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

MRC Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Journal of Bone and Mineral Research (Impact Factor: 6.83). 07/2009; 9(7):1115-22. DOI: 10.1002/jbmr.5650090720
Source: PubMed


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 approximately 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 approximately 900 bp actin fragment that was coamplified in the same reaction mixture or amplified separately in a duplicate sample of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)

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    • "whereas less active osteoclasts retain type 2 morphology. Supporting this assumption are data showing that the morphology of rabbit osteoclasts changes with the cell activity: less active osteoclasts are flattened and spread, whereas more active osteoclasts are compact, rounded, and less spread, having a greater height relative to attached surface (Asotra et al., 1994). Further, mammalian osteoclasts share morphological features with both type 1 and 2 scale osteoclasts, as they appear asymmetric, with several cell processes but are generally lacking folds on the cell surface (Helfrich & Mieremet, 1988; Jones & Boyde, 1994; Muller-Mai et al., 1990). "
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    ABSTRACT: The morphology of scale osteoclasts in rainbow trout Oncorhynchus mykiss was characterized by light and scanning electron microscopy, and the effects of oestradiol-17β-treatment and sexual maturation on scale osteoclast morphology were investigated. The cells associated with resorption cavities could be distinguished morphologically as two types: symmetrical, compact cells lacking or having only a few cell processes, termed type 1 cells, and asymmetrical cells covered with folds and having several cell processes, termed type 2 cells. In adult sexually maturing fish, where scale resorption was high, type 1 cells were predominant. In juveniles and spawned adults where scale resorption was assumed to be relatively low, mostly type 2 cells were present. Oestradiol 17-β-treatment of juvenile rainbow trout increased the osteoclast activity, but did not affect the osteoclast morphology. Using light microscopy, the majority of the cells observed in, and closely associated with, the resorption cavities were mononucleated in both maturing and spawned fish. Occasionally, bi- and multinucleated osteoclasts were observed in the maturing, but not in the spawned fish. Light microscopic enzyme-histochemistry showed that the majority of the mononucleated cells, as well as the bi- and multinucleated ones, were tartrate resistant acid phosphatase positive in both groups of fish, thus implying that both type 1 and type 2 cells were osteoclasts. It is thus apparent that scale resorption in rainbow trout is carried out by two morphologically distinct osteoclast populations, representing different stages of osteoclast activity and/or different stages of osteoclast differentiation.
    Full-text · Article · Mar 2005 · Journal of Fish Biology
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    ABSTRACT: We have examined parameters of bone metabolism in a new mutation, microphthalmia blanc (mib), in the rat exhibiting a skeletal sclerosis at birth that improves with age. There were no significant differences in the rate of bone formation during the first postnatal month except a temporary reduction in mutants at 3 weeks that coincided with compromised nutrition at weaning. At birth the ruffled border in mutant osteoclasts was absent or poorly developed and mRNA analyses of mutant bone compared to normal bone showed significant reductions in the messages for the osteoclast-specific genes carbonic andydrase II and tartrate-resistant ATPase. These distinctive ultrastructural and molecular differences were not present 1 month later. These data show that the transient osteopetrosis in mib rats results from a perinatal reduction in ultrastructural and enzymatic features of active osteoclasts and is not complicated by elevations in bone formation. The molecular basis for both the production and resolution of these abnormalities deserves further study.
    No preview · Article · Jun 1995 · Bone
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