particularly branching morphogenesis and alveolarization. Branching morphogenesis occurs during the pseudoglandular stage (E9.5-16.6 in mice, approximately 16 weeks of gestation in humans), and promotes the formation of a tree-like bronchial system through interactions between epithelial and mesenchymal cells [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e][\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e][\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Alveolarization occurs during the last stages of lung development, specifically during the alveolar stage (P5\u0026ndash;P30 in mice, around 20 week of gestation in humans, continuing postnatally), involving alveoli formation and maturation, as well as ATI and ATII epithelial cell differentiation. ATII cells play a critical role in producing, secreting, and recycling pulmonary surfactant, which is essential for normal lung function, reducing alveolar surface tension and preventing alveolar collapse.\u003c/p\u003e \u003cp\u003eHistological analysis of embryo and newborn \u003cem\u003eEGFR\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice lungs revealed reduced tubules and increased interstitial mesenchyme during embryonic development, as well as poorly formed, collapsed alveoli characterized by reduced airspace and thickened septa, compared to control mice[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e][\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. \u003cem\u003eIn vitro\u003c/em\u003e (e.g., in lung explant cultures), EGF treatment enhances branching complexity and number, indicating EGFR\u0026rsquo;s positive regulatory role in epithelial proliferation and migration [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e][\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Similarly, our histological analysis of lung tissues from WT and \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice revealed reduced bronchiole formation and increased cellular density during the embryonic stage. Consequently, the \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e adult mice displayed abnormal bronchiole maturation with increased epithelial cell layers. This atypical bronchial hyperplasia is a hallmark of inflammation and pulmonary fibrosis, contributing to respiratory distress conditions like asthma and chronic obstructive pulmonary disease (COPD)[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Furthermore, persistent hypercellularity and altered alveolar structure with thickened walls indicated impaired alveolarization in \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice.\u003c/p\u003e \u003cp\u003eTo characterize Sorcin\u0026rsquo;s role in lung development, we investigated the molecular pathway and markers involved in branching morphogenesis and alveolarization. The highly coordinated interplay of growth factors and transcription factors, such as FGF10[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e][\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e][\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] and SOX9 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], regulates cellular proliferation within tubules, new bud formation, and branching morphogenesis. The surfactant genes \u003cem\u003eSP-B\u003c/em\u003e and \u003cem\u003eSP-C\u003c/em\u003e encode components of pulmonary surfactant, produced by ATII cells, essential for postnatal alveolar function. Consistently, \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice showed significantly reduced FGF10 and SOX9 levels during embryonic development (E13.5) compared to controls, suggesting impaired branching morphogenesis. As expected, SP-B and SP-C expression was not altered during the embryonic period, given that ATII differentiation occurs later, during the last step of lung development. Since FGF10 also drives normal and physiological formation of alveoli [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] and the pool of SOX9\u003csup\u003e+\u003c/sup\u003e progenitors represents the precursor of ATI and ATII cells [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], their reduction suggested alterations in mature lung structures as well. At 3 weeks, \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice exhibited significant decreases in SP-B, SP-C, and ABCA3 (the lipid surfactant transporter in LBs), indicating defective alveolarization, including surfactant trafficking and secretion. These molecular results were consistent with the observed lower lipid surfactant accumulation in \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice, demonstrating impaired secretory pathways and ATII function. Increased glycogen deposits within ATII cells indicated cell immaturity or damage.\u003c/p\u003e \u003cp\u003eATII cells play a critical role in producing and secreting pulmonary surfactant via LB fusion. The surfactant secretion pathway in ATII cells involves the translocation of the precursor proteins pro-SP-B and pro-SP-C from the ER to the Golgi apparatus, followed by their trafficking to small vesicles and MVBs. Within these compartments, SP-B and SP-C undergo maturation before fusing with LBs. The early endosome pathway, regulated by RAB proteins (including the RAB5 family isoforms RAB5a, RAB5b, and RAB5c), sorts these precursors, directing them to the appropriate intracellular compartments for further processing, including small vesicles and MVBs. Huang et al. showed that a negative variant of RAB5c led to altered early endosome that failed the fusion with proSP-B- or proSP-C-containing nascent sorting vesicles, impairing surfactant protein processing and trafficking, and causing interstitial lung disease [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Consistent with this, we observed reduced SP-B and RAB5c protein levels in 3-week-old \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice. Disruption in early endosomal trafficking can impair proper SP-B delivery and processing, potentially leading to surfactant deficiencies and respiratory dysfunction, highlighting the critical role of Sorcin in surfactant secretion.\u003c/p\u003e \u003cp\u003eAs noted, impaired EGFR signaling is associated with branching morphogenesis and alveolarization defects. Accordingly, both E13.5 and 3-week-old \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice showed decreased EGFR and impairment of its downstream pathway, including RAS protein, compared to WT. These data reinforce the relationship between Sorcin and the EGFR signaling pathway previously demonstrated in our lung adenocarcinoma \u003cem\u003ein vitro\u003c/em\u003e study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Calcium oscillations between ER and cytosol are essential for muscle contraction [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In airway smooth muscle (ASM), force generation begins with the myosin light chain kinase (MLCK) phosphorylation and spreads via actin filament polymerization. Alterations in the contractile apparatus and mechanotransduction pathways can lead to asthma, a chronic inflammatory disease of the airways [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Increased cytosolic Ca\u0026sup2;⁺ activates MLCK, leading to ASM contraction and airway narrowing [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Many experimental asthma mouse models show airway remodeling and hyperresponsiveness, including increased ASM contractility due to actin cytoskeletal remodeling and cell proliferation [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Mahan et al. demonstrated that SERCA2 downregulation leads to airway remodeling, highlighting the importance of calcium homeostasis - including the calcium release from ER- for normal lung function. ASM of asthmatics patients showed reduced SERCA2 expression [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Accordingly, 3-week and 3-month-old \u003cem\u003eSRI⁻\u003c/em\u003e\u003csup\u003e\u003cem\u003e/\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e⁻\u003c/em\u003e mice showed increased f-actin filaments and α-smooth muscle actin expression around the peribronchial region, suggesting cytoskeletal airway remodeling and excessive bronchial contraction, likely due to altered intracellular calcium homeostasis.\u003c/p\u003e \u003cp\u003eAsthma symptoms worsen in obese patients, which often exhibit increased airway-associated adipose tissue [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e][\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. This fat accumulation may contribute to increased bronchial wall thickness and inflammation [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], potentially explaining the high adipose tissue infiltration observed during lung development in our \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study provides novel insights into Sorcin\u0026rsquo;s role in fetal lung development, demonstrating its impact on branching morphogenesis and alveolarization, including surfactant production and secretion. Additionally, altered intracellular calcium levels may directly affect EGFR signaling and bronchial contractility, leading to airway cytoskeletal remodeling and adipose tissue accumulation, which can exacerbate airway obstruction. These combined changes may contribute to respiratory disorder development.\u003c/p\u003e \u003cp\u003eThis study also reveals novel key cell signaling mechanisms regulating fetal lung development, providing a basis for innovative strategies to enhance lung maturation in clinical conditions where development is compromised. Additionally, it addresses critical challenges by identifying approaches to prevent surfactant-related disorders, such as respiratory distress syndrome, ultimately contributing to improved therapeutic outcomes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABCA3: ATP-binding cassette sub-family A member 3\u003c/p\u003e\n\u003cp\u003e\u0026alpha;-SMA: alpha-smooth muscle actin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eASM: airway smooth muscle\u003c/p\u003e\n\u003cp\u003eATI: alveolar type I\u003c/p\u003e\n\u003cp\u003eATII: alveolar type II\u003c/p\u003e\n\u003cp\u003eCOPD: chronic obstructive pulmonary disease.\u003c/p\u003e\n\u003cp\u003eER: Endoplasmic Reticulum\u003c/p\u003e\n\u003cp\u003eEGFR: Epidermal growth factor receptor\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFFPE: Formalin-fixed paraffin-embedded\u003c/p\u003e\n\u003cp\u003eKO: Knockout\u003c/p\u003e\n\u003cp\u003eLBs: \u0026nbsp;Lamellar bodies\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLVCC: L-type voltage-gated calcium channel\u003c/p\u003e\n\u003cp\u003eMLCK: Myosin light chain kinase\u003c/p\u003e\n\u003cp\u003eMVBs: Multivesicular bodies\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNCX: Na+-Ca2+ exchanger\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNSCLC: Non-small-cell lung carcinoma\u003c/p\u003e\n\u003cp\u003ePAS: Periodic acid-Schiff\u003c/p\u003e\n\u003cp\u003ePMCA: Plasma membrane Ca2+ pump\u003c/p\u003e\n\u003cp\u003eRDS: Respiratory distress syndrome\u003c/p\u003e\n\u003cp\u003eRYRs: Ryanodine receptors\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSERCA: Sarco/endoplasmic reticulum Ca2+ pump\u003c/p\u003e\n\u003cp\u003eSP-B: Surfactant protein B\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSP-C: Surfactant protein C\u003c/p\u003e\n\u003cp\u003eSRI: Sorcin\u003c/p\u003e\n\u003cp\u003eTTF-1: Thyroid transcription factor-1\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCT, LDA, AI, APA, AB, GM, performed the experiments and contributed to data analysis. CT, SM, GC and FF contributed to the experiment designs and wrote the manuscript. CT, GC, FF, and VP conceived or designed the experiments. LT, ML and AI contributed to the discussion and data analysis. All authors read and approved the final manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research leading to these results received funding from: PRIN_2022 Prot. 2022WB59LB - PRIN_2022PNRR Prot. P2022C948R - European Union - Next Generation EU, Mission 4, Component 2, CUP B93D21010860004 - CN 00000041 to FF; CNCCS s.c.a.r.l. (National Collection of Chemical Compounds and Screening Center, www.cnccs.it); \u0026ldquo;Potentiating the Italian Capacity for Structural Biology Services in Instruct-ERIC\u0026rdquo;, acronym \u0026ldquo;ITACA.SB\u0026rdquo; (Project No. IR0000009, CUP B53C22001790006), funded by the European Union\u0026rsquo;s NextGenerationEU under the MUR call 3264/2021 PNRR M4/C2/L3.1.1; PNRR PE8 Age-IT., co-funding from Next Generation EU [DM 1557 11.10.2022], in the context of the National Recovery and Resilience Plan, Investment PE8 \u0026ndash; Project Age-It: \u0026ldquo;Ageing Well in an Ageing Society\u0026rdquo;; Project PRIN 2022 MUR 2022HYF8KS to GC.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reason- able request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll procedures were approved by the Italian Ministry for Health and were conducted in accordance with the US National Institutes of Health (NIH) guidelines (Approval number: 605/2023-PR). Written informed consent was not applicable because this study was conducted on mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI, the undersigned, give my consent for the publication.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge H\u0026egrave;ctor H. Valdivia and Carmen R. Valdivia (Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI 48109, USA) for providing \u003cem\u003eSRI⁻\u003csup\u003e/\u003c/sup\u003e⁻\u003c/em\u003e mice.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNogee LM, Dunbar AE, Wert SE, et al (2001) A mutation in the surfactant protein C gene associated with familial interstitial lung disease. 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Eur Respir J 15:600\u0026ndash;16. https://doi.org/10.1034/j.1399-3003.2000.15.29.x\u003c/li\u003e\n\u003cli\u003eYan F, Gao H, Zhao H, et al (2018) Roles of airway smooth muscle dysfunction in chronic obstructive pulmonary disease. J Transl Med 16:262. https://doi.org/10.1186/s12967-018-1635-z\u003c/li\u003e\n\u003cli\u003eTang DD (2015) Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling. Respir Res 16:134. https://doi.org/10.1186/s12931-015-0296-1\u003c/li\u003e\n\u003cli\u003eMahn K, Hirst SJ, Ying S, et al (2009) Diminished sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) expression contributes to airway remodelling in bronchial asthma. Proc Natl Acad Sci U S A 106:10775\u0026ndash;80. https://doi.org/10.1073/pnas.0902295106\u003c/li\u003e\n\u003cli\u003eWang CJ, Smith JT, Lu D, et al (2023) Airway-associated adipose tissue accumulation is increased in a kisspeptin receptor knockout mouse model. Clin Sci (Lond) 137:1547\u0026ndash;1562. https://doi.org/10.1042/CS20230792\u003c/li\u003e\n\u003cli\u003eMiethe S, Karsonova A, Karaulov A, Renz H (2020) Obesity and asthma. J Allergy Clin Immunol 146:685\u0026ndash;693. https://doi.org/10.1016/j.jaci.2020.08.011\u003c/li\u003e\n\u003cli\u003eElliot JG, Donovan GM, Wang KCW, et al (2019) Fatty airways: implications for obstructive disease. Eur Respir J 54:1900857. https://doi.org/10.1183/13993003.00857-2019\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":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"lung development, Sorcin KO, alveolarization, branching morphogenesis, airway remodeling","lastPublishedDoi":"10.21203/rs.3.rs-6297074/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6297074/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSorcin, a key calcium-sensing protein, regulates calcium concentration within the endoplasmic reticulum (ER), promoting apoptosis resistance and ER stress. It also modulates downstream signaling pathways of the epidermal growth factor receptor (EGFR), influencing cellular migration and invasion in non-small-cell lung carcinoma (NSCLC) cell lines. For this purpose, this study investigates the relationship between Sorcin and EGFR expression during lung development at the physiological level.\u003c/p\u003e \u003cp\u003eOur study was conducted on WT and Sorcin Knock-out (\u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u0026minus;/\u0026minus;\u003c/em\u003e\u003c/sup\u003e) mice, where we performed various analyses, including histological examination, gene and protein expression analysis, and confocal microscopy. Our findings reveal that \u003cem\u003eSRI\u003c/em\u003e\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice, compared to wild-type controls, exhibit: 1) impaired alveolarization and abnormal development of bronchi and bronchioles, as observed in histological sections; 2) decreased expression of genes encoding branching morphogenesis markers (e.g., FGF10) and surfactant proteins (e.g., SP-B, SP-C and ABCA3), as shown by real-time PCR; 3) increased glycogen content decreased lipid droplets, indicative of type II pneumocyte immaturity and impaired surfactant lipid production; 4) reduced levels of EGFR, RAS and RAB5C proteins, consistent with defects in lung maturation and surfactant protein recycling, as demonstrated by Western blot analysis; and 5) increased expression of phalloidin, α-smooth muscle actin and vimentin, suggesting increased bronchial thickening associated with airway tissue remodeling.\u003c/p\u003e \u003cp\u003eCollectively, these data reveal a novel role for Sorcin in lung alveolarization, pulmonary surfactant production, and airway remodeling associated with bronchial contractility, supporting its involvement in respiratory diseases such as respiratory distress syndrome (RDS), asthma and chronic obstructive pulmonary disease (COPD).\u003c/p\u003e","manuscriptTitle":"Sorcin regulates alveolarization and airway tissue remodeling during lung morphogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-15 08:51:11","doi":"10.21203/rs.3.rs-6297074/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2025-04-29T04:28:27+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-04-05T04:11:20+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-27T11:31:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-27T03:41:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-03-24T12:00:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cellular-and-molecular-life-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"life","sideBox":"Learn more about [Cellular and Molecular Life Sciences](https://link.springer.com/journal/18)","snPcode":"18","submissionUrl":"https://www.editorialmanager.com/life/default2.aspx","title":"Cellular and Molecular Life Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d3f7bc79-9e13-45ec-912b-a37005823c9d","owner":[],"postedDate":"April 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-03T16:08:52+00:00","versionOfRecord":{"articleIdentity":"rs-6297074","link":"https://doi.org/10.1007/s00018-025-05870-y","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-10-28 15:57:49","publishedOnDateReadable":"October 28th, 2025"},"versionCreatedAt":"2025-04-15 08:51:11","video":"","vorDoi":"10.1007/s00018-025-05870-y","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05870-y","workflowStages":[]},"version":"v1","identity":"rs-6297074","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6297074","identity":"rs-6297074","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}