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 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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ABSTRACT: Early osteoclast precursors, in the form of murine bone marrow macrophages (BMMs), while expressing no detectable avb3 integrin, contain abundant avb5 and attach to matrix in an av integrin-depen- dent manner. Furthermore, avb5 expression by osteoclast precursors progressively falls as they assume the resorptive phenotype. We find the osteoclastogenic agent, tumor necrosis factor-a, (TNF) down-reg- ulates avb5 expression by BMMS via attenuation of b5 messenger RNA (mRNA) t1/2. Using BMMs from TNF receptor knockout mice we establish the p55 receptor transmits the b5 suppressive effect. The functional implications of TNF-mediated avb5 down-regulation are underscored by the capacity of an av inhibitory peptide mimetic to prevent spreading by BMMs expressing abundant avb5 while failing to impact those in which the integrin has been diminished by TNF. Finally, b5 mRNA in BMMs of wild-type mice administered lipopoly- saccharide (LPS) progressively falls with time of in vivo treatment. Alternatively, b5 mRNA does not decline in BMMs of LPS-treated mice lacking both TNF receptors, documenting down-regulation of the b5 integrin subunit, in vivo, is mediated by TNF. Thus, matrix at- tachment of osteoclast precursors and mature osteoclasts are gov- erned by distinct av integrins which are differentially regulated by specific cytokines. (Endocrinology 141: 284 -290, 2000)Endocrinology 01/2000; 141(1):284-290. · 4.72 Impact Factor
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ABSTRACT: Bone diseases such as osteoporosis are mainly caused by upregulated activity of osteoclasts. The present study was designed to examine the effects of light-emitting diode (LED) irradiation on the formation and activity of multinucleated osteoclasts, specifically "round-shaped" osteoclast cells (ROC) in different cell types derived from mouse. After 635-nm LED irradiation, the cell viability was evaluated by MTT assay. The amount of total tartrate-resistant acid phosphatase (TRAP) + osteoclast and the number of ROC cells were also estimated by TRAP solution assay and TRAP staining, respectively. Actin rings were stained with rhodamine-conjugated phalloidin, and resorption assay was performed by dentin slices. In addition, gene expression levels between the control and irradiation groups were evaluated by RT-PCR. In a morphological analysis, the formation of ROC was significantly inhibited by 635-nm LED irradiation in the different cell types. Actin rings were seen at cell peripheries in most ROC cells of the control group, but patches containing disorganized actin were found in the irradiation group. Both the number of ROCs and bone resorption activity were much lower in the irradiation group than in the control group. Also, the gene expression levels involved in actin ring formation such as integrin β3 and c-Src decreased in RT-PCR analysis. Overall, 635-nm LED therapy may play a pivotal role in regulating bone remodeling, and it may prove to be a valuable tool to prevent bone loss in osteoporosis and other resorptive bone diseases.Lasers in Medical Science 06/2013; · 2.42 Impact Factor
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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.Journal of Fish Biology 03/2005; 54(3):669 - 684. · 1.73 Impact Factor