Upregulation of MKP-7 in response to rosiglitazone treatment ameliorates lipopolysaccharide-induced destabilization of SIRT1 by inactivating JNK

Abstract

Silent mating type information regulation 2 homolog 1 (SIRT1), a NAD-dependent deacetylase, mediates cellular processes involved in gene silencing and aging. The regulation of lifespan by SIRT1 has been extensively investigated, but less is known about the mechanisms associated with its cellular turnover during inflammatory responses. In this study, we found that peroxisome proliferator-activated receptor (PPAR) γ is associated with SIRT1 stability in murine macrophage RAW 264.7 cells exposed to lipopolysaccharide (LPS). Activation of PPARγ by rosiglitazone, a specific ligand of PPARγ, rescues LPS-induced destabilization of SIRT1, with a concomitant decrease in phosphorylation of residue Ser-46, which is targeted by JNK-1 to promote proteasome-mediated degradation of SIRT1. The rosiglitazone-mediated increase in SIRT1 stability is accompanied by upregulation of mitogen-activated protein kinase phosphatase (MKP)-7, a JNK-specific phosphatase. These effects are significantly influenced by ablation or ectopic expression of PPARγ, indicating that PPARγ is directly involved in the regulation of SIRT1 stability. Furthermore, gain of MKP-7 function mimicked the effect of rosiglitazone on LPS-induced destabilization and ubiquitination of SIRT1. These results indicate that PPARγ-dependent upregulation of MKP-7 improves the stability of SIRT1 by inactivating JNK during inflammatory responses of LPS-activated macrophages.

Introduction

Silent mating type information regulation 2 homolog 1 (SIRT1) plays pivotal roles in diverse metabolic and physiological processes including cellular senescence, stress resistance, mitochondrial biogenesis, energy metabolism, and inflammatory responses [1]. As a NAD⁺-dependent class III protein deacetylase, SIRT1 coordinates complex gene expression programs by deacetylating its target proteins, which include histone proteins, transcription factors, and co-regulators [1], [2], [3], [4]. SIRT1 was recently shown to transcriptionally repress various inflammation-related genes by deacetylating transcription factors such as peroxisome proliferator-activated receptor (PPAR) γ, PPAR coactivator-1α (PGC-1α), forkhead box O3 (FOXO3), nuclear factor kappa B (NF-κB), and activator protein 1 (AP-1) [5], [6], [7]. The level and activity of SIRT1 are inversely correlated to inflammation conditions, emphasizing the importance of SIRT1 in the control of cellular inflammatory responses [8]. These effects of SIRT1 are further supported by the observation that synthetic ligands for PPARγ inhibit the release of high mobility group box 1 (HMGB1), a late proinflammatory mediator, via upregulation of SIRT1 [9]; specifically, SIRT1 induced by PPARγ ligand deacetylates its substrate HMGB1, thereby forming an anti-inflammatory complex with HMGB1 and allowing cells to bypass the response to lipopolysaccharide (LPS)-triggered inflammation [10]. In addition to regulation of SIRT1 expression, several lines of evidence suggest that post-translational modification may also govern the SIRT1 level by modulating the protein’s stability [11], [12]. For example, in obese mice, c-Jun N-terminal kinase 1 (JNK1) phosphorylates SIRT1 at Ser-46, resulting in its degradation by the proteasome [12]. By contrast, in cancer cells JNK2 exerts the opposite effect on SIRT1 stability by phosphorylating it at Ser-27 [11]. Mouse double minute 2 homolog (MDM2) E3 ligase and ubiquitin-specific protease (USP)-22 also regulate SIRT1 stability via ubiquitination and deubiquitination, respectively [13], [14]. These findings suggest that the cellular level of SIRT1 is determined not only by transcriptional regulation of the SIRT1 gene, but also by post-translational modifications that influence the protein’s stability.

PPARγ, a member of the PPAR nuclear receptor family of ligand-dependent nuclear receptors [15], [16], forms a heterodimer with the retinoid X receptor (RXR) to recognize the PPAR-response element (PPRE) located in the promoters of target genes. Transcriptional regulation of select target genes by PPARγ leads to pleiotropic effects on lipid and glucose homeostasis, adipogenesis, and inflammation [15], [16], [17]. PPARγ has been proposed to exert its anti-inflammatory effects via trans-suppression of inflammatory genes by negatively interfering with the NF-κB, AP-1, and STAT (signal transducer and activator of transcription)-1 signaling pathways via a mechanism that is independent of DNA binding [18], [19]. The anti-inflammatory effects of PPARγ are supported by the observation that the synthetic PPARγ activator rosiglitazone prevents the LPS-triggered release of HMGB1 in a mouse model of endotoxemia, thereby improving survival [20].

On the other hand, recent studies reported that ligand-activated PPARγ exerts its (patho)physiological activities by regulating stabilities of its target proteins post-translationally, rather than at the transcriptional level [21], [22], [23], [24], [25]. Indeed, PPARγ-mediated stabilization or destabilization of target proteins plays an important role in the differentiation of pancreatic beta-cell and vascular smooth muscle cells (VSMCs), proliferation of cancer and VSMCs, and conversion of white to brown fat [21], [22], [23], [24], [26]. Therefore, we hypothesized that PPARγ modulates diverse cellular functions through its transcriptional and/or post-translational activity. Here, we show that, in murine macrophage RAW 264.7 cells, ligand-activated PPARγ rescued LPS-triggered destabilization of SIRT1 by inactivating JNK signaling through upregulation of mitogen-activated protein kinase phosphatase (MKP)-7.