Piezo1 selectively enhances TGF-β1-induced IgA class switching by B cells

Piezo1 selectively enhances TGF-β1-induced IgA class switching by B cells | 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 Piezo1 selectively enhances TGF-β1-induced IgA class switching by B cells Yoonji Jung, Younghwan Han, Jaeku Kang, Seong-Lan Yu, Seok-Rae Park This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5552251/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted 5 You are reading this latest preprint version Abstract Piezo1 is a mechanosensitive cationic channel that regulates Ca 2+ influx, gene transcription, and cell migration. Recent studies suggest that Piezo1 affects regulatory T cells differentiation and is critical in B cell responses to membrane-presented antigens. However, the role of Piezo1 in B cells function is not completely elucidated. This study investigated the role of Piezo1 in IgA class switching and Ab production by mouse B cells using qRT-PCR, flow cytometric analysis, and isotype-specific ELISA. The Piezo1 agonist Yoda1 selectively upregulated TGF-β1-induced germline α transcripts (GLTα) /post-switch α transcripts (GLTα) expression, surface IgA expression, and IgA production. Conversely, the Piezo1 inhibitor OB-1 reduced IgA class switching. TGF-β1-induced IgA class switching and IgA production decreased in Piezo1 knockdown B cells. Additionally, Piezo1 enhanced TGF-β1-induced Smad3 phosphorylation. These results demonstrate that Piezo1 selectively enhances TGF-β1-induced IgA class switching via Smad3 phosphorylation, leading to IgA production in B cells. B cells Piezo1 IgA Class switching Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Piezo1 is a mechanosensitive cation channel expressed in several tissues. Since its discovery in 2010, it is known that this channel is activated by mechanical pressure on the cell membrane [ 1 ]. It can detect mechanical forces on its own and regulate the influx of various ions, such as Ca 2+ [ 2 , 3 ], to control biological activities, including lymphatic valve formation [ 4 ] and blood pressure [ 5 ]. In addition, Piezo1 upregulates cartilage degradation and inflammation in temporomandibular joint osteoarthritis through Smad3 phosphorylation [ 6 ], recruits Rab5c, and activates TGF-β signaling to promote hepatocellular carcinoma progression and epithelial-mesenchymal transition [ 7 ]. Recent studies have shown that Piezo1 contributes to several immune cell responses. In macrophages, Piezo1 can affect macrophage activation and IL-4/STAT6 and IFN-γ/NF-κB pathway to reduce wound healing and increase inflammation [ 8 ]. Piezo1-specific deletion in macrophages reduces mouse liver fibrosis by modulating several factors such as cathepsin S and MHC class II [ 9 ]. Piezo1 also enhances T cell activation by increasing T cell receptor signaling through actin rearrangement [ 10 ]. In a multiple sclerosis mouse model, Piezo1 influences regulatory T cell differentiation by regulating TGF-β signaling [ 11 ]. Furthermore, Piezo1 is critical for the response of B cells to membrane-presented antigens [ 12 ]. IgG-, IgA-, and IgE-producing cells are generated via Ig class switch recombination after the activation of B cells expressing IgM and IgD. B cell activation is initiated when CD40L secreted from activated T cells binds to CD40 on B cells or stimuli such as LPS stimulate B cells [ 13 ]. Initial Ab genes contain a gene producing µ immediately downstream of the V(D)J region, leading to IgM production. Cytokines such as IL-4, TGF-β1, and IFN-γ induce the expression of germline transcripts (GLTs), leading to the production of various types of Abs. IL-4 induces GLTε and GLTγ1 via STAT6, TGF-β1 induces GLTα and GLTγ2b via Smad3, and IFN-γ induces GLTγ2a (or GLTγ2c) and GLTγ3 in mouse B cells [ 14 ]. Cytokine stimulation induces activation-induced cytidine deaminase to convert cytidine in the switch region to uracil, ultimately leading to a double-strand break [ 15 ]. After class switching is completed, the GLTµ promoter binds to each constant region gene and continues to be active, generating post-switch transcripts (PSTs) [ 16 ]. Thus, GLTs and PSTs can be used as indicators of class switching. Several studies have indicated that Piezo1 is associated with immune cells. However, the role of Piezo1 in Ig class switching and Ab production is yet to be elucidated. In this study examined whether Piezo1 is involved in Ig class switching and Ab production using mouse B cells treated with the Piezo1 agonist Yoda1 [ 3 ], Piezo1 inhibitor OB-1 [ 17 ], and Piezo1 small interfering RNA (siRNA). Results suggested that Piezo1 selectively enhances TGF-β1-induced IgA class switching in B cells. Materials and Methods Animals C57BL/6 mice were purchased from DBL (Chungbuk, Republic of Korea) and maintained on an 8:16 h light/dark cycle in an animal environmental control chamber; 8- to 12-week-old mice were used. Cell culture and reagents Mouse splenic resting B cells were purified as described previously [ 18 ]. After staining resting B cells with anti-B220-PE (BD Biosciences, San Jose, CA, USA) and anti-CD43-FITC (eBioscience, San Jose, CA, USA), the purity of resting B cells (CD43 − B220 + ; ≥95%) was assessed by CytoFLEX (Beckman Coulter, Pasadena, CA, USA). The mouse B cell line CH12F3-2A (mouse mature B cell line; IgM + IgD + ) [ 19 ] was provided by Dr. T. Honjo (Kyoto University, Kyoto, Japan). Cells were cultured at 37°C in a humidified CO 2 incubator (Forma Scientific, Marietta, OH, USA) in RPMI-1640 medium (WelGENE) supplemented with 50 µM 2-merchaptoethanol, 10% fetal bovine serum (FBS; WelGENE), and 1% Antibiotic-Antimycotic (Gibco, Grand Island, NY, USA). Cells were treated with LPS (Sigma-Aldrich, St. Louis, MO, USA), rhTGF-β1 (R&D Systems, Minneapolis, MN, USA), rmIL-4 (Miltenyi Biotec), rmIFN-γ (Miltenyi Biotec) with the Piezo1 agonist Yoda1 (MCE, Monmouth Junction, NJ, USA) or the inhibitor OB-1 (Tocris, Bristol, UK) according to each purpose. Yoda1 is a small chemical molecule that lowers the mechanical threshold for activating the Piezo1 channel [ 20 ]. Conversely, OB-1 is a well-known Piezo1 inhibitor that blocks the gating properties of Piezo1 and prevents its activity [ 17 ]. In this study, we used concentrations of 0.5 µM for Yoda1 and 0.2 µM for OB-1, considering cell viability (Supplementary Fig. 1). LY2109761 (MCE), a selective inhibitor of TGF-β type I/II receptors [ 21 ], was used to block TGF-β receptor signaling. BAPTA (MCE), a calcium chelator [ 22 ], was used to inhibit Ca²⁺ signaling. Cell viability assay Cell viability was determined using the EZ-Cytox cell viability assay (DaeilLab Service Co., Ltd., Seoul, Korea) according to the manufacturer’s instructions [ 23 ]. Transfection of CH12F3-2A cells with siRNA ON-TARGETplus mouse Piezo1 siRNA-SMARTpool (L-061455-00-0005; Horizon Discovery, Cambridge, UK) and ON-TARGETplus Non-targeting Pool (D-001810-10-05; Horizon Discovery) were used as knock down mouse Piezo1 and control, respectively. A total of 1 × 10 7 cells for CH12F3-2A were suspended in 1 ml RPMI-1640 medium (serum and antibiotics free). The final volume of the mixture was adjusted to 500 µL in a cuvette (gap: 0.4 cm). Cells were exposed to a single pulse at 950 µF and 300 V using a Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA). After transfection, cells were rested at room temperature for 15 min and diluted into the culture medium (RPMI-1640 medium), including 10% FBS and LPS. Cells were incubated at 37°C in a CO 2 incubator. RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) RNA isolation and RT-PCR were performed as described previously [ 23 ]. PCR primers (Supplementary Table 1) were synthesized by Bioneer (Daejeon, Korea). PCR for β-actin was performed in parallel to normalize cDNA concentrations within each set of samples. Flow cytometric analysis Surface Ig staining was performed using anti-mouse IgA-FITC, anti-mouse IgG2b-FITC, anti-mouse IgG1-FITC, anti-mouse IgE-FITC, anti-mouse IgG2c-FITC, or anti-mouse IgG3-FITC along with anti-mouse IgM-PE (eBioscience) in the dark for 30 min at 4°C. The stained cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter). Isotype-specific enzyme-linked immunosorbent analysis (ELISA) Antibodies produced in B cell cultures were detected using isotype-specific ELISA, as described previously. The colorimetric reactions were measured at 450 nm with an absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) [ 23 ]. Western blot analysis Cell lysates were prepared in lysis buffer (radioimmunoprecipitation assay buffer; [iNtRON Biotechnology, Seongnam, Korea]) with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich) mixture. Proteins were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Dogen, Seoul, Korea). The membranes were incubated with a specific primary antibody [phospho-Smad3 rabbit mAb, Smad3 rabbit mAb (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-Rab5 mAb (Abcam, Cambridge, UK), anti-β-actin mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. The membranes probed with horseradish peroxidase (HRP)-conjugated secondary antibodies [donkey anti-rabbit IgG-HRP secondary Ab (SouthernBiotech, Birmingham, AL), mouse IgG1κ Ab (Santa Cruz Biotechnology) detected with Amersham Biosciences ECL Prime Western Blotting Detection Reagent (Amersham Biosciences, Roosendaal, The Netherlands)]. Intracellular Ca 2+ influx measurements CH12F3-2A and purified mouse splenic B cells were pretreated with the Zombie Red Fixable Viability Kit (Biolegend, San Diego, CA, USA) and 1 mM EDTA solution in calcium-free Hanks’ balanced salt solution. Cells were incubated for 30 min at 37°C in the dark with 3 µM Fluo-4 AM (Invitrogen, Waltham, MA, USA). The samples were analyzed using flow cytometry (CytoFLEX, Beckman Coulter). CFSE-based B cell proliferation assay B cell proliferation was assessed using the CellTrace™ CFSE Cell Proliferation Kit (eBioscience) according to the manufacturer’s instructions. Splenic B cells were washed with serum-free Hank’s Balanced Salt Solution (Invitrogen) and resuspended at a density of 1×10⁶ cells/ml. An equal volume of CFSE (carboxyfluorescein succinimidyl ester) was added to achieve a final concentration of 5 µM, and the cells were incubated for 20 min at 37°C in the dark. The labeling reaction was quenched by washing the cells with RPMI 1640 medium supplemented with 10% FBS. Labeled cells were subsequently diluted in complete RPMI medium and cultured in the presence or absence of LPS, rhTGF-β1, and Yoda1 to investigate their roles in B cell proliferation and IgA class switching. Cells were harvested at the indicated time points and analyzed by flow cytometry to assess CFSE dilution as a measure of cell proliferation. Statistical analysis Statistical differences between experimental groups were determined by analysis of variance, and the p-values were calculated using unpaired two-tailed Student’s t-tests to consider statistical significance. Results Effects of the Piezo1 agonist and Piezo1 inhibitor on Ig class switching and Ab production by B cells First, this study examined whether Piezo1 is expressed in mouse B cells. Piezo1 expression was confirmed in CH12F3-2A and splenic B cells (Supplementary Fig. 2). The mouse macrophage cell line RAW 264.7 was used as a positive control. To evaluate the effect of Piezo1 on Ig class switching by B cells, the Piezo1 agonist Yoda1 was used. Yoda1 enhanced Ca 2+ influx in B cells (Supplementary Fig. 3). GLTs and PSTs expressions were analyzed in cytokine-stimulated cells. Yoda1 upregulated only TGF-β1-induced GLTα expression, whereas TGF-β1-induced GLTγ2b and other cytokine-stimulated GLTs (GLTε, GLTγ1, GLTγ2c, and GLTγ3) did not change significantly (Fig. 1 A). Moreover, TGF-β1-induced PSTα expression was selectively upregulated in B cells treated with Yoda1. Next, flow cytometry and ELISA were used to evaluate the surface Igs expression and Igs production. Yoda1 enhanced surface IgA-expressing cells and IgA secretion (Fig. 1 B and C). Similarly, levels of other Igs did not change. To investigate whether Piezo1-mediated, TGF-β1-induced IgA class switching is linked to B cell proliferation, we performed a CFSE-based cell proliferation assay. TGF-β1 significantly inhibited LPS-induced B cell proliferation. When B cells were additionally treated with Yoda1 under LPS/TGF-β1 stimulation, their proliferation was slightly reduced; however, this reduction was not statistically significant (Supplementary Fig. 4A). These results indicate that the enhancement of TGF-β1-induced IgA class switching by Yoda1 is not due to changes in B cell proliferation. In addition, Yoda1 increased TGF-β1-induced surface IgA expression at each B cell division (Supplementary Fig. 4B). Following the experiments with Yoda1, the Piezo1 inhibitor OB-1 was utilized to further investigate the role of Piezo1 on Ig class switching. OB-1 selectively downregulated TGF-β1-induced GLT/PSTα expression (Fig. 2 A). OB-1 also selectively reduced surface IgA expression and IgA production in contrast to the effect of Yoda1 (Fig. 2 B and C). Furthermore, this study investigated the effect of Piezo1 on TGF-β1-induced IgA class switching in CH12F3-2A, a mouse B cell line characterized by IgA class switching upon stimulation with TGF-β1 [ 19 ]. Yoda1 enhanced TGF-β1-induced GLTα expression and PSTα expression (Fig. 3 A). Flow cytometry and ELISA demonstrated that Yoda1 increased surface IgA expression and IgA production (Fig. 3 B and C). In contrast, OB-1 selectively downregulated TGF-β1-induced GLTα and PSTα expression, surface IgA expression, and IgA production (Fig. 3 D–F). These results suggest that Piezo1 selectively upregulates IgA class switching and IgA production without affecting other Igs class switching and production. To explore the physiological relevance of these findings in vivo, we examined the expression of Piezo1 in Peyer’s patches (PP), the major site for IgA class switching. We compared Piezo1 expression levels in B cells from the spleen and PP using qRT-PCR and flow cytometry. PP B cells exhibited higher Piezo1 mRNA expression than unstimulated splenic B cells (Supplementary Fig. 5A). Consistently, the Piezo1 protein level was also elevated in PP B cells compared to unstimulated splenic B cells (Supplementary Fig. 5B). Piezo1 knockdown downregulates TGF-β1-induced IgA class switching To elucidate whether upregulation of TGF-β1-induced IgA class switching is directly mediated by Piezo1, siRNA-mediated knockdown of Piezo1 was performed in CH12F3-2A cells. This study examined how TGF-β1-induced GLTα and PSTα changed. Piezo1 siRNA effectively reduced Piezo1 mRNA and protein expression (Supplementary Fig. 6). Piezo1 knockdown significantly decreased GLTα and PSTα expression (Fig. 4 A). In addition, surface IgA expression and IgA production were diminished (Fig. 4 B and C), demonstrating that Piezo1 enhances TGF-β1-induced IgA switching and IgA production. Piezo1 enhances TGF-β1-induced Smad3 phosphorylation via the TGF-β receptor by upregulating Rab5c expression In TGF-β1-induced IgA class switching, Smad3 is a critical signal transducer and an essential transcription factor. When stimulated by TGF-β1, Smad3 is phosphorylated by activated TGF-β1 receptors, and phosphorylated Smad3 activates GLTα transcription [ 24 , 25 ]. Therefore, this study examined the levels of phosphorylated Smad3 after Yoda1/OB-1 treatment and Piezo1 knockdown. Yoda1 upregulated TGF-β1-induced Smad3 phosphorylation in mouse splenic B cell and CH12F3-2A cells (Fig. 5 A and B). Conversely, OB-1 decreased Smad3 phosphorylation (Fig. 5 C and D). Moreover, Piezo1 knockdown downregulated TGF-β1-induced Smad3 phosphorylation (Fig. 5 E), indicating that Piezo1 upregulates Smad3 phosphorylation to enhance TGF-β1-induced IgA class switching. To further elucidate the mechanism of Piezo1-mediated, TGF-β1-induced IgA class switching, we assessed the expression of TGF-β1-induced GLTα/PSTα following treatment with the TGF-β receptor inhibitor LY2109761, in the presence or absence of Yoda1. LY2109761 decreased Yoda1-mediated, TGF-β1-induced GLTα/PSTα expression (Fig. 6 A). This result suggests that Piezo1 upregulates TGF-β1-induced IgA class switching via TGF-β receptor. Previous studies have reported that Piezo1 activates TGF-β signaling through the recruitment of Rab5c in hepatocellular carcinoma [ 7 ]. Rab5c is a key regulator of early endosome trafficking and has also been implicated in enhancing TGF-β receptor-mediated signaling pathway [ 26 ]. Therefore, we examined whether Piezo1-mediated, TGF-β1-induced IgA class switching is related to Rab5c. Yoda1 increased the expression of Rab5c, whereas Piezo1 knockdown reduced Rab5c expression (Fig. 6 B). In this context, Yoda1 enhanced TGF-β1-induced GLTα/PSTα expression, whereas Piezo1 knockdown diminished both TGF-β1-induced GLTα/PSTα expression and the enhancing effect of Yoda1. Taken together, these findings suggest that Piezo1 enhances TGF-β1-induced Smad3 phosphorylation via the TGF-β receptor by upregulating Rab5c expression, thereby facilitating IgA class switching in B cells. Discussion Piezo1 is a channel that has been studied in various cell types and has shown to significantly influence their functions [ 8 , 27 ]. Thus, Piezo1 may also influence B cells and cytokines. This study investigated the relationship between Piezo1 and Ig class switching and Ab production in B cells. According to the results, Piezo1 substantially increased TGF-β1-induced IgA class switching and IgA production by increasing TGF-β1-induced Smad3 phosphorylation without significant effects on other isotypes. Piezo1 may affected the TGF-β1 receptor. As noted earlier, Piezo1 has been reported to activate TGF-β signaling through the recruitment of Rab5c, a member of the Rab5 subfamily, in hepatocellular carcinoma [ 7 ]. Rab5 is a small GTPase that regulates the early stages of clathrin-mediated endosome formation [ 28 – 30 ]. Clathrin-mediated endosomes internalize the TGF-β receptor, a process required for receptor-mediated Smad2/3 phosphorylation. Thus, Rab5c-mediated endocytosis facilitates Smad activation and downstream transcriptional responses via TGF-β signaling [ 26 , 31 ]. Therefore, TGF-β1-induced IgA class switching involving Piezo1 may be enhanced by promoting TGF-b receptor internalization through Rab5c. Our experimental data partially support this possibility, as we observed increased Rab5c expression and Smad3 phosphorylation following Piezo1 activation. Nevertheless, additional studies are needed to better understand the underlying mechanisms. Calmodulin-dependent protein kinases (CaMKs) can affect TGF-β1-induced IgA class switching. CaMK is a family of protein kinases activated in response to calcium signals [ 32 ]. CaMK is expressed in B cells, but its role is unknown. Some studies showed that CaMKs can promote TGF-β signaling-induced Smad2/3 phosphorylation in pluripotent stem cell-derived chondrocytes [ 33 ]. Thus, it is possible that CaMKs are activated by Piezo1-mediated Ca²⁺ influx to upregulate Smad3 phosphorylation. We examined whether CaMK affects TGF-β1-induced IgA class switching by using the CaMK inhibitor KN-93. KN-93 did not affect TGF-β1-induced GLTα/PSTα expression in B cells (data not shown). Also, to determine whether Ca²⁺ levels affect TGF-β1-induced IgA class switching, we treated B cells with the calcium chelator BAPTA. BAPTA had no effect on TGF-β1-induced GLTα/PSTα expression, either in the absence or presence of Yoda1 (Supplementary Fig. 7). These results suggest that Piezo1-mediated, TGF-β1-induced IgA class switching is independent of calcium signaling. TGF-β1 induces IgA and IgG2b class switching through Smad3 signaling in mouse B cells. However, Piezo1 did not significantly change TGF-β1-induced GLTγ2b transcription and IgG2b class switching. This may be due to TGF-β differentially regulating GLTα and GLTγ2b transcription. Previous studies indicated that Runx3 overexpression increased the activity of the GLTα promoter but had no effect on the GLTγ2b promoter [ 34 ]. Hence, Piezo1 may interact with Runx3 to influence only the GLTα promoter. To investigate whether Piezo1 selectively influences GLTα promoter through Runx3, Piezo1 knockdown and overexpression of Runx3 could be used to assess its effects on class switching. Furthermore, the transcription factor CREB/ATF cooperates with Smad3/4/Runx3 to promote GLTα transcription [ 24 , 35 ], and Piezo1 may further enhance GLTα transcription by reinforcing CREB/ATF activity. Thus, further investigation is needed to clarify how Piezo1 selectively enhances only TGF-β1-induced GLTα transcription without affecting TGF-β1-induced GLTγ2b transcription. Peyer’s patches, as the major mucosal inductive site for IgA class switching in vivo, exhibited higher Piezo1 expression than unstimulated splenic B cells. This may provide preliminary evidence supporting a potential role for Piezo1 in IgA class switching under physiological conditions. However, all data presented in this study were obtained from in vitro experiments, which may not fully reflect the in vivo environment, including cell–cell interactions, immune responses, and tissue-specific environments. Therefore, future studies using animal models will be essential to validate our findings. In conclusion, Piezo1 selectively reinforces TGF-β1-induced IgA class switching by enhancing TGF-β receptor/Smad3 signaling, thereby promoting IgA production in B cells. As a key Ab in mucosal immunity, IgA plays a central role in host defense at mucosal surfaces. Although further investigation is warranted, our findings support the potential of Piezo1 as a therapeutic target for modulating IgA responses and treating mucosal immunity-related disorders. Declarations Funding This research was funded by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), grant number NRF-2017R1A6A1A03015713. This work was also supported in part by Konyang University Myunggok Research Fund of 2023. Conflicts of Interests The authors declare that they have no conflicts of interest. Author information Authors and Affiliations Department of Microbiology, Konyang University College of Medicine, Daejeon 35365, Korea Yoonji Jung, Seok-Rae Park Priority Research Center, Myunggok Medical Research Institute, Konyang University College of Medicine, Daejeon 35365, Korea Younghwan Han, Jaeku Kang, Seong-Lan Yu, Seok-Rae Park Department of Pharmacology, Konyang University College of Medicine, Daejeon 35365, Korea Jaeku Kang Contributions Seok-Rae Park contributed to the study conceptualization. Data curation was performed by Yoonji Jung and Seok-Rae Park. Formal analysis was conducted by Yoonji Jung, Younghwan Han, and Seok-Rae Park. Funding acquisition was managed by Seok-Rae Park. Investigation was carried out by Yoonji Jung, Younghwan Han, Seong-Lan Yu, and Seok-Rae Park. Resources were provided by Seok-Rae Park and Jaeku Kang. Supervision of the study was conducted by Seok-Rae Park. The first draft of the manuscript was written by Yoonji Jung and Seok-Rae Park, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Corresponding author Correspondence to Seok-Rae Park. Data availability The data supporting the findings of this study are detailed within the article and its supplementary materials. All datasets can be obtained from the corresponding author on reasonable request. Ethics approval Animals were cared for in accordance with the institutional guidelines of the Institutional Animal Care and Use Committee of Konyang University (approval no. 23-24-A-01). 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J Biol Chem 274(44):31577–31582 Supplementary Files Piezo1IgASupplrevisedSRP.docx Cite Share Download PDF Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Cellular and Molecular Life Sciences → Version 1 posted Reviewers agreed at journal 17 Apr, 2025 Reviewers invited by journal 17 Apr, 2025 Editor assigned by journal 17 Apr, 2025 First submitted to journal 17 Apr, 2025 Editorial decision: Minor Revision 13 Jan, 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5552251","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":444405873,"identity":"4536c6a3-32fc-4262-8ccf-de53a47886f4","order_by":0,"name":"Yoonji Jung","email":"","orcid":"","institution":"Konyang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yoonji","middleName":"","lastName":"Jung","suffix":""},{"id":444405874,"identity":"12a12639-7c52-40ae-afbf-3c111f75f3cf","order_by":1,"name":"Younghwan Han","email":"","orcid":"","institution":"Konyang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Younghwan","middleName":"","lastName":"Han","suffix":""},{"id":444405875,"identity":"9d210198-50b0-489f-8a82-303cc2f450a3","order_by":2,"name":"Jaeku Kang","email":"","orcid":"","institution":"Konyang University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jaeku","middleName":"","lastName":"Kang","suffix":""},{"id":444405876,"identity":"dd6736b3-8630-446c-bfdf-2494d29f2490","order_by":3,"name":"Seong-Lan Yu","email":"","orcid":"","institution":"Myunggok Medical Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Seong-Lan","middleName":"","lastName":"Yu","suffix":""},{"id":444405877,"identity":"737a56e2-2ce9-4219-99fe-861c3cb18c95","order_by":4,"name":"Seok-Rae Park","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYNCCAzYGSDw2orSkka7lMAla5GefPfi54sx5Y37pHsPPBb8Y5Pkb2NI+4NNicC4vWfLMjdtmknPOGEvP7GMwnHGA7fAMvFp4eAwkGz7ctjG4kWMgzdvDwLiBgb0Zv8N6eIx/Nnw4B9Ji/BuoxZ6gFoYzPGaSDTcOmAG1mEnz/GBI3MDAdhivDoMzfGmWDWeSjSVnpJVZ8zZIJM84zJZMwGG8h282HLMz7JdI3nyb54+NbX97mzF+hzHwwBgcBgyMbRIMDMwENCBpYX/AwPCHoPJRMApGwSgYgQAAnHpGTSUs+xwAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1407-4495","institution":"Konyang University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Seok-Rae","middleName":"","lastName":"Park","suffix":""}],"badges":[],"createdAt":"2024-11-30 04:49:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5552251/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5552251/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00018-025-05789-4","type":"published","date":"2025-06-19T15:57:41+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80901315,"identity":"be86901e-8869-4c01-8418-79ffdfd0517a","added_by":"auto","created_at":"2025-04-18 13:30:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1118411,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Yoda1 on IgA class switching and IgA production by splenic B cells\u003c/p\u003e\n\u003cp\u003eSplenic B cells were purified from the mouse spleen and cultured with the indicated stimuli (Yoda1, 0.5 μM; TGF-β1, 0.2 ng/mL; IL-4, 0.5 ng/mL; IFN-γ, 5 ng/mL) in the presence of LPS (12.5 μg/mL) (n = 3). (A) After 2.5 days of culture, RNA was isolated. GLTs/PSTs were measured by qRT-PCR. Graphs depict the relative GLT/PST cDNA fold change normalized to β-actin expression. (B) After 4.5 days of culture, B cells were isolated, and surface Ig was measured by flow cytometry. (C) After 6.5 days of culture, supernatants were harvested, and Ig production was measured by isotype-specific ELISA. Data are the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; NS, not significant\u003c/p\u003e","description":"","filename":"OnlineFig1revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/6c3621608f46144b4a9f63e2.png"},{"id":80901314,"identity":"8ee981e8-ba96-45cc-a9d9-549721e84d95","added_by":"auto","created_at":"2025-04-18 13:30:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1051464,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of OB-1 on class switching and Ig production by splenic B cells\u003c/p\u003e\n\u003cp\u003eResting B cells were purified from the mouse spleen and cultured with the indicated stimuli (OB-1, 0.2 μM; TGF-β1, 0.2 ng/mL; IL-4, 0.5 ng/mL; IFN-γ, 5 ng/mL) in the presence of LPS (12.5 μg/mL) (n = 3). (A) After 2.5 days of culture, RNA was isolated. GLTs/PSTs were measured by qRT-PCR. Graphs show the relative GLT/PST cDNA normalized to β-actin expression. (B) After 4.5 days of culture, B cells were isolated, and surface Ig was measured by flow cytometry. (C) After 6.5 days of culture, supernatants were harvested, and Ig production was measured by isotype-specific ELISA. Data are the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; NS, not significant\u003c/p\u003e","description":"","filename":"OnlineFig2revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/9aa8570c378a426a0b476cb3.png"},{"id":80900624,"identity":"81bf02df-e275-4ac8-b2a9-ef6fcae3447b","added_by":"auto","created_at":"2025-04-18 13:22:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":660410,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Yoda1 and OB-1 on class switching and Ig production by B cell line\u003c/p\u003e\n\u003cp\u003eCH12F3-2A cells were cultured with the indicated stimuli (Yoda1, 0.5 μM; OB-1, 0.2 μM; TGF-β1, 0.5 ng/mL) in the presence of LPS (12.5 μg/mL). (A and D) After 1.5 days of culture, RNA was isolated. GLTα/PSTα were measured by qRT-PCR. Graphs show the relative GLTα/PSTα cDNA normalized to β-actin expression. (B and E) After 3 days of culture, B cells were isolated, and surface IgA/IgM was measured by flow cytometry. (C and F) After 4 days of culture, supernatants were harvested, and IgA/IgM production was measured by isotype-specific ELISA. Data are the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; NS, not significant\u003c/p\u003e","description":"","filename":"OnlineFig3revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/e8dee48060af52b9b423b634.png"},{"id":80900629,"identity":"12dd57e6-a040-47cf-b9d9-d0e4a66cd46e","added_by":"auto","created_at":"2025-04-18 13:22:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":486663,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Piezo1 knockdown on IgA class switching\u003c/p\u003e\n\u003cp\u003eCH12F3-2A was transfected with siRNA by electroporation. Cells were treated with the indicated stimuli (Yoda1, 0.5 μM; TGF-β1, 0.5 ng/mL) in the presence of LPS (12.5 μg/mL).\u003cbr\u003e\n(A) After 1.5 days of culture, RNA was isolated. GLTα/PSTα levels were measured by qRT-PCR. Graphs illustrate relative GLTα/PSTα cDNA levels normalized to the expression of β-actin. (B) After 3 days of culture, B cells were isolated, and surface IgA/IgM was measured by flow cytometry. (C) After 4 days of culture, supernatants were harvested, and IgA/IgM production was measured by isotype-specific ELISA. Data are the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001\u003c/p\u003e","description":"","filename":"OnlineFig4revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/e69d241eb9fc8f6c9d986f5d.png"},{"id":80900628,"identity":"0dbedda5-0eb1-4696-bb8a-9dfdbf37ee98","added_by":"auto","created_at":"2025-04-18 13:22:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":422542,"visible":true,"origin":"","legend":"\u003cp\u003ePiezo1 enhances TGF-β1-induced Smad3 phosphorylation\u003c/p\u003e\n\u003cp\u003eSplenic B cells were purified from the mouse spleen and cultured with the indicated stimuli [Yoda1, 0.5 µM (A); OB-1, 0.2 μM (B); TGF-β1, 0.2 ng/mL)] in the presence of LPS (12.5 µg/mL). After 24 h of culture, western blots for p-Smad3, Smad3, and β-actin were performed. CH12F3-2A was cultured (C and D) or transfected with siRNA before (E) with the indicated stimuli [Yoda1, 0.5 µM (C); OB-1, 0.2 μM (D); TGF-β1, 0.5 ng/mL) in the presence of LPS (12.5 µg/mL). After 3 h of culture, western blots for p-Smad3, Smad3, and β-actin was performed. Data are expressed as the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; NS, not significant\u003c/p\u003e","description":"","filename":"OnlineFig5revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/99cebf710e1847d43def466d.png"},{"id":80900642,"identity":"e7d962c1-0b50-4016-bab1-40fc5b614a12","added_by":"auto","created_at":"2025-04-18 13:22:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":389970,"visible":true,"origin":"","legend":"\u003cp\u003ePiezo1 may facilitate TGF-β1-induced IgA switching via TGF-b receptor signaling by upregulating Rab5c expression\u003c/p\u003e\n\u003cp\u003e(A) Resting B cells were purified from the mouse spleen and cultured with the indicated stimuli (Yoda1, 0.5 µM; TGF-β1, 0.2 ng/mL; LY2109761, 0.2 µM) in the presence of LPS (12.5 µg/mL) (n = 3). After 2.5 days of culture, RNA was isolated and GLTa/PSTa were measured by qRT-PCR. Graphs show the relative GLTa/PSTa cDNA normalized to β-actin expression. (B, upper panel) Splenic B cells were cultured with the indicated stimuli (Yoda1, 0.5 μM; TGF-β1, 0.2 ng/mL). After 24 hours, western blotting was performed for Rab5c and β-actin. After 2.5 days, RNA was isolated and GLTα/PSTα expression was analyzed by RT-PCR. (B, lower panel) CH12F3-2A was transfected with siRNA by electroporation and treated with the indicated stimuli (Yoda1, 0.5 μM; TGF-β1, 0.5 ng/mL) in the presence of LPS (12.5 μg/mL). After 3 h of culture, western blotting was performed for Rab5c and β-actin. After 1.5 days, RNA was isolated and GLTα/PSTα levels were measured by RT-PCR. Data represent the mean ± SEM from three independent experiments. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001; NS, not significant\u003c/p\u003e","description":"","filename":"OnlineFig6revised.png","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/c6a3ba2aa91a8c84eabc9aa9.png"},{"id":85231466,"identity":"f1238719-fff8-4640-83fe-f74989c167fd","added_by":"auto","created_at":"2025-06-23 16:08:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7172524,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/87defbf0-e115-43c3-a9d8-70197291459e.pdf"},{"id":80901316,"identity":"87ff89a3-0ee2-4d1b-ab7e-dfe6f40fc653","added_by":"auto","created_at":"2025-04-18 13:30:16","extension":"docx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":8213365,"visible":true,"origin":"","legend":"","description":"","filename":"Piezo1IgASupplrevisedSRP.docx","url":"https://assets-eu.researchsquare.com/files/rs-5552251/v1/1f5a201b0ffeaef5ba35b9b1.docx"}],"financialInterests":"","formattedTitle":"Piezo1 selectively enhances TGF-β1-induced IgA class switching by B cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePiezo1 is a mechanosensitive cation channel expressed in several tissues. Since its discovery in 2010, it is known that this channel is activated by mechanical pressure on the cell membrane [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It can detect mechanical forces on its own and regulate the influx of various ions, such as Ca\u003csup\u003e2+\u003c/sup\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], to control biological activities, including lymphatic valve formation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and blood pressure [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In addition, Piezo1 upregulates cartilage degradation and inflammation in temporomandibular joint osteoarthritis through Smad3 phosphorylation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], recruits Rab5c, and activates TGF-β signaling to promote hepatocellular carcinoma progression and epithelial-mesenchymal transition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies have shown that Piezo1 contributes to several immune cell responses. In macrophages, Piezo1 can affect macrophage activation and IL-4/STAT6 and IFN-γ/NF-κB pathway to reduce wound healing and increase inflammation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Piezo1-specific deletion in macrophages reduces mouse liver fibrosis by modulating several factors such as cathepsin S and MHC class II [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Piezo1 also enhances T cell activation by increasing T cell receptor signaling through actin rearrangement [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In a multiple sclerosis mouse model, Piezo1 influences regulatory T cell differentiation by regulating TGF-β signaling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Furthermore, Piezo1 is critical for the response of B cells to membrane-presented antigens [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIgG-, IgA-, and IgE-producing cells are generated via Ig class switch recombination after the activation of B cells expressing IgM and IgD. B cell activation is initiated when CD40L secreted from activated T cells binds to CD40 on B cells or stimuli such as LPS stimulate B cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Initial Ab genes contain a gene producing \u0026micro; immediately downstream of the V(D)J region, leading to IgM production. Cytokines such as IL-4, TGF-β1, and IFN-γ induce the expression of germline transcripts (GLTs), leading to the production of various types of Abs. IL-4 induces GLTε and GLTγ1 via STAT6, TGF-β1 induces GLTα and GLTγ2b via Smad3, and IFN-γ induces GLTγ2a (or GLTγ2c) and GLTγ3 in mouse B cells [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Cytokine stimulation induces activation-induced cytidine deaminase to convert cytidine in the switch region to uracil, ultimately leading to a double-strand break [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. After class switching is completed, the GLT\u0026micro; promoter binds to each constant region gene and continues to be active, generating post-switch transcripts (PSTs) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, GLTs and PSTs can be used as indicators of class switching.\u003c/p\u003e \u003cp\u003eSeveral studies have indicated that Piezo1 is associated with immune cells. However, the role of Piezo1 in Ig class switching and Ab production is yet to be elucidated. In this study examined whether Piezo1 is involved in Ig class switching and Ab production using mouse B cells treated with the Piezo1 agonist Yoda1 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], Piezo1 inhibitor OB-1 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and Piezo1 small interfering RNA (siRNA). Results suggested that Piezo1 selectively enhances TGF-β1-induced IgA class switching in B cells.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eC57BL/6 mice were purchased from DBL (Chungbuk, Republic of Korea) and maintained on an 8:16 h light/dark cycle in an animal environmental control chamber; 8- to 12-week-old mice were used.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell culture and reagents\u003c/h3\u003e\n\u003cp\u003eMouse splenic resting B cells were purified as described previously [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. After staining resting B cells with anti-B220-PE (BD Biosciences, San Jose, CA, USA) and anti-CD43-FITC (eBioscience, San Jose, CA, USA), the purity of resting B cells (CD43\u003csub\u003e\u0026minus;\u003c/sub\u003eB220\u003csup\u003e+\u003c/sup\u003e; \u0026ge;95%) was assessed by CytoFLEX (Beckman Coulter, Pasadena, CA, USA). The mouse B cell line CH12F3-2A (mouse mature B cell line; IgM\u003csup\u003e+\u003c/sup\u003eIgD\u003csup\u003e+\u003c/sup\u003e) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] was provided by Dr. T. Honjo (Kyoto University, Kyoto, Japan). Cells were cultured at 37\u0026deg;C in a humidified CO\u003csub\u003e2\u003c/sub\u003e incubator (Forma Scientific, Marietta, OH, USA) in RPMI-1640 medium (WelGENE) supplemented with 50 \u0026micro;M 2-merchaptoethanol, 10% fetal bovine serum (FBS; WelGENE), and 1% Antibiotic-Antimycotic (Gibco, Grand Island, NY, USA). Cells were treated with LPS (Sigma-Aldrich, St. Louis, MO, USA), rhTGF-β1 (R\u0026amp;D Systems, Minneapolis, MN, USA), rmIL-4 (Miltenyi Biotec), rmIFN-γ (Miltenyi Biotec) with the Piezo1 agonist Yoda1 (MCE, Monmouth Junction, NJ, USA) or the inhibitor OB-1 (Tocris, Bristol, UK) according to each purpose. Yoda1 is a small chemical molecule that lowers the mechanical threshold for activating the Piezo1 channel [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Conversely, OB-1 is a well-known Piezo1 inhibitor that blocks the gating properties of Piezo1 and prevents its activity [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In this study, we used concentrations of 0.5 \u0026micro;M for Yoda1 and 0.2 \u0026micro;M for OB-1, considering cell viability (Supplementary Fig.\u0026nbsp;1). LY2109761 (MCE), a selective inhibitor of TGF-β type I/II receptors [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], was used to block TGF-β receptor signaling. BAPTA (MCE), a calcium chelator [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], was used to inhibit Ca\u0026sup2;⁺ signaling.\u003c/p\u003e\n\u003ch3\u003eCell viability assay\u003c/h3\u003e\n\u003cp\u003eCell viability was determined using the EZ-Cytox cell viability assay (DaeilLab Service Co., Ltd., Seoul, Korea) according to the manufacturer\u0026rsquo;s instructions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eTransfection of CH12F3-2A cells with siRNA\u003c/h3\u003e\n\u003cp\u003eON-TARGETplus mouse Piezo1 siRNA-SMARTpool (L-061455-00-0005; Horizon Discovery, Cambridge, UK) and ON-TARGETplus Non-targeting Pool (D-001810-10-05; Horizon Discovery) were used as knock down mouse Piezo1 and control, respectively. A total of 1 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e cells for CH12F3-2A were suspended in 1 ml RPMI-1640 medium (serum and antibiotics free). The final volume of the mixture was adjusted to 500 \u0026micro;L in a cuvette (gap: 0.4 cm). Cells were exposed to a single pulse at 950 \u0026micro;F and 300 V using a Gene Pulser II electroporation system (Bio-Rad Laboratories, Hercules, CA, USA). After transfection, cells were rested at room temperature for 15 min and diluted into the culture medium (RPMI-1640 medium), including 10% FBS and LPS. Cells were incubated at 37\u0026deg;C in a CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003ch3\u003eRNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eRNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)\u003c/div\u003e \u003cp\u003eRNA isolation and RT-PCR were performed as described previously [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. PCR primers (Supplementary Table\u0026nbsp;1) were synthesized by Bioneer (Daejeon, Korea). PCR for β-actin was performed in parallel to normalize cDNA concentrations within each set of samples.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e \u003cp\u003eSurface Ig staining was performed using anti-mouse IgA-FITC, anti-mouse IgG2b-FITC, anti-mouse IgG1-FITC, anti-mouse IgE-FITC, anti-mouse IgG2c-FITC, or anti-mouse IgG3-FITC along with anti-mouse IgM-PE (eBioscience) in the dark for 30 min at 4\u0026deg;C. The stained cells were analyzed by flow cytometry (CytoFLEX, Beckman Coulter).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eIsotype-specific enzyme-linked immunosorbent analysis (ELISA)\u003c/h3\u003e\n\u003cp\u003eAntibodies produced in B cell cultures were detected using isotype-specific ELISA, as described previously. The colorimetric reactions were measured at 450 nm with an absorbance microplate reader (BioTek Instruments, Inc., Winooski, VT, USA) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eWestern blot analysis\u003c/h3\u003e\n\u003cp\u003eCell lysates were prepared in lysis buffer (radioimmunoprecipitation assay buffer; [iNtRON Biotechnology, Seongnam, Korea]) with protease inhibitor (Sigma-Aldrich) and phosphatase inhibitor (Sigma-Aldrich) mixture. Proteins were subjected to SDS-PAGE and transferred onto a nitrocellulose membrane (Dogen, Seoul, Korea). The membranes were incubated with a specific primary antibody [phospho-Smad3 rabbit mAb, Smad3 rabbit mAb (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-Rab5 mAb (Abcam, Cambridge, UK), anti-β-actin mAb (Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. The membranes probed with horseradish peroxidase (HRP)-conjugated secondary antibodies [donkey anti-rabbit IgG-HRP secondary Ab (SouthernBiotech, Birmingham, AL), mouse IgG1κ Ab (Santa Cruz Biotechnology) detected with Amersham Biosciences ECL Prime Western Blotting Detection Reagent (Amersham Biosciences, Roosendaal, The Netherlands)].\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular Ca\u003csup\u003e2+\u003c/sup\u003e influx measurements\u003c/h2\u003e \u003cp\u003eCH12F3-2A and purified mouse splenic B cells were pretreated with the Zombie Red Fixable Viability Kit (Biolegend, San Diego, CA, USA) and 1 mM EDTA solution in calcium-free Hanks\u0026rsquo; balanced salt solution. Cells were incubated for 30 min at 37\u0026deg;C in the dark with 3 \u0026micro;M Fluo-4 AM (Invitrogen, Waltham, MA, USA). The samples were analyzed using flow cytometry (CytoFLEX, Beckman Coulter).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eCFSE-based B cell proliferation assay\u003c/h2\u003e \u003cp\u003eB cell proliferation was assessed using the CellTrace\u0026trade; CFSE Cell Proliferation Kit (eBioscience) according to the manufacturer\u0026rsquo;s instructions. Splenic B cells were washed with serum-free Hank\u0026rsquo;s Balanced Salt Solution (Invitrogen) and resuspended at a density of 1\u0026times;10⁶ cells/ml. An equal volume of CFSE (carboxyfluorescein succinimidyl ester) was added to achieve a final concentration of 5 \u0026micro;M, and the cells were incubated for 20 min at 37\u0026deg;C in the dark. The labeling reaction was quenched by washing the cells with RPMI 1640 medium supplemented with 10% FBS. Labeled cells were subsequently diluted in complete RPMI medium and cultured in the presence or absence of LPS, rhTGF-β1, and Yoda1 to investigate their roles in B cell proliferation and IgA class switching. Cells were harvested at the indicated time points and analyzed by flow cytometry to assess CFSE dilution as a measure of cell proliferation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical differences between experimental groups were determined by analysis of variance, and the p-values were calculated using unpaired two-tailed Student\u0026rsquo;s t-tests to consider statistical significance.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eEffects of the Piezo1 agonist and Piezo1 inhibitor on Ig class switching and Ab production by B cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFirst, this study examined whether Piezo1 is expressed in mouse B cells. Piezo1 expression was confirmed in CH12F3-2A and splenic B cells (Supplementary Fig.\u0026nbsp;2). The mouse macrophage cell line RAW 264.7 was used as a positive control. To evaluate the effect of Piezo1 on Ig class switching by B cells, the Piezo1 agonist Yoda1 was used. Yoda1 enhanced Ca\u003csup\u003e2+\u003c/sup\u003e influx in B cells (Supplementary Fig.\u0026nbsp;3). GLTs and PSTs expressions were analyzed in cytokine-stimulated cells. Yoda1 upregulated only TGF-β1-induced GLTα expression, whereas TGF-β1-induced GLTγ2b and other cytokine-stimulated GLTs (GLTε, GLTγ1, GLTγ2c, and GLTγ3) did not change significantly (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Moreover, TGF-β1-induced PSTα expression was selectively upregulated in B cells treated with Yoda1. Next, flow cytometry and ELISA were used to evaluate the surface Igs expression and Igs production. Yoda1 enhanced surface IgA-expressing cells and IgA secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and C). Similarly, levels of other Igs did not change. To investigate whether Piezo1-mediated, TGF-β1-induced IgA class switching is linked to B cell proliferation, we performed a CFSE-based cell proliferation assay. TGF-β1 significantly inhibited LPS-induced B cell proliferation. When B cells were additionally treated with Yoda1 under LPS/TGF-β1 stimulation, their proliferation was slightly reduced; however, this reduction was not statistically significant (Supplementary Fig.\u0026nbsp;4A). These results indicate that the enhancement of TGF-β1-induced IgA class switching by Yoda1 is not due to changes in B cell proliferation. In addition, Yoda1 increased TGF-β1-induced surface IgA expression at each B cell division (Supplementary Fig.\u0026nbsp;4B). Following the experiments with Yoda1, the Piezo1 inhibitor OB-1 was utilized to further investigate the role of Piezo1 on Ig class switching. OB-1 selectively downregulated TGF-β1-induced GLT/PSTα expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). OB-1 also selectively reduced surface IgA expression and IgA production in contrast to the effect of Yoda1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). Furthermore, this study investigated the effect of Piezo1 on TGF-β1-induced IgA class switching in CH12F3-2A, a mouse B cell line characterized by IgA class switching upon stimulation with TGF-β1 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Yoda1 enhanced TGF-β1-induced GLTα expression and PSTα expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Flow cytometry and ELISA demonstrated that Yoda1 increased surface IgA expression and IgA production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C). In contrast, OB-1 selectively downregulated TGF-β1-induced GLTα and PSTα expression, surface IgA expression, and IgA production (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD\u0026ndash;F). These results suggest that Piezo1 selectively upregulates IgA class switching and IgA production without affecting other Igs class switching and production. To explore the physiological relevance of these findings in vivo, we examined the expression of Piezo1 in Peyer\u0026rsquo;s patches (PP), the major site for IgA class switching. We compared Piezo1 expression levels in B cells from the spleen and PP using qRT-PCR and flow cytometry. PP B cells exhibited higher Piezo1 mRNA expression than unstimulated splenic B cells (Supplementary Fig.\u0026nbsp;5A). Consistently, the Piezo1 protein level was also elevated in PP B cells compared to unstimulated splenic B cells (Supplementary Fig.\u0026nbsp;5B).\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePiezo1 knockdown downregulates TGF-β1-induced IgA class switching\u003c/h2\u003e \u003cp\u003eTo elucidate whether upregulation of TGF-β1-induced IgA class switching is directly mediated by Piezo1, siRNA-mediated knockdown of Piezo1 was performed in CH12F3-2A cells. This study examined how TGF-β1-induced GLTα and PSTα changed. Piezo1 siRNA effectively reduced Piezo1 mRNA and protein expression (Supplementary Fig.\u0026nbsp;6). Piezo1 knockdown significantly decreased GLTα and PSTα expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In addition, surface IgA expression and IgA production were diminished (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C), demonstrating that Piezo1 enhances TGF-β1-induced IgA switching and IgA production.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePiezo1 enhances TGF-β1-induced Smad3 phosphorylation via the TGF-β receptor by upregulating Rab5c expression\u003c/h2\u003e \u003cp\u003eIn TGF-β1-induced IgA class switching, Smad3 is a critical signal transducer and an essential transcription factor. When stimulated by TGF-β1, Smad3 is phosphorylated by activated TGF-β1 receptors, and phosphorylated Smad3 activates GLTα transcription [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Therefore, this study examined the levels of phosphorylated Smad3 after Yoda1/OB-1 treatment and Piezo1 knockdown. Yoda1 upregulated TGF-β1-induced Smad3 phosphorylation in mouse splenic B cell and CH12F3-2A cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and B). Conversely, OB-1 decreased Smad3 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D). Moreover, Piezo1 knockdown downregulated TGF-β1-induced Smad3 phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), indicating that Piezo1 upregulates Smad3 phosphorylation to enhance TGF-β1-induced IgA class switching. To further elucidate the mechanism of Piezo1-mediated, TGF-β1-induced IgA class switching, we assessed the expression of TGF-β1-induced GLTα/PSTα following treatment with the TGF-β receptor inhibitor LY2109761, in the presence or absence of Yoda1. LY2109761 decreased Yoda1-mediated, TGF-β1-induced GLTα/PSTα expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). This result suggests that Piezo1 upregulates TGF-β1-induced IgA class switching via TGF-β receptor. Previous studies have reported that Piezo1 activates TGF-β signaling through the recruitment of Rab5c in hepatocellular carcinoma [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rab5c is a key regulator of early endosome trafficking and has also been implicated in enhancing TGF-β receptor-mediated signaling pathway [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Therefore, we examined whether Piezo1-mediated, TGF-β1-induced IgA class switching is related to Rab5c. Yoda1 increased the expression of Rab5c, whereas Piezo1 knockdown reduced Rab5c expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). In this context, Yoda1 enhanced TGF-β1-induced GLTα/PSTα expression, whereas Piezo1 knockdown diminished both TGF-β1-induced GLTα/PSTα expression and the enhancing effect of Yoda1. Taken together, these findings suggest that Piezo1 enhances TGF-β1-induced Smad3 phosphorylation via the TGF-β receptor by upregulating Rab5c expression, thereby facilitating IgA class switching in B cells.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePiezo1 is a channel that has been studied in various cell types and has shown to significantly influence their functions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Thus, Piezo1 may also influence B cells and cytokines. This study investigated the relationship between Piezo1 and Ig class switching and Ab production in B cells. According to the results, Piezo1 substantially increased TGF-β1-induced IgA class switching and IgA production by increasing TGF-β1-induced Smad3 phosphorylation without significant effects on other isotypes.\u003c/p\u003e \u003cp\u003ePiezo1 may affected the TGF-β1 receptor. As noted earlier, Piezo1 has been reported to activate TGF-β signaling through the recruitment of Rab5c, a member of the Rab5 subfamily, in hepatocellular carcinoma [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Rab5 is a small GTPase that regulates the early stages of clathrin-mediated endosome formation [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Clathrin-mediated endosomes internalize the TGF-β receptor, a process required for receptor-mediated Smad2/3 phosphorylation. Thus, Rab5c-mediated endocytosis facilitates Smad activation and downstream transcriptional responses via TGF-β signaling [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, TGF-β1-induced IgA class switching involving Piezo1 may be enhanced by promoting TGF-b receptor internalization through Rab5c. Our experimental data partially support this possibility, as we observed increased Rab5c expression and Smad3 phosphorylation following Piezo1 activation. Nevertheless, additional studies are needed to better understand the underlying mechanisms.\u003c/p\u003e \u003cp\u003eCalmodulin-dependent protein kinases (CaMKs) can affect TGF-β1-induced IgA class switching. CaMK is a family of protein kinases activated in response to calcium signals [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. CaMK is expressed in B cells, but its role is unknown. Some studies showed that CaMKs can promote TGF-β signaling-induced Smad2/3 phosphorylation in pluripotent stem cell-derived chondrocytes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, it is possible that CaMKs are activated by Piezo1-mediated Ca\u0026sup2;⁺ influx to upregulate Smad3 phosphorylation. We examined whether CaMK affects TGF-β1-induced IgA class switching by using the CaMK inhibitor KN-93. KN-93 did not affect TGF-β1-induced GLTα/PSTα expression in B cells (data not shown). Also, to determine whether Ca\u0026sup2;⁺ levels affect TGF-β1-induced IgA class switching, we treated B cells with the calcium chelator BAPTA. BAPTA had no effect on TGF-β1-induced GLTα/PSTα expression, either in the absence or presence of Yoda1 (Supplementary Fig.\u0026nbsp;7). These results suggest that Piezo1-mediated, TGF-β1-induced IgA class switching is independent of calcium signaling.\u003c/p\u003e \u003cp\u003eTGF-β1 induces IgA and IgG2b class switching through Smad3 signaling in mouse B cells. However, Piezo1 did not significantly change TGF-β1-induced GLTγ2b transcription and IgG2b class switching. This may be due to TGF-β differentially regulating GLTα and GLTγ2b transcription. Previous studies indicated that Runx3 overexpression increased the activity of the GLTα promoter but had no effect on the GLTγ2b promoter [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Hence, Piezo1 may interact with Runx3 to influence only the GLTα promoter. To investigate whether Piezo1 selectively influences GLTα promoter through Runx3, Piezo1 knockdown and overexpression of Runx3 could be used to assess its effects on class switching. Furthermore, the transcription factor CREB/ATF cooperates with Smad3/4/Runx3 to promote GLTα transcription [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], and Piezo1 may further enhance GLTα transcription by reinforcing CREB/ATF activity. Thus, further investigation is needed to clarify how Piezo1 selectively enhances only TGF-β1-induced GLTα transcription without affecting TGF-β1-induced GLTγ2b transcription.\u003c/p\u003e \u003cp\u003ePeyer\u0026rsquo;s patches, as the major mucosal inductive site for IgA class switching in vivo, exhibited higher Piezo1 expression than unstimulated splenic B cells. This may provide preliminary evidence supporting a potential role for Piezo1 in IgA class switching under physiological conditions. However, all data presented in this study were obtained from in vitro experiments, which may not fully reflect the in vivo environment, including cell\u0026ndash;cell interactions, immune responses, and tissue-specific environments. Therefore, future studies using animal models will be essential to validate our findings.\u003c/p\u003e \u003cp\u003eIn conclusion, Piezo1 selectively reinforces TGF-β1-induced IgA class switching by enhancing TGF-β receptor/Smad3 signaling, thereby promoting IgA production in B cells. As a key Ab in mucosal immunity, IgA plays a central role in host defense at mucosal surfaces. Although further investigation is warranted, our findings support the potential of Piezo1 as a therapeutic target for modulating IgA responses and treating mucosal immunity-related disorders.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (MEST), grant number NRF-2017R1A6A1A03015713. This work was also supported in part by Konyang University Myunggok Research Fund of 2023.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDepartment of Microbiology, Konyang University College of Medicine, Daejeon 35365, Korea\u003c/p\u003e\n\u003cp\u003eYoonji Jung, Seok-Rae Park\u003c/p\u003e\n\u003cp\u003ePriority Research Center, Myunggok Medical Research Institute, Konyang University College of Medicine, Daejeon 35365, Korea\u003c/p\u003e\n\u003cp\u003eYounghwan Han, Jaeku Kang, Seong-Lan Yu, Seok-Rae Park\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacology, Konyang University College of Medicine, Daejeon 35365, Korea\u003c/p\u003e\n\u003cp\u003eJaeku Kang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeok-Rae Park contributed to the study conceptualization. Data curation was performed by Yoonji Jung and Seok-Rae Park. Formal analysis was conducted by Yoonji Jung, Younghwan Han, and Seok-Rae Park. Funding acquisition was managed by Seok-Rae Park. Investigation was carried out by Yoonji Jung, Younghwan Han, Seong-Lan Yu, and Seok-Rae Park. Resources were provided by Seok-Rae Park and Jaeku Kang. Supervision of the study was conducted by Seok-Rae Park. The first draft of the manuscript was written by Yoonji Jung and Seok-Rae Park, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to Seok-Rae Park.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data supporting the findings of this study are detailed within the article and its supplementary materials. All datasets can be obtained from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimals were cared for in accordance with the institutional guidelines of the Institutional Animal Care and Use Committee of Konyang University (approval no. 23-24-A-01).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCoste B et al (2010) Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 330(6000):55\u0026ndash;60\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoste B et al (2012) Piezo proteins are pore-forming subunits of mechanically activated channels. Nature 483(7388):176\u0026ndash;181\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSyeda R et al (2016) Piezo1 Channels Are Inherently Mechanosensitive. 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J Biol Chem 274(44):31577\u0026ndash;31582\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"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":"B cells, Piezo1, IgA, Class switching","lastPublishedDoi":"10.21203/rs.3.rs-5552251/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5552251/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePiezo1 is a mechanosensitive cationic channel that regulates Ca\u003csup\u003e2+\u003c/sup\u003e influx, gene transcription, and cell migration. Recent studies suggest that Piezo1 affects regulatory T cells differentiation and is critical in B cell responses to membrane-presented antigens. However, the role of Piezo1 in B cells function is not completely elucidated. This study investigated the role of Piezo1 in IgA class switching and Ab production by mouse B cells using qRT-PCR, flow cytometric analysis, and isotype-specific ELISA. The Piezo1 agonist Yoda1 selectively upregulated TGF-β1-induced germline α transcripts (GLTα) /post-switch α transcripts (GLTα) expression, surface IgA expression, and IgA production. Conversely, the Piezo1 inhibitor OB-1 reduced IgA class switching. TGF-β1-induced IgA class switching and IgA production decreased in Piezo1 knockdown B cells. Additionally, Piezo1 enhanced TGF-β1-induced Smad3 phosphorylation. These results demonstrate that Piezo1 selectively enhances TGF-β1-induced IgA class switching via Smad3 phosphorylation, leading to IgA production in B cells.\u003c/p\u003e","manuscriptTitle":"Piezo1 selectively enhances TGF-β1-induced IgA class switching by B cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 13:22:10","doi":"10.21203/rs.3.rs-5552251/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-04-17T13:07:05+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-17T13:04:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-17T12:01:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cellular and Molecular Life Sciences","date":"2025-04-17T04:17:42+00:00","index":"","fulltext":""},{"type":"decision","content":"Minor Revision","date":"2025-01-13T16:58:24+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":"1571e69e-a6dc-44b2-8273-a7475757735b","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-23T16:04:31+00:00","versionOfRecord":{"articleIdentity":"rs-5552251","link":"https://doi.org/10.1007/s00018-025-05789-4","journal":{"identity":"cellular-and-molecular-life-sciences","isVorOnly":false,"title":"Cellular and Molecular Life Sciences"},"publishedOn":"2025-06-19 15:57:41","publishedOnDateReadable":"June 19th, 2025"},"versionCreatedAt":"2025-04-18 13:22:10","video":"","vorDoi":"10.1007/s00018-025-05789-4","vorDoiUrl":"https://doi.org/10.1007/s00018-025-05789-4","workflowStages":[]},"version":"v1","identity":"rs-5552251","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5552251","identity":"rs-5552251","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}