Esophageal Cancer Related Gene-4 Is a Choroid Plexus-Derived Injury Response Gene: Evidence for a Biphasic Response in Early and Late Brain Injury

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24–72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6–8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation

Esophageal cancer related gene-4 is a choroid plexus-derived injury response gene: evidence for a biphasic response in early and late brain injury.

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24-72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6-8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation

Ecrg4 gene expression decreases after CNS injury.

<p>Cortical stab wound decreased gene expression levels in the CP as determined by <i>in situ</i> hybridization. The ophthalmic knife penetrated the rat dorsal cortex to transect the corpus callosum 3 mm deep and lateral to the right lateral ventricle and Ecrg4 gene expression evaluated by binding of an Ecrg4 mRNA antisense probe in the contralateral CP in intact rat brains (Panel A), 24 hrs (Panel B) 72 hrs (Panel C) and 7 days (Panel D) post-injury. A synthetic, negative control mRNA probe was generated from the pSPT18-Neo plasmid control backbone and was used to assess background signal in Control (Panel E) and 7 day (Panel F) sections. Images representative of n = 3 rats.</p

Augurin (ECRG4(31–148)) localizes to the cell surface.

<p>Panel A: Immunohistochemical evidence for tethering at the cell surface: The immunoreactive augurin recognized by this antibody in CP extracts was identified as the product of Ecrg4 by immunoblotting and demonstrating the presence of a 14 kDa protein ECRG4(31–148) processed by cleavage of the 30 amino acid leader sequence as we determined previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024609#pone.0024609-Mirabeau1" target="_blank">[13]</a>. Among periventricular cells in the brain, polarized foci of punctated augurin staining were observed at the extracellular ventricular face of the choroid plexus epithelial cell layer (red, arrow) in reference to nuclear counterstain (blue, DAPI). A granular staining pattern is observed in the cytoplasm if the CPe cell layer, which is indicative of regulated vesicular secretion to the cell surface. Panel B: Detection of immunoreactive augurin in rat CSF: Immunoblotting of rat CSF showed a 14 kDa band (arrow) that corresponds to the predicted molecular weight of augurin (ECRG4(31–148)). Panel C: Biochemical evidence for cell surface tethering of augurin: Expression of Ecrg4 in the cultured CPe cells was detected neither by Western blot (left panel) nor by RT-PCR of untransduced cells (not shown). Thus to study Ecrg4 in vitro it was necessary to use an adenovirus vector containing the human Ecrg4 ORF. Protein was extracted from CPe cells 48 hours after no transduction or transduction with AD_GFP or AD_ECRG4. The molecular weight of Ecrg4-derived peptide fragments expressed in whole cell lysates was determined by Western blotting in the left panel. Cell-surface proteins were fractionated by precipitation of Neutravidin-binding of biotinylated proteins on the cell surface (center panel) followed by Western blot analysis. The peptide form secreted from cells was detected by direct Western blotting of conditioned medium (right panel).</p

A biphasic model for augurin activity and CNS dysinhibition.

<p>Normal choroid plexus epithelia express Ecrg4 and contain significant 14 kDa augurin protein that is in an inhibitory conformation. The unprocessed peptide is contained in 3 compartments (1) intracellular vesicles (i.e. punctate intracellular staining in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024609#pone-0024609-g001" target="_blank">Figure 1</a>) (2) near the ventricular cell surface (i.e. polarized apical staining of cells in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024609#pone-0024609-g001" target="_blank">Figure 1</a> and (3) tethered at the cell surface (i.e. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024609#pone-0024609-g001" target="_blank">Figure 1B</a>). Upon injury, the sudden release and processing at the cell surface is followed by release from intracellular stores as a “panic signal” to indicate systemic injury. The initial pro-inflammatory response then shuts down Ecrg4 gene expression for at least 1 to 3 days post-injury. Therefore, during the initial phase of injury, its normal, constitutively inhibitory functions are absent and so repair cells proliferate. As gene expression returns, quiescence is restored and homeostasis re-established.</p

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