Kynurenine Promotes RANKL-Induced Osteoclastogenesis In Vitro by Activating the Aryl Hydrocarbon Receptor Pathway

Abstract

There is increasing evidence of the involvement of the tryptophan metabolite kynurenine (KYN) in disrupting osteogenesis and contributing to aging-related bone loss. Here, we show that KYN has an effect on bone resorption by increasing osteoclastogenesis. We have previously reported that in vivo treatment with KYN significantly increased osteoclast number lining bone surfaces. Here, we report the direct effect of KYN on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis in Raw 264.7 macrophage cells, and we propose a potential mechanism for these KYN-mediated effects. We show that KYN/RANKL treatment results in enhancement of RANKL-induced osteoclast differentiation. KYN drives upregulation and activation of the key osteoclast transcription factors, c-fos and NFATc1 resulting in an increase in the number of multinucleated TRAP+ osteoclasts, and in hydroxyapatite bone resorptive activity. Mechanistically, the KYN receptor, aryl hydrocarbon receptor (AhR), plays an important role in the induction of osteoclastogenesis. We show that blocking AhR signaling using an AhR antagonist, or AhR siRNA, downregulates the KYN/RANKL-mediated increase in c-fos and NFATc1 and inhibits the formation of multinucleated TRAP + osteoclasts. Altogether, this work highlights that the novelty of the KYN and AhR pathways might have a potential role in helping to regulate osteoclast function with age and supports pursuing additional research to determine if they are potential therapeutic targets for the prevention or treatment of osteoporosis.

Keywords: AhR; Kynurenine; NFATc1; c-fos; osteoclast.

Figure 1

Kynurenine (KYN) treatment induces RANKL-mediated osteoclast differentiation of Raw 264.7. KYN treatment (10 μM or 25 μM) for 48 h does not affect viability of Raw 264.7 cells either alone (A) or with co-treatment of RANKL (B). Data are presented as mean ± SD (n = 6–12). Experiments were independently repeated three times for each condition, and a one-way ANOVA test was applied. (C) Representative images of TRAP stained Raw 264.7 cells treated with KYN at concentration of 10 μM or 25 μM for 5 days with no RANKL (upper panel) or with RANKL addition (lower panel). (D) The total number of TRAP+ multinucleated cells per well in each condition. Data are presented as mean ± SD (n = 4–6). Experiments were independently repeated three times for each condition, and a one-way ANOVA test was applied. Images were taken at 10X objective magnification with scale bar indicated. TRAP+ multinucleated cells were counted blindly.

Figure 2

Kynurenine (KYN) treatment induces RANKL-mediated osteoclast bone resorption activity of Raw 264.7 cells. (A) Representative images of resorption lacunae formed when an equal number of Raw 264.7 cells were seeded with KYN at concentrations of 10 or 25 μM for 48 h without RANKL addition (Left panel) or with RANKL (Right panel). (B) The percentage of resorption area per total area in each condition. Data are presented as mean ± SD. Experiments were independently repeated two times for each condition. ANOVA analysis was used. Images were taken at 10X objective magnification with scale bar indicated.

Figure 3

Kynurenine (KYN) treatment upregulates the expression of osteoclast differentiation markers c-fos and NFATc1. mRNA expression level of c-fos (A) or NFATc1 (B) in Raw 264.7 cells treated with KYN (10 or 25 μM) in presence of RANKL for 24 h. Data are presented relative to RANKL-only (0 μM KYN) control. Data are representative of three independent experiments and presented as mean ± SD. One-way ANOVA test was applied. Representative Western blotting of c-fos (C) or NFATc1 (D) proteins in Raw 264.7 cells treated with KYN (10 or 25 μM) in presence of RANKL for 24 h. (E) Densitometric analysis of c-fos protein expression in (C). (F) Densitometric analysis of NFATc1 protein expression in (D). Data are expressed as c-fos/β-actin or NFATc1/β-actin ratio relative to RANKL-only (0 μM KYN) control. Data are representative of three independent experiments and presented as mean ± SD. One-way ANOVA test was applied. (G) Immunocytochemistry analysis of Raw 264.7 cells treated with KYN (10 μM or 25 μM) in presence of RANKL for 24 h using NFATc1 (red) antibody. Nuclei were visualized using DAPI (blue). Images are representative of two independent experiments. Images were taken at 40X objective magnification with scale bar indicated. (H) Representative Western blotting of cytoplasmic and nuclear fractions of NFATc1 in Raw 264.7 cells treated with KYN (10 or 25 μM) in the presence of RANKL for 24 h. Histone H3 was used as nuclear marker and GAPDH was used as cytoplasmic marker. (I) Densitometric analysis of nuclear NFATc1 localization. Data are expressed as NFATc1/Histone H3 ratio relative to RANKL-only (0 μM KYN) control. Data are representative of three independent experiments and presented as mean ± SD. A one-way ANOVA test was applied.

Figure 4

Blocking AhR signaling attenuates KYN/RANKL regulation of osteoclast differentiation in Raw 264.7 cells via inhibition of c-fos and NFATc1. (A) mRNA expression level of AhR in Raw 264.7 cells in the absence or presence of RANKL for 24 h. Data are presented relative to—RANKL control. Data are representative of two independent experiments and presented as mean ± SD. Unpaired t-test was applied. (B) mRNA expression level of CYP1B1 in Raw 264.7 cells treated with KYN (10 or 25 μM) in presence of RANKL for 24 h. Data are presented relative to RANKL-only (0 μM KYN) control. Data are representative of three independent experiments and presented as mean ± SD. A one-way ANOVA test was applied. (C) mRNA expression level of CYP1B1 in Raw 264.7 cells treated with RANKL/KYN at concentration of 10 μM or 25 μM KYN for 24 h in presence or absence of DMF (10 μM). Data are presented relative to RANKL-only (0 μM KYN) control. Data are representative of two independent experiments and presented as mean ± SD. A two-way ANOVA test was applied. (D) Representative images of TRAP stained Raw 264.7 cells treated with RANKL/KYN at concentration of 10 or 25 μM for 5 days with no DMF added (upper panel) or with DMF (10 μM) (lower panel). (E) The total number of TRAP+ multinucleated cells per well in each condition. Data are presented as mean ± SD (n = 5–6). Experiments were repeated three times for each condition. A two-way ANOVA test was applied. Images were taken at 10X objective magnification with scale bar indicated. TRAP+ multinucleated cells were counted blindly. (F,G) Representative Western blotting of c-fos and NFATc1 proteins expression in Raw 264.7 cells treated with RANKL/KYN (10 or 25 μM) for 24 h in the presence or absence of DMF (10 μM). (H,I) Densitometric analysis of c-fos and NFATc1 proteins expression in (F,G). Data are expressed as c-fos/β-actin or NFATc1/β-actin ratio relative to RANKL-only (0 μM KYN) control. Data are representative of three independent experiments and presented as mean ± SD. A two-way ANOVA test was applied. (J) mRNA expression level of AhR in Raw 264.7 cells transfected with NT siRNA or AhR siRNA before treating with RANKL/KYN (10 or 25 μM) and collected 48 h post-transfection. Data are presented relative to NT siRNA control for each condition. Unpaired t-test was applied. (K) Representative Western blotting of AhR protein expression in Raw 264.7 cells transfected with NT siRNA or AhR siRNA before treating with RANKL/KYN (10 or 25 μM) and collected 72 h post-transfection. (L) Densitometric analysis of AhR protein expression in (K). Data are expressed as AhR/β-actin relative to NT siRNA control for each condition. Unpaired t-test was applied. Data are representative of two independent experiments and presented as mean ± SD. (M,N) Representative Western blotting of c-fos and NFATc1 proteins expression in Raw 264.7 cells transfected with NT siRNA or AhR siRNA before treating with RANKL/KYN (10 or 25 μM) and collected 48 h (M) or 72 h (N) post-transfection. Data are representative of two independent experiments.

Figure 5

Kynurenine Induces Osteoclastogenesis Via the AhR Signaling Pathway. In the presence of RANKL, KYN induces Raw 264.7 cells to undergo osteoclastogenesis. This is mediated by KYN binding to AhR which translocates to the nucleus. Based on the literature the ligand bound to AhR forms a transcription factor complex with ARNT [12]. This complex binds to the Xenobiotic responsive elements in the promotor regions of target genes, which here include c-fos, NFATc1 and CYP1B1 [43,45,46,47]. The down-stream consequence is osteoclast differentiation and activation of osteoclast bone resorption. These effects can be blocked by pharmacologic inhibition of AhR with the DMF, or genetic inhibition with AhR siRNA. This suggests that inhibiting KYN levels, blocking KYN binding of AhR, or blocking KYN/AhR signaling may be potential therapeutic approaches to limit the role of osteoclast activity in age-associated bone loss or osteoporosis.