Abstract
BackgroundThe ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.MethodA20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.ResultsIn normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.ConclusionThese results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1118-7) contains supplementary material, which is available to authorized users.
Highlights
The ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc)
A20 is detected in skin fibroblasts and its basal and inducible expression is suppressed by transforming growth factor-ß (TGF-ß) While constitutive A20 expression is low in most normal cell types, A20 was detectable in cultured human fibroblasts in the absence of stimulation [7]
To investigate the cellular mechanisms underlying suppression of A20 by TGF-ß, we examined the effect of SB43542, a potent and Expression of A20 is rapidly and transiently induced by the prototypic TLR4 ligand LPS, and one of the best characterized roles of A20 is negative regulation of TLR signaling in an inhibitory feedback loop [6]
Summary
The ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts. In order to avoid injury resulting from aberrant or sustained innate immune responses, a number of cellular mechanisms have evolved that negatively regulate TLR signaling [3, 6]. Best studied of these to date is the ubiquitin-editing enzyme A20 (TNFAIP3), a direct downstream target of TLR signaling, which is rapidly and transiently induced by lipopolysaccharide (LPS), and negatively regulates both TLR-dependent and TLR-independent inflammatory responses [7]
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