Epidemiological and clinical studies have increasingly shown that fine particulate matter (PM2.5) is associated with a number of pathological respiratory diseases, such as bronchitis, asthma, and chronic obstructive pulmonary disease, which share the common feature of airway inflammation induced by particle exposure. Thus, understanding how PM2.5 triggers inflammatory responses in the respiratory system is crucial for the study of PM2.5 toxicity. In the current study, we found that exposing human bronchial epithelial cells (immortalized Beas-2B cells and primary cells) to PM2.5 collected in the winter in Wuhan, a city in southern China, induced a significant upregulation of VEGFA (vascular endothelial growth factor A) production, a signaling event that typically functions to control chronic airway inflammation and vascular remodeling. Further investigations showed that macroautophagy/autophagy was induced upon PM2.5 exposure and then mediated VEGFA upregulation by activating the SRC (SRC proto-oncogene, non-receptor tyrosine kinase)-STAT3 (signal transducer and activator of transcription 3) pathway in bronchial epithelial cells. By exploring the upstream signaling events responsible for autophagy induction, we revealed a requirement for TP53 (tumor protein p53) activation and the expression of its downstream target DRAM1 (DNA damage regulated autophagy modulator 1) for the induction of autophagy. These results thus extend the role of TP53-DRAM1-dependent autophagy beyond cell fate determination under genotoxic stress and to the control of proinflammatory cytokine production. Moreover, PM2.5 exposure strongly induced the activation of the ATR (ATR serine/threonine kinase)-CHEK1/CHK1 (checkpoint kinase 1) axis, which subsequently triggered TP53-dependent autophagy and VEGFA production in Beas-2B cells. Therefore, these findings suggest a novel link between processes regulating genomic integrity and airway inflammation via autophagy induction in bronchial epithelial cells under PM2.5 exposure.

Two catalytic subunits of the IKK complex, IKKα and IKKβ, trigger NF-κB activation as well as NF-κB-independent signaling events under both physiological and pathological conditions. Here we identified the NF-κB-unrelated cytoprotective function of IKKα in promoting autophagy by triggering p53 transactivation and upregulation of its downstream autophagic mediator, DRAM1, in the arsenite-treated hepatoma cells, which responses depended on IKKα kinase activity. Furthermore, IKKα triggered p53/DRAM1-dependent autophagy by inducing CHK1 activation and CHK1/p53 interaction. Interestingly, after provoking autophagy, IKKα could be specifically recognized by the autophagic machinery via directly binding with LC3B, resulting in selective degradation of IKKα by autophagy. Unexpectedly, the selectivity of autophagic sequestration towards IKKα was mediated by novel mechanism independent of the classical LC3-interacting regions (LIRs) within IKKα, while C-terminal arm of LIR was involved in mediating IKKα/LC3B interaction. Taken together, we conclude that IKKα attenuates arsenite-induced apoptosis by inducing p53-dependent autophagy, and then selective feedback degradation of IKKα by autophagy contributes to the cytotoxic response induced by arsenite.

Our previous studies revealed that GADD45α is a liable protein, which undergoes MDM2-dependent constitutive ubiquitination and degradation in resting HepG2 hepatoma cells. Arsenite exposure induces ribosomal stress responses mediated by the ribosomal protein S7, which can block MDM2 activity and result in GADD45α accumulation and cell apoptosis. In the present study, we found that one of the catalytic subunits of IκB kinase (IKK), IKKβ, exerted a novel IKKα- and NF-κB-independent function in stabilizing MDM2 and therefore contributed to ubiquitination-dependent degradation of GADD45α in resting HepG2 cells. Arsenite stimulation induced transactivation of p53, which formed a complex with its downstream target, Ets-1, and then synergistically repressed IKKβ transcription, reduced MDM2 stability, and ultimately removed the inhibitory effect of MDM2 on GADD45α induction. In addition, DAPK1 functioned as an upstream protein kinase triggering p53/Ets-1-dependent IKKβ and MDM2 reduction and GADD45α accumulation, thus promoting apoptosis in HepG2 cells. Subsequent studies further revealed that the activation of the DAPK1/p53/Ets-1/IKKβ/MDM2/GADD45α cascade was a common signaling event in mediating apoptosis of diverse cancer cells induced by arsenite and other tumor therapeutic agents. Therefore, we conclude that data in the current study have revealed a novel role for IKKβ in negatively regulating GADD45α protein stability and the contribution of p53-dependent IKKβ reduction to mediating cancer cell apoptosis.

Our previous studies reveal that GADD45α is a liable proapoptotic protein, which undergoes MDM2 dependent constitutive ubiquitination and degradation in resting cancer cells. Under chemotherapeutic agents (such as arsenite, 5-Fu, VP-16) exposure, DAPK1 functions as novel p53 kinase, which induces phosphorylation of p53 at Ser15 and transactivates p53 target, Ets-1, to synergistically repress IKKβ dependent MDM2 stability, and ultimately removes the inhibitory effect of MDM2 on GADD45α, resulting in GADD45α accumulation and cell apoptosis. In the current study, we observed a strong induction of ISG20L1 expression in several cancer cell lines under exposure of arsenite and other chemotherapeutic agents. Surprisingly, although originally identified as a transcriptional target of p53, ISG20L1 induction was not controlled by p53. Instead, ISG20L1 functioned as upstream activator of p53 by interacting with DAPK1 and play an essential role in determining the ability of DAPK1/p53 complex formation and the subsequent activation of Ets-1/IKKβ/MDM2/GADD45α cascade. Therefore, our findings have revealed novel function of ISG20L1 in mediating cancer cell apoptosis induced by chemotherapeutic agents via modulating activation of the DAPK1/p53 dependent cell death pathway.