During characterization of the osteocyte-like murine long bone osteocyte-Y4 (MLO-Y4) cell line, comparison was made with antigen-presenting cells of the immune system known as dendritic cells. It was observed that the MLO-Y4 osteocyte-like cells express CD40 antigen and MHC class I antigen, but they are negative for a series of other dendritic cells markers (DEC-205, CD11b, CD11c, CD86, and MHC class II) and immune cell markers [CD45, CD3, CD4, B220, Gr-1, and CD40 ligand (CD40L)]. RT-PCR results showed expression of CD40 mRNA and lack of CD40L mRNA expression. Like MLO-Y4 osteocyte cells, both primary osteoblasts and the osteoblast-like cell lines MC3T3, OCT-1, and 2T3 were shown to express CD40 antigen by fluorescence-activated cell sorting. Because CD40L has been shown to function as an antiapoptotic factor in dendritic cells, it was reasoned that this molecule may have a similar function in bone cells. In three different assays for apoptosis, including trypan blue exclusion, changes in nuclear morphology, and fluorescence-activated cell sorting staining for annexin V/propidium iodide, CD40L significantly inhibited apoptosis of MLO-Y4 cells induced by dexamethasone, TNFα, or etoposide. CD40L also inhibited dexamethasone and TNFα-induced apoptosis in the osteoblast cell lines, OCT1 and MC3T3-E1. These data support the hypothesis that CD40L preserves viability of osteoblasts and osteocytes against a wide variety of apoptotic factors independent of signaling or transcriptional mechanisms. Because osteocyte cell death appears to result in bone loss, these studies have important implications for the treatment of bone loss due to glucocorticoid excess and/or to osteoporosis in general.
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