Rac1 and Rac2 in Osteoclastogenesis: A Cell Immortalization Model

Abstract

Cell lines generated from primary cells with a particular gene deletion are useful for examining the function of the specific deleted genes and provide the opportunity to genetically rescue the lost genes using standard gene transfection techniques. In the present study, bone marrow monocytes from wild-type (WT), Rac1 null, and Rac2 null mice were primed with macrophage colony-stimulating factor and soluble receptor activator of NF-κB ligand to generate preosteoclasts. This was followed by transduction of a retrovirus containing simian virus 40 large T-antigen and a neomycin-resistant cassette. Seven to 19 immortalized cell lines from each genotype were established. Among them, WT2, Rac1 null-D9, and Rac2 null-A2 were characterized to verify that osteoclastogenesis and osteoclast functions were identical to the parental primary cells. Results showed that immortalized WT2 cells were able to differentiate into mature, multinucleated, functional, tartrate-resistant acid phosphatase-positive osteoclasts. Immortal Rac1 null cells, as with their primary cell counterparts, displayed a severe defect in osteoclastogenesis and function. Transfection of the Rac1 gene into Rac1 null cells was sufficient to rescue osteoclastogenesis. We believe this method of generating immortalized preosteoclasts will provide a key tool for studying the signaling mechanisms involved in osteoclastogenesis.