IL-36 Induces Bisphosphonate-Related Osteonecrosis of the Jaw-Like Lesions in Mice by Inhibiting TGF-β-Mediated Collagen Expression

Abstract

Long-term administration of nitrogen-containing bisphosphonates can induce detrimental side effects such as bisphosphonate-related osteonecrosis of the jaw (BRONJ) in human. Although inflammation is known to be associated with BRONJ development, the detailed underlying mechanism remains unknown. Here, we report that the pro-inflammatory cytokine IL-36α is, in part, responsible for the BRONJ development. We found a notably higher level of IL-36α and lower level of collagen in the BRONJ lesions in mice. We also found that IL-36α remarkably suppressed TGF-β-mediated expression of Collα1 and α-Sma via the activation of Erk signaling pathway in mouse gingival mesenchymal stem cells. When IL-36 signaling was abrogated in vivo, development of BRONJ lesions was ameliorated in mice. Taken together, we showed the pathologic role of IL-36α in BRONJ development by inhibiting collagen expression and demonstrated that IL-36α could be a potential marker and a therapeutic target for the prevention and treatment of BRONJ. © 2016 American Society for Bone and Mineral Research.

Keywords: BISPHOSPHONATE; COLLAGEN; Erk; IL-36; OSTEONECROSIS OF THE JAW; TGF-β.

Conflict of interest statement

Additional Supporting Information may be found in the online version of this article.

Disclosures

All authors state that they have no conflicts of interest.

© 2016 American Society for Bone and Mineral Research.

Figures

Fig. 1

Microarray gene profiling identifies overexpressed IL-36 family members in ONJ-like lesions in mice. (A) A graphic representation of timeline used in this study. (B) Clinical illustration of the osteomucosal tissues before and after removal at the tooth-extracted areas photographed 2 weeks after tooth extraction. (C) A graphic representation of the microarray profiling that are differentially expressed more than fivefold were represented (n = 3 per group). (D) The ingenuity pathway analysis (IPA) of functional pathways that are associated with ONJ lesions. (E) Lists of genes involved in biosynthesis of steroids. (F) List of genes involved in role of osteoblasts, osteoclasts, and chondrocytes in rheumatoid arthritis. The unhealed osteomucosal tissues at the tooth-extracted sites were harvested and subjected mRNA isolation, cDNA synthesis, and qRT-PCR for (G) IL-18, (H) IL-36α, (I) IL-36β, (J) IL-36γ, and (K) FDPS. The results are from two independent non-BRONJ and BRONJ lesions. (L) Staining for H&E and IHC staining for IL-36α at the tooth-extracted sites in vehicle (Veh)- or Zometa (ZOL)-treated mice. Scale bar = 100 µm. ELISA for (M) IL-18 and (N) IL-36α from the serum obtained from Veh- or ZOL-treated mice 2 weeks after tooth extraction. *p<0.05; **p<0.01; ***p<0.001. Results represent the means ± SD performed in triplicate unless otherwise indicated.

Fig. 2

IL-36α inhibits expression of collagen and α-Sma in GMSCs. (A) List of genes associated with connective tissues (eg, collagenous and non-collagenous proteins) from the microarray data. (B) qRT-PCR of Il-6 in gingival mesenchymal stem cells (GMSCs) treated with recombinant IL-36α, IL-36β, and IL-36γ for 4 days. (C) qRT-PCR of Col1a1, Col1a2, and Col3a1 in GMSCs treated with recombinant IL-36α, IL-36β, and IL-36γ for 4 days. (D) Western blotting of IL-1Rrp2 after knocking down with control siRNA (CTLsi) or IL-1Rrp2 siRNA (IL-1Rrp2si) in GMSCs. (E) qRT-PCR of Col1a1, Col1a2, and Col3a1 in GMSCs with IL-1Rrp2 knockdown and IL-36a treatment. (F) Western blotting of Col1a1 and α-Sma in GMSCs treated with IL-36α and TGF-β1 for 2 days. (G) ELISA for collagen type I from supernatants obtained from GMSCs treated with IL-36α and TGF-β1. (H) qRT-PCR of Col1a1, Col1a2, and α-Sma in GMSCs treated with recombinant IL-36α and TGF-β1 for 2 days. *p<0.05; **p<0.01; ***p<0.001. Results represent the means ± SD performed in triplicate.

Fig. 3

IL-36α inhibits nuclear translocation of activated Smad complex upon TGF-β1 treatment by activating the Erk signaling pathway. (A, C) Western blotting for p-Smad2 and Smad2 from the whole cell lysates, the nuclear extracts, and the cytoplasmic extracts in GMSCs treated with IL-36α for 6 hours and TGF-β1 for 2 hours. (D, E) Chromatin immunoprecipitation (ChIP) assay using IgG or Smad2 antibody followed by qRT-PCR using primers specific to the promoter regions of Col1a1 and α-Sma. The values were normalized to the internal control. (F) Western blotting for indicated antibodies in GMSCs treated with IL-36α or TNF-α for 6 hours. (G, H) qRT-PCR analysis for Col1a1 and α-Sma in GMSCs treated with U0126 (10 µM) and IL-36α (10 ng/mL) for 6 hours before adding TGF-β1 (10 ng/mL). Cells were harvested after 2 days. (I, J) Western blotting against p-Smad2 and Smad2 from the nuclear and cytoplasmic extracts. (K) PCR for genomic IL-1Rrp2 in GMSCs obtained from IL-1Rrp2+/+ or IL-1Rrp2−/− mice. (L, M) Western blotting for p-Smad2 from the nuclear and cytoplasmic extracts in IL-1Rrp2+/+ or IL-1Rrp2−/− GMSCs treated with IL-36α for 6 hours followed by TGF-β1 for 2 hours. (N) Quantification of the Western blotting (L) of p-Smad2 normalized to the Lamin B1 using ImageJ. *p<0.05; **p<0.01; ***p< 0.001. Results represent the means ± SD performed in triplicate.

Fig. 4

Anti-IL-1Rrp2 neutralizing antibody rescues ONJ lesions in mice. (A) A graphic representation of the timeline used in this study. (B) ELISA for TRAP 5b from the serum obtained from mice at the end of the experiments. (C) Percent of mice with exposed bone was assessed (n = 8–10 per group). (D) Clinical illustrations of the osteomucosal tissues at the tooth-extracted areas (left panels) and µCT scans of the maxillae taken from the occlusal views (middle panels) and the angled views (right panels). (E) H&E-stained tissues at tooth-extracted sites. Scale bar = 100 µm. (F, G) The numbers of empty lacunae per mm2 and the percentage of necrotic bones were quantified. *p<0.05; **p<0.01; ***p< 0.001.

Fig. 5

Proposed model. TGF-β1 binds TGF-β receptor and causes phosphorylation of Smad2/3, which in turns translocates into the nucleus and activates the target genes such as Col1a1 or α-Sma. IL-36α binds to IL-36 receptor complex and activates the ERK signaling pathway. Activated Erk inhibits translocation of TGF-β1-activated Smad complex, thereby suppressing the Smad-mediated activation of gene expression.