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. 2004 Jul;114(2):206-13.
doi: 10.1172/JCI15797.

The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL-induced signaling

Michael W-H Wang  1 , Shi Wei, Roberta Faccio, Sunao Takeshita, Pablo Tebas, William G Powderly, Steven L Teitelbaum, F Patrick Ross
Affiliations

The HIV protease inhibitor ritonavir blocks osteoclastogenesis and function by impairing RANKL-induced signaling

Michael W-H Wang et al. J Clin Invest. 2004 Jul.

Abstract

Highly active antiretroviral therapy (HAART), which includes HIV protease inhibitors (PIs), has been associated with bone demineralization. To determine if this complication reflects accelerated resorptive activity, we studied the impact of two common HIV PIs, ritonavir and indinavir, on osteoclast formation and function. Surprisingly, we find that ritonavir, but not indinavir, inhibits osteoclast differentiation in a reversible manner and also abrogates bone resorption by disrupting the osteoclast cytoskeleton, without affecting cell number. Ritonavir given in vivo completely blunts parathyroid hormone-induced osteoclastogenesis in mice, which confirms that the drug is bone sparing. In keeping with its antiresorptive properties, ritonavir impairs receptor activator of nuclear factor kappaB ligand-induced (RANKL-induced) activation of NF-kappaB and Akt signaling pathways, both critical to osteoclast formation and function. In particular, ritonavir is found to inhibit RANKL-induced Akt signaling by disrupting the recruitment of TNF receptor-associated factor 6/c-Src complex to lipid rafts. Thus, ritonavir may represent a bone-sparing PI capable of preventing development of osteopenia in patients currently on HAART.

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Figures

Figure 1
Figure 1
Osteoclastogenesis is impaired by ritonavir but not indinavir. (A) Osteoclasts were generated from bone marrow macrophages stimulated with RANKL and M-CSF for 4 days in the presence of the indicated doses of ritonavir or indinavir. TRAP solution assay quantitation of osteoclast formation shows that the IC50 for ritonavir is near 10 μg/ml. In contrast, cultures exposed to indinavir show no inhibition or enhancement of osteoclast formation. (B) Representative fields of TRAP-stained osteoclasts in the presence of control medium, indinavir (10 μg/ml), and ritonavir (10 μg/ml). Magnification, ×100. (C) Ritonavir dose dependently suppresses osteoclast gene markers determined by RT-PCR analysis of osteoclasts on day 4 culture.
Figure 2
Figure 2
Osteoclastogenic arrest by ritonavir is reversible. (A) Osteoclasts were generated from bone marrow macrophages as in Figure 1 and quantitated by TRAP solution assay. The presence of ritonavir (20 μg/ml) completely suppresses osteoclast formation, while normalization of osteoclast number follows withdrawal of the PI on day 3, 4, and 5. + Ritonavir, continuous ritonavir exposure; – Ritonavir, ritonavir withdrawal. (B) TRAP-stained osteoclasts on day 7 of culture following persistent exposure to ritonavir (left panel) and withdrawal of ritonavir on day 5 (right panel). Magnification, ×200.
Figure 3
Figure 3
Osteoclast function is impaired by ritonavir. (A) Osteoclasts, generated on whale dentine slices for 3 days by treatment with RANKL and M-CSF, were exposed to control medium, ritonavir (10 μg/ml), or indinavir (10 μg/ml) for an additional 2 days. TRAP-stained dentine slices show no change in osteoclast number with exposure to PIs (top panels). Following cell removal, Coomassie blue staining of dentine slices show decreased bone pits with ritonavir treatment (bottom panels). Magnification, ×100. (B) Ritonavir, but not indinavir, decreases pit number (per 0.36 mm2), percentage of pit area, and pit depth (in micrometers) by half (each P < 0.01).
Figure 4
Figure 4
Ritonavir blocks PTH-induced osteoclast formation in vivo. (A) Osteoclast number was determined from TRAP-stained histologic sections of calvariae from mice stimulated with PTH or vehicle and intraperitoneally injected with ritonavir or vehicle. Ritonavir abrogates the osteoclast increase stimulated by PTH (n = 3 mice per group; P < 0.05). Cal. inj., calvarial injection; i.p. inj., intraperitoneal injection; Veh, vehicle. (B) Representative fields of TRAP-stained sections of calvariae show PTH injection fails to induce osteoclast formation in ritonavir-treated mice despite a robust PTH-dependent stromal cell response. Magnification, ×250.
Figure 5
Figure 5
Ritonavir inhibits RANKL-induced NF-κB activation. (A) Bone marrow macrophages pretreated with ritonavir (20 μg/ml) or vehicle for 1 hour were stimulated with RANKL for the indicated time points, and protein lysates were prepared for IκBα immunoblot. Ritonavir pretreatment impairs RANKL-induced degradation of IκBα. (B) Bone marrow macrophages pretreated with ritonavir (20 μg/ml) for the indicated time were stimulated with RANKL for 15 minutes and evaluated for NF-κB activation. Ritonavir inhibits NF-κB activation as assessed by EMSA after 1 hour and after 18 hours of pretreatment (Pretx). Numbers below lanes indicate relative band intensity. (C) Cell lysates prepared as in A and immunoblotted for phospho-IκBα (p-IκBα) and IκBα reveal similar levels of phospho-IκBα intensity, irrespective of ritonavir pretreatment, despite the failure of ritonavir pretreatment to decrease total IκBα levels. β-Actin immunoblots confirm similar amounts of cell extracts were analyzed.
Figure 6
Figure 6
Ritonavir inhibits RANKL-induced Akt activation. (A) RAW 264.7 cells pretreated with ritonavir (2 μg/ml) for 1 hour and stimulated with RANKL (+RANKL) for the indicated time points. Phospho-Akt (p-Akt), total Akt (Akt), and phospho-FKHR (p-FKHR) immunoblots were performed on cell extracts. Immunoblots reveal impaired Akt and FKHR phosphorylation with ritonavir pretreatment. (B) Osteoclasts were stimulated with either RANKL or M-CSF for the indicated time points, and cell extracts were immunoblotted for Akt activation. Ritonavir inhibits only RANKL-induced Akt activation but not that stimulated by M-CSF. Total Akt immunoblots confirm that similar amounts of cell extracts were analyzed. +V, vehicle pretreatment; +R, ritonavir pretreatment.
Figure 7
Figure 7
Introduction of PI3K-CA restores RANKL-induced phosphorylation of Akt and osteoclast actin ring formation in the presence of ritonavir. (A) Retroviral transduction of either vector or PI3K-CA (p110a-CA) into bone marrow macrophages was followed by 3 days of culture in selection media, M-CSF, and RANKL. After starvation (3 hours) and pretreatment (1 hour) with either vehicle or ritonavir, cells were stimulated with RANKL for 15 minutes. Immunoblots reveal restoration of RANKL-induced Akt phosphorylation when PI3K-CA is introduced. As expected, total PI3K is enhanced as a result of transduction (p110α blot). TRAF6 Western blots act as a loading control. (B) Percentage of osteoclasts with intact actin rings after ritonavir exposure is quantitated. (C) Osteoclasts, retrovirally transduced with either vector or PI3K-CA, were generated on glass coverslips. After 4 days, cells were exposed to various doses of ritonavir for 2 hours, then processed for immunofluorescence microscopy for β-actin. Dose-dependent disruption of the characteristic actin ring of the osteoclast cytoskeleton is observed in vector but not PI3K-CA–transduced cells.
Figure 8
Figure 8
RANKL-induced recruitment of c-Src and TRAF6 to lipid raft component is inhibited by ritonavir treatment. Preosteoclasts generated after 3 days of culture with RANKL and M-CSF were starved for 3 hours, followed by pretreatment with either vehicle or ritonavir (1 hour). Cells were then stimulated with RANKL for 5 minutes, followed by lipid raft isolation. Note the continued recruitment of TRAF2 to lipid rafts with ritonavir treatment, indicating that the inhibition is specific to TRAF6 and c-Src.
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References

    1. Manolagas SC. Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. Endocr. Rev. 2000;21:115–137. - PubMed
    1. Teitelbaum SL. Bone resorption by osteoclasts. Science. 2000;289:1504–1508. - PubMed
    1. Suda T, et al. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr. Rev. 1999;20:345–357. - PubMed
    1. Ross FP. RANKing the importance of measles virus in Paget’s disease. J. Clin. Invest. 2000;105:555–558. - PMC - PubMed
    1. Paton NIJ, Macallan DC, Griffin GE, Pazianas M. Bone mineral density in patients with human immunodeficiency virus infection. Calcif. Tissue Int. 1997;61:30–32. - PubMed

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