Stroke is the second leading cause of mortality and the most common cause of long-term disability worldwide (Mathers et al. 2009). Over 15 million people suffer from stroke each year, and approximately 80–85% of these cases are ischemic stroke. Accumulating evidence from the past two decades has suggested that the oxidative stress associated with the excessive production of reactive oxygen species (ROS) has a profound effect on ischemic stroke pathogenesis (El Kossi and Zakhary 2000; Kelly et al. 2008; Lei et al. 2011). ROS have direct cellular effects, such as lipid peroxidation, protein denaturation, and DNA and RNA damage, which result in tissue destruction and cell death. ROS also act in several signal transduction pathways, including intrinsic and extrinsic caspase activation and nuclear factor kappa B (NF-κB) activation, which may lead to excessive cell apoptosis and inflammatory gene expression (Chan 2001; Allen and Bayraktutan 2009). In recent years, continuous efforts have been made to modulate oxidative stress after ischemic stroke. Although some free radical scavenging agents and radical trapping agents have shown therapeutic potential in animal models, they have failed in clinical trials. In the present study, we found that the deletion of the signal regulatory protein (SIRP) family member SHPS-1 inhibits oxidative damage and mitigates brain injury after ischemic stroke.
SHPS-1, which is also known as SIRP α or SIRPA, is a transmembrane protein that consists of three domains: an immunoglobulin (Ig)-like extracellular domain, a transmembrane domain, and an intracellular domain (Yamao et al. 1997; Oshima et al. 2002). The intracellular domain of SHPS-1 contains two immunoreceptor tyrosine-based inhibition motifs (ITIMs) with four tyrosine residues, which can be phosphorylated by growth factors and integrins that mediate cell adhesion to extracellular matrix proteins (Galbaugh et al. 2010; Kapoor and O’Rourke 2010; Shen et al. 2010). The intracellular tyrosine-phosphorylated sites of SHPS-1 then bind to and activate two Src homology-2 (SH2) domain-containing protein tyrosine phosphatases, SHP-1 and SHP-2. The activated SHP-1 and SHP-2 act on multiple signaling molecules and modulate downstream signal transduction via dephosphorylation.
The MAPK, JAK/STAT, and PI3K-Akt pathways have been reported to be modulated by SHP-1 or SHP-2 under diverse biological states. Akt, an important member of Arg-directed kinases, plays a central role in mediating critical cellular responses including cell survival, metabolism, angiogenesis, and transcriptional regulation. GSK3β is a direct substrate of Akt, which can be directly phosphorylated by activated Akt at Ser 9. Previous studies have demonstrated that Akt activation plays an important role in the expression and activation of nuclear factor-E2-related factor 2 (Nrf2) (Martin et al. 2004; Bak et al. 2012). Nrf2 is an important transcription factor that induces the expression of a number of genes including those that encode for several antioxidant enzymes, and it plays a physiological role in the regulation of oxidative stress.
SHPS-1 is predominantly expressed in neurons, dendritic cells, and in macrophages, and previous studies have shown that SHPS-1 is involved in a variety of physiological processes, including the regulation of immune cells, the self-recognition of red blood cells, macrophage multinucleation, vascular smooth muscle proliferation, neuronal development, and survival (Ikeda et al. 2006; Mitsuhashi et al. 2008; Sobota et al. 2008). To date, studies have not examined the role of SHPS-1 in ischemic stroke or the impact of SHPS-1 on oxidative stress after cerebral ischemia. Identifying the functional role and the mechanisms of SHPS-1 in the stroke pathological process may provide potential treatment targets for ischemic stroke.
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This study suggests that SHPS-1 plays an important role in the pathological process of focal cerebral ischemia. In the SHPS-1 MT mice, the infarct volume was decreased, neurological function was improved, neuronal apoptosis was reduced, the activities of caspase 3 and caspase 8 were inhibited, oxidative injury was attenuated and the expression levels of the antioxidant genes Nrf2 and HO-1 were up-regulated. The phosphorylation of SHP-1 and SHP-2 was also inhibited, and the activity of Akt was enhanced in the SHPS-1 MT mice.
SHPS-1 is a ubiquitously expressed receptor-type transmembrane glycoprotein that is abundantly expressed in the brain (Yamao et al. 1997; Oshima et al. 2002). Previous studies (Stofega et al. 2000; Ikeda et al. 2006) have demonstrated that SHPS-1 plays a key role in the negative regulation of tyrosine kinase-coupled cellular responses that are induced by cell adhesion, growth factors and/or insulin. SHPS-1 has been shown to be involved in various biological functions, such as cell migration, phagocytosis, and mast and dendritic cell activation. In the brain, SHPS-1 has been identified as a neural adhesion molecule that participates in brain-derived neurotrophic factor (BDNF)-mediated neuronal survival via Akt activation (Araki et al. 2000). In addition, transfection with wild-type and mutant SHPS-1 both have been shown to enhance Akt activation in neurons (Araki et al. 2000). CD47 is a ligand of SHPS-1, Koshimizu et al. and Xing et al. suggested that the activation of CD47 by its activating peptide induces oxidative injury and cytotoxicity in cultured neurons (Koshimizu et al. 2002; Xing et al. 2009); however, previous studies have not shown that SHPS-1 is involved in oxidative stress.
Oxidative stress has been identified as an important factor in the pathological process of stroke because it has direct effects on cellular injury and activates downstream signaling pathways, which may aggravate the post-ischemic injury (El Kossi and Zakhary 2000; Kelly et al. 2008; Lei et al. 2011). In the present study, the biomarkers of oxidative damage (i.e., 8OHdG and 4HNE) were markedly decreased in the brains of the ischemic SHPS-1 MT mice, which suggested that oxidative damage is mitigated by the SHPS-1 mutation. The severity of oxidative stress depends on the balance between antioxidants and pro-oxidants. We estimated the expression of the antioxidant genes Nrf2 and HO-1. Nrf2 is an important transcription factor that regulates the expression of a large number of antioxidant genes (Venugopal and Jaiswal 1996; Solis et al. 2002; Kweon et al. 2006), and HO-1 is a target gene of Nrf2 that plays a protective role in cerebral ischemic injury via an anti-oxidative mechanism (Kweon et al. 2006; Aztatzi-Santillán et al. 2010; Kim et al. 2010). The mRNA and protein levels of Nrf2 and HO-1 were markedly up-regulated in the brains of the ischemic SHPS-1 MT mice, which indicated that the SHPS-1 mutation mitigated oxidative stress, likely through the up-regulation of an anti-oxidative mechanism.
The number of TUNEL-positive neurons decreased, and the protein levels of cleaved caspase 3 and caspase 8 were down-regulated in the SHPS-1 knockout mice in this study. TUNEL detects DNA fragmentation which appeared in programmed cell death. Caspases, or cysteine-aspartic proteases or cysteine-dependent aspartate-directed proteases are a family of cysteine proteases that play essential roles in programmed cell death, necrosis, and inflammation. Caspase 8 is involved in the programmed cell death induced by Fas and various apoptotic stimuli. Activated caspase 8 cleaves and activates downstream effector caspases, such as caspase 1, caspase 3, caspase 6, and caspase 7. Caspase 3 ultimately elicits the morphological hallmarks of apoptosis, including DNA fragmentation and cell shrinkage. Several studies have demonstrated that oxidative stress-induced neural injury is mediated by caspase 8 and caspase 3 (Russell et al. 2002; Wang et al. 2002). In the present study, the inhibition of neural injury appeared to be attributed to the mitigation of oxidative stress in the SHPS-1 MT mice.
We observed that the phosphorylation of SHP-1 and SHP-2 was inhibited after cerebral ischemia in the SHPS-1 MT mice. SHP-1 and SHP-2 are SH2 domain-containing non-transmembrane protein tyrosine phosphatases. When SHPS-1 is activated by growth factors, cell adhesion or other stimuli, the phosphorylated cytoplasmic region binds to and activates SHP-1 and SHP-2. SHP-1 and SHP-2 have been implicated in several signaling pathways, such as the MAPK, JAK/STAT, and PI3K-Akt pathways (Dubois et al. 2006; Pandey et al. 2009; Won et al. 2011). In addition, SHP-1 and SHP-2 have been shown to interact with a variety of signaling intermediates, and they dephosphorylate associated signaling molecules, which negatively regulate the local signal pathways. In the present study, higher levels of Akt phosphorylation were detected in the SHPS-1 MT mice, which was likely because of decreased activity of SHP-1 and SHP-2 (Zhang et al. 2002; Lodeiro et al. 2011).
In previous studies, it has been demonstrated that the PI3K/Akt signaling pathway is involved in the expression and activation of Nrf2 and that the induction of Nrf2 protein expression contributes to the transcriptional activity of Nrf2 (Martin et al. 2004; Bak et al. 2012). Not only does it affect the accumulation of Nrf2, but the PI3K/Akt signaling pathway also contributes to the activation of Nrf2 by promoting transfer of cytoplasmic Nrf2 to the nucleus (Wang et al. 2008). GSK-3β is a downstream signaling molecule of the PI3K/Akt pathway whose activity can be inhibited by Akt-mediated phosphorylation at Ser9. A previous study demonstrated that GSK-3β down-regulated Nrf2 activity; thus, phosphorylation of GSK-3β can increase the activity of Nrf2 (Rojo et al. 2008). In the present study, the mRNA level and protein level of Nrf2 were up-regulated in SHPS-1 MT mice, the expression of Nrf2 protein was more concentrated in the nucleus in SHPS-1 MT mice, the mRNA, and protein level of Nrf2 targeted gene HO-1 were also up-regulated in SHPS-1 MT mice. The increasing expression and activity of Nrf2 is probably attributed to the phosphorylation of Akt/GSK-3β.
In conclusion, this study suggested that the SHPS-1 deficiency protects the brain from acute ischemic injury. Oxidative stress was mitigated and neural injury was inhibited in SHPS-1 MT mice. In addition, we demonstrated that Akt was activated in the brains of ischemic SHPS-1 MT mice. The activated Akt signaling may account for the up-regulation of Nrf2 and HO-1 and may eventually lead to the decline of oxidative stress. These findings may provide a new therapeutic target for the treatment of acute ischemic stroke.