Inhibition of intravacuolar acidification by antisense RNA decreases osteoclast differentiation and bone resorption in vitro

The role of proton transport and production in osteoclast differentiation w as studied in vitro by inhibiting the transcription/translation of carbonic anhydrase II (CA II) and vacuolar H+-ATPase (V-ATPase) by antisense RNA mo lecules. Antisense RNAs targeted against CA II, or the 16 kDa or 60 kDa sub unit of V-ATPase were used to block the expression of the specific proteins . A significant decrease in bone resorption rate and TRAP-positive osteocla st number was seen in rat bone marrow cultures and fetal mouse metacarpal c ultures after antisense treatment. Intravacuolar acidification in rat bone marrow cells was also significantly decreased after antisense treatment. Th e CA II antisense RNA increased the number of TRAP-positive mononuclear cel ls, suggesting inhibition of osteoclast precursor fusion. Antisense molecul es decreased the number of monocytes and macrophages, but increased the num ber of granulocytes in marrow cultures. GM-CSF, IL-3 and IL-6 were used to stimulate haematopoietic stem cell differentiation. The 16 kDa V-ATPase ant isense RNA abolished the stimulatory effect of GM-CSF, IL-3 and IL-6 on TRA P-positive osteoclast formation, but did not affect the formation of monocy tes and macrophages after IL-3 treatment, or the formation of granulocytes after IL-6 treatment. These results suggest that CA II and V-ATPase are nee ded, not only for the actual resorption, but also for osteoclast formation in vitro.