This study explores the role of the calmodulin- and Ca2+-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aα and Aβ cDNA from a rabbit osteoclast library. Sequence analysis revealed an ∼95 and 86% homology between the amino acid and the nucleotide sequences, respectively, of the two isoforms. The two rabbit isoforms also showed significant homology with the mouse, rat, and human homologs. In situ RT-PCR showed evidence of high levels of expression of calcineurin Aα mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aα expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 × 10-7 M) and FK506 (5 × 10-9 and 5 × 10-7 M). We then constructed a fusion protein comprising calcineurin Aα and TAT, a 12-amino acid-long arginine-rich sequence of the human immunodeficiency virus protein. Others have previously shown that the fusion of proteins to this sequence results in their receptor-less transduction into cells, including osteoclasts. Similarly, unfolding of the TAT-calcineurin Aα fusion protein by shocking with 8 M urea resulted in its rapid influx, within minutes, into as many as 90% of all freshly isolated rat osteoclasts, as was evident on double immunostaining with anti-calcineurin Aα and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aα-positive osteoclasts revealed a concentration-dependent (10-200 nM) attenuation of bone resorption in the absence of cell cytotoxicity or changes in cell number. TAT-hemaglutinin did not produce significant effects on bone resorption or cell number. The study suggests the following: 1) the 61-kDa protein phosphatase calcineurin Aα can be effectively tranduced into osteoclasts by using the TAT-based approach, and 2) the transduced protein retains its capacity to inhibit osteoclastic bone resorption.
|Original language||English (US)|
|Journal||American Journal of Physiology - Renal Physiology|
|Issue number||3 53-3|
|Publication status||Published - Mar 1 2003|
- Calcium channel
- Gene cloning
ASJC Scopus subject areas