Molecular cloning, expression, and function of osteoclastic calcineurin Aalpha.
This study explores the role of the calmodulin- and Ca(2+)-sensitive phosphatase calcineurin A in the control of bone resorption by mature osteoclasts. We first cloned full-length calcineurin Aalpha and Abeta cDNA from a rabbit osteoclast library. Sequence analysis revealed an approximately 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 Aalpha mRNA in freshly isolated rat osteoclasts. Semiquantitative analysis of staining intensity revealed no significant difference in calcineurin Aalpha expression in cells treated with vehicle vs. those treated with the calcineurin (activity) inhibitors cyclosporin A (8 x 10(-7) M) and FK506 (5 x 10(-9) and 5 x 10(-7) M). We then constructed a fusion protein comprising calcineurin Aalpha 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 Aalpha 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 Aalpha and anti-TAT antibodies. Pit assays performed with TAT-calcineurin Aalpha-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 Aalpha 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.
Sun
Li
L
Mount Sinai Bone Program and Division of Endocrinology, Mount Sinai School of Medicine, New York, 10029, USA. li.sun@mssm.edu
Moonga
Baljit S
BS
Lu
Min
M
Zaidi
Neeha
N
Iqbal
Jameel
J
Blair
Harry C
HC
Epstein
Solomon
S
Abe
Etsuko
E
Troen
Bruce R
BR
Huang
Christopher L-H
CL
Zaidi
Mone
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001681:Biological Assay; D001862:Bone Resorption / Q000378:metabolism; D019703:Calcineurin / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D015696:Gene Products, tat / Q000235:genetics; D008969:Molecular Sequence Data; D010010:Osteoclasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
D015696:Gene Products, tat; D020033:Protein Isoforms; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D019703:Calcineurin