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The Dysregulated Galectin Network Activates NF-?B to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes.

ABSTRACT: This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Exposure of 2D and 3D cultures to an in vivo-like galectin mixture (Gal-1 and Gal-8: 5 µg/ml, Gal-3: 1 µg/ml) was followed by the assessment of pellet size, immunohistochemical matrix staining, and/or quantification of MMP-1, -3, and -13. Application of inhibitors of NF-?B activation probed into the potential of intervening with galectin-induced matrix degradation. Galectin profiling revealed maintained dysregulation of Gal-1, -3, and -8 in pellet cultures, resembling the OA situation in situ. The presence of the galectin mixture promoted marked reduction of pellet size and loss of collagen type II-rich extracellular matrix, accompanied by the upregulation of MMP-1, -3, and -13. Inhibition of p65-phosphorylation by caffeic acid phenethyl ester effectively alleviated the detrimental effects of galectins, resulting in downregulated MMP secretion, reduced matrix breakdown and augmented pellet size. This study suggests that the dysregulated galectin network in OA cartilage leads to extracellular matrix breakdown, and provides encouraging evidence of the feasible inhibition of galectin-triggered activities. OA chondrocyte pellets have the potential to serve as in vitro disease model for further studies on galectins in OA onset and progression.

SUBMITTER: Pichler KM

PROVIDER: S-EPMC7881967 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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The Dysregulated Galectin Network Activates NF-κB to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes.

Pichler K M KM Weinmann D D Schmidt S S Kubista B B Lass R R Martelanz L L Alphonsus J J Windhager R R Gabius H J HJ Toegel S S

Calcified tissue international 20201113 3

This work aimed to study the dysregulated network of galectins in OA chondrocyte pellets, and to assess whether their recently discovered activity as molecular switches of functional biomarkers results in degradation of extracellular matrix in vitro. Scaffold-free 3D pellet cultures were established of human OA chondrocytes. Expression and secretion of galectin(Gal)-1, -3, and -8 were monitored relative to 2D cultures or clinical tissue sections by RT-qPCR, immunohistochemistry and ELISAs. Expos ...[more]

PMID: 33185768

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Project description:IntroductionHypoxia is considered to be a positive influence on the healthy chondrocyte phenotype and cartilage matrix formation. However, hypoxia-inducible factors (HIFs) have been implicated in the pathogenesis of osteoarthritis (OA). Thus, we assessed whether healthy and OA chondrocytes have distinct responses to oxygen, particularly with regard to hypertrophy and degradation during redifferentiation.MethodsMonolayer-expanded healthy and OA chondrocytes were redifferentiated for 14 days in pellet cultures under standard (20% oxygen) or hypoxic (2% oxygen) conditions. Cartilage matrix gene expression, matrix quality and quantity, degradative enzyme expression and HIF expression were measured.ResultsIn hypoxia, both healthy and OA chondrocytes had higher human collagen type II, ?1 gene (COL2A1), and aggrecan (ACAN) expression and sulfated glycosaminoglycan (sGAG) accumulation, concomitant with lower human collagen type X, ?1 gene (COL10A1), and human collagen type I, ?1 gene (COL1A1), expression and collagen I extracellular accumulation. OA chondrocytes had significantly lower sGAGs/DNA than healthy chondrocytes, but only in high oxygen conditions. Hypoxia also caused significantly greater sGAG retention and hyaluronic acid synthase 2 (HAS2) expression by OA chondrocytes. Both healthy and OA chondrocytes had significantly lower expression of matrix metalloproteinases (MMPs) MMP1, MMP2, MMP3 and MMP13 in hypoxia and less active MMP2 enzyme, consistent with lower MMP14 expression. However, aggrecanase (ADAMTS4 and ADAMTS5) expression was significantly lowered by hypoxia only in healthy cells, and COL10A1 and MMP13 remained significantly higher in OA chondrocytes than in healthy chondrocytes in hypoxic conditions. HIF-1? and HIF-2? had similar expression profiles in healthy and OA cells, increasing to maximal levels early in hypoxia and decreasing over time.ConclusionsHypoxic culture of human chondrocytes has long been acknowledged to result in increased matrix accumulation, but still little is known of its effects on catabolism. We show herein that the increased expression of matrix proteins, combined with decreased expression of numerous degradative enzymes by hypoxia, minimizes but does not abolish differences between redifferentiated healthy and OA chondrocytes. Hypoxia-induced HIF expression is associated with hypertrophic marker and degradative enzyme downregulation and increased measures of redifferentiation in both healthy and OA chondrocytes. Therefore, though HIFs may be involved in the pathogenesis of OA, conditions that promote HIF expression in vitro promote matrix accumulation and decrease degradation and hypertrophy, even in cells from OA joints.

Project description:BackgroundCheckpoint blockade therapies targeting programmed death ligand 1 (PD-L1) and its receptor programmed cell death 1 promote T cell-mediated immune surveillance against tumors and have been associated with significant clinical benefit in cancer patients. The long-stranded non-coding RNA HOTAIR is highly expressed and associated with metastasis in a variety of cancer types and promotes tumor metastasis at least in part through association with the PRC2 complex that induces redirection to hundreds of genes involved in tumor metastasis. Here, we report that HOTAIR is an activator lncRNA of the NF-κB pathway and demonstrate that its apparent upregulation promotes inflammatory signaling and immune escape in glioma cells.MethodsBioinformatics analysis was used to elucidate the relationship between HOTAIR and NF-κB pathway in HOTAIR knockdown glioma cells. At the cytological level, protein hybridization and immunofluorescence were used to detect the response of proteins in the NF-κB signaling pathway to HOTAIR regulation. ChIP and ChIRP experiments identified HOTAIR target genes. Animal experiments verified alterations in inflammation and immune escape following HOTAIR knockdown and activity inhibition.ResultsHOTAIR activated the expression of proteins involved in NF-κB, TNFα, MAPK and other inflammatory signaling pathways. In addition, HOTAIR induced various proteins containing protein kinase structural domains and promoted the enrichment of proteins and complexes of important inflammatory signaling pathways, such as the TNFα/NF-κB signaling protein complex, the IκB kinase complex, and the IKKA-IKKB complex. In addition, HOTAIR aberrantly activated biological processes involved in glioma immune responses, T-cell co-stimulation and transcription initiation by RNA polymerase II. HOTAIR facilitated the induction of IκBα phosphorylation by suppressing the expression of the NF-κB upstream protein UBXN1, promoting NF-κB phosphorylation and nuclear translocation. In vivo, reduction of HOTAIR decreased PD-L1 protein expression, indicating that cells are more likely to be targeted by immune T cells.ConclusionIn conclusion, our results provide convincing evidence that lncRNA HOTAIR drives aberrant gene transcription and immune escape from tumor cells through the NF-κB pathway.

Dataset Information

The Dysregulated Galectin Network Activates NF-?B to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes.

Publications

The Dysregulated Galectin Network Activates NF-κB to Induce Disease Markers and Matrix Degeneration in 3D Pellet Cultures of Osteoarthritic Chondrocytes.

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