Bcl-2 family proteins are implicated as essential regulators in tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. Bim(L), a BH3-only member of Bcl-2 family, can directly or indirectly activate the proapoptotic Bax and the subsequent mitochondrial apoptotic pathway. However, the molecular mechanism of Bim(L) activating Bax activation during TNFalpha-induced apoptosis is not fully understood. In this study, the role of Bim(L) in Bax activation during TNFalpha-induced apoptosis was investigated in differentiated PC12 and MCF7 cells, with real-time single-cell analysis. The experimental results show that Bax translocated to mitochondria and cytochrome c (Cyt c) released from mitochondria after TNFalpha treatment. Furthermore, SP600125 (specific inhibitor of JNK) could inhibit the Cyt c release from mitochondria. Co-immunoprecipitation results show that, the interaction between Bcl-x(L) and Bax decreased after TNFalpha treatment, while that between Bcl-x(L) and Bim(L) increased. Bax did not co-immunoprecipitate with Bim(L) before or after the TNFalpha treatment. In addition, the increased interaction between Bim(L) and Bcl-x(L) was dynamically monitored by using fluorescence resonance energy transfer (FRET) technique. Most importantly, there was no evidence of Bim(L) redistribution to mitochondria until cell apoptosis. By comprehensively analyzing these data, it is concluded that Bim(L) displaces Bcl-x(L) in the mitochondria and promotes Bax translocation during TNFalpha-induced apoptosis.
Backgrounds
Heterogeneous ribonucleoproteins (hnRNPs) are involved in the metastasis-related network. Our previous study demonstrated that hnRNP K is associated with epithelial-to-mesenchymal transition (EMT) in A549 cells. However, the precise molecular mechanism of hnRNP K involved in TGF-β1-induced EMT remains unclear. This study aimed to investigate the function and mechanism of hnRNP K interacted with microtubule-associated protein 1B light chain (MAP 1B-LC1) in TGF-β1-induced EMT.
Methods
Immunohistochemistry was used to detect the expression of hnRNP K in non-small-cell lung cancer (NSCLC). GST-pull down and immunofluorescence were performed to demonstrate the association between MAP 1B-LC1 and hnRNP K. Immunofluorescence, transwell assay and western blot was used to study the function and mechanism of the interaction of MAP 1B-LC1 with hnRNP K during TGF-β1-induced EMT in A549 cells.
Results
hnRNP K were highly expressed in NSCLC, and NSCLC with higher expression of hnRNP K were more frequently rated as high-grade tumors with poor outcome. MAP 1B-LC1 was identified and validated as one of the proteins interacting with hnRNP K. Knockdown of MAP 1B-LC1 repressed E-cadherin downregulation, vimentin upregulation and actin filament remodeling, decreased cell migration and invasion during TGF-β1-induced EMT in A549 cells. hnRNP K increased microtubule stability via interacting with MAP 1B-LC1 and was associated with acetylated ɑ-tubulin during EMT.
Conclusion
hnRNP K can promote the EMT process of lung cancer cells induced by TGF-β1 through interacting with MAP 1B-LC1. The interaction of MAP 1B/LC1 with hnRNP K may improve our understanding on the mechanism of TGF-β1-induced EMT in lung cancer.
Electronic supplementary material
The online version of this article (10.1186/s12885-019-6119-x) contains supplementary material, which is available to authorized users.
Differentiated PC12 cells have been used widely as a model for the analysis of neuronal degeneration. Some evidences showed that differentiated PC12 cells were more sensitive than naïve PC12 against apoptosis stimuli. However, the apoptosis mechanism of both types of PC12 cells was not fully known. In this study, the signaling pathways involved in tumor necrosis factor-a (TNFa)-induced apoptosis in living differentiated and naïve PC12 cells were investigated using confocal microscope for the first time. Our results showed that during TNFa-induced apoptosis, Bax translocation to mitochondria and cytochrome C (Cyt c) release from mitochondria were observed in differentiated PC12 cells, but not in naïve PC12 cells. Furthermore, the mRNA levels of bim, c-Jun N-terminal protein kinase 1 and 2 (JNK1 and JNK2) increased noticeably in differentiated PC12 cells. The apoptosis induced by TNFa was inhibited by Z-IETD-fmk (specific inhibitor of caspase-8) but not SP600125 (specific inhibitor of JNK) in naïve PC12 cells. While in differentiated PC12 cells, the process of apoptosis could only be inhibited effectively by Z-IETD-fmk and SP600125 cotreatment, and SP600125 inhibited the Bax translocation to mitochondria implying that JNK mediated activation of Bax. The experimental data strongly demonstrated that TNFa induced apoptosis through JNK/ Bax-dependent pathway in differentiated, but not naïve PC12 cells.
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