Upregulation of DSCR1 (RCAN1 or Adapt78) in the peri-infarct cortex after experimental stroke

Abstract

Down syndrome candidate region 1 (DSCR1; also known as RCAN1 or Adapt78) has been shown to be induced by calcium overload and oxidative stress which are included in the pathogenic hallmarks of the ischemic diseases. After ischemic stroke, inflammatory responses play an important role in the exacerbation of neuronal loss. In this study, we investigated the expression pattern of DSCR1 in the mouse cortex after transient middle cerebral artery occlusion (MCAO). Then, in vitro studies were taken to address whether inflammatory mediators could induce DSCR1. Male C57BL/6 mice were subjected to transient MCAO for 35 min and sacrificed at 6, 24, and 72 h after the reperfusion. The expression of DSCR1 began to increase in layer VI of the peri-infarct cortex at 24 h and was prominently enhanced at 72h after transient MCAO. Moreover, real-time reverse transcriptase–polymerase chain reaction and immunohistochemistry showed that the induction of the DSCR1 isoform 4 (DSCR1-4) mRNA preceded the expression of the DSCR1 protein. In in vitro studies, tumor necrosis factor α and interleukin-1β (IL-1β) were found to induce strong upregulation of DSCR1-4 mRNA. Furthermore, western blot analysis revealed that overexpression of DSCR1-4 in SK-N-SH neuroblastoma cells attenuated IL-1β-induced cyclooxygenase 2 and intercellular adhesion molecule 1 expression. These results demonstrate upregulation of DSCR1 in the mouse peri-infarct cortex following transient MCAO. In addition, our results suggest that inflammatory mediators such as TNFα and IL-1β can induce DSCR1-4 transcription, which may be associated with the alleviation of inflammatory processes.

Introduction

Neuronal tissue is highly vulnerable to ischemic injury resulting from the disturbance of blood supply. In the penumbra surrounding the ischemic core, cell survival is poorly maintained by hypoperfusion from a collateral blood supply (Ginsberg, 2003). Energy depletion after ischemic insult causes excessive glutamate release, which in turn initiates calcium influx. Subsequently, the calcium-associated activation of second messenger systems triggers the expression of a large number of pro-inflammatory genes. These various inflammatory mediators, such as tumor necrosis factor α (TNFα), interleukin-1β (IL-1β), or inflammation-related enzymes, including inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), are known to exacerbate the secondary events that amplify cerebral ischemic injury (Dirnagl et al., 1999).

The human Down syndrome candidate region 1 (DSCR1; also called RCAN1 or Adapt78) gene was first isolated from chromosome 21 (Hattori et al., 2000). This gene consists of seven exons, and alternative first exon choices generate four different transcripts (DSCR1-1 through DSCR1-4). Among them, DSCR1-1 and DSCR1-4 comprise the majority of the observed forms. DSCR1-4, in particular, is unique among DSCR1 variants because it is a [Ca²⁺]_i-responsive gene and can regulate calcineurin activity by a negative feedback mechanism (Cano et al., 2005, Minami et al., 2004, Yang et al., 2000). For example, the transcriptional regulation through multiple calcineurin/nuclear factors of activated T cells (NFAT) binding sites is exclusive for the expression of DSCR1-4 (Yang et al., 2000). Moreover, depolarization of neurons and the stimulation of endothelial cells with vascular endothelial growth factor or Ca²⁺ ionophore induce the transcription of DSCR1-4 through the calcineurin/NFAT pathway (Cano et al., 2005, Minami et al., 2004). Efforts to elucidate the functional roles of DSCR1 have also been carried out. Several reports suggest that DSCR1 attenuates the inflammatory response and angiogenesis (Hesser et al., 2004, Kim et al., 2006, Minami et al., 2004). Furthermore, a previous study has shown that DSCR1 has protective effects against oxidative stress (Ermak et al., 2002), whereas a recent study claims that DSCR1 increases neuronal cell death in response to the same stimulus (Porta et al., 2007a, Porta et al., 2007b). However, the role of DSCR1 in ischemic insults has not been investigated to date.

Thus, given that DSCR1-4 is inducible by elevation of [Ca²⁺]_i in neuronal cells, we were interested in the potential role of this protein during ischemic insult. Here, we report for the first time that transcription of DSCR1-4 is increased in the peri-infarct cortex of the mouse brain, using the transient middle cerebral artery occlusion (MCAO) model. IL-1β and TNFα treatment strongly induced expression of DSCR1-4 in brain cells. Overexpression of DSCR1-4 significantly attenuated IL-1β-induced expression of inflammation-associated genes.