Background/Purpose: Fibronectin fragments (FN-fs) are increased in the synovial fluid of osteoarthritis patients and have a potent chondrolytic effect. However, little is known about the cellular receptors and signaling mechanisms that are mediated by FN-fs. Here we investigated whether the 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) regulates cartilage metabolism through Toll-like receptor-2 (TLR-2) signaling pathway in human articular chondrocytes.

Methods: Human articular chondrocytes were enzymatically isolated from articular cartilage and cultured in monolayer. In order to investigate whether 29-kDa FN-f induces MMPs production through TLR-2, human chondrocytes were transfected with TLR-2 expression plasmid or small interfering RNAs (siRNAs) targeting TLR-2 and Myeloid differentiation factor 88 (MyD88). In 29-kDa FN-f-stimulated chondrocytes, the relative levels of mRNA for matrix metalloproteinase 1 (MMP-1), MMP-3, and MMP-13 were analyzed by real-time quantitative reverse transcription-polymerase chain reaction. Protein expression levels of MMP-1 and MMP-3 and the regulatory effect of TLR-2 on 29-kDa FN-f-mediated signaling pathways were assessed by immunoblotting. MMP-13 production was measured by ELISA assay. Association of 29-kDa FN-f with human chondrocytes through TLR-2 was evaluated by fluorescence microscopic analysis.

Results: The expression levels of TLR-2, 3, 4, and 5 in TLR family were remarkably elevated in OA cartilage compared to normal cartilage. But, TLR-1 expression of normal and OA cartilages was not significantly different. When human chondrocytes were stimulated with various fibronectin fragments, TLR-2 expression was highly increased by 29-kDa FN-f stimulation. Knockdown of TLR-2 expression using siTLR-2 significantly suppressed 29-kDa FN-f-induced MMPs production in human normal and OA chondrocytes. Conversely, overexpression of TLR-2 enhanced 29-kDa FN-f-stimulated MMPs production. Moreover, we found that knockdown of MyD88, a downstream adaptor in TLR-2 signaling pathways, led to marked reduction of MMPs production induced by 29-kDa FN-f. In addition, 29-kDa FN-f-mediated phosphorylation of IkBa and p38 was apparently inhibited by transfection of siTLR-2. However siTLR-2 treatment did not affect 29-kDa FN-f-induced activation of JNK and ERK. Notably, fluorescence microscopic analysis showed direct interaction between TLR-2 and 29-kDa FN-f in human chondrocytes.

Conclusion: MyD88-dependent TLR-2 signaling pathway plays an important role in 29-kDa FN-f-stimulated procatabolic responses of human chondrocytes. Modulation of TLR-2-mediated signaling may be as a potential therapeutic strategy for the prevention of cartilage degradation in OA.

---

« Back to 2012 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/fibronectin-fragment-induces-procatabolic-effects-through-tlr-2-signaling-pathway-in-human-articular-chondrocytes/