Osteoblastic Cell Response to Acidic Conditions

Cell culture experiments were conducted with UMR106 rat osteoblastic osteosarcoma cells (CRL-1661; ATCC, Manassas, VA, USA) cultured in Dulbecco’s Modified Eagle Medium (DMEM) high glucose (Gibco, Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Life Technologies) under standard culture conditions. Cells were pretreated with 10 nM 1,25(OH)₂D₃ (Tocris, Bristol, UK) for 24 h (6-well format; 2 × 10⁵ cells/well). Twenty-four hours later, they were treated with the indicated concentration of L-lactic acid or Sodium (Na⁺)-L-lactate (sodium chloride as vehicle control; Sigma–Aldrich, Schnelldorf, Germany; 24 h), nuclear factor kappa-light-chain enhancer of activated B-cells (NFκB) inhibitors withaferin A (Tocris; 500 nM, 24 h) or wogonin (Sigma; 100 µM, 24 h), or with vehicle only. withaferin A and wogonin are potent inhibitors of NFκB signaling [55 (link)–57 (link)] that is a major enhancer of Fgf23 gene expression [58 (link)]. In further series of experiments, UMR106 cells were treated with 22.8 mM formic acid (Carl Roth, Karlsruhe, Germany), 10 mM acetic acid (Carl Roth), or water for 24 h and, pH of supernatants was measured.