Carbonic anhydrase II mRNA expression in individual osteoclasts under “resorbing” and “nonresorbing” conditions

Abstract

Rabbit osteoclasts can be transformed from a nonresorbing state to a resorbing state by transferring them from culture medium at pH 7.5 to one at pH 6.5. We evaluated whether expression of mRNA for carbonic anhydrase (CA‐II) could be used as an indicator of the state of activity of individual osteoclasts. A cDNA probe to rabbit carbonic anhydrase II (CA‐II) was prepared and used for in situ hybridization analysis of osteoclasts isolated from neonatal rabbit long bones. Quantitation by grain counting revealed heterogeneity within the osteoclast population: osteoclasts with a “compact” (rounded, less spread) morphology expressed higher levels of CA‐II mRNA than “spread” osteoclasts with similar numbers of nuclei. When maintained at pH 6.5 for 6 h, the level of CA‐II mRNA was increased significantly in osteoclasts of both morphologies compared with those in parallel cultures maintained at pH 7.5. These results were confirmed by quantitating CA‐II mRNA using the polymerase chain reaction (PCR). Oligonucleotide primers specific for rabbit CA‐II were synthesized and used to amplify CA‐II cDNA transcribed from mRNA prepared from single or small numbers (one to eight cells) of osteoclasts that were collected with a micromanipulator. This generated a ∼510 bp PCR product, corresponding to the predicted size of the CA‐II fragment encompassed by the primers. For quantitation, CA‐II mRNA levels were compared with the levels of a ∼900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture. The ratio of CA‐II mRNA expression to actin mRNA expression was significantly increased in osteoclasts cultured at pH 6.5 for 6 h compared with osteoclasts maintained at pH 7.5 (1.89 + 0.12 versus 0.98 + 0.06, n = 39, mean + SEM, of all assays combined; P < 0.001). Our results demonstrate that CA‐II mRNA expression is upregulated in osteoclasts in the resorptive state. The methods used provide a novel molecular approach for analyzing osteoclast activity with assays that are applicable to single cells and obviate the problem of osteoclast impurity, allowing investigation of osteoclast heterogeneity.