Molecular hydrogen suppresses activated Wnt/β-catenin signaling

Molecular hydrogen (H2) is effective for many diseases. However, molecular bases of H2 have not been fully elucidated. Cumulative evidence indicates that H2 acts as a gaseous signal modulator. We found that H2 suppresses activated Wnt/β-catenin signaling by promoting phosphorylation and degradation οf β-catenin. Either complete inhibition of GSK3 or mutations at CK1- and GSK3-phosphorylation sites of β-catenin abolished the suppressive effect of H2. H2 did not increase GSK3-mediated phosphorylation of glycogen synthase, indicating that H2 has no direct effect on GSK3 itself. Knock-down of adenomatous polyposis coli (APC) or Axin1, which form the β-catenin degradation complex, minimized the suppressive effect of H2 on β-catenin accumulation. Accordingly, the effect of H2 requires CK1/GSK3-phosphorylation sites of β-catenin, as well as the β-catenin degradation complex comprised of CK1, GSK3, APC, and Axin1. We additionally found that H2 reduces the activation of Wnt/β-catenin signaling in human osteoarthritis chondrocytes. Oral intake of H2 water tended to ameliorate cartilage degradation in a surgery-induced rat osteoarthritis model through attenuating β-catenin accumulation. We first demonstrate that H2 suppresses abnormally activated Wnt/β-catenin signaling, which accounts for the protective roles of H2 in a fraction of diseases.

The cells were then vortexed and centrifuged at 5000 x g for 2 min. Supernatant was removed, and the pellet was washed with buffer A and resuspended in buffer C containing 50 mM HEPES-KOH (pH 7.8), 420 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 2% glycerol, 10 mM sodium pyrophosphate, 1 g/l aprotinin, 1 g/l leupeptin, 1 g/l pepstatin A, 1 mM PMSF, 1 mM sodium orthovanadate, and the Phosphatase Inhibitor Cocktail. The pellet was vortexed and incubated at 4 °C on a rotating shaker for 30 min. The pellet was then centrifuged at at 20,600 x g at 4 º C for 15 min. The supernatant was collected as nuclear fraction.

In vivo experiments with fed and starved mice
Eight-week-old C57BL/6J mice were purchased from Japan SLC. Mice were randomly divided into 4 groups: free access to degassed water and food before euthanasia (fed control group), free access to hydrogen water and food before euthanasia (fed+H2 group), free access to degassed water and starved before euthanasia (starved control group), and free access to hydrogen water and starved before euthanasia (starved+H2 group). Mice had free access to either degassed water or H2 water for 10 d. Mice had free access to food up to euthanasia or were starved for 18 h before euthanasia. The liver was isolated, flash-frozen in liquid nitrogen, and stored at -80 °C until further applications. For protein extraction, liver tissues were homogenized and lysed in RIPA buffer (Thermo Scientific Pierce, 89900) followed by centrifugation for 15 min at 20,600 x g at 4 °C.

In vivo ubiquitination assay
Cells were harvested by a lysis buffer containing 2% SDS, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0) with 2 mM sodium orthovanadate, 1 mM PMSF, 50 mM NaF, 1 g/l pepstatin A, 1mM DTT and the Protease Inhibitor Cocktail, and boiled for 10 min. Samples were then diluted in a dilution buffer (1: 10) containing 10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% TritonX-100, incubated at 4 °C for 30 min on a rotating shaker, and centrifuged at 20,000 x g for 30 min. The total protein concentrations of the supernatants were measured by Pierce 3 660 nm Protein Assay Reagent, and 500 g protein were incubated with anti--catenin antibody at 4 °C on a rotating shaker overnight, followed by addition of Dynabeads Protein G (Invitrogen) for another 1 h. Proteinantibody-bound beads were washed with a washing buffer [10 mM Tris-HCl (pH 8.0), 150 M NaCl, 1 mM EDTA, 1% NP-40] and boiled for 5 min with standard 2 Laemmli buffer. The immunoprecipitates were separated by SDS-PAGE and immunoblotted with anti-ubiquitin and anti--catenin antibody, as stated in Supplementary Table   S1.

Co-immunoprecipitation
Cells were lysed in a buffer containing 20 mM Tris pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 1mM DTT, 50 mM NaF, 1 mM PMSF, 1 mM sodium orthovanadate, 1 g/l pepstatin A, and the Phosphatase Inhibitor Cocktail. After centrifugation for 15 min at 20,600 x g at 4 °C , supernatants were collected and protein concentrations were measured by Pierce 660 nm Protein Assay Reagent. Lysates with the same amounts of proteins (750-1000 g) were incubated with 1.0-1.5 g of an antibody against Axin1 (05-1579, Millipore) or control mouse IgG (sc-2025, Santa Cruz Biotechnology) overnight at 4 °C . Dynabeads protein G (Invitrogen) were blocked with 1 g/ml BSA for 1 h at 4 °C , added to the lysates with indicated antibody, and incubated for 1 h.

Alcian blue staining
ATDC5 cells were seeded in a 12-well plate, and were differentiated into chondrocytes with insulintransferrin-selenite (ITS, Invitrogen) for 21 days. Cells were treated with either 50% control CM, 50% Wnt3a CM, or 2 M BIO with 10% H2 or 10% N2 gas for 48 h. Then, cells were fixed with methanol for 30 min at -20 °C, and stained overnight with 0.5% Alcian Blue 8 GX (Sigma) in 1N HCl. The stained cells were lysed in 200 l or 400 l of 6 M guanidine HCl for 6 h at room temperature. The optical density of the extracted Alcian blue was measured at 630 nm using PowerScan 4 (DS Pharma Biomedical).

Cell proliferation assay
Proliferation of ATDC5 cells was estimated by the BrdU cell proliferation ELISA kit (Roche) according to the manufacturer's recommendations. Briefly, ATDC5 cells were seeded at 510 3 /well in a 96-well plate and were incubated with 10% H2 or 10% N2 gas for 48 h. Then, cells were labeled with 10 M BrdU for 2 h at 37 °C, fixed and incubated with anti-BrdU antibody for 90 min at room temperature. After incubating the lysate with the substrate solution for 10 min, the reaction was stopped by H2SO4. Absorbance was measured at 450 nm using an absorbance microplate reader (Sunrise Remote, Tecan).

Immunofluorescence staining
For immunofluorescence staining, the paraffin-embedded sections were first deparaffinized and rehydrated. Then, the sections were unmasked by 10 mM sodium citrate buffer (pH 6.0) at 95 °C for 15 min and were incubated with 3% H2O2 for 15 min to inactivate endogenous peroxidases. Sections were blocked with 5% goat serum in TBS-T for 1 h at room temperature followed by overnight incubation with antibodies against -   (a) L cells were treated with 2 µM BIO with 10% H2 or 10% N2 gas for 24 h. Western blots are shown with densitometry of phospho-glycogen synthase (p-GS)/glycogen synthase (GS) (n = 3). No statistical difference by Student's t-test.

(b)
Knock-down efficiencies of endogenous APC by two siRNAs were assessed by qRT-PCR in L cells (n = 3).