Age-related increase of kynurenine enhances miR29b-1-5p to decrease both CXCL12 signaling and the epigenetic enzyme Hdac3 in bone marrow stromal cells

Abstract

Mechanisms leading to age-related reductions in bone formation and subsequent osteoporosis are still incompletely understood. We recently demonstrated that kynurenine (KYN), a tryptophan metabolite, accumulates in serum of aged mice and induces bone loss. Here, we report on novel mechanisms underlying KYN's detrimental effect on bone aging. We show that KYN is increased with aging in murine bone marrow mesenchymal stem cells (BMSCs). KYN reduces bone formation via modulating levels of CXCL12 and its receptors as well as histone deacetylase 3 (Hdac3). BMSCs responded to KYN by significantly decreasing mRNA expression levels of CXCL12 and its cognate receptors, CXCR4 and ACKR3, as well as downregulating osteogenic gene RUNX2 expression, resulting in a significant inhibition in BMSCs osteogenic differentiation. KYN's effects on these targets occur by increasing regulatory miRNAs that target osteogenesis, specifically miR29b-1-5p. Thus, KYN significantly upregulated the anti-osteogenic miRNA miR29b-1-5p in BMSCs, mimicking the up-regulation of miR-29b-1-5p in human and murine BMSCs with age. Direct inhibition of miR29b-1-5p by antagomirs rescued CXCL12 protein levels downregulated by KYN, while a miR29b-1-5p mimic further decreased CXCL12 levels. KYN also significantly downregulated mRNA levels of Hdac3, a target of miR-29b-1-5p, as well as its cofactor NCoR1. KYN is a ligand for the aryl hydrocarbon receptor (AhR). We hypothesized that AhR mediates KYN's effects in BMSCs. Indeed, AhR inhibitors (CH-223191 and 3',4'-dimethoxyflavone [DMF]) partially rescued secreted CXCL12 protein levels in BMSCs treated with KYN. Importantly, we found that treatment with CXCL12, or transfection with an miR29b-1-5p antagomir, downregulated the AhR mRNA level, while transfection with miR29b-1-5p mimic significantly upregulated its level. Further, CXCL12 treatment downregulated IDO, an enzyme responsible for generating KYN. Our findings reveal novel molecular pathways involved in KYN's age-associated effects in the bone microenvironment that may be useful translational targets for treating osteoporosis.

Keywords: AhR; CXCL12; Hdac3; Kynurenine; miR-29b-1-5p.

Fig. 1

Aging and kynurenine downregulate CXCL12 axis. (A) CXCl12 protein levels in murine plasma from 3, 6, 12, 18, 24, and 29 months-old mice (n = 10). (B) CXCl12 protein levels in murine bone marrow interstitial fluid from 3, 6, 12, 18, 24, and 29 months-old mice (n = 10 mice per group). (C) Kynurenine level (μM) in cell culture media of BMSCs isolated from 6 and 18 months old mice (n = 3). (D,E) mRNA levels of CXCL12 (D) and CXCR4 (E) in BMSCs isolated from 6, 11, and 27 months-old mice (n = 4 for 6 and 11 months old mice and n = 7 for 27 months old mice). (F) CXCL12 protein levels in BMSCs isolated from pooled 6 months-old mice after treatment with 200 μM kynurenine for 48 h (n = 4). (G-I) mRNA levels of CXCL12 (F), and CXCR4 (G) and ACKR3 (H) after treatment with 50 and 200 μM kynurenine for 6 h (n = 3). Data presented as mean ± SD. Data analysis was done using one way ANOVA for panels A, B, D, E, F, G, H, and I, and using unpaired t-test for panel C. Data presented as mean ± SD. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2

Kynurenine inhibits BMSCs osteogenic differentiation. (A) Optical density of alizarin red osteogenic assay staining and corresponding wells of BMSCs isolated from pooled 6 months-old mice after treatment with different doses of kynurenine (10, 50, and 200 μM) for 21 days (n = 6). (B) Alkaline Phosphatase (ALP) activity relative to control in BMSCs isolated from pooled 6 months-old mice treated with different doses of kynurenine (10, 50, and 200 μM) for 7 days (n = 6) (C) mRNA levels of Runx2 in BMSCs isolated from young (6–8 months-old) and old (22–24 months-old) mice, in osteogenic differentiation medium with or without 200 μM kynurenine for 7 days (n = 3 mice per group). (D) Protein levels of Runx2 in BMSCs isolated from 6 months mice after incubation in osteogenic differentiation medium with or without 200 μM kynurenine for 7 days. Data presented as mean ± SD. Data analysis was done using one way ANOVA for panels A, B, and C, and using unpaired t-test for panel D. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 3

Kynurenine upregulates pro-aging and CXCL12-targeting miR-29b-1-5p. (A,B) miR-29b-1-5p (A) and miR-29b-1-3p (B) levels in BMSCs isolated from 11 versus 27 months old (n = 4 for 11 months old mice and n = 7 for 27 months old mice). (C,D) miR-29b-1-5p levels (C) and miR-29b-1-3p levels (D) in BMSCs isolated from pooled 6 months-old mice after treatment with 200 μM kynurenine for 1, 3, 6, 24 and 48 h (n = 3). (E,F) CXCL12 levels in BMSCs isolated from 6 months old mice after treatment with 200 μM kynurenine with or without transfection with miR-29b-1-5p inhibitor (E) or miR-29b-1-5p mimic (F) (n = 8 for inhibitor and n = 4 for mimic). (G) Luciferase activity of CXCL12 reporter plasmids (wild type and mutant) after transfection with miR-29b-1-5p mimic relative to luciferase activity of wild type CXCL12 reporter plasmid after transfection with miRNA mimic control for 24 h (n = 3). Data presented as mean ± SD. Data analysis was done using one way ANOVA for panels E, F amd G, using two way ANOVA for panels C and D, and using unpaired t-test for panels A and B. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 4

Kynurenine downregulates Hdac3 and its cofactors. (A,B) mRNA expression levels of (A) Hdac3 and (B) NcoR1 in BMSCs isolated directly from young and old mice and cultured in osteogenic differentiation medium with or without 200 μM kynurenine for 7 days (n = 3). (C,D) mRNA expression levels of Hdac3 in BMSCs isolated from pooled 6 months-old mice and transfected with either (C) miR-29b-1-5p mimic or (D) miR-29b-1-5p inhibitor compared to their respective controls (n = 3). (E) Luciferase activity of Hdac3 reporter plasmids (wild type and mutant) after transfection with miR-29b-1-5p mimic relative to luciferase activity of wild type Hdac3 reporter plasmid after transfection with miRNA mimic control for 24 h (n = 3). (F,G) Protein levels of Hdac3 (F) and H4 acetylation (G) in BMSCs isolated from pooled 6 months-old mice after incubation in osteogenic differentiation medium with or without 200 μM kynurenine for 7 days. Data presented as mean ± SD. Data analysis was done using one way ANOVA for panel E, using two way ANOVA for panels A and B, and using unpaired t-test for panels C, D, F, and G. *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 5

AhR antagonist inhibits kynurenine-induced CXCL12 downregulation. CXCL12 protein levels in BMSCs isolated from pooled 6-month-old mice after treatment for 48 h with 100 or 200 μM kynurenine with or without 10 μM of AhR-antagonist 3′,4′-Dimethoxyflavone (DMF) (n = 3). Data presented as mean ± SD. Data analysis was done using unpaired t-test. **P < 0.01.

Fig. 6

CXCL12 regulates mRNA levels of IDO-1 and AhR. (A,B) mRNA expression levels of (A) IDO-1, and (B) AhR after treatment with CXCL12 (200 ng/mL) for 24 and 6 h respectively (n = 4). Data presented as mean ± SD. Data analysis was done using one way ANOVA for panel C, and using unpaired t-test for panels A and B. *P < 0.05, ***p < 0.001, ****p < 0.0001.

Fig. 7

Proposed pathway of kynurenine effects on BMSCs. Aging upregulates activity of IDO1 which converts tryptophan to kynurenine. Kynurenine is then taken up by BMSCs and binds to cytoplasmic AhR. Upon binding, ligand-bound AhR is transported into the nucleus where it forms a complex with Aryl hydrocarbon receptor nuclear translocator (ARNT). The newly formed complex acts as a transcription factor and downregulates Hdac3 and CXCL12 transcription while upregulating miR-29b-1-5p. Hence, the overall effect of kynurenine and AhR complex is shifting from osteogenic to adipogenic differentiation in BMSCs. CXCL12 acts via a feedback regulation mechanism to downregulate IDO1 and AhR mRNA levels, while miR-29b-1-5p upregulates AhR mRNA level.