DJ-1 facilitates the interaction between STAT1 and its phosphatase, SHP-1, in brain microglia and astrocytes: A novel anti-inflammatory function of DJ-1
Introduction
Parkinson's disease (PD), the second-most common progressive neurodegenerative movement disorder, is caused by the selective loss of dopaminergic neurons in the substantia nigra pars compacta (Baba et al., 1998, Damier et al., 1999). A central focus of PD studies has been to determine how dopaminergic neurons die and how these cells might be protected. An important contributor to the viability of neurons is the neuronal microenvironment, which is maintained by astrocytes and microglia. Accordingly, brain inflammation mediated by astrocytes and microglia has been considered a risk factor for PD (Drouin-Ouellet and Cicchetti, 2012; for review, see Hirsch et al., 1998).
Mutations of DJ-1, also called Parkinson protein 7 (PARK7), were identified in an early onset autosomal-recessive form of PD (Bonifati et al., 2003). The first described DJ-1 mutants have a large deletion and an L166P substitution caused by a missense point mutation (Bonifati et al., 2003). Additional mutants, including M26I, E64D and E163K, have subsequently been found (Abou-Sleiman et al., 2003, Hering et al., 2004). Although first identified as an oncogene (Nagakubo et al., 1997), DJ-1 has since been shown to possess multiple functions. It positively regulates expression of anti-oxidant enzymes through stabilization of Nrf2 (Clements et al., 2006); modulates cell death through interaction with and regulation of localization, stability, and transcriptional activity of apoptosis-associated proteins, including Daxx, Bcl-XL, and p53 (Fan et al., 2008, Junn et al., 2005, Ren et al., 2011); and acts as a chaperone to suppress fibrillation of α-synuclein (Shendelman et al., 2004).
In an effort to reveal how DJ-1 is associated with PD, researchers have developed DJ-1-knockout (KO) or mutant mice. However, these animals show subtle PD-like symptoms with no loss of nigrostriatal dopaminergic neurons (Chen et al., 2005, Kim et al., 2005, Kitada et al., 2009, Pham et al., 2010). Similarly, animal models of other PD-related genes, including PINK1 (PTEN-induced putative kinase 1) and parkin, show no dopaminergic neuronal loss (Gispert et al., 2009, Goldberg et al., 2003, Kitada et al., 2009). These observations suggest that genetic defects in PD-related genes alone are not sufficient for manifestation of the PD phenotype; thus, cooperation of certain additional factors may be required.
DJ-1 is expressed in astrocytes and microglia (Bader et al., 2005, Bandopadhyay et al., 2004, Kotaria et al., 2005), suggesting that a DJ-1 mutation could alter the function of these cells. Accordingly, it has been reported that DJ-1-deficient astrocytes exhibit impaired neuroprotection whereas those that overexpress DJ-1 exhibit enhanced neuroprotective function (Mullett and Hinkle, 2009, Yanagida et al., 2009). It has recently been reported that lipopolysaccharide (LPS)-induced expression of pro-inflammatory mediators is increased in DJ-1-KO astrocytes (Waak et al., 2009). However, the detailed mechanisms by which DJ-1 exerts its anti-inflammatory functions are unknown.
STATs (signal-transducers and activators of transcription) are important signaling molecules that activate brain inflammation induced by interferon-gamma (IFN-γ) and gangliosides (Kim et al., 2002, Shuai et al., 1993a). Activation and deactivation of STATs are regulated by phosphorylation and dephosphorylation of tyrosine residues of STATs (Shuai et al., 1993b). SHP-1 (Src-homology 2 [SH2] domain-containing protein tyrosine phosphatase-1), a member of a family of cytosolic non-receptor protein tyrosine phosphatases (Neel, 1993, Tsui et al., 1993) containing N-terminal SH2 domains (Neel, 1993), negatively regulates STAT signaling pathways induced by interferons. Thus, an SHP-1 deficiency enhances the levels of phosphorylated STAT1/2 in macrophages in response to stimulation with INF-α/β or IFN-γ (Christophi et al., 2009, David et al., 1995). SHP-1 has strong anti-inflammatory roles, as evidenced by the fact that IFN-γ- and LPS-induced iNOS (inducible nitric oxide synthase) expression is higher in SHP-1-deficient macrophages than in wild-type (WT) cells (Christophi et al., 2009, Hardin et al., 2006). It has been reported that astrocytes and microglia express SHP-1 and that astrocytes and microglia prepared from SHP-1-deficient mouse brains show enhanced inflammatory responses to IFN-γ and LPS (Massa and Wu, 1996, Massa and Wu, 1998, Zhao et al., 2006).
In this study, we found that DJ-1 exerts anti-inflammatory actions in astrocytes and microglia by facilitating the interactions of SHP-1 with p-STAT1 and STAT, thereby preventing excessive and prolonged phosphorylation of STAT1. Therefore, loss of DJ-1 function enhances brain inflammation, which may cause brain damage and, in turn, increase the risk of PD. Since brain inflammation is associated with injury, DJ-1 mutants may not produce any detectable defects in the intact brain, but may become prominent in the injured brain where the anti-inflammatory functions of DJ-1 are required.
Section snippets
DJ-1-deficient mice
The DJ-1-KO mice used in this study were a generous gift from Dr. UJ Kang (Chicago University, Chicago, IL, USA). DJ-1-KO mice were previously generated by deleting a 9.3-kb region of genomic DNA containing the first five exons and part of the promoter region of the DJ-1 gene (Chen et al., 2005).
Cell culture
Primary astrocytes and microglia were cultured from the cortex of DJ-1-KO, heterozygous (HT), and WT mice brains. In brief, cortexes were removed and triturated in Dulbecco's modified Eagle medium
Anti-inflammatory functions of DJ-1 in IFN-γ-treated astrocytes and microglia
Inflammation is considered a risk factor for PD (Drouin-Ouellet and Cicchetti, 2012, Hirsch et al., 1998), and astrocytes and microglia mediate inflammatory responses in the injured brain (Jeong et al., 2010, Kim et al., 2011). Accordingly, we examined INF-γ-induced inflammatory responses in these cells cultured from wild-type (WT; DJ-1+/+), heterozygous (HT; DJ-1+/−), and knockout (KO; DJ-1−/−) mice by analyzing expression of IRF-1 (interferon regulatory factor-1), the transcription factor
Discussion
It has been reported that DJ-1 is a multifunctional protein. The current study adds yet another property to the DJ-1 functional repertoire, demonstrating that DJ-1 serves as a scaffold protein that acts through binding to p-STAT1, STAT1, and SHP-1 to facilitate the interaction of SHP-1 with p-STAT1 and STAT1. In so doing, as shown in the diagram, DJ-1 down-regulates STAT1 activation and attenuates inflammatory responses of astrocytes and microglia (Fig. 7). Therefore, DJ-1 deficiency increases
Acknowledgment
This work was supported by a KOSEF NRL Program Grant funded by the Korean government (MEST) (grant no. 2-2008025-0), and KOSEF, through the Chronic Inflammatory Disease Research Center (CIDRC) at Ajou University (grant no. NRF-2012R1A5A2051429) to E. Joe.
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