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Accelerated bone resorption leading to osteopenia and osteoporosis has been noted in human immunodeficiency virus (HIV) seropositive, treatment-naive patients, but it may be greatly increased in incidence in those receiving highly active anti-retroviral therapies that incorporate certain protease inhibitors (PI). The pathophysiology of these processes is unclear. We have documented the induction of the primary cytokine responsible for osteoclast differentiation and bone resorption, the receptor activator of nuclear factor κB ligand (RANKL), in T cells exposed to soluble HIV-1 envelope glycoprotein gp120. Using a murine osteoclast precursor cell line as well as primary human osteoclast precursors, we demonstrate that pharmacologic levels of two PIs that are linked clinically to osteopenia, ritonavir and saquinavir, abrogate a physiological block to RANKL activity, interferon-γ-mediated degradation of the RANKL signaling adapter protein, TRAF6 (tumor necrosis factor receptor-associated protein 6) in proteasomes. In contrast, indinavir and nelfinavir, PIs that may promote or stabilize bone formation in vivo, had no impact on this system. These findings offer a molecular basis for the acceleration of bone resorption by certain PIs and provide the first example of clinically useful drugs that can interfere with the cross-talk between RANKL and interferon-γ via the proteasome. They also suggest a novel therapeutic approach to HIV osteopenia through modulation of these two molecules. Accelerated bone resorption leading to osteopenia and osteoporosis has been noted in human immunodeficiency virus (HIV) seropositive, treatment-naive patients, but it may be greatly increased in incidence in those receiving highly active anti-retroviral therapies that incorporate certain protease inhibitors (PI). The pathophysiology of these processes is unclear. We have documented the induction of the primary cytokine responsible for osteoclast differentiation and bone resorption, the receptor activator of nuclear factor κB ligand (RANKL), in T cells exposed to soluble HIV-1 envelope glycoprotein gp120. Using a murine osteoclast precursor cell line as well as primary human osteoclast precursors, we demonstrate that pharmacologic levels of two PIs that are linked clinically to osteopenia, ritonavir and saquinavir, abrogate a physiological block to RANKL activity, interferon-γ-mediated degradation of the RANKL signaling adapter protein, TRAF6 (tumor necrosis factor receptor-associated protein 6) in proteasomes. In contrast, indinavir and nelfinavir, PIs that may promote or stabilize bone formation in vivo, had no impact on this system. These findings offer a molecular basis for the acceleration of bone resorption by certain PIs and provide the first example of clinically useful drugs that can interfere with the cross-talk between RANKL and interferon-γ via the proteasome. They also suggest a novel therapeutic approach to HIV osteopenia through modulation of these two molecules. Loss of bone mineral density (BMD) 1The abbreviations used are: BMDbone mineral densityHIVhuman immunodeficiency virusPIprotease inhibitorRANKLreceptor activator of nuclear factor κB ligandIFNinterferonTNFtumor necrosis factorTRAF6TNF receptor-associated protein 6OPGosteoprotegerinFBSfetal bovine serumPBMCperipheral blood mononuclear cellsPBSphosphate-buffered salineTRAPtartrate-resistant acid phosphataseJNKc-Jun NH2-terminal kinaseMNCmultinucleated osteoclast-like cells. leading to osteopenia has been observed among HIV seropositive patients naive to antiretroviral therapy, but it is infrequent and of unclear clinical significance (1Knobel H. Guelar A. Vallecillo G. Nogues X. Diez A. AIDS. 2001; 15: 807-808Crossref PubMed Scopus (168) Google Scholar, 2Lawal A. Engelson E.S. Wang J. Heymsfield S.B. Kotler D.P. AIDS. 2001; 15: 278-280Crossref PubMed Scopus (55) Google Scholar, 3McGowan, I., Cheng, A., Coleman, S., Johnson, A., and Genant, H. (2001) 8th Conference Retrov. Opportun. 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T. Y. Y. K. T. Res. 1998; PubMed Scopus Google Scholar). this we for in cells in of in the of human factor cells were medium from control or cell cultures was 264.7 cells or cells from were for to with from control or The by of the was cell of the medium was with medium to tartrate-resistant acid phosphatase to cells were for in medium by 6 of in medium study the effect of HIV PIs and IFN-γ on osteoclast 264.7 cells were for 6 in the of RANKL and concentrations of IFN-γ and PIs. The medium was was activity as in acid phosphatase as a The was to the and activity was at For cells were in for by in the of and for at as N. Y. Y. S. K. E. E. T. Res. 2000; PubMed Scopus Google Scholar). 264.7 murine cells or cells from human were osteologic discs (BD in of and exposed to cell for from to The medium was were with and in and the of resorption was by as N. Y. Y. S. K. E. E. T. Res. 2000; PubMed Scopus Google Scholar). For formation in whale dentine were used that the with cells was The medium was and was to the for The dentine slices were by with for and with were of TRAF6 by and 264.7 cells at were for with in medium with were and for with RANKL and IFN-γ in the or of ritonavir or was anti-TRAF6 and protein were by and were by For 264.7 cells were in the of RANKL and IFN-γ with or PIs in for Cell were prepared in and and of TRAF6 was by the with anti-TRAF6 of HIV-1 gp120 in the of or cells in HIV+ patients, it is that HIV of T cells be in RANKL in However, of T cells from the of two normal to levels of recombinant HIV-1 gp120 with that in in patients in induction of RANKL protein RANKL protein was by gp120 in from normal We the activity of RANKL in two for osteoclast the osteoclast precursor murine line 264.7 and precursor cells from human cell can osteoclast-like by cells and resorption of dentine We first precursor cells from human and 264.7 with of medium from control or exposed to gp120 for 2, A and in the of 2, A and the multinucleated cells cells in both of osteoclast by from formation of resorption on dentine The of the formation of from medium 2, A and with precursor cells exposed to from cultures a in the formation of multinucleated The RANKL of the active factor in cultures was by of cell induction In monoclonal and a monoclonal data the effect of gp120 on the induction of activity in A activity was in with the as a of osteoclastogenesis with the on osteoclast the evaluation of osteoclast formation by multinucleated cell formation and by activity 2, and gp120 is known to of by V. 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D. J. for Scholar), the PI block to of osteoclastogenesis was a in osteoclast differentiation by and levels of IFN-γ the ritonavir block to control of RANKL activity at levels of IFN-γ The concentrations of IFN-γ had no effect on the of these cells. The in of the IFN-γ effect between and its effect on the ritonavir block to TRAF6 degradation as by of the the cell of relative to the the pathophysiology of of osteopenia and osteoporosis S.L. Science. 2000; Scopus Google Scholar). RANKL osteoclast differentiation from bone precursor cells in the of a H. Sarosi I. Tan H.L. Timms E. Capparelli C. S. G. A. W. A. Dunstan C.R. D.L. Boyle W.J. Nature. 1999; PubMed Scopus Google Scholar, D.L. Dunstan C.R. Kelley M. R. S. T. G. M. Elliott R. Colombero A. Tan H.L. G. Sullivan J. Davy E. N. D. W. P. Boyle W.J. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). of the RANKL in knockout mice to RANKL activity and osteoporosis in the of in or in bone N. Sarosi I. Dunstan C.R. S. J. 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Fakruddin et al. (Sat,) studied this question.