Decreased bone resorption, osteoclast differentiation, and expression of vacuolar H+-ATPase in antisense DNA-treated mouse metacarpal and calvaria cultures ex vivo

Expression and function of vacuolar H+-ATPase, a key enzyme in bone resorpt ion, were monitored in antisense DNA-treated bone organ cultures ex vivo. A novel fluoroimmunoassay was used to quantitate mRNA levels after treatment with various antisense, sense, or random DNA oligonucleotides, Conventiona l slot blots and in vitro translation experiments were used to monitor the efficiency of the antisense molecules, In cell cultures, the used antisense molecules were transported into osteoclasts and a population of mononuclea r cells, A significant decrease in bone resorption and in the expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPa se was seen after antisense treatment. Also, osteoclast differentiation was decreased in antisense-treated mouse metacarpal cultures. These data show that the proper function of V-ATPase in osteoclasts requires expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPas e, Antisense DNA molecules can be used to inhibit osteoclast differentiatio n and function in tissue cultures, in which the physical and chemical cellu lar environment resembles that in vivo. However, more studies are needed to learn if antisense DNA molecules can be used for inhibiting bone resorptio n also in vivo.