Abstract
We investigated the regulatory roles of USP2 in mRNA accumulation of proinflammatory cytokines in macrophage-like cells after stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS). Human macrophage-like HL-60 cells, mouse macrophage-like J774.1 cells, and mouse peritoneal macrophages demonstrated negative feedback to USP2 mRNA levels after LPS stimulation, suggesting that USP2 plays a significant role in LPS-stimulated macrophages. USP2 knockdown (KD) by short hairpin RNA in HL-60 cells promoted the accumulation of transcripts for 25 of 104 cytokines after LPS stimulation. In contrast, limited induction of cytokines was observed in cells forcibly expressing the longer splice variant of USP2 (USP2A), or in peritoneal macrophages isolated from Usp2a transgenic mice. An ubiquitin isopeptidase-deficient USP2A mutant failed to suppress LPS-induced cytokine expression, suggesting that protein ubiquitination contributes to USP2-mediated cytokine repression. Although USP2 deficiency did not accelerate TNF receptor-associated factor (TRAF) 6-nuclear factor-κB (NF-κB) signaling, it increased the DNA binding ratio of the octamer binding transcription factor (Oct)-1 to Oct-2 in TNF, CXCL8, CCL4, and IL6 promoters. USP2 decreased nuclear Oct-2 protein levels in addition to decreasing the polyubiquitination of Oct-1. In summary, USP2 modulates proinflammatory cytokine induction, possibly through modification of Oct proteins, in macrophages following TLR4 activation.
Highlights
Inflammation is the basis for a wide variety of diseases
In contrast to IL6 expression, the mRNA levels of both USP2A and USP2B significantly decreased as a result of the LPS treatment (Figure 1(a))
LPS decreased Usp2a and Usp2b transcripts in the mouse macrophage-like J774.1 cells and peritoneal macrophages (Figures 1(b) and 1(c)), while IL6 was upregulated in these cells
Summary
Inflammation is the basis for a wide variety of diseases. In addition to canonical inflammatory diseases, such as inflammatory bowel disease and septic shock, it constitutes a pathological basis for atherosclerosis, type 2 diabetes, and carcinogenesis [1,2,3]. Macrophages are the predominant effector cells in terms of number and cellular function. They abundantly express toll-like receptors (TLRs), which recognize and bind to specific pathogen-associated molecular patterns (PAMPs). There are 10–15 TLR genes encoding surface and intracellular TLR proteins [4]. Of the surface TLRs, TLR4 responds to bacterial lipopolysaccharide (LPS) and participates in antibacterial
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