한양대학교 류마티즘연구원

Objective: 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.

Methods: Kynurenine sera level was 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.

Results: Kyn sera level was significantly elevated in the AS group compared to the HC. In addition, Kyn sera level was 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-osteoprogenitors interrupted the RANKL-mediated osteoclastogenesis of mouse osteoclast precursor such as TRAP-positive osteoclast formation, NFATc1 expression, and osteoclast differentiation markers.

Conclusion: 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.