Proteomic Dynamics Reveal Cell-Cycle and Rho GTPase Remodeling Associated with Transient Senescence Traits in Human...

Chronic inflammatory signaling contributes to cartilage degeneration across multiple joint diseases, yet the molecular consequences of sustained cytokine exposure remain incompletely understood. We investigated how prolonged interleukin-1β (IL-1β) stimulation remodels the chondrocyte proteome and whether these changes are associated with senescence-associated traits. Primary human articular chondrocytes were exposed to IL-1β (10 ng/mL) for up to four days. Time-resolved data-independent acquisition (DIA) proteomics was integrated with immunofluorescence, quantitative PCR, multiplex metalloproteinase profiling, BrdU incorporation, growth-curve analysis, and senescence-associated β-galactosidase assays. Sustained IL-1β induced extensive time-dependent proteomic remodeling, with early inflammatory and extracellular matrix responses followed by alterations in cell-cycle regulation and cytoskeletal organization. Prolonged stimulation was associated with persistent downregulation of CDK4, Cyclin D1, DNA replication-associated proteins, and Rho GTPase-associated components, accompanied by actin cytoskeletal remodeling. These molecular changes were associated with impaired proliferation, increased senescence-associated β-galactosidase activity, and transient modulation of p21. Following cytokine withdrawal, BrdU incorporation showed partial recovery. Together, these findings indicate that sustained IL-1β progressively reshapes the chondrocyte cellular state through coordinated remodeling of proliferative, cytoskeletal, and metalloprotease programs while showing some degree of proliferative plasticity under the conditions tested.