Characterization of the osteoclast vacuolar H(+)-ATPase B-subunit

During bone resorption, osteoclasts acidify the extracellular bone resorbing compartment via a vacuolar H(+)-ATPase (V-ATPase), which resides in the ruffled-border membrane. In an effort to characterize the composition of the osteoclast V-ATPase catalytic domain, we have isolated a cDNA clone that encodes the V-ATPase B-subunit from a cDNA library constructed from highly purified chicken osteoclasts. Comparison of the predicted amino-acid sequence with the published sequences of isoforms of V-ATPase B-subunits from other sources revealed that the chicken osteoclast B-subunit is brain type and not kidney type. Furthermore, only clones encoding the brain type isoform of subunit B could be generated by PCR from a cDNA library prepared from human osteoclastoma osteoclast-like cells. Northern blot analysis revealed that two B-subunit mRNAs, approx. 1.7 and 3.5 kb in length, are expressed in chicken bone marrow mono-nuclear cells, brain and kidney, although the relative amounts of these two transcripts were different in each tissue. In brain, the 3.5-kb mRNA was predominantly expressed. In bone marrow cells, the levels of the 1.7-kb mRNA were higher than in other tissues and expression of this message was increased by 1,25-dihydroxyvitamin D-3, suggesting that this mRNA is specifically upregulated during osteoclast differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)