p38 mitogen-activated protein kinase inhibits USP22 transcription in HeLa cells

Abstract

Abstract. Elevated expression of ubiquitin-specific processing enzyme 22 (USP22) was identified in multiple types of human cancers, and was correlated with tumorigenesis and progression. Despite an increase in the numbers of studies in the physiological function of USP22, little is known regarding the regulation of its expression. The 5' flanking sequence of the USP22 gene was recently characterized. In the present study, USP22 transcription was regulated by p38 mitogen-activated protein kinase (MAPK). Treatment of human cervical carcinoma (HeLa) cells with SB203580, an inhibitor of p38 MAPK, enhanced basal USP22 promoter activity and mRNA abundance. Transfection of MAPK kinase 6 (MKK6), an upstream activator of p38 MAPK, resulted in a 40% decrease in USP22 mRNA, while the dominant negative MKK6 increased the transcription level of the USP22, similar to SB203580. Dual luciferase report assays showed that mutations of the Sp1 binding site ahead of the transcription start site abolished the promoting effect of the USP22 promoter by SB203580. Cisplatin, the activator of p38 MAPK, also suppressed USP22 expression. This suppression was blocked by SB203580. In conclusion, p38 MAPK acts as an upstream negative regulator of USP22 transcription in HeLa cells. IntroductionUbiquitin-specific processing enzyme 22 (USP22) is a novel deubiquitinating enzyme that can cleave ubiquitin (Ub) from Ub-conjugated protein substrates (1). As the subunit of the human SAGA coactivator complex, USP22 is linked to the regulation of gene transcription by deubiquitinating histones H2A and H2B (2,3). In addition, USP22 deubiquitinates intracellular protein, including the shelterin protein telomeric repeat binding factor 1 (4), the histone deacetylase sirtuin 1 (5) and the far upstream element-binding protein 1 fructose-1,6-bisphosphatase 1 (4), and therefore performs an extensive physiological function. A murine study showed that USP22 also regulates embryonic stem cell differentiation (6). In humans, the USP22 gene is located on chromosome 17, consists of 14 exons, and is transcribed and produced broadly across various tissues (7). Of note, elevated levels of USP22 have been identified in numerous types of human cancer, including colorectal (8) lung (9) and breast cancer (10). USP22 has been indicated in tumorigenesis. Deletion of USP22 leads to the accumulation of cells in the G 1 phase of the cell cycle (2). For these reasons, USP22 is a putative cancer stem cell marker. Reducing the rate of USP22 expression may be a suitable target for cancer therapy (11). However, the mechanisms that lead to USP22 transcriptional activation, particularly in the human tumor cells, remain unknown.Previously, USP22 transcription was activated by mitogen stimulation or viral infection in normal T and B lymphocytes (12), suggesting the regulation of USP22 gene expression occurs mainly at the transcriptional level. However, the mechanism in which signal transduction pathways regulate USP22 transcription is unclear. It is well-known that activation of the mito...

Pirarubicin reduces USP22 expression by inhibiting CREB‑1 phosphorylation in HeLa cells

“…Our previous study revealed that transcription factors SP1 and CREB-1 bound to the USP22 promoter and regulated the expression of USP22 (18). In addition, certain chemotherapeutic drugs, including cisplatin and TSA, downregulate the expression of USP22 and trigger apoptosis in HeLa cells (19,20). In the current study, treatment with THP decreased the expression of USP22 in HeLa cells in a dose-and time-dependent manner.…”

“…Transfection and dual luciferase assays. The USP22 promoter and its mutant constructs were generated as previously described (18,19). HeLa cells were cultured in 24-well plates and transfected with 0.8 µg pGL3-basic construct, P-2828 (-2828/+52), P-595 (-595/+52), or P-210 (-21/+52) promoter regions together with 0.2 µg pRL-TK (Promega Corporation, Madison, WI, USA) using Lipofectamine ® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.…”

“…Subsequently, cells were lysed and used for dual luciferase assays using Dual-Luciferase ® Reporter Assay System (Promega Corporation) according to the manufacturer's protocols. In addition, cells were transfected with plasmids: P-210, P-210/MYB mutant (mut), P-210/CREB mut, P-210/SP1 mut (0.5 µg for each), with or without 0.5 µg plasmid for CREB-1 expression [pCMV-CREB (Beyotime Institute of Biotechnology)] as previously described (19). The empty vector pCMV-control (0.5 µg) was used as the blank group.…”

“…In our previous study, the 3.0-kb USP22 promoter was cloned to identify the basic activity region containing motifs for the binding of cyclic AMP-responsive element-binding protein (CREB), transcriptional activator MYB and E3 ubiquitin-protein ligase SP1 (SP1), regulating the USP22 promoter activity (18). Previous studies indicated that chemotherapeutic drug cisplatin (19) or histone deacetylase inhibitor trichostatin A (TSA) (20) induce cancer cell apoptosis by inhibiting the expression of USP22. THP induces cancer cell apoptosis by modulating the expression of several regulators of proliferation and apoptosis (5).…”

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“…Moreover, recent studies show that USP22 deubiquitinase activity can raise protein levels of Sirt1 by reversing this ubiquitination [ 92 , 93 , 94 , 95 ]. The proximal promoter of the USP22 gene contains an Sp1-binding site, and Sp1 binding suppresses the gene’s transcription [ 96 , 97 ]. Activated p38 MAP kinase can confer a phosphorylation on Sp1 that expedites its binding to its response elements and has been found to inhibit Sp1 expression at the transcriptional level [ 97 , 98 ].…”