Dataset Information

Basal and IL-1β enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKβ NF-κB activating kinases.

ABSTRACT: IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKβ KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1β stimulated conditions. We find that in their response to IL-1β stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1β dependent increases. Our results suggest that IKKα could be a novel OA disease target.

SUBMITTER: Olivotto E

PROVIDER: S-EPMC8568921 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Basal and IL-1β enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKβ NF-κB activating kinases.

Olivotto Eleonora E Minguzzi Manuela M D'Adamo Stefania S Astolfi Annalisa A Santi Spartaco S Uguccioni Mariagrazia M Marcu Kenneth B KB Borzì Rosa Maria RM

Scientific reports 20211104 1

IKKα and IKKβ are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotax ...[more]

PMID: 34737366

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Project description:The inhibitor-κB kinase-nuclear factor-κB (IKK-NF-κB) and epidermal growth factor receptor-activator protein-1 (EGFR-AP1) pathways are often co-activated and promote malignant behavior, but the underlying basis for this relationship is unclear. Resistance to inhibitors of IKKβ or EGFR is observed in head and neck squamous cell carcinomas (HNSCC). Here, we reveal that both IKKα and β contribute to nuclear activation of canonical and alternate NF-κB/REL family transcription factors, and overexpression of signal components that enhance co-activation of the EGFR-AP1 pathway. We observed that IKKα and IKKβ exhibit increased protein expression, nuclear localization, and phosphorylation in HNSCC tissues and cell lines. Individually, IKK activity varied among different cell lines, but overexpression of both IKKs induced the strongest NF-κB activation. Conversely, siRNA knock down of both IKKs significantly decreased nuclear localization and phosphorylation of canonical RELA and IκBα and alternative p52 and RELB subunits. Knock down of both IKKs more effectively inhibited NF-κB activation, broadly modulated gene expression and suppressed cell proliferation and migration. Global expression profiling revealed that NF-κB, cytokine, inflammatory response and growth factor signaling are among the top pathways and networks regulated by IKKs. Importantly, IKKα and IKKβ together promoted the expression and activity of transforming growth factor α, EGFR and AP1 transcription factors cJun, JunB and Fra1. Knock down of AP1 subunits individually decreased 8/15 (53%) of IKK-targeted genes sampled and similarly inhibited cell proliferation and migration. Mutations of NF-κB and AP1-binding sites abolished or decreased IKK-induced interleukin-8 (IL-8) promoter activity. Compounds such as wedelactone with dual IKK inhibitory activity and geldanomycins that block IKKα/β and EGFR pathways were more active than IKKβ-specific inhibitors in suppressing NF-κB activation and proliferation and inducing cell death. We conclude that IKKα and IKKβ cooperatively activate NF-κB and EGFR/AP1 networks of signaling pathways and contribute to the malignant phenotype and the intrinsic or acquired therapeutic resistance of HNSCC.

Project description:BackgroundWith the rapid growth of the elderly population, the incidence of osteoarthritis (OA) increases annually, which has attracted extensive attention in public health. The roles of dietary intake in controlling joint disorders are perhaps one of the most frequently posed questions by OA patients, while the information about the interaction between dietary intake and OA based on scientific research is limited. α-Chaconine is the richest glycoalkaloid in eggplants such as potatoes. Previous evidence suggests that α-Chaconine is a toxic compound to nervous and digestive systems with potentially severe and fatal consequences for humans and farm animals, but its effect on OA development remains obscure.ObjectiveTo determine whether α-Chaconine deteriorates OA progression through sensory innervation and chondrocyte pyroptosis via regulating nuclear factor-κB (NF-κB) signaling, providing evidence for a possible linkage between α-Chaconine and OA progression.MethodsWe established a mouse OA model by destabilization of medial meniscus (DMM) surgery and then intra-articular injection of 20 or 100 μM α-Chaconine into the OA mice for 8 and 12 weeks. The severity of OA progression was evaluated by histological staining and radiographic analyses. The expressions of matrix metabolic indicators, Col2, Mmp3, and Mmp13, as well as pyroptosis-related proteins, Nlrp3, Caspase-1, Gsdmd, IL-1β, IL-18, were determined by immunohistochemistry. And the changes in sensory nerve ingrowth and activity of NF-κB signaling were determined by immunofluorescence.ResultsWe found that α-Chaconine could exacerbate mouse OA progression, resulting in subchondral sclerosis, osteophyte formation, and higher OARSI scores. Specifically, α-Chaconine could augment cartilage matrix degradation and induce chondrocyte pyroptosis and nerve ingrowth. Mechanistical analysis revealed that α-Chaconine stimulated NF-κB signaling by promoting I-κB α phosphorylation and p65 nuclear translocation.ConclusionCollectively, our findings raise the possibility that α-Chaconine intake can boost chondrocyte pyroptosis and nerve ingrowth to potentiate OA progression by activating NF-κB signaling.

Dataset Information

Basal and IL-1β enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKβ NF-κB activating kinases.

Publications

Basal and IL-1β enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKβ NF-κB activating kinases.

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