Mice
Wild-type C57BL/6N mice were purchased from SLC Japan, Inc. (Hamamatsu, Japan). Piezo2-GFP-IRES-Cre mice (Piezo2tm1.1(cre)Apat (MGI:5574478)) and Piezo2+/− mice (Piezo2tm2.2Apat (MGI:5574479))22 were kind gifts from Prof. Keiko Nonomura and Prof. Ardem Patapoutian. All the mice were housed in the specific pathogen-free animal facility at Saga University with a 12-h light cycle and free access to water and food (CE-2; CLEA Japan, Inc., Tokyo, Japan). All animal experimental procedures were approved by the Animal Care and Use Committee of Saga University with No. 28–030–6, G2020–01–07, A2020–009–1, and A2023–026–0. We have complied with all relevant ethical regulations for animal use. The care of the animals was in accordance with institutional guidelines. Age-matched male and female mice aged 6–9 weeks were used for experiments. All mice were randomly selected for experiments.
Allergic asthma model
The allergic asthma model was generated as previously described40. The mice were intraperitoneally sensitized with 50 μg of ovalbumin (OVA) (A5503, Sigma‒Aldrich, St. Louis, MO, USA) containing 2 μl of aluminum gel (Alum) (LG-6000, LSL, Japan) diluted in 200 µl of phosphate-buffered saline (PBS) (10 ml/kg body weight) on days 0, 7, and 14 (Fig. 1A). The mice were intranasally challenged with 2.5 mg/ml OVA solution for four consecutive days. PBS- or Alum- and PBS-sensitized mice were used as controls (Fig. 1A). For Yoda1-treated mice, we intraperitoneally injected Yoda1 (SML1558, Sigma, St. Louis, MO) into PBS- or OVA+Alum (O + A)-sensitized mice at days 1–5 and 8–12 (Fig. 5A). Yoda1 was dissolved in 40 mM DMSO as a stock, diluted in 5% ethanol and administered intraperitoneally at a dose of 5 µmol/kg body weight.
Histology and histochemistry
The mice were deeply anaesthetized with a mixture of midazolam (4 mg/kg, 042824; Maruishi), butorphanol tartrate (5 mg/kg, VETLI5; Meiji Seika Pharma), and hydrochloric acid medetomidine (0.3 mg/kg, 9021; Kyoritsu Seiyaku) and then intracardially perfused with heparinized 0.1 M PBS, followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The lungs, femurs, and tibias were removed and postfixed overnight at 4 °C. Femoral or tibial tissues were decalcified in 10% ethylenediaminetetraacetic acid for ten days. After cryoprotection with 20% sucrose overnight, the tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek Japan, Tokyo, Japan). Five-micron-thick lung sections were cut with an FSE cryostat (Thermo Fisher Scientific, Waltham, MA, US) and placed on microslide glass (Frontier FRC-01, Matsunami Glass Ind., Ltd., Kishiwada, Japan). The sections were stained with periodic acid-Schiff (PAS) and Giemsa solutions. For alkaline phosphatase (ALP) staining, longitudinal 5 µm thick sections of bones were incubated with 5% N,N-dimethylformamide (045-02916; Wako Pure Chemical Industries), 1 mg/ml naphthol AS-MX phosphate (N4875-1G; Sigma‒Aldrich), and 1 mg/ml fast blue BB salt (066-05461; Wako Pure Chemical Industries) in 0.1 mM Tris-HCl (pH 9.2) at 37 °C for 30 min. TRAP staining was performed using an acid phosphatase leukocyte kit (387 A; Sigma‒Aldrich). Images were taken with an Axio Imager M2 microscope (Carl Zeiss) or a Nanozoomer S60 microscope (Hamamatsu Photonics K.K., Shizuoka, Japan).
Microcomputed tomography analysis
The right femurs and tibias were removed from the surrounding soft tissues and stored in 70% ethanol at 4 °C. The distal femurs or proximal tibias were scanned with a SkyScan 1272 microcomputed tomography (µCT) system (Bruker, Kontich, Belgium) and analyzed using CTan software (version 1.20.8.0, Bruker). The primary spongiosa region was extracted by segmenting the epiphysis, growth plate, and diaphysis from the distal femur or proximal tibia. The interface between the growth plate and the metaphysis was established as the reference plane, with the primary spongiosa region defined as the area extending 150 µm (for the femur) or 120 µm (for the tibia) from this reference plane towards the diaphysis. The trabecular bone volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), trabecular bone pattern factor (Tb.Pf), and structure model index (SMI) were analyzed. 3D-rendered images of the VOI were produced by CTvox software (version 3.3.1, Bruker).
Antibody generation
The guinea pig polyclonal anti-mouse Piezo1 antibody was raised against a peptide corresponding to mouse Piezo1 (residues 1759 to 1773: VLRRYENKPYFPPRI). A rabbit polyclonal anti-mouse Piezo2 antibody was raised against a peptide corresponding to mouse Piezo2 (residues 401 to 413: SMTQDDYKPSDGL). Both antibodies were affinity-purified with the peptide. HEK293 cells transfected with either mouse Piezo1 or Piezo2 (kind gifts from Prof. Makoto Tominaga and Dr. Yasunori Takayama) were used to assess antibody specificity. Immunoblotting revealed that the protein fractions of the membrane or cytosol of the transfected cells reacted with Piezo1 or Piezo2, respectively (Supplementary Fig. 3A). Immunofluorescent controls involved the use of no primary or secondary antibody or the replacement of the primary antibody with preabsorbed with the control peptide at 10−4–10−6 M (Supplementary Fig. 3B).
Immunofluorescence staining
Longitudinal serial femoral sections at a thickness of 5 μm were permeabilized with 0.3% Triton X-100 diluted in PBS for 10 min. After incubation in 5% normal donkey serum, 1% bovine serum albumin, or 0.05% sodium azide for 45 min at room temperature, the sections were incubated with primary antibodies overnight at 4 °C. For mouse antibodies, samples were processed with Mouse-on-Mouse (M.O.M.) mouse Ig blocking reagent (BMK-2202, Vector Laboratories, Burlingame, CA, US) for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: Piezo1 (1 µg/ml), Piezo2 (1 µg/ml), Osterix (1:4000, ab22552, Abcam), type I collagen (1 µg/ml)41, podoplanin (1:1000, AF3244, R&D Systems), semaphorin 3 A (1 µg/ml, ab23393, Abcam), Piezo1 (1:400, 15939-1, Proteintech), Piezo1 (1:400, NBP1-78446SS, Novus), KDEL (1:200, M181-3, MBL), GM130 (1:200, BD 610822, BD Pharmingen), calnexin (1:3000, ab22595, Abcam), CD63 (1:600, BD 564221, BD Pharmingen), Lamp-1 (1:200, BD 553792, BD Pharmingen) and EEA1 (1:200, M176-3, MBL). After primary antibody incubation, the sections were washed and incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:200, 711-545-152, Jackson ImmunoResearch Laboratories), Alexa Fluor 594-conjugated donkey anti-guinea pig IgG (1:200, 706-585-148, Jackson ImmunoResearch Laboratories), Alexa Fluor 594-conjugated donkey anti-rat IgG (1:200, A21209, Thermo Fisher Scientific), and DyLight 649-conjugated donkey anti-mouse IgG (1:400, ab96878, Abcam). Filamentous actin was stained with Phalloidin-iFluor647 Reagent (1:1000, ab176759, Abcam) for 30 min. Nuclei were counterstained with 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (1:1000, 340-07971, Dojindo Laboratories) for 5 min. The sections were mounted, and images were taken with a benchtop spinning disc confocal microscope BC43 (Oxford Instruments) with a Plan-Apochromat 20× objective (Nikon, Japan) or a confocal scanning microscope LSM800 with an Airyscan module (Carl Zeiss, Oberkochen, Germany) with a Plan-Apochromat 20× objective or Plan-Apochromat 63× oil immersion objective. Z stack images were taken in 16-bit mode using unidirectional scanning, with a digital zoom of 2.0, a field of view of 50 × 50 µm, and pixel scaling of (x, y, z) 29 × 29 × 160 nm. Superresolution images with XY resolution of ~120 nm and Z resolution of ~350 nm42 were obtained by Airyscan processing of the raw images with automatically determined superresolution parameters using ZEN Blue 3.5 software (Carl Zeiss). For Fig. 4A, Supplementary Fig. 4A–C, and Supplementary Fig. 5B–E, the raw images were processed using automatic parameters with additional 0.5 manual adjustments per channel. For Fig. 4C, images with XY resolution 90 nm and Z resolution of 270 nm43 were obtained by Airyscan Joint Deconvolution conducted at 5 iterations for DAPI, 10 iterations for Piezo2, and 20 iterations for Col1 signals. We confirmed that the present configuration showed little or no chromatic aberration by imaging 0.2-µm-diameter Tetraspeck microspheres (Invitrogen).
Correlative confocal laser scanning light microscopy (CLSM)–electron microscopy (EM)
The CLSM–EM method was performed according to the previous methods with slight modifications44,45,46. Serial 50-μm-thick sections were cut from decalcified femur using a vibrating microtome (Dosaka TTK-3000W). After cryoprotection with 25% sucrose in PBS, the sections were quickly frozen in liquid nitrogen vapor and rapidly thawed with 25% sucrose in PBS. The sections were incubated with 5% normal donkey serum (Jackson ImmunoResearch Laboratories) and 0.1% sodium azide in PBS for 1 h, followed by incubation with anti-Piezo1 (1:300) or anti-Piezo2 (1:2000) antibody at 4 °C for 4 days. The incubating medium did not contain any detergent, such as Triton-X100. After being rinsed several times in PBS, the sections were incubated with biotinylated anti-guinea pig IgG (1:250; Jackson ImmunoResearch Laboratories) or biotinylated anti-rabbit IgG (1:250; Jackson ImmunoResearch Laboratories) for Piezo1 and Piezo2 labeling, respectively, at 4 °C overnight, followed by incubation with Rhodamine red-conjugated streptavidin (1:500, Jackson ImmunoResearch) at 4 °C overnight. Hoechst33342 (1:1000) and phalloidin 647 (1:1000) were added to the solution during the last 30 min of incubation. Immunostained sections were rinsed several times, mounted in Vectashield (Vector Laboratories), and examined using a confocal laser-scanning light microscope (CLSM; model C2, Nikon) with a 60× objective (Plan Apo, ND = 1.40, Nikon). After CLSM, the coverslips were gently removed, and the sections were subjected to the ABC method (Vector Laboratories) using the DAB peroxidase reaction to visualize Piezo1 or Piezo2 antibodies. After the reaction, the sections were rinsed and treated with 1% OsO4 in 0.1 M phosphate buffer, en bloc staining with 1.5% uranyl acetate, dehydration in ethanol, and embedding in Epon-Araldite. The Piezo1- or Piezo2-immunolabelled osteoblasts on the trabecular or cortical bone observed by CLSM were examined by EM (HT7700, Hitachi).
Western blot analysis
Western blot analysis was performed as previously described47. Briefly, protein extracts from tissues were lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% NP-40) containing protease inhibitor (Nacalai Tesque, Kyoto, Japan) and phosphatase inhibitor (PhosSTOP; Roche, Basel, Switzerland) for 10 min on ice. The protein extracts were subsequently centrifuged at 14,000 rpm at 4 °C for 20 min. The supernatants were collected, and the protein concentration was quantified with a Pierce BCA protein assay kit (23225, Thermo Fisher Scientific). Proteins were separated on 4–15% Mini-PROTEAN TGX gels (Bio-Rad Laboratories, Hercules, CA, US) and transferred to PVDF membranes (Immobilon P, Merck Millipore, Burlington, MA, US). The membranes were blocked with 5% bovine serum albumin for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. The primary antibodies used were as follows: Piezo1 (1 µg/ml), Piezo2 (1 µg/ml), Osterix (1:4000, ab22552, Abcam), type I collagen (1 µg/ml), semaphorin 3 A (1 µg/ml, ab23393, Abcam), GAPDH (1:2000, sc25778, Santa Cruz Biotechnology), TBP (TATA-binding protein, 1:2000, #8515, Cell Signaling Technology), and pan-actin (1:2000, 12748, Cell Signaling Technology). After being washed three times with Tris-buffered saline with 0.1% Tween 20, the PVDF membranes were incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies used were donkey anti-rabbit IgG (1:4000, NA934, GE Healthcare) and goat anti-guinea pig IgG (1:4000, 106-035-003, Jackson ImmunoResearch Laboratories). Immunoreactive bands were visualized using ECL Prime Western blotting detection reagent (RPN2232, GE Healthcare, Chicago, IL, US) and analyzed via a FUSION-FX7 imaging system (Vilber, Collégien, France).
Image analysis
The trabecular region of the distal femur extending 1.0 mm from the growth plate towards the diaphysis was measured. The osteoblast surface/bone surface (Ob. S/BS) from the ALP-stained images and the osteoclast surface/bone surface (Oc. S/BS) from the TRAP-stained images were quantified using ImageJ software (v2.14.0). The intensity of immunofluorescence and the number of Osx-positive cells and Piezo1- or Piezo2-positive surfaces per bone surface were measured by ImageJ. The intensity profile of immunofluorescence and the colocalization of immunoreactivities were determined with ZEN Blue 3.5 (Carl Zeiss).
Statistics and reproducibility
All the experiments were conducted at least three times independently under the same conditions. For data measurements, individual mice were used as experiment units. Graphical presentation was performed using JMP Pro 15 (SAS Institute Inc.) or GraphPad Prism 9.5.1 (GraphPad Software Inc.). Statistical differences were analyzed by one-way ANOVA with Tukey’s post hoc test for three groups using JMP Pro 15. The data are shown as the median with interquartile range (IQR) without exclusion, and differences were considered significant at p < 0.05.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.