0114: A Rare Variant in the extl3 Gene Is Associated with Altered Wnt Signaling Pathway in a Novel Mouse Model of Axial Spondyloarthritis

Background/Purpose: The ossification processes observed in ankylosing spondylitis (AS) remain incompletely understood. Substantial evidence from both human and animal studies implicates the Wnt signaling pathway in the excessive endochondral ossification characteristic of AS. Through linkage analysis, we identified a rare variant in the EXTL3 gene within a family affected by a severe familial form of AS. EXTL3 is a member of the exostosin gene family and is essential for heparan sulfate biosynthesis.


We generated a mouse model heterozygous for the Extl3 mutation (Extl3^mut/+) that develops osteoarticular lesions of the sacroiliac joint (SIJ), resembling those found in axial spondyloarthritis (axSpA), along with altered trabecular bone structure in the spine and SIJ. These abnormalities are accompanied by accelerated chondrocyte differentiation, disrupted cartilage architecture in the SIJ, and impaired osteoblast differentiation and mineralization.

Objective
To investigate the role of Wnt signaling and the effects of its modulation in this mouse model of axSpA.

Methods: Immunohistochemical analysis of Tcf1 was performed on SIJs from 6-month-old Extl3^mut/+ mice and wild-type littermates (Extl3^mut/+). In vitro, primary osteoblast and chondrocyte cultures were established at various differentiation stages. RNA sequencing, qPCR, and Western blot (WB) analyses were conducted at different time points.
To confirm the involvement of Wnt signaling, three Wnt pathway inhibitors—Dkk-1, sFRP2, and PKF 118-310—targeting different components of canonical and non-canonical pathways were applied to osteoblast and chondrocyte cultures. The aforementioned analyses were repeated following inhibitor treatment.

Results: Tcf1 immunostaining was markedly increased in chondrocyte columns and subchondral bone of Extl3^mut/+SIJs. RNA-seq revealed downregulation of Wnt pathway inhibitors Dkk-1 and Sclerostin at late stages of both chondrocyte and osteoblast differentiation. qPCR analysis demonstrated overexpression of Wnt pathway components, including

β-catenin

and Tcf7. Accelerated chondrocyte differentiation appeared to involve Wnt3, Wnt4, Wnt5a, and Wnt5b.
Overexpression of Tcf1 in Extl3^mut/+ osteoblast cultures was confirmed by Western blot at day 21 (terminal differentiation), and by immunohistochemistry in SIJ cartilage and subchondral bone. Notably, Wnt pathway inhibition, particularly with Dkk-1, reversed the observed phenotypes, including delayed osteoblast and accelerated chondrocyte differentiation.

Conclusion: Our findings demonstrate that the Extl3 mutation disrupts Wnt signaling in both osteoblasts and chondrocytes. These results align with prior studies in mouse models and human data, reinforcing the critical role of Wnt signaling in the pathological ossification associated with AS. This work highlights potential therapeutic targets aimed at modulating ossification mechanisms in AS.