Fig 2: MMP12 functions as an effector molecule in the process of RON-mediated bladder cancer cell migration and matrix invasion.A J82 cells were transduced with siRNA targeting MMP12 and an overexpression plasmid for RON. Western blot analysis was utilized to confirm the expression of MMP12. B The migratory capacity was assessed through a wound-healing assay. C The invasive potential was evaluated using a trans-well assay. D In total, 5637 cells were transduced with an overexpression plasmid for MMP12 and a shRON plasmid. Western blot analysis was employed to validate the expression of MMP12. E The migratory ability was assessed through a wound-healing assay. F The invasion ability was evaluated by trans-well assay. The scale bar above is all 1 µm. Actin-Tracker Red-555 and DAPI were utilized for staining the cytoskeleton and nucleus, respectively, with representative images displayed in Fig. (G) and (H). The scale bar measures 500 µm. I, J Western blot analysis was used to confirm the expression of N-cadherin and vimentin. All experiments above were repeated three times (n = 3).

Fig 3: RON modulates the expression of MMP12 via inhibiting ubiquitination of the transcription factor HIF-2α.A Co-immunoprecipitation was employed to demonstrate the lack of direct interaction between RON and MMP12. B–D The mRNA expression levels of HIF-2α in 5637-shRON, 5637-NC, J82-oeRON, and J82-NC cells were quantified using quantitative real-time polymerase chain reaction (qRT-PCR), and confirmed by immunofluorescence staining and western blot analysis, respectively. E Transfection of 5637-shRON cells with a HIF-2α plasmid and J82-oeRON cells with HIF-2α-targeting siRNA was performed, followed by western blot analysis to assess the expression levels of HIF-2α and MMP12. F J82-oeRON cells were subjected to a 24-h period of starvation, followed by washing with ice-cold PBS to remove basal phosphorylation. Subsequently, the cells were stimulated with the RON-specific ligand MSP for 30 min, and western blot analysis was performed to assess the phosphorylation levels of ERK and JNK. G, H J82-oeRON cells were treated with varying concentrations of ERK inhibitors or JNK inhibitors for 24 h, after which the cells were harvested to evaluate the expression of HIF-2α and MMP12 by western blot. I J82-oeRON cells were subjected to treatment with or without 20 µM JNK inhibitors for a duration of 24 h, followed by the addition of 200 nM bortezomib 8 h prior to cell collection. Subsequently, the cells underwent immunoprecipitation using an anti-HIF-2α antibody, and the ubiquitination level of HIF-2α was assessed through immunoblotting with an anti-ubiquitin antibody. All experiments above were repeated three times (n = 3).

Fig 4: Illustration depicting the regulatory pathway involving hsa-miR-659-3p, RON, HIF-2α, and MMP12 in the context of bladder cancer cell migration and invasion.The inhibition of miR-659-3p resulted in the prevention of RON 3’UTR binding and subsequent RON mRNA degradation in the context of cancer progression. The overexpression of RON can lead to its activation through spontaneous phosphorylation, initiating downstream activation of the JNK signaling pathway, which in turn inhibits HIF-2α ubiquitination and stabilizes its expression. HIF-2α serves as a transcription factor for MMP12, promoting its expression and ultimately enhancing the migration and invasion of bladder cancer cells.

Fig 5: Proposed model of extracellular Ca2+ induced MET release: Our results support a model in which human monocyte‐derived macrophages release METs in response to extracellular calcium, a process that requires PLC, PAD2 activity, activity of the NLRP3 inflammasome, ROS and plasma membrane rupture. In LPS‐primed macrophages, extracellular calcium‐induced activation of the calcium‐sensing receptor triggers NLRP3 inflammasome assembly secondary to PLC activation and in the later stages GSDMD cleavage and proinflammatory IL‐1b release. Simultaneously, a PLC‐dependent increase in intracellular calcium may activate PAD2, which catalyzes histone citrullination and promotes MET formation. MET release is promoted by the release of mito‐ROS. NLRP3 seems to modulate histone citrullination through an unknown mechanism. Decondensed chromatin consists of citrullinated histone H3, MPO, ASC specks, and MMP12, and is released into the extracellular space by plasma membrane rupture.