SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-β1 via ROS/p38 MAPK/STAT3 pathway

Abstract

SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-β1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-β1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-β1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-β1 promoter plasmids indicated that TGF-β1 promoter region between −175 to −60, the Egr-1 binding site, was responsible for TGF-β1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-β1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-β1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-β1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-β1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.

SARS coronavirus papain-like protease up-regulates the collagen expression through non-Samd TGF-β1 signaling

“…Our prior study demonstrated SARS-CoV PLpro triggering the TGF-β1 production in vitro and in vivo that linked with up-regulating the expression of pro-fibrotic proteins (vimentin and glial fibrillary acidic protein) (Li et al, 2016b). This study assesses possible effects and mechanisms of SARS-CoV PLpro-induced TGF-β1 upregulation on the expression of Type I collagen.…”

“…Human alveolar basal epithelial A549 cells grew in Dulbecco's Modified Eagle's Medium (HyClone Laboratories) and were transfected with control vector pcDNA3.1/His C (Invitrogen), or pSARS-PLpro containing SARS-CoV PLpro gene, as described in our prior reports (Li et al, 2011(Li et al, , 2012(Li et al, , 2016a(Li et al, , 2016b. In addition, pSARS-PLpro (H273A) that had the alanine substitution for histidine at position 273 by Ala within PLpro gene was constructed using PCR-based sitedirected mutagenesis with a mutated primer pair (5′-GGTAACT ATC-AGTGTGGTGCTTACACTCATATAACTGCTAAG-3′ and 5′-CTTAGCAGT TATATGAGTGTAAGCACCACACTGATAGTTACC-3′).…”

“…To measure the expression of type I collagen, TGF-β1, and vimentin in transfected cells, total RNAs extracted from transfected cells 2 days post transfection and mouse lung tissues were analyzed using two-step real time RT-PCR with SYBR Green I, as described in our prior reports (Li et al, 2016b). Primer pairs included (1) 5′-GTTCGTGACCGTGAC-CTCG-3′ and 5′-TCTTGTCCTTGGGGTTCTTGC-3′ for human type I collagen, (2) 5′-GAGCGGAGAGTACTGGATCG-3′ and 5′-TACTCGAAC-GGGAATCCATC-3′ for mouse type I collagen, (3) 5′-GGCCTTTCCTGC-TTCTCATGG-3′ and 5′-CCTTGCTGTACTGCGTGTCC-3′ for human TGF-β1, (4) 5′-TCTCTGAGGCTGCCAACCG-3′ and 5′-CGAAGGTGACGAGC-CATTTCC-3′ for human vimentin, (5) 5′-CAGAACAGCCTCCCGAATG-3′ and 5′-TGCTACGCTCACTCCATTAC-3′ for human Rac1, (6) 5′-AGCC-ACATCGCTCAGACAC-3′ and 5′-GCCCAATACGACCAA ATCC-3′ for human GAPDH, and (7) 5′-TGAGGCCGGTGCTGAGTATGTCG-3′ and 5′-CCACAGTCTTCTGGGTGGCAGTG-3′ for mouse GAPDH.…”

“…Among SARS-CoV proteins, the nucleocapsid induces a Smad3dependent induction of TGF-β1 expression (Zhao et al, 2008); spike protein stimulates the IL-8 up-regulation in lung cells (Chang et al, 2004); NSP1 provokes the expression of CCL5, CXVL10, and CCL3 (Law et al, 2007); PLpro elavates the production of TGF-β1 and pro-fibrotic markers via ubiquitin proteasome, p38 MAPK, and ERK1/2-mediated signaling (Li et al, 2012). Recently, SARS-CoV PLpro notably initiates ROS/p38 MAPK/STAT3 pathway to activate Egr-1 dependent expression of TSP-1, TGF-β1 and vimentin in vitro and in vivo (Li et al, 2016b). Therefore, PLpro becomes a virulent factor in SARS pathogenesis.…”

Early urinary biomarkers of renal tubular damage by a high‐salt intake independent of blood pressure in normotensive rats

“…Until now, it has been widely accepted that a relatively high level of ROS causes redox imbalance, which induces cell apoptosis or necrosis under a wide variety of physiological and pathological conditions . Recent studies have proven that ROS can activate MAPKs, and apoptotic cell death induced by ROS is mediated by the p38MAPK pathway in lung and renal cells. Interestingly, in the kidney of CKD rats, the levels of ROS were markedly increased, the activities of anti‐oxidative enzymes, SOD and GSH‐Px, were significantly decreased, and ROS could induce apoptosis …”