Abstract
We investigated the NF-kappaB transcription factor in osteoclast-like cells. Osteoclast-like cells were differentiated from mouse bone marrow cells in co-culture with mouse calvaria-derived primary osteoblasts in the presence of 1alpha,25-dihydroxyvitamin D3 and prostaglandin E2 in collagen gel-coated dishes. We enriched osteoclast-like cells from the co-cultures by Pronase treatment. When the enriched osteoclast-like cells were treated with phorbol 12-myristate 13-acetate, interleukin-1 (IL-1), calcitonin, or macrophage colony-stimulating factor, only IL-1 activated an NF-kappaB-like factor, which specifically bound to a kappaB motif DNA sequence, as detected by an electrophoretic mobility shift assay. IL-1 also activated NF-kappaB induction in osteoblasts. However, the NF-kappaB-like factor induced by IL-1-stimulated osteoclast-like cells is of smaller molecular size than the factor in osteoblasts, as shown by an electrophoretic mobility shift assay. The NF-kappaB activity of osteoclast-like cells was recognized completely by antibodies against the p50 subunit, and only partially by antibodies against the p65 subunit of NF-++kappaB. Antibodies against c-Rel, Rel B, and p52 did not recognize the NF-kappaB-like factor. These results suggest that IL-1 activates an NF-kappaB-like factor in osteoclast-like cells, which contains p50 and p65-related proteins.
Highlights
We investigated the NF-B transcription factor in osteoclast-like cells
Mouse bone marrow cells were co-cultured with the primary osteoblasts, which were derived from mouse calvaria, in the presence of 10Ϫ8 M 1␣,25(OH)2 D3 and 10Ϫ6 M prostaglandin E2 (PGE2)
The cell suspensions were reinoculated into plastic dishes (Fig. 1A), and after 10 h adherent cells were treated with Pronase E to remove the weakly adhered osteoblasts and other bone marrow cells
Summary
We investigated the NF-B transcription factor in osteoclast-like cells. Osteoclast-like cells were differentiated from mouse bone marrow cells in co-culture with mouse calvaria-derived primary osteoblasts in the presence of 1␣,25-dihydroxyvitamin D3 and prostaglandin E2 in collagen gel-coated dishes. A method where osteoclast-like cells were induced from cultured bone marrow cells under a certain set of conditions was developed [1,2,3]. 1 The abbreviations used are: MNC, multinucleated cell(s); TRAP, tartrate-resistant acid phosphatase; CT, calcitonin; NF-B, nuclear factor B; IL, interleukin; PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethanesulfonyl fluoride; EMSA, electrophoretic mobility shift assay; OCL, osteoclast-like cell(s); POB, primary osteoblast(s); DTT, dithiothreitol; MMP, matrix metalloproteinase; 1␣,25(OH)2 D3, 1␣,25-dihydroxyvitamin D3.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
