Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

Abstract

Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.

Transforming growth factor-β1-mediated cardiac fibrosis

“…But RTV had no direct impact on autophagy, at least in adipocytes [38]. Another PI, atazanavir, actually triggered autophagy in those cells [44], consistent with the lack of association of atazanavir with cardiac fibrosis or enhanced TRAF6 activity in our system [14, and unpublished observations]. Given the RTV-induced cardiac fibrosis seen in rodent models, and persistent cardiac fibrosis in patients on ART, we hypothesize that this secondary pathway does not predominate.…”

“…1C), but levels of key proinflammatory cytokines are not normalized, and may still have pathologic consequences [7,40]. As noted above, we demonstrated that RTV enhanced RANKL signaling via inhibition of TRAF6 degradation in the immunoproteasome [6,13,14], resulting in increased monocyte differentiation into bone-resorbing osteoclasts [11]. TRAF6 also mediates TGF-β1 signaling [29-31], so that in our opinion RTV should facilitate TGF-β activity in a manner similar to RANKL.…”

“…In our opinion, certain ART regimens could also facilitate cytokine activity via inhibition of physiologic regulators of inflammatory signaling, regardless of their effects on cytokine levels . As one example, we found that RTV abrogates a physiologic block to the activity of RANKL, the primary mediator of osteoclast differentiation, via inhibition of degradation of the nuclear signaling adaptor TNF receptor associated factor (TRAF)-6 [6,13,14]. This led to accelerated osteoclast formation in HIV+ women receiving RTV, compared to those on non-RTV based regimens [11].…”

The protease inhibitors and HIV-associated bone loss

“…NFV and IDV have been shown to alter osteoblast gene expression leading to a decrease in osteoblastic phenotype including a decrease in bone alkaline phosphatase activity and calcium deposition [65], while an increase in senescence of human MSCs when exposed to ATV and LPV leads to a decrease in differentiation to osteoblasts [59], which is consistent with clinical observations [26, 40]. Additional studies have demonstrated that RTV, at serum concentrations achieved with standard dosing for PI-boosting, increases the differentiation of peripheral blood mononuclear cells (PBMCs) into osteoclasts by upregulating growth factors and suppressing transcripts of antagonists in vitro [64, 66, 67]. A greater effect was observed in bone turnover markers (BTMs) and osteoclast differentiation from PBMCs in sera obtained from women on RTV-containing regimens compared with sera from HIV-infected women on other cART regimens as well as from HIV-uninfected women [66].…”

A novel ameloblastoma cell line (AM-3) secretes MMP-9 in response to Wnt-3a and induces osteoclastogenesis

“…29,41 Recently, protease inhibitor treatment for HIV was shown to induce Wnt-5a and accelerated RANK-L dependent-osteoclastogenesis and osteoporosis. 42,43 However, the physiological roles of high expression of Wnt-5a in AM-1 and AM-3 cells remain unclear.…”