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

Abstract

Piezo1 is a mechanosensitive cationic channel that regulates Ca²⁺ 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.

Keywords: B cells; Class switching; IgA; Piezo1.

Fig. 1

Effect of Yoda1 on IgA class switching and IgA production by splenic B cells Splenic 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 show 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 < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Fig. 2

Effect of OB-1 on class switching and Ig production by splenic B cells Resting 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 < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Fig. 3

Effect of Yoda1 and OB-1 on class switching and Ig production by B cell line CH12F3-2 A 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 < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Fig. 4

Effect of Piezo1 knockdown on IgA class switching CH12F3-2 A 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) (A) After 1.5 days of culture, RNA was isolated. GLTα/PSTα levels were measured by qRT-PCR. Graphs show the 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 < 0.05; **p < 0.01; ***p < 0.001

Fig. 5

Piezo1 enhances TGF-β1-induced Smad3 phosphorylation Splenic 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. CH12F3-2 A 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 were performed. Data are the mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant

Fig. 6

Piezo1 may facilitate TGF-β1-induced IgA switching via TGF-b receptor signaling by upregulating Rab5c expression (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, left panel) Splenic B cells were cultured with the indicated stimuli (Yoda1, 0.5 µM; TGF-β1, 0.2 ng/mL). After 24 h, 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, right panel) CH12F3-2 A 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. (C–F) CH12F3-2 A was transfected with pCMV-SPORT6-Rab5c 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). After 1.5 days of culture, RNA was isolated. GLTα/PSTα levels were measured by qRT-PCR and the relative GLTα/PSTα cDNA levels normalized to the expression of β-actin (C). After 3 days of culture, B cells were isolated, and surface IgA/IgM was measured by flow cytometry (D). After 4 days of culture, supernatants were harvested, and IgA/IgM production was measured by isotype-specific ELISA (E). After 3 h of culture, western blots for p-Smad3, Smad3, and β-actin were performed (F). Data are the mean ± SEM from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant; OE, overexpression