Mechanosensitive piezo1 calcium channel activates connexin 43 hemichannels through PI3K signaling pathway in bone

Cell culture and generation of Rosa26 and Cx43 KD MLO-Y4 cell line

Osteocytic MLO-Y4 cells derived from murine long bones were provided by Dr. Lynda Bonewald at Indiana University School of Medicine. Lentiviral-mediated CRISPR/Cas9 genome editing technology using lentiCRISPRv2:Cx43-sgRNA or ROSA26-sgRNA was applied to generate Cx43 KD and Rosa26 control MLO-Y4 cells as previously described [26]. These cells were cultured on collagen-coated (rat tail collagen type I, 0.15 mg/ml) surfaces and were grown in medium (α-MEM, HCO3−) supplemented with 2.5% fetal bovine serum and 2.5% bovine calf serum and incubated in a 5% CO2 incubator at 37 °C as described previously [27].

Fluid flow shear stress and in vitro dye uptake assay

FFSS experiments were conducted as previously described [28]. Briefly, FFSS was created by parallel plate flow chambers separated by a gasket of defined thickness with gravity-driven fluid flow using a peristaltic pump. Shear stress levels of 2, 4, 8, and 16 dynes/cm2 were generated, and a cell surface area of 5 cm2 was used for dye uptake assay. MLO-Y4 cells were pre-incubated with Cx43(E2) antibody, generated and affinity-purified as previously described [29], 10Panx (#33-481, Tocris Bioscience, Bristol, UK), Dooku1 (#6568, Tocris Bioscience, Bristol, UK), BAPTA-AM (#B1205, Invitrogen, Carlsbad, CA, USA), apyrase (#A6237-100UN, Sigma-Aldrich, St. Louis, MO, USA), and LY294002 (#NC9642062, Cayman Chemical, Ann Arbor, MI, USA). The cells were then subjected to FFSS (2, 4, 8, and 16 dynes/cm2, 10 min), followed by dye uptake assay. In dye uptake assay, cells were incubated with 10 μM ethidium bromide (EtBr) (Mw: 394 Da) and FITC-Dextran (Mw: 10 kDa) at room temperature (RT) for 5 min and FITC-Dextran was used to detect the apoptotic and unhealthy cells. The FITC-Dextran-positive cells were excluded for quantification of dye uptake level. The treatment with Yoda1 (#5586, Tocris Bioscience, Bristol, UK) was performed in the presence of the dyes. Cells were rinsed with PBS and fixed with 2% paraformaldehyde, and the mean intensity of the nuclear region was determined using NIH ImageJ software (NIH, Bethesda, MD, USA) [8].

Live cell calcium imaging

MLO-Y4 cells were cultured at a total of 2.5 × 105 cells on a collagen-coated 35 mm culture dish with an inner 25-mm diameter circle glass slide 48 h prior to live Ca2+ imaging. Before imaging capturing, cells were incubated with Cx43(E2) antibody, 10Panx, Dooku1, BAPTA-AM, oATP (#71997-40-5, Sigma), and apyrase (#9000–95-7, Sigma) at RT for 30 min, followed with Fluo-4-AM (#F14201, Invitrogen), a Ca2+ indicator, at 37 ℃ for 40 min. The culture medium was then rinsed and replaced with recording medium (α-MEM, 10 mM HEPES, PH 7.4), and the glass slide was placed in the Coverslip Cell Chamber (#SC15012, AirekaCells, HK, China). The chamber was placed on the objective stage of the microscope and set still before capturing images. Ca2+ signaling (488 nm) of the MLO-Y4 cells was imaged using a Nikon sweptfield laser scanning confocal microscope (Nikon, Tokyo, Japan) with a 40X objective. The entire imaging process lasted 600 s, and each image was taken with a 2-s interval, and Yoda1 was loaded at 240 s. For sequential inhibition of Cx43 HCs or Panx1 Channel, Cx43(E2) antibody or 10Panx was added at 360 s. The BAPTA-AM was added at 360 s. For the recording of Ca2+ signal in mechanically loaded MLO-Y4 cells, the cells cultured on coverslips were then placed in the FFSS chamber and connected with the fluid circulation system, and live calcium images were captured at 900 s with FFSS (8 dynes/cm2) stimulation applied at 300 s and lasted for 300 s. The entire process lasted for 900 s with 3 phases (300 s each) rest, stimulation, and recovery.

Cell surface staining

MLO-Y4 cells cultured on collagen-coated glass slides were subjected to FFSS (16 dynes/cm2) for 1 h or Yoda1 for 30 min and rinsed 3 times with cold (4 ℃) DPBS. The live cells on the slides were incubated with rabbit polyclonal anti-Cx43(E2) antibody (1:100 dilution) in recording medium supplemented with 2.5% fetal bovine serum and 2.5% bovine calf serum at 4 ℃ for 90 min. After rinsing 3 times with cold DPBS supplemented with 1 mM Ca2+, cells were fixed with 2% paraformaldehyde for 10 min, followed by blocking with donkey blocking buffer (2% donkey serum, 2% fish skin gelatin, 0.025% Triton X-100 and 1% bovine serum albumin in PBS). The cells were then incubated with 1:100 dilution mouse monoclonal anti-Piezo1 antibody (clone 2–10 ThermoFisher Scientific, Waltham, MA, USA) at 4 ℃, overnight. The labeling by primary antibodies was performed by sequential incubation of secondary antibodies first with donkey anti-mouse Alexa Fluor 488-conjugated (#NC0241229, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h and then with donkey anti-rabbit Alexa 594 (#NC0303478, Jackson ImmunoResearch, West Grove, PA, USA) for 1 h. Slides were mounted and sealed for microscopic analysis. Confocal sectional images were taken with an optical microscope (BZ- X710, KEYENCE, Itasca, IL, USA) using the appropriate filters.

Preparation of cell lysates and Western blot

LY-294002/Yoda1 treated MLO-Y4, Cx43 KDa, and Rosa26 MLO-Y4 cells were collected and lysed with lysis buffer (5 mM Tris, 5 mM EDTA/EGTA, 0.6% sodium dodecyl sulfate (SDS) and proteinase inhibitors) and then ruptured by pipetting through a 20-gauge needle. Cell lysates with Cx43 KD or Rosa26 control were ultracentrifuged at 45,000 g for 45 min at 4 ℃ to prepare crude membrane extracts. Protein concentration of protein lysates was determined by Micro BCA Protein Kit (Thermo Scientific, Rockford, IL, USA). 30 µg proteins were boiled in SDS sample loading buffer (250 mM Tris–HCl, pH 6.8, 10% SDS, 30% (v/v) glycerol, 10 mM DTT, 0.05% (w/v) bromophenol blue), subjected to 10% SDS–polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. Membranes were blocked with donkey serum blocking buffer at RT for 1 h, then incubated with a 1:1000 dilution of rabbit polyclonal anti-Akt antibody (#9272, Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal anti- phosphorylated-AKT Ser473 antibody (#9271, Cell Signaling Technology) (1:300 dilution) for LY-294002/Yoda1 treated MLO-Y4 cell samples or incubated with 1:500 dilution of rabbit polyclonal anti-Cx43(CT) antibody or 1:150 of mouse monoclonal anti-Piezo1 antibody (#MA5-32876, Invitrogen) for Cx43 KD and Rosa26 samples. Primary antibodies were detected with goat anti-rabbit IgG conjugated IRDye® 800CW and goat anti-mouse IgG conjugated IRDye® 680RD (1:15 000 dilutions) using a Licor Odyssey Infrared Imager (Lincoln, NE, USA). The band intensity was quantified by densitometry using NIH ImageJ software.

Immunoprecipitation

MLO-Y4 cell lysates were resuspended in immunoprecipitation (IP) buffer (0.1 M NaCl, 15 mM EDTA, 15 mM EGTA 0.02% Na azide, 0.02 M Na borate, 10 mM NEM, 2 mM PMFS, pH 8.5). Lysates were precleared with protein A/G plus-agarose beads (NC9371547, Santa Cruz, CA, USA) and pelleted by centrifugation at 7000 rpm at 4 ℃ for 5 min. Supernatants (precleared lysates) were then incubated with rabbit polyclonal anti-Piezo1 antibody (15,939–1-AP, Proteintech, Rosemont, IL, USA) overnight at 4 ℃, followed by precipitation of the complexes after incubation with protein A/G plus-agarose beads at 4 ℃. Immunoprecipitants were separated on 10% SDS–polyacrylamide gel and immunoblotted with mouse monoclonal anti-Cx43 (SAB4200730, Sigma) (1:200 dilution) or mouse monoclonal anti-Piezo1 antibody (MA5-32,876, Invitrogen) (1:500 dilution).

Tibia compression and in vivo dye uptake assay

We subjected 15-week-old male C57Bl/6 mice to mechanical loading through tibial compression using a loading setup established in our laboratory [16, 30, 31]. Mice were subjected to tibial loading at a frequency of 2 Hz using a haversine waveform for 600 cycles with a constant force of 8.86 N at the left tibia. 15-week-old male C57Bl/6 mice were injected intraperitoneally with Cx43(M1) antibody, control mouse IgG, 5 mg/kg Dooku1, or vehicle DMSO as control 4 h prior to intravenous tail injection of 200 mg/kg Evans Blue. Mice were subjected to intraperitoneal injection of 2.5 mg/kg Yoda1, or cyclic tibia compression 30 min after the injection of the dye, and perfusion with 4% paraformaldehyde and tissue collected 2 h or 1 h post Yoda1 administration or mechanical loading. Mice were housed in a temperature-controlled room with a 12-h light/12-h dark cycle and under specific pathogen-free conditions in the Institutional Lab Animal Research facility at the University of Texas Health Science Center at San Antonio (UTHSCSA). All described animal protocols were reviewed and approved by UTHSCSA Institutional Animal Care and Use Committee (IACUC), in accordance with policies dictated by the Office of Animal Welfare at the NIH, USA.

Statistical analysis

GraphPad Prism8 statistics software (GraphPad, San Diego, CA, USA) was used for statistical analysis. Student-Newman Keul’s test and One-way ANOVA were used for two or multiple comparison analyses. Two-way ANOVA with post hoc Tukey test was used to assess the difference between multiple groups with two variables. Data were shown as mean ± SEM, and asterisks indicate the degree of significant differences compared with the controls, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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