Stretch-induced Expression of CYR61 Increases the Secretion of IL-8 in A549 Cells via the NF-κβ/Iκβ Pathway

Mechanical ventilation （MV） with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, resulting in ventilator-induced lung injury （VILI）. The mechanisms of the injurious effects of MV and the genetic susceptibility remain unclear. VILI-related genes such as cysteine-rich angiogenic inducer 61 （Cyr61） have been demonstrated to play a detrimental role in the aggressive ventilation strategies. In the present study, we investigated the involvement of Cyr61 in the VILI and the underlying mechanism. A549 cells were exposed to cyclic stretch of varying durations and then the mRNA and protein levels of Cyr61 were measured by real-time PCR and Western blotting, respectively. Additionally, after exposure ofA549 cells to cyclic stretch for 5 min to 1 h, the expression levels of nuclear factor kappaB （NF-κβ） and IL-8 were detected by ELISA and Western blotting. Thereafter, Cyr61 expression was depressed in A549 cells with the siRNA pGenesill. 1-Cyr61-3 before the cyclic stretch, and IL-8 secretion and the activation of NF- κB pathways were probed by ELISA and Western blotting, respectively. Moreover, a NF- κB inhibitor （PDTC） and an activator （TNF） were used before mechanical stretch. Realtime PCR and ELISA were performed to detect the mRNA and protein of IL-8, respectively. The results showed that the mechanical cyclic stretch led to increased Cyr61 expression at mRNA and protein levels in A549 cells. Additionally, cyclic stretch also mobilized NF- κB from the cytoplasm to the nucleus and increased IL-8 secretion in A549 cells. The inhibition of Cyr61 blocked the NF-κB activation and IL-8 secretion in response to cyclic stretch. Inhibition of NF-κB attenuated the mRNA and protein expression of IL-8 in A549 cells transfected with Cyr61 siRNA. It was suggested that Cyr61/NF-κB signaling pathway mediates the upregulation of IL-8 in response to cyclic stretch in A594 cells. These findings support the hypothesis that Cyr61 plays a critical role in acute lung inflammation triggered by mechanical strain.

Dynamic phosphorylation of CENP-N by CDK1 guides accurate chromosome segregation in mitosis

In mitosis,accurate chromosome segregation depends on the kinetochore,a supermolecular machinery that couples dynamic spin-dle microtubules to centromeric chromatin.However,the structure–activity relationship of the constitutive centromere-associated network(CCAN)during mitosis remains uncharacterized.Building on our recent cryo-electron microscopic analyses of human CCAN structure,we investigated how dynamic phosphorylation of human CENP-N regulates accurate chromosome segregation.Our mass spectrometric analyses revealed mitotic phosphorylation of CENP-N by CDK1,which modulates the CENP-L–CENP-N interaction for accurate chromosome segregation and CCAN organization.Perturbation of CENP-N phosphorylation is shown to prevent proper chromosome alignment and activate the spindle assembly checkpoint.These analyses provide mechanistic insight into a previously undefined link between the centromere–kinetochore network and accurate chromosome segregation.

Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics

Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.

Mps1 dimerization and multisite interactions with Ndc80 complex enable responsive spindle assembly checkpoint signaling

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules,which is monitored by the spindle assembly checkpoint(SAC)signaling.As an upstream factor of SAC,the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling.However,the underlying molecular mechanism remains elusive.Here,we demonstrated that the multisite interactions between Mps1 and Ndc80 complex(Ndc80C)govern Mps1 kinetochore targeting.Importantly,we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C.We further identified that Mps1 C-terminal fragment,which contains the protein kinase domain and C-tail,enhances Mps1 kinetochore localization.Mechanistically,Mps1 C-terminal fragment mediates its dimerization.Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1,leading to aberrant mitosis due to compromised SAC function.Taken together,our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.