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This Article Full Text Full Text (PDF) All Versions of this Article: 288/5/E1011    most recent 00534.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via ISI Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Gilbert, L. C. Articles by Nanes, M. S. Search for Related Content PubMed PubMed Citation Articles by Gilbert, L. C. Articles by Nanes, M. S.

The p55 TNF receptor mediates TNF inhibition of osteoblast differentiation independently of apoptosis

Division of Endocrinology and Metabolism, Department of Medicine, Emory University School of Medicine and Veterans Affairs Medical Center, Atlanta, Georgia

Submitted 2 November 2004 ; accepted in final form 23 December 2004

After menopause, increased tumor necrosis factor- (TNF-) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and -glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55^+/+/p75^–/– MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55^–/– MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with >4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

osteoblast; p75; tumor necrosis factor receptor 1; tumor necrosis factor receptor 2; bone; osteoporosis; tumor necrosis factor; apoptosis