Mechanism of autophagy induced by activation of the AMPK/ERK/mTOR signaling pathway after TRIM22-mediated DENV-2 infection of HUVECs

Abstract

Background Dengue virus type 2 (DENV-2) was used to infect primary human umbilical vein endothelial cells (HUVECs) to examine autophagy induced by activation of the adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR) signaling pathway following tripartite motif-containing 22 (TRIM22)-mediated DENV-2 infection to further reveal the underlying pathogenic mechanism of DENV-2 infection. Methods Quantitative real-time polymerase chain reaction (qRT-PCR) was used to screen putative interference targets of TRIM22 and determine the knockdown efficiency. The effect of TRIM22 knockdown on HUVEC proliferation was determined using the CCK8 assay. Following TRIM22 knockdown, transmission electron microscopy (TEM) was used to determine the ultrastructure of HUVEC autophagosomes and expression of HUVEC autophagy and AMPK pathway-related genes were measured by qRT-PCR. Moreover, HUVEC autophagy and AMPK pathway-related protein expression levels were determined by western blot analysis. Cell cycle and apoptosis were assessed by flow cytometry (FCM) and the autophagosome structure of the HUVECs was observed by TEM. Results Western blot results indicated that TRIM22 protein expression levels increased significantly 36 h after DENV-2 infection, which was consistent with the proteomics prediction. The CCK8 assay revealed that HUVEC proliferation was reduced following TRIM22 knockdown (P < 0.001). The TEM results indicated that HUVEC autolysosomes increased and autophagy was inhibited after TRIM22 knockdown. The qRT-PCR results revealed that after TRIM22 knockdown, the expression levels of antithymocyte globulin 7 (ATG7), antithymocyte globulin 5 (ATG5), Beclin1, ERK, and mTOR genes decreased (P < 0.01); however, the expression of AMPK genes (P < 0.05) and P62 genes (P < 0.001) increased. FCM revealed that following TRIM22 knockdown, the percentage of HUVECs in the G2 phase increased (P < 0.001) along with cell apoptosis. The effect of TRIM22 overexpression on HUVEC autophagy induced by DENV-2 infection and AMPK pathways decreased after adding an autophagy inhibitor. Conclusions In HUVECs, TRIM22 protein positively regulates autophagy and may affect autophagy through the AMPK/ERK/mTOR signaling pathway. Autophagy is induced by activation of the AMPK/ERK/mTOR signaling pathway following TRIM22-mediated DENV-2 infection of HUVECs.

Multiple receptor tyrosine kinases regulate dengue infection of hepatocytes

“…Of note, IGF1R and ERBB2 drug combinations are already being investigated in the context of cancer (Chakraborty et al, 2015;McDermott et al, 2017). Additionally, both IGF1R and ERBB2 interact with other host factors known to play a role in dengue infection or pathogenesis, such as phosphoinositide 3-kinase (PI3K)/ AKT (Liu et al, 2014;Chen et al, 2017;Okamoto et al, 2017;Airo et al, 2018;Cuartas-Loṕez et al, 2018;Lahon et al, 2021;Carter et al, 2022), proto-oncogene tyrosine-protein kinase Src (Chu and Yang, 2007;Chu and Yang, 2008;Vincetti et al, 2015;Kumar et al, 2016), and mitogen-activated protein kinases (ERK) (Shyu et al, 2010;He et al, 2012;de Oliveira et al, 2020;Wu et al, 2022;Chen et al, 2023;Liang et al, 2023). Thus, the activity of ERBB2 and IGF1R during DENV infection should be further investigated to understand their utility as dual targets of dengue therapeutics.…”

“…To further assess the potential of RTKs as regulators of DENV infection, we examined if the seven predicted RTKs interacted with other KiR predictions or with kinases previously shown to be important for DENV infection (Chu and Yang, 2007;Ceballos-Olvera et al, 2010;Shyu et al, 2010;Chang et al, 2012;Le Sommer et al, 2012;Chen et al, 2013;de Wispelaere et al, 2013;Li et al, 2013;Nagila et al, 2013;Yeh et al, 2013;Liu et al, 2014;Sreekanth et al, 2014;Callaway et al, 2015;Kumar et al, 2016;Sreekanth et al, 2016;Noppakunmongkolchai et al, 2016;Duran et al, 2017;Jordan and Randall, 2017;Limjindaporn et al, 2017;Smith et al, 2017;Airo et al, 2018;Barbachano-Guerrero et al, 2020;Cuartas-Loṕez and Gallego-Goḿez, 2020;de Oliveira et al, 2020;Kong et al, 2020;Lahon et al, 2021;Carter et al, 2022;Puerta-Guardo et al, 2022;Rahman et al, 2022;Sinha et al, 2022;Wu et al, 2022;Kao et al, 2023). We utilized the kinase-substrate phosphorylation database PhosphoSitePlus ® (Hornbeck et al, 2015) to identify interacting kinases and substrates of the predicted RTKs and then visualized these interactions using Cytoscape 3.9.1 (Figure 1D).…”