Acidosis environment promotes osteoclast formation by acting on the last phase of preosteoclast differentiation: A study to elucidate the action points of acidosis and search for putative target molecules

Acidosis promoted tartaric acid-resistant acid phosphatase-positive multinuclear cell (TRAP + MNC) or osteoclast formation. Large osteoclast or TRAP + LMNC formation was observed far more in an acidosis environment than in a physiologically neutral environment. One of the major action points of acidosis was determined to be located in the last phase of preosteoclast differentiation using a co-culture system and a soluble RANKL-dependent bone marrow cell culture system. On-going osteoclast formation in an acidosis environment markedly deteriorated when the medium was replaced with physiologically neutral medium within the first 6 h; however, bone marrow cells previously stimulated in an acidosis environment for 9 h differentiated into TRAP + LMNC in pH 7.4 medium. Messenger RNA (mRNA) expression levels of DC-STAMP, a key molecule in cell fusion, and NFATc1 did not increase in the acidosis environment compared with those under physiologically neutral conditions. Ruthenium red, a general TRP antagonist, deteriorated acidosis-promoted TRAP + LMNC formation. 4-Alpha-PDD, a TRPV4-specific agonist, added in the last 21 h of preosteoclast differentiation, potentiated TRAP + LMNC formation in a mild acidosis environment, showing synergism between TRPV4 activation and acidosis. RN1734, a TRPV4-specific antagonist, partly inhibited acidosis-promoted TRAP + LMNC formation. We thus narrowed down the major action points of acidosis in osteoclast formation and elucidated the characteristics of this system in detail. Our results show that acidosis effectively uses TRPV4 to drive large-scale cell fusion and also utilizes systems independently of TRPV4.