Calcineurin stimulates the expression of inflammatory factors in RAW 264.7 cells by interacting with proteasome subunit alpha type 6

https://doi.org/10.1016/j.bbrc.2011.03.071Get rights and content

Abstract

Calcineurin is the only Ca2+-dependent serine/threonine-specific protein phosphatase and is considered a potential regulator of many intracellular signaling events. In this study we identified a novel interaction between calcineurin and the 20S proteasome subunit PSMA6 that increased intracellular proteasomal activity. Using RAW 264.7 macrophage cells, we demonstrated that expression of inflammatory factors was induced by calcineurin, and suppressed by the calcineurin inhibitor FK506. We also found that these calcineurin-activated processes result from activation of NF-κB, and that the interaction of calcineurin with PSMA6 stimulates transcription by NF-κB via degradation of IκB by the ubiquitin–proteasome pathway. These findings indicate that calcineurin is required for expression of inflammatory factors, and reveal a novel process of calcineurin-mediated activation of NF-κB.

Highlights

CN binds to PSMA6 and regulates proteasomal activity via this interaction. ► CN induces the expression of inflammatory factors. ► CN increases NF-κB activity via proteasome-mediated degradation of IκB. ► These findings reveal a novel process of CN-mediated activation of NFκB.

Introduction

Calcineurin (CN), a Ca2+-dependent serine/threonine-specific protein phosphatase, is directly regulated by calcium and calmodulin (CaM). CN consists of a catalytic subunit (CNA, 60 KD) and a regulatory subunit (CNB, 19 KD) [1], [2]. CNA contains a regulatory subunit-binding domain, a CaM-binding domain (CBD) and an auto-inhibitory domain (AI). Deletion mutants lacking the CBD and AI have constitutive phosphatase activity [3]. The key roles of CN have been demonstrated by using the immunosuppressant drugs cyclosporinA (CsA) and tacrolimus (FK506), which from inhibitory complexes by binding to their respective cytoplasmic receptors, cyclophilin and FKBP [4]. CN participates in a variety of physiological and pathological processes, including T-cell activation, apoptosis, ion channel regulation, neurodegenerative disease, and cardiac myocyte hypertrophy [5], [6], [7], [8], and is therefore seen as a regulator of many intracellular signaling events.

Protein degradation is essential for many intracellular processes, and proteasomes play a critical role in degradation. The 26S proteasome, a multi-subunit enzyme complex, is a major cellular non-lysosomal protease [9]. Proteasome subunit alpha type 6 (PSMA6) is one of the alpha-type subunits of the 20S proteasome core complex. It not only participates in the ubiquitin–proteasome pathway (UPP) [10], but also interacts with intracellular proteins such as HIV-1 Tat, PLK1, CUL1, RBX1 and SLX1B, which are implicated in HIV replication, cell proliferation and cell cycle control, tumor growth, apoptosis, stress responses and DNA repair and recombination [11], [12], [13], [14]. Moreover PSMA6 protein is significantly reduced in level in the renal cortex of diabetic rats, and is thought to be involved in the initiation and progression of diabetic nephropathy [15]. Recent studies have shown that elevated levels of PSMA6 increase the risk of myocardial infarction, and inhibition of PSMA6 expression in cultured cells decreases inflammation [16], [17], [18]. These findings have led to increased interest in PSMA6.

Nuclear transcription factor κB (NF-κB) is a major and essential activator that regulates the expression of genes related to inflammation, such as interleukin-6, interleukin-12, interleukin-1β and tumor necrosis factor α [19], [20], [21]. In unstimulated cells, NF-κB activity is inhibited by binding to a family of inhibitors, called IκBs, and activation of NF-κB is initiated by signal-induced degradation of IκB proteins [22]. Degradation occurs primarily via the ubiquitin–proteasome pathway.

In the present study we found that CN stimulated immune responses and NF-κB activation in RAW 264.7 macrophages. We also showed that it increased the proteolytic activity of proteasomes by interacting directly with PSMA6, and stimulated the transcriptional activity of NF-κB via ubiquitin–proteasome pathway-dependent degradation of IκB family proteins.

Section snippets

Materials

Anti-calcineurin (rabbit IgG), anti-IκBα (rabbit IgG) and anti-IκBβ (rabbit IgG) were from Cell Signaling Technology (Massachusetts, USA). Anti-PSMA6 (mouse IgG) and anti-20S proteasome (mouse IgG) were purchased from Santa Cruz Biotechnology (San Diego, USA). Anti-β-actin (mouse IgG) was from Proteintech Group (Chicago, USA) and anti-GST (mouse IgG) and anti-HA were purchased from Zhongshan Golden Bridge Biotechnology (Beijing, China).

Cell culture

RAW 264.7 cells were grown in Dulbecco’s modified Eagle’s

CN interacts with proteasome subunit α type 6

The primary aim of our study was to identify new substrates of CN. For this purpose, we performed binding assays with yeast expressing the BD–CN protein and an AD-library. This led to the identification of a cDNA clone encoding the 20S proteasome subunit alpha type 6 (NM_002791.1). Further study showed that CN interacted specifically with that proteasomal subunit (Fig. 1A).

To confirm this interaction, we performed GST pull-down assays using the catalytic subunit of CN, and GST–PSMA6 fusion

Discussion

CN has a much narrower substrate range than other phosphatases, and there has been no previous report of any interaction between CN and proteasomal subunits. In this study, we demonstrate a novel interaction between CN and PSMA6, a subunit of the 20S proteasome. Our findings indicate that PSMA6 interacts with CN both in vivo and in vitro, and that the N-terminal domain of PSMA6 is important for this interaction. Subunits of the proteasome complex interact with many proteins, and a large

Acknowledgment

This work was supported by the National Natural Science Foundation of China.

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