HuR (Elavl1) and HuB (Elavl2) Stabilize Matrix Metalloproteinase-9 mRNA During Seizure-Induced Mmp-9 Expression in Neurons

Matrix metalloproteinase-9 (Mmp-9) is involved in different general and cell-type–specific processes, both in neuronal and non-neuronal cells. Moreover, it is implicated in an induction or progression of various human disorders, including diseases of the central nervous system. Mechanisms regulating activity-driven Mmp-9 expression in neurons are still not fully understood. Here, we show that stabilization of Mmp-9 mRNA is one of the factors responsible for the neuronal activity-evoked upregulation of Mmp-9 mRNA expression in hippocampal neurons. Furthermore, we demonstrate that the molecular mechanism related to this stabilization is dependent on the neuronal seizure-triggered transiently increased binding of the mRNA stability-inducing protein, HuR, to ARE1 and ARE4 motifs of the 3′UTR for Mmp-9 mRNA as well as the stably augmented association of another mRNA-stabilizing protein, HuB, to the ARE1 element of the 3′UTR. Intriguingly, we demonstrate further that both HuR and HuB are crucial for an incidence of Mmp-9 mRNA stabilization after neuronal activation. This study identifies Mmp-9 mRNA as the first HuB target regulated by mRNA stabilization in neurons. Moreover, these results are the first to describe an existence of HuR-dependent mRNA stabilization in neurons of the brain

Epigenetics of Epileptogenesis-Evoked Upregulation of Matrix Metalloproteinase-9 in Hippocampus

<div><p>Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms–DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45β to Mmp-9 proximal gene promoter <i>in vivo</i>. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal <i>Mmp-9</i> promoter chromatin <i>in vivo</i>. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in <i>Neurospora</i>. The study identifies <i>MMP-9</i> as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of <i>MMP-9</i> expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.</p></div

MMP-9 mRNA upregulation is accompanied by robust <i>MMP-9</i> promoter demethylation in hippocampi of epileptic patients.

<p><b>(A) <i>MMP-9 mRNA expression is substantially increased in the hippocampi of adult and pediatric epileptic patients</i></b>. For each RT-qPCR analysis, equal amounts of RNA samples isolated from control, epileptic adult and pediatric patients’ hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; <i>n</i> = 6 for adults, <i>n</i> = 3–4 for pediatric patients). (<b>B) <i>MMP-9 proximal promoter methylation is strongly diminished in vivo in the hippocampi of epileptic patients</i></b>. <i>MMP-9</i> proximal promoter methylation level was evaluated by qPCR using DNA samples obtained by the immunoprecipitation of methylated DNA (MeDIP) coming from hippocampi of control as well as adult and pediatric epileptic patients. Data is presented as fold change in the methylation status. Values are means ± SEM (*, <i>p</i> < 0.05; ***, <i>p</i> < 0.001; <i>n</i> = 6 for adults, <i>n</i> = 3–4 for pediatric patients). <b>(C) <i>MMP-9 proximal promoter is hydroxymethylated at similar level in vivo in epileptic and control patients</i></b>. Level of <i>MMP-9</i> proximal promoter hydroxymethylation was evaluated using qPCR with DNA samples obtained from immunoprecipitation of hydroxymethylated DNA (hMeDIP) coming from hippocampi of control and epileptic patients. Data is presented as fold change in hydroxymethylation status. Values are means ± SEM (<i>n</i> = 6).</p

<i>Mmp-9</i> proximal promoter is progressively demethylated during PTZ-induced epileptogenesis in the rat hippocampus.

<p><b>(A) <i>Schematic presentation of our experimental approach to a detailed evaluation of the rat Mmp-9 proximal promoter methylation</i>.</b><i>In vivo</i> methylation status of ~1000 bp fragment of the <i>Mmp-9</i> proximal promoter was analyzed using the methylated DNA immunoprecipitation method (MeDIP). Methylation of CpG sites localized in the -1263/-685 and the -269/-1 bp fragments of the proximal <i>Mmp-9</i> promoter (sites 1–5 and 9–18 in the scheme) were analyzed <i>in vitro</i> by bisulfite sequencing. The methylation status of CpG sites situated in the -684/-270 bp fragment of the proximal <i>Mmp-9</i> promoter (sites 6–8 at the scheme) were evaluated <i>in vitro</i> using methylation-specific PCR (MSP). Additionally, to provide a control for the consistency of bisulfite sequencing results and data obtained by MSP, the -98/-97bp CpG site of the <i>Mmp-9</i> promoter was evaluated using both these procedures (compare middle panel in Fig 3C and 3D). (<b>B) <i>Mmp-9 proximal promoter is demethylated during epileptogenesis in vivo</i>.</b> Methylation level was revealed by MeDIP and presented as % of input. (<b>C) <i>Mmp-9 proximal promoter is progressively demethylated in most of its CpG sites during epileptogenesis in vitro</i>.</b> Methylation of CpG sites localized in -1263/-685 and -269/-1 bp fragments of <i>Mmp-9</i> promoter was evaluated using bisulfite sequencing. Graph data is presented as % of the total number of methylated and unmethylated CpG sites found in all examined clones in these regions (left panel) or as % of the total number of methylated and unmethylated CpG sites detected in the particular CpG site in the promoter fragments in all analyzed clones (central panel). Right panel presents results of DNA methylation at CpG sites located in the -684/-270 bp fragment of <i>Mmp-9</i> promoter using MSP. Data is presented as % of unmethylated DNA. (<b>D) <i>The -98/-97bp CpG site of Mmp-9 promoter is gradually demethylated during epileptogenesis in vitro</i>.</b> Methylation level was analyzed by MSP (presented as % of unmethylated DNA). (<b>E) <i>DNA hydroxymethylation of rat hippocampal Mmp-9 proximal promoter remains unchanged during epileptogenesis in vivo</i>.</b> Hydroxymethylation was assessed by hMeDIP and data is presented as % of input. In Fig 3B, 3C (right panel), 3D and 3E values are means ±SEM and <i>n</i> = 4. In Fig 3B and 3C (right panel) **, <i>p</i><0.01; ***, <i>p</i><0.001. In Fig 3D *, <i>p</i><0.05; ***, <i>p</i><0.001.</p

YY1- and PRC2-related repression controls epileptogenesis-induced MMP-9 upregulation in the hippocampus.

<p><b>(A) <i>YY1 is expressed on a constant level during epileptogenesis in the rat hippocampus</i>.</b> Equal amounts (20 μg) of nuclear cell lysates obtained from the unstimulated (control) as well as PTZ-kindled (partially kindled, fully kindled) rat hippocampi were analyzed by Western blot with anti-YY1 antibody. As a loading control, TATA-binding protein (TBP) was used. Representative cropped Western blot analyses are shown. <b>(B) <i>YY1 binds to the proximal Mmp-9 promoter in the unstimulated rat hippocampus</i>, <i>and dissociates from it early during hippocampal epileptogenesis in vivo</i>.</b> DNA samples were obtained by chromatin immunoprecipitation with anti-YY1 antibody from the unstimulated (control), and the PTZ-kindled (partially kindled, fully kindled) rat hippocampi. The <i>Mmp-9</i> proximal promoter content was evaluated by qPCR. Control ChIP reaction was performed using isotype antibody. Values are means ± SEM (***, <i>p</i> < 0.001; <i>n</i> = 3). <b>(C) <i>The expression of histone H3 trimethylated on lysine 27 (H3K27me3) is decreased and histone H2A monoubiquitylated on lysine 119 (H2AK119ub1) is increased during PTZ-induced kindling</i>.</b> Equal amounts (20 μg) of the nuclear cell lysates were analyzed by Western blot. As a loading control, histone H3 was used. (<b>D) <i>During epileptogenesis in the rat hippocampus</i>, <i>PRC2-related transcriptionally repressive histone modification H3K27me3 is progressively decreased in chromatin of the proximal Mmp-9 promoter in vivo</i>.</b> DNA samples were chromatin immunoprecipitated using anti-H3K27me3 antibody and analyzed by qPCR for <i>Mmp-9</i> proximal promoter content. Values are means ±SEM (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; <i>n</i> = 4). (<b>E) <i>In the rat hippocampus</i>, <i>the PRC1-related transcriptionally repressive histone modification H2AK119ub1 is not present in the chromatin of the proximal Mmp-9 promoter in vivo in the unstimulated rats as well as in rats developing epilepsy</i>.</b> DNA samples were chromatin immunoprecipitated with anti-H2AK119ub1 antibody and analyzed by qPCR for <i>Mmp-9</i> proximal promoter content. Values are means ± SEM (<i>n</i> = 4). <b>(F) <i>Histone H3 is strongly and stably enriched in the chromatin of the proximal MMP-9 promoter in vivo during epileptogenesis in the rat hippocampus</i>.</b> DNA samples were chromatin immunoprecipitated with anti-histone H3 antibody and analyzed by qPCR for <i>Mmp-9</i> proximal promoter content. Values are means ± SEM (**, <i>p</i> < 0.01; <i>n</i> = 4).</p

Coordinated action of Dnmt3a, Dnmt3b and Gadd45β regulates the epileptogenesis-evoked Mmp-9 promoter demethylation in hippocampus.

<p><b>(A) <i>Dnmt3a and Dnmt3b</i>, <i>but not Dnmt1</i>, <i>bind to the proximal Mmp-9 promoter in the unstimulated rat hippocampus</i>, <i>and are gradually dissociating from it during the hippocampal epileptogenesis in vivo</i>.</b> DNA samples were obtained by chromatin immunoprecipitation with anti-Dnmt3a (left graph), anti-Dnmt3b (central graph), or anti-Dnmt1 (right graph) antibodies from the unstimulated (control), and the PTZ-kindled (partially kindled, fully kindled) rat hippocampi. <i>Mmp-9</i> proximal promoter content was evaluated by qPCR. Control ChIP reaction was performed using isotype antibody. Values are means ± SEM (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; <i>n</i> = 4). (<b>B) <i>The regulator of neuronal activity–dependent DNA demethylation Gadd45β</i>, <i>but not its functional and structural homologue Gadd45α</i>, <i>increasingly binds to the Mmp-9 proximal promoter during epileptogenesis in the rat hippocampus in vivo</i>.</b> DNA samples were obtained by chromatin immunoprecipitation with anti-Gadd45α (left graph) or anti-Gadd45β (right graph) antibodies from naive (control) as well as from the PTZ-treated (partially kindled, fully kindled) rat hippocampi. <i>Mmp-9</i> proximal promoter content was evaluated by qPCR. Control ChIP reaction was performed using isotype antibody. Values are means ± SEM (*, <i>p</i> < 0.05; **, <i>p</i> < 0.01; <i>n</i> = 4).</p

Model of the epileptogenesis-evoked upregulation of Mmp-9 expression in the hippocampus.

<p><b>(A)</b> In the control rat hippocampus, Mmp-9 proximal promoter is bound by YY1, which nucleates the DNA methyltransferases, Dnmt3a and Dnmt3b, as well as PRC2, leading to the strong promoter DNA methylation and simultaneous trimethylation on lysine 27 of histone H3 in the surrounding chromatin. (<b>B)</b> In partially kindled rat hippocampus, YY1 dissociates out of the gene promoter, leading to a partial removal from the chromatin region other silencing proteins Dnmt3a, Dnmt3b, and PRC2. Concomitantly, DNA-demethylation-related protein Gadd45β and the activating histone mark H3S10ph start to accumulate in the chromatin. Consequently, it induces partial demethylation of the Mmp-9 gene promoter and leads to a reduction in its chromatin of the repressive chromatin mark H3K27me3. These complex molecular events lead to a moderate stimulation of the Mmp-9 gene expression. <b>(C)</b> In the fully kindled rat hippocampus, Mmp-9 expression is highly upregulated as a consequence of a much more profound proximal promoter demethylation (due to a complete dissociation from its chromatin of Dnmt3a and Dnmt3b as well as a strongly increased accumulation of Gadd45β), disappearance of the PRC2-related repression with a complete removal of H3K27me3, and a significant increase in the abundance of H3S10ph in the chromatin region.</p

Mmp-9 upregulation during epilepsy development is strictly dependent on epileptogenesis-evoked demethylation of its gene promoter.

<p>To block epilepsy development, we used dizocilpine (the NMDA receptor antagonist displaying anticonvulsant activity). 0.1 mg/kg of dizocilpine or saline was intraperitoneally injected to rats 30 min before each PTZ dose administration (30 mg/kg). (<b>A) <i>Dizocilpine treatment effectively suppresses the development of PTZ-evoked epilepsy in rats</i></b>. Animals were observed up to 2 h after each PTZ injection and seizures were scored according to a modified Racine’s scale. Values are means ± SEM (*, <i>p</i><0.05; <i>n</i> = 11). (<b>B) <i>PTZ kindling-evoked upregulation of the Mmp-9 mRNA expression is fully inhibited by dizocilpine administration in the rat hippocampus</i>.</b> Dizocilpine administration suppresses the PTZ kindling–evoked augmentation in the hippocampal Mmp-9 mRNA expression, whereas repeated PTZ treatment without dizocilpine injections leads to significant upregulation of Mmp-9 mRNA level. For RT-qPCR analysis equal amounts of RNA isolated from naive (control), PTZ-treated (saline + PTZ), and dizocilpine-treated (dizocilpine + PTZ) rat hippocampi were used. Data is presented as fold change in mRNA expression. Values are means ± SEM (*, <i>p</i><0.05; ***, <i>p</i><0.001; <i>n</i> = 8). <b>(C) <i>Dizocilpine treatment completely inhibits the PTZ kindling-dependent Mmp-9 proximal promoter demethylation in the rat hippocampus</i></b>. <i>Mmp-9</i> proximal promoter methylation level was evaluated using qPCR in DNA samples obtained by immunoprecipitation of methylated DNA from naive (control), PTZ-treated (saline+PTZ), and dizocilpine-treated (dizocilpine+PTZ) rat hippocampi. Data is presented as a percent of input. Values are means ± SEM (*, <i>p</i><0.05; <i>n</i> = 5).</p