Abstract
Osteoclasts require colony-stimulating factor-1 (CSF-1) for formation, survival, fusion and differentiation. Extracellular protons are the most powerful activators of mammalian osteoclasts. We investigated whether extracellular pH effects on osteoclast fusion and differentiation is induced by CSF-1 treatment.
Osteoclasts were isolated from the tibiae and femurs of 1-day-old rats according to the method described by Chambers. After adherence for 1 hr at 37°C onto plastic coverslips, contaminating cells were removed by washing. The obtained osteoclasts were then incubated in the presence or absence of 500 pM recombinant human (rh) CSF-1 (a gift from Chiron Corporation, USA) in Iscove's modified Dulbecco's medium without bicarbonate, buffered with 25 mM Hepes at pH 6.8-7.5, with 15% FBS, at atmospheric pressure. We confirmed that CSF-1 stimulates the fusion and actin-ring formation of osteoclasts.
The maximal effect of CSF-1 on fusion of osteoclasts was found at pH 7.0. Whereas, CSF-1 stimulated actin-ring formation at lower pH, i. e., pH 6.8 or 7.0. Ten μM hexamethylene amiloride blocked both of these effects completely, suggesting that the activation of Na+/H+ exchanger may be essential to the CSF-1-induced fusion and differentiation of osteoclasts.
