Rojo-Garcia, A. V. | bioRxiv Science

bioRxiv2026

FERMT2 expression in human articular chondrocytes is regulated by hypoxia-associated transcriptional programs

Objective: Kindlin-2, encoded by FERMT2, is a focal adhesion protein essential for cartilage homeostasis and mechanotransduction. While loss of Kindlin-2 in mice induces osteoarthritis (OA)-like pathology, the upstream mechanisms regulating its expression in human cartilage remain unknown. Methods: A bioinformatics pipeline was applied to the FERMT2 promoter to predict transcriptional regulators, followed by network and enrichment analyses. Predicted candidates were validated by siRNA knockdown in the human C28/I2 chondrocyte cell line and in primary OA chondrocytes. Pathway enrichment highlighted hypoxia-related regulators, which were further tested by hypoxia mimetic treatment (IOX2) and culture under low oxygen (1% O2). Gene expression changes were quantified by qPCR, and effects on extracellular matrix (ECM) markers were assessed. Results: In silico analysis identified 21 candidate transcription factors, of which GATA1, MEF2A, and RBPJ were validated as regulators of FERMT2. Silencing of GATA1 and MEF2A reduced FERMT2 expression, whereas RBPJ knockdown increased FERMT2 in both C28/I2 cells and primary OA chondrocytes. However, RBPJ silencing also reduced ACAN and increased COL1A1, suggesting detrimental effects on ECM homeostasis. Enrichment analysis revealed a strong association between FERMT2 regulation and hypoxia pathways, supported by conserved hypoxia response elements in the promoter. Experimentally, both IOX2 and 1% O2 significantly upregulated FERMT2 expression in primary chondrocytes. Hypoxia also increased anabolic ECM genes and reduced catabolic enzymes, but these effects occurred independently of Kindlin-2. Conclusions: This study identifies hypoxia as a novel regulator of kindlin-2 expression in human cartilage, providing new insight into the upstream control of this molecule in osteoarthritis. While additional transcription factors such as GATA1, MEF2A, and RBPJ contribute to FERMT2 regulation, only hypoxia consistently enhanced FERMT2 expression without adverse ECM effects.

molecular biology↗

bioRxiv2023

Mechanical loading rescues mechanoresponsiveness in a human osteoarthritis explant model despite Wnt activation

ObjectivesOptimizing rehabilitation strategies for osteoarthritis necessitates a comprehensive understanding of chondrocytes mechanoresponse in both health and disease, especially in the context of the interplay between loading and key pathways involved in osteoarthritis development, like canonical Wnt signaling. This study aims to elucidate the role of Wnt signaling in the mechanoresponsiveness of healthy and osteoarthritic human cartilage. MethodsWe used an ex-vivo model involving short-term physiological mechanical loading of human cartilage explants. First, the loading protocol for subsequent experiments was determined. Next, loading was applied to non-OA explants with or without Wnt activation with CHIR99021. Molecular read-outs of anabolic, pericellular matrix and matrix remodeling markers were used to assess the effect of Wnt on cartilage mechanoresponse. Finally, the same set-up was used to study the effect of loading in cartilage from patients with established OA. ResultsOur results confirm that physiological loading maintains expression of anabolic genes in non-OA cartilage, but indicate a deleterious effect of Wnt activation in the chondrogenic mechanoresponsiveness. This suggests that loading-induced regulation of cartilage markers occurs downstream of canonical Wnt signaling. Interestingly, our study highlighted contrasting mechanoresponsiveness in the model of Wnt activation and the established OA samples, with established OA cartilage maintaining its mechanoresponsiveness, and mechanical loading rescuing the chondrogenic phenotype. ConclusionThis study provides insights into the mechanoresponsiveness of human cartilage in both non-OA and OA conditions. These findings hold the potential to contribute to the development of strategies that optimize the effect of dynamic compression by correcting OA pathological cell signaling.

molecular biology↗