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

Authors

  • S. Asotra Ph.D.,

    Corresponding author
    1. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
    • MRC Group in Periodontal Physiology Faculty of Dentistry 1 King's College Circle University of Toronto Toronto, Ontario, Canada M5S 1A8
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  • A.K. Gupta,

    1. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • J. Sodek,

    1. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • J.E. Aubin,

    1. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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  • J. N. M. Heersche

    1. MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada
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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.

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