Abnormal kynurenine level contributes to the pathological bone features of ankylosing spondylitis

Abstract

Abstract

Abnormal kynurenine level contributes to the pathological bone features of ankylosing spondylitis

Chanhyeok Jeon Department of Translational Medical Science Graduate School of Biomedical Science & Engineering Hanyang University

Ankylosing spondylitis (AS) exhibits paradoxical bone features typically characterized by new bone formation and systemic bone loss. Although abnormal kynurenine (Kyn), a tryptophan metabolite, has been closely linked to the disease activity of AS, the distinct role of its pathological bone features remains unknown.

Kynurenine serum levels were collected from healthy control (HC; n = 22) and AS (n = 87) patients and measured by ELISA. In the AS group, we analyzed and compared the Kyn level based on the modified stoke ankylosing spondylitis spinal score (mSASSS), MMP13, and OCN. Under osteoblast differentiation, the treatment with Kyn in AS-osteoprogenitors conducted cell proliferation, alkaline phosphatase activity, bone mineralization-related alizarin red s (ARS), von kossa (VON), hydroxyapatite (HA) staining, and mRNA expression markers (ALP, RUNX2, OCN, and OPG) for bone formation. TRAP and F-actin staining was used for osteoclast formation of mouse osteoclast precursors.

Kyn serum levels were significantly elevated in the AS group compared to the HC. In addition, Kyn serum levels were correlated with mSASSS (r = 0.03888, p = 0.067), MMP13 (r = 0.0327, p = 0.093), and OCN (r = 0.0436, p = 0.052). During osteoblast differentiation, treatment with Kyn exhibited no difference in cell proliferation and alkaline phosphate (ALP) activity for bone matrix maturation but promoted ARS, VON, and HA staining for bone mineralization. Interestingly, osteoprotegerin (OPG) and OCN expressions of AS-osteoprogenitors were augmented in the Kyn treatment during differentiation. In growth medium, Kyn treatment of AS-osteoprogenitors resulted in induction of OPG mRNA, protein expression, and Kyn-response genes (AhRR, CYP1b1, and TIPARP). Secreted OPG proteins were observed in the supernatant of AS-osteoprogenitors treated with Kyn. Notably, the supernatant of Kyn-treated AS-osteoprogenitor interrupted the RANKL-mediated osteoclastogenesis of mouse osteoclast precursor such as TRAP-positive osteoclast formation, NFATc1 expression, and osteoclast differentiation markers.

Our results revealed that elevated Kyn level increased the bone mineralization of osteoblast differentiation in AS and decreased RANKL-mediated osteoclast differentiation by inducing OPG expression. Out study have implication for potential coupling factors linking osteoclast and osteoblast where abnormal Kyn level could be involved in pathological bone features of AS.|국문요지

강직척추염에서 kynurenine에 의한 뼈 항상성의 OPG-RANKL 조절 기전 연구

전찬혁 임상의과학과 한양대학교 의생명공학전문대학원

강직척추염은 과도한 조골세포의 활성으로 인한 뼈 항상성의 불균형을 임상적 특징으로 갖고 있다. Tryptophan의 대사물질인 kynurenine(Kyn)은 강직척추염 환자에서 높은 수준을 유지하고 질병 활성과의 상관관계가 보고되었다. 하지만 Kyn이 뼈 항상성을 조절하는 정확한 메커니즘은 아직 알려지지 않았다. 따라서 본 연구는 강직척추염 환자에서 증가된 Kyn이 뼈 항상성을 조절하는 기전을 규명하고자 한다.

강직척추염 환자의 Kyn의 혈청 수준은 건강 대조군에 비해 높은 수준의 증가를 보였다. 강직척추염에서 Kyn의 높은 수준은 혈청 내 mSASSS, MMP13, OCN 단백질과 양의 상관관계를 확인하였다. 다음으로 강직척추염 환자로부터 얻어진 osteoprogenitors에서 Kyn에 의한 골 분화와 세포증식의 변화를 확인하였다. 첫 번째, Kyn 처리는 osteoprogenitors의 세포증식에 영향을 주지 않는 것을 MTT assay를 통해 확인하였다. 또한 growth medium 조건의 Kyn 처리는 OPG와 Kyn 반응 유전자(AhRR, CYP1b1 그리고 TIPARP)의 mRNA와 단백질의 발현량을 증가시켰다. 상층액에서 분비된 OPG 단백질량은 Kyn처리 조건에서 증가되었다. 흥미롭게도 Kyn으로 처리된 osteoporgenotrs의 배양액의 처리는 마우스 파골 전구세포의 분화과정 동안 TRAP 양성 세포, NFATc1 발현과 파골세포 분화의 마커의 감소를 나타냈다.

결론적으로, 강직척추염에서 증가된 Kyn은 조골세포 분화의 뼈 무기질화를 증가시켰고, OPG 발현 증가를 유도함으로써 RANKL 매개 파골세포 분화를 감소시켰다. 본 연구는 강직척추염에서 증가된 Kyn이 뼈 항상성을 조절함으로써 비정상적인 뼈 형성에 기여하는 기전을 보여주었다.