Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL expression through Ets-2

Abstract

Objective. To investigate why bisphosphonates are less effective at preventing focal bone loss in rheumatoid arthritis (RA) patients than in those with generalized osteoporosis, and the mechanisms involved.Methods. The response of osteoclasts to alendronate (ALN) in tumor necrosis factor-transgenic (TNFTg) mice that develop erosive arthritis and in wild-type littermates was studied. TNF-Tg and wild-type mice were given ALN, and the osteoclast numbers in the inflamed joints and in the long bones were compared. The expression levels of Bcl-xL in the osteoclasts of TNF-Tg and wild-type mice were examined by immunostaining. The effect of overexpression of Bcl-xL and Ets-2 proteins on ALN-induced osteoclast apoptosis was determined using an in vitro osteoclast survival assay and retrovirus transfer approach.Results. ALN reduced osteoclast numbers in the metaphyses by 97%, but by only 46% in the adjacent inflamed joints. Bcl-xL expression was markedly higher in osteoclasts in the joints than in those in the metaphyses of TNF-Tg mice. Bcl-xL or Ets-2 overexpression protected osteoclasts from ALN-induced apoptosis, and TNF stimulated Bcl-xL and Ets-2 expression in osteoclasts. Overexpression of Ets-2 increased Bcl-xL messenger RNA in osteoclasts, while a dominant-negative form of the Ets-2 blocked the protective effect of Bcl-xL or TNF on ALN-induced apoptosis.Conclusion. The reduced efficacy of bisphosphonates to stop bone erosion in the inflamed joints of RA patients may result from local high levels of TNF up-regulating Ets-2 expression in osteoclasts, which in turn stimulates Bcl-xL expression in them and reduces their susceptibility to bisphosphonate-induced apoptosis.

Tumor Necrosis Factor-α Increases Circulating Osteoclast Precursor Numbers by Promoting Their Proliferation and Differentiation in the Bone Marrow through Up-regulation of c-Fms Expression

“…Because M-CSF is constitutively expressed in the bone microenvironment in vivo, and TNF␣ stimulates osteoblasts and T lymphocytes to up-regulate their expression of M-CSF (16,17), TNF␣-induced c-Fms expression must work through both M-CSF-dependent and -independent mechanisms. We have reported previously that TNF␣ promotes mature osteoclast survival (33). However, we did not observe clear changes in OCP apoptosis.…”

VEGF-C, a Lymphatic Growth Factor, Is a RANKL Target Gene in Osteoclasts That Enhances Osteoclastic Bone Resorption through an Autocrine Mechanism

“…After confirmation of the VEGF-C sequence by DNA sequencing, VEGF-C was cloned into the EcoRI site of the pMX-IRES-GFP vector provided by Dr. Matsuo Koichi. The retrovirus vector was transfected into the Plat-E retroviral packaging cell line, and viral supernatant was collected 48 h later for infection as we described previously (31,33). pMX-GFP plasmid was used as control vector.…”

“…The cells were then cultured with 1:20 diluted conditioned medium containing M-CSF (from an M-CSF-producing cell line) (34) and RANKL with or without additional treatment to form mature osteoclasts. Alternatively, the cells were cultured with M-CSF for 3 days to generate OCPs, as we described previously (33), and then infected with retroviral supernatants and cultured with M-CSF and RANKL on bone slices to form mature osteoclasts. The cells were fixed and stained for tartrate-resistant acid (TRAP) activity to identify osteoclasts as TRAP ϩ cells containing at least three nuclei and then removed and stained with 0.5% toluidine blue to visualize resorption pits for bone resorption activity, as we described previously (31,33).…”

The antiapoptotic protein Bcl-xL negatively regulates the bone-resorbing activity of osteoclasts in mice

“…Pioneering work by Roodman and coworkers demonstrated that the targeting of Bcl-xL and SV40 large T antigen to cells of the osteoclast lineage in transgenic mice under the control of the TRAP promoter immortalized osteoclast precursors (19). Zhang et al reported that TNF-α inhibited alendronate-induced apoptosis of osteoclasts by stimulating Bcl-xL expression (20). On the other hand, Jimi et al (21) reported that Bcl-2 and Bcl-xL in osteoclasts were not upregulated by RANKL treatment.…”