Transduction of TAT fusion proteins into osteoclasts and osteoblasts.
It has been difficult to transduce primary cultures of bone cells with proteins of interest. Here, we report the development and validation of a new technology for transduction of osteoblasts and osteoclasts with peptides and moderately sized proteins. Fusion proteins between TAT, an 11 amino acid Arg-rich sequence derived from the HIV protein, and either hemagglutinin or calcineurin Aalpha were synthesized and purified. Exposure of osteoblasts and osteoclasts in primary culture to either TAT-HA or TAT-calcineurin Aalpha resulted in a rapid (within 10 min) intracellular movement of the fusion protein evident on co-immunostaining. Almost 99% of cells were transduced and the fusion protein was retained in approximately 50% of the cells for up to 5 days. TAT did not abolish the functionality of calcineurin Aalpha; the fusion protein stimulated osteoblast differentiation and inhibited osteoclastic resorption. We expect that our studies will provide a firm basis for the future development of TAT fusion proteins for critical molecules involved in bone cell differentiation and function.
Dolgilevich
Svetlana
S
Mount Sinai Bone Program and Department of Medicine, Mount Sinai School of Medicine, and Bronx Veterans Affairs Medical Center GRECC, One Gustave L. Levy Place, PO 1055, Endocrinology, New York, NY 10029, USA.
Zaidi
Neeha
N
Song
Jianbo
J
Abe
Etsuko
E
Moonga
Baljit S
BS
Sun
Li
L
D000818:Animals; D001692:Biological Transport / Q000502:physiology; D019703:Calcineurin / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D015696:Gene Products, tat / Q000235:genetics* / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D010010:Osteoclasts / Q000166:cytology / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D014161:Transduction, Genetic
D015696:Gene Products, tat; D006388:Hemagglutinins; D011993:Recombinant Fusion Proteins; D019703:Calcineurin