Rhythmic Regulation of DNA Methylation Factors and Core-Clock Genes in Brain Structures Activated by Cocaine or Sucrose: Potential Role of Chromatin Remodeling.

The circadian system interacts with the mesocorticolimbic reward system to modulate reward and memory in a time-of-day dependent manner. The circadian discrimination of reward, however, remains difficult to address between natural reinforcers and drugs of abuse. Circadian rhythms control cocaine sensitization and conversely cocaine causes long-term alteration in circadian periodicity in part through the serotonergic neurotransmission. Since neural circuits activated by cocaine and natural reinforcers do not completely overlap, we compared the effect of cocaine with that of sucrose, a strong reinforcer in rodents, by using passive chronic administration. The expression of fifteen genes playing a major role in DNA methylation (Dnmts, Tets), circadian rhythms (Clock, Bmal1, Per1/2, Cry1/2, Rev-Erbß, Dbp1), appetite, and satiety (Orexin, Npy) was analyzed in dopamine projection areas like the prefrontal cortex, the caudate putamen, and the hypothalamus interconnected with the reward system. The corresponding proteins of two genes (Orexin, Per2) were examined by IHC. For many factors controlling biological and cognitive functions, striking opposite responses were found between the two reinforcers, notably for genes controlling DNA methylation/demethylation processes and in global DNA methylation involved in chromatin remodeling. The data are consistent with a repression of critical core-clock genes by cocaine, suggesting that, consequently, both agents differentially modulate day/night cycles. Whether observed cocaine and sucrose-induced changes in DNA methylation in a time dependent manner are long lasting or contribute to the establishment of addiction requires further neuroepigenetic investigation. Understanding the mechanisms dissociating drugs of abuse from natural reinforcers remains a prerequisite for the design of selective therapeutic tools for compulsive behaviors.

Epigenetic Regulation of Circadian Clocks and Its Involvement in Drug Addiction.

Based on studies describing an increased prevalence of addictive behaviours in several rare sleep disorders and shift workers, a relationship between circadian rhythms and addiction has been hinted for more than a decade. Although circadian rhythm alterations and molecular mechanisms associated with neuropsychiatric conditions are an area of active investigation, success is limited so far, and further investigations are required. Thus, even though compelling evidence connects the circadian clock to addictive behaviour and vice-versa, yet the functional mechanism behind this interaction remains largely unknown. At the molecular level, multiple mechanisms have been proposed to link the circadian timing system to addiction. The molecular mechanism of the circadian clock consists of a transcriptional/translational feedback system, with several regulatory loops, that are also intricately regulated at the epigenetic level. Interestingly, the epigenetic landscape shows profound changes in the addictive brain, with significant alterations in histone modification, DNA methylation, and small regulatory RNAs. The combination of these two observations raises the possibility that epigenetic regulation is a common plot linking the circadian clocks with addiction, though very little evidence has been reported to date. This review provides an elaborate overview of the circadian system and its involvement in addiction, and we hypothesise a possible connection at the epigenetic level that could further link them. Therefore, we think this review may further improve our understanding of the etiology or/and pathology of psychiatric disorders related to drug addiction.

Regulation of Brain DNA Methylation Factors and of the Orexinergic System by Cocaine and Food Self-Administration.

Inhibitors of DNA methylation and orexin type-1 receptor antagonists modulate the neurobiological effects driving drugs of abuse and natural reinforcers by activating common brain structures of the mesolimbic reward system. In this study, we applied a self-administration paradigm to assess the involvement of factors regulating DNA methylation processes and satiety or appetite signals. These factors include Dnmts and Tets, miR-212/132, orexins, and orx-R1 genes. The study focused on dopamine projection areas such as the prefrontal cortex (PFCx) and caudate putamen (CPu) and in the hypothalamus (HP) that is interconnected with the reward system. Striking changes were observed in response to both reinforcers, but differed depending on contingent and non-contingent delivery. Expression also differed in the PFCx and the CPu. Cocaine and food induced opposite effects on Dnmt3a expression in both brain structures, whereas they repressed both miRs to a different extent, without affecting their primary transcript in the CPu. Unexpectedly, orexin mRNAs were found in the CPu, suggesting a transport from their transcription site in the HP. The orexin receptor1 gene was found to be induced by cocaine in the PFCx, consistent with a regulation by DNA methylation. Global levels of 5-methylcytosines in the PFCx were not significantly altered by cocaine, suggesting that it is rather their distribution that contributes to long-lasting behaviors. Together, our data demonstrate that DNA methylation regulating factors are differentially altered by cocaine and food. At the molecular level, they support the idea that neural circuits activated by both reinforcers do not completely overlap.

Differential regulation of MeCP2 and PP1 in passive or voluntary administration of cocaine or food.

Cocaine exposure induces changes in the expression of numerous genes, in part through epigenetic modifications. We have initially shown that cocaine increases the expression of the chromatin remodeling protein methyl-CpG binding protein 2 (MeCP2) and characterized the protein phosphatase-1Cß (PP1Cß) gene, as repressed by passive i.p. cocaine injections through a Mecp2-mediated mechanism involving de novo DNA methylation. Both proteins being involved in learning and memory processes, we investigated whether voluntary cocaine administration would similarly affect their expression using an operant self-administration paradigm. Passive and voluntary i.v. cocaine intake was found to induce Mecp2 and to repress PP1Cß in the prefrontal cortex and the caudate putamen. This observation is consistent with the role of Mecp2 acting as a transcriptional repressor of PP1Cß and shows that passive intake was sufficient to alter their expression. Surprisingly, striking differences were observed under the same conditions in food-restricted rats tested for food pellet delivery. In the prefrontal cortex and throughout the striatum, both proteins were induced by food operant conditioning, but remained unaffected by passive food delivery. Although cocaine and food activate a common reward circuit, changes observed in the expression of other genes such as reelin and GAD67 provide new insights into molecular mechanisms differentiating neuroadaptations triggered by each reinforcer. The identification of hitherto unknown genes differentially regulated by drugs of abuse and a natural reinforcer should improve our understanding of how two rewarding stimuli differ in their ability to drive behavior.

Cocaine represses protein phosphatase-1Cß through DNA methylation and Methyl-CpG Binding Protein-2 recruitment in adult rat brain.

Repeated cocaine exposure induces epigenetic factors such as DNA methyl-binding proteins, indicating that resulting changes in gene expression are mediated by alterations in brain DNA methylation. While the activity of protein phosphatase type-1 (PP1) is involved in cocaine effects and in brain plasticity, the expression of the PP1Cß catalytic subunit gene was identified here as modulated by cocaine. Its expression was induced together with that of PP1Cγ in the brain of Methyl-CpG Binding Protein-2 (Mecp2) mutant mice, whereas PP1Cα expression was not affected, illustrating a different regulation of PP1C isoforms. Repeated cocaine administration was found to increase DNA methylation at the PP1Cß gene together with its binding to Mecp2 in rat caudate putamen, establishing a link between two genes involved in cocaine-related effects and in learning and memory processes. Cocaine also increased DNMT3 expression, resulting in PP1Cß repression that did not occur in the presence of DNMT inhibitor. Cocaine-induced PP1Cß repression was observed in several brain structures, as evaluated by RT-qPCR, immunohistochemistry and Western blot, but did not occur after a single cocaine injection. Our data demonstrate that PP1Cß is a direct MeCP2-target gene in vivo. They suggest that its repression may participate to behavioral adaptations triggered by the drug.

Histone deacetylase inhibitors upregulate MMP11 gene expression through Sp1/Smad complexes in human colon adenocarcinoma cells.

MMP-11 (stromelysin-3) is a matrix metalloproteinase associated with tumor progression and poor prognosis. Its expression was initially described exclusively in stromal cells surrounding tumors, but more recently it has also been detected in macrophages and hepatocarcinoma cells. Here we show MMP-11 expression in human epithelial colon adenocarcinoma cell lines (Caco-2, HT-29 and BCS-TC2). Treatment of BCS-TC2 cells with butyrate and trichostatin A (TSA) (histone deacetylase inhibitors) increases MMP11 promoter activity and protein expression. Using electrophoretic mobility shift assay (EMSA) and supershift assays, we demonstrate for the first time that Sp1 is able to bind to the GC-boxes within the MMP11 proximal promoter region; this binding has been confirmed by chromatin immunoprecipitation. Sp1 is involved in MMP11 basal expression and it is essential for the upregulation of transcription by histone deacetylase inhibitors as deduced from mutant constructs lacking the Sp1 sites and by inhibition of its binding to the promoter with mithramycin. This regulation requires the formation of Sp1/Smad2 heterocomplexes, which is stimulated by an increase in the acetylation status of Smad after butyrate or TSA treatments. We have also found that ERK1/2-mitogen-activated protein kinase (MAPK), but not p38-MAPK or JNK, is involved in the upregulation of MMP11 by HDAC inhibitors.

CDKL5 is a brain MeCP2 target gene regulated by DNA methylation.

Rett syndrome and its "early-onset seizure" variant are severe neurodevelopmental disorders associated with mutations within the MECP2 and the CDKL5 genes. Antidepressants and drugs of abuse induce the expression of the epigenetic factor MeCP2, thereby influencing chromatin remodeling. We show that increased MeCP2 levels resulted in the repression of Cdkl5 in rat brain structures in response to cocaine, as well as in cells exposed to serotonin, or overexpressing MeCP2. In contrast, Cdkl5 was induced by siRNA-mediated knockdown of Mecp2 and by DNA-methyltransferase inhibitors, demonstrating its regulation by MeCP2 and by DNA methylation. Cdkl5 gene methylation and its methylation-dependent binding to MeCP2 were increased in the striatum of cocaine-treated rats. Our data demonstrate that Cdkl5 is a MeCP2-repressed target gene providing a link between genes the mutation of which generates overlapping symptoms. They highlight DNA methylation changes as a potential mechanism participating in the long-term plasticity triggered by pharmacological agents.

Inhibition of histone deacetylases in rats self-administering cocaine regulates lissencephaly gene-1 and reelin gene expression, as revealed by microarray technique.

Injection of the histone deacetylase inhibitor trichostatin A (TsA) to rats has been shown to decrease their motivation to self-administer cocaine. In the present study, we investigated alterations in gene expression patterns in the anterior cingulate cortex and nucleus accumbens of rats self-administering cocaine and treated with TsA. Using oligonucleotide microarrays, we identified 722 probe sets in the cortex and 136 probe sets in the nucleus accumbens that were differentially expressed between vehicle and TsA-treated rats that self-administered cocaine. Microarray data were validated by real-time PCR for seven genes. Using immunohistochemistry, we further investigated the expression of Lis1 and reelin genes, because (i) they were similarly regulated by TsA at the mRNA level; (ii) they belong to the same signal transduction pathway; (iii) mutations within both genes cause lissencephaly. Cocaine self-injection was sufficient to activate the two genes at both the mRNA and protein levels. TsA treatment was found to up-regulate both Lis1 and reelin protein expression in the cortex and to down-regulate it in the nucleus accumbens of rats self-administering cocaine. The data suggest that the two proteins contribute to establish neurobiological mechanisms underlying brain plasticity whereby TsA lowers the motivation for cocaine.

Fluoxetine and cocaine induce the epigenetic factors MeCP2 and MBD1 in adult rat brain.

Once bound to methylated CpG sites, methyl-CpG-binding protein 2 (MeCP2) is thought to silence transcription of downstream genes by recruiting a histone deacetylase (HDAC). Mutations within the MeCP2 gene have been found to cause Rett syndrome, a disorder of arrested neuronal development. Using immunohistochemistry, we found that Mecp2, as well as the methyl-CpG-binding protein MBD1, were significantly induced in normal adult rat brain after repeated injections of fluoxetine or cocaine for 10 days (one injection per day). Mecp2 was not induced by repeated injections of 1-(2-bis(4-fluorphenyl)-methoxy)-ethyl)-4-(3-phenyl-propyl)piperazine (GBR-12909) or nortriptyline. Together, the data indicate that the serotonergic system is predominantly involved. Using real-time reverse transcription-polymerase chain reaction experiments, MBD1 mRNA and both Mecp2_e1 and Mecp2_e2 transcripts were found to be induced by fluoxetine. Induction of the methylbinding proteins was accompanied with enhanced HDAC2 labeling intensity and mRNA synthesis in response to fluoxetine. In tandem, acetylated forms of histone H3 were found to be decreased. The effect was characterized in three serotonin projection areas, the caudate-putamen, the frontal cortex, and the dentate gyrus subregion of hippocampus. Our data highlight GABAergic neurons as major target cells expressing Mecp2 in response to the serotonin-elevating agents and suggest that serotonin signaling enhances gene silencing in postmitotic neurons.

Catechins prevent vascular smooth muscle cell invasion by inhibiting MT1-MMP activity and MMP-2 expression.

OBJECTIVE: Regular consumption of green tea is associated with a reduced risk of mortality due to coronary diseases and cancer. The present study examined whether a green tea extract (GTE) inhibits activation of matrix metalloproteinase-2 (MMP-2), a major collagenase involved in vascular remodeling of atherosclerotic plaques, in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: The expression of MMP-2 was assessed by Northern and Western blot analyses in human aortic VSMCs. MMP-2 activity was evaluated by zymography, membrane-type1-MMP (MT1-MMP, MMP-14) activity by an enzymatic assay, and cell invasion by a modified Boyden chamber assay. The thrombin-induced activation of secreted MMP-2 was abolished by GTE and the green tea polyphenols (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG). GTE reduced the expression of MMP-2 mRNA and protein. GTE, EGCG and ECG directly inhibited cell-associated MT1-MMP activity, the physiological activator of MMP-2, in a reversible manner. Thrombin-stimulated VSMCs invasion was abolished by EGCG and ECG, and reduced by GTE. CONCLUSIONS: GTE inhibits thrombin-induced VSMCs invasion most likely by preventing MMP-2 expression and its activation by a direct inhibition of MT1-MMP. The ability of green tea to prevent cell invasion and matrix degradation might contribute to its protective effect on atherosclerosis and cancer.

Tumor cell-mediated induction of the stromal factor stromelysin-3 requires heterotypic cell contact-dependent activation of specific protein kinase C isoforms.

Stromelysin-3 (ST3, MMP-11) has been shown to be strongly overexpressed in stromal fibroblasts of most invasive human carcinomas. However, the molecular mechanisms leading to ST3 expression in nonmalignant fibroblasts remain unknown. The aim of the present study was to analyze the signaling pathways activated in normal pulmonary fibroblasts after their interaction with non-small cell lung cancer (NSCLC) cells and leading to ST3 expression. The use of selective signaling pathway inhibitors showed that conventional and novel protein kinase Cs (PKC) were required for ST3 induction, whereas Src kinases exerted a negative control. We observed by both conventional and real time confocal microscopy that green fluorescent protein-tagged PKCalpha and PKCepsilon, but not PKCdelta, transfected in fibroblasts, accumulate selectively at the cell-cell contacts between fibroblasts and tumor cells. In agreement, RNAi-mediated depletion of PKCalpha and PKCepsilon, but not PKCdelta significantly decreased co-culture-dependent ST3 production. Finally, a tetracycline-inducible expression model allowed us to confirm the central role of these PKC isoforms and the negative regulatory function of c-Src in the control of ST3 expression. Altogether, our data emphasize signaling changes occurring in the tumor microenvironment that may define new stromal targets for therapeutic intervention.

Clinicopathological significance of stromelysin-3 expression in colorectal cancer.

OBJECTIVE: Stromelysin-3 (ST3) is a member of the matrix metalloproteinases and suggested to play a role in tissue remodeling observed in growth and metastasis of tumors. ST3 overexpression in breast cancer is associated with a worse outcome. Our aims were to analyze ST3 expression in primary colorectal tumors and metastases, and further to identify relationships of the expression to clinicopathological factors. MATERIALS AND METHODS: ST3 expression was immunohistochemically analyzed in 200 primary colorectal adenocarcinomas and 36 corresponding lymph node metastases. RESULTS: Scoring was performed by counting the percentages of positive cells and the percentages of positive areas. One hundred and one (51%) cases showed < or = 5% positive cells and 99 (49%) >5% positive cells. One hundred and two (51%) cases showed < or = 30% positive area and 98 (49%) >30% positive area. ST3 expression determined by both scoring methods was individually related to females, distally located tumors, infiltrative growth pattern and microsatellite stability. No relationship was found with age, Dukes' stage, differentiation and survival. CONCLUSIONS: These results suggest that ST3 protein was more involved in the pathway of colorectal cancer development in females, distal locations, infiltrative growth patterns and microsatellite stability.

Red wine polyphenolic compounds strongly inhibit pro-matrix metalloproteinase-2 expression and its activation in response to thrombin via direct inhibition of membrane type 1-matrix metalloproteinase in vascular smooth muscle cells.

BACKGROUND: Regular consumption of moderate amounts of red wine is associated with a reduced risk of coronary disease. Matrix metalloproteinases (MMPs) that participate in extracellular matrix degradation have been involved in atherosclerotic plaque growth and instability. The present study examined whether red wine polyphenolic compounds (RWPCs) inhibit activation of MMP-2, a major gelatinase, in vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Expression of pro-MMP-2 was assessed by Western and Northern blot analyses; MMP-2 activity was assessed by zymography and cell invasion by a modified Boyden's chamber assay. High levels of pro-MMP-2 and low levels of MMP-2 activity were found in conditioned medium from unstimulated VSMCs. Thrombin induced cell-associated pro-MMP-2 protein expression and MMP-2 activity in conditioned medium of VSMCs. The stimulatory effect of thrombin on MMP-2 activation was prevented by RWPCs in a concentration-dependent and reversible manner. Thrombin markedly increased cell-associated membrane type 1 (MT1)-MMP activity, the physiological activator of pro-MMP-2, and this response was not affected by RWPCs. However, addition of RWPCs directly to MT1-MMP abolished its metalloproteinase activity in a reversible manner. Finally, matrix invasion of VSMCs was stimulated by thrombin, and this response was prevented by RWPCs as efficiently as a broad-spectrum MMP inhibitor. CONCLUSIONS: The present findings demonstrate that RWPCs effectively inhibit thrombin-induced matrix invasion of VSMCs, most likely by preventing the expression and activation of MMP-2 via direct inhibition of MT1-MMP activity. The inhibitory effect of RWPCs on the activation of pro-MMP-2 and matrix degradation might contribute to their beneficial effects on the cardiovascular system.

Matrix metalloproteinases are differentially expressed in adipose tissue during obesity and modulate adipocyte differentiation.

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling, a process that takes place during obesity-mediated adipose tissue formation. Here, we examine expression profiles and the potential role of MMPs and their tissue inhibitors (TIMPs) in adipose tissue remodeling during obesity. Expression patterns are studied by Northern blot and real-time PCR in two genetic models of obesity (ob/ob and db/db mice) and in a diet-induced model of obesity (AKR mice). Of the MMPs and TIMPs studied, mRNA levels for MMP-2, MMP-3, MMP-12, MMP-14, MMP-19, and TIMP-1 are strongly induced in obese adipose tissues compared with lean tissues. In contrast, MMP-7 and TIMP-3 mRNAs are markedly decreased in obesity. Interestingly, enzymatic activities of MMP-12 and of a new identified adipocyte-derived 30-kDa metalloproteinase are enhanced in obese adipose tissue fractions, demonstrating that MMP/TIMP balance is shifted toward increased matrix degradation in obesity. Finally, we analyze the modulation of MMP-2, MMP-19, and TIMP-1 during 3T3-L1 preadipocyte differentiation, and we explore the effect of inhibition of MMP activity on in vitro adipogenesis. We find that the synthetic MMP inhibitor BB-94 (Batimastat) decreases adipose conversion of 3T3-L1 and primary rat preadipocytes. BB-94 represses differentiation without affecting mitotic clonal expansion but prevents the early expression of CCAAT/enhancer-binding protein beta, a transcription factor that is thought to play a major role in the adipogenic program. Such findings support a role for the MMP/TIMP system in the control of proteolytic events and adipogenesis during obesity-mediated fat mass development.

Alternative splicing and promoter usage generates an intracellular stromelysin 3 isoform directly translated as an active matrix metalloproteinase.

Human stromelysin 3 (ST3) is a matrix metalloproteinase (MMP) that has been implicated in cancer progression and in various tissue remodeling processes. Unlike most MMPs, ST3 is characterized by a distinct substrate specificity and a specific regulation and is not directly involved in extracellular matrix degradation. In the present study, we have identified an additional ST3 gene promoter that is accessible to nuclear factors such as C/EBP and retinoic acid receptors. This human specific promoter is inducible and controls the expression of a novel ST3 transcript called the beta-ST3 that is expressed in cultured cells and in placenta. This transcript encodes a 40-kDa ST3 isoform that lacks both the signal peptide common to all secreted MMPs and the prodomain that normally maintains enzyme latency. Consistent with the lack of a signal peptide, the beta-ST3 was found to be intracellular. The relative amount of the extracellular alpha-ST3 isoform was about 20-fold higher than that of the intracellular ST3 isoforms, as estimated by Western blot analysis. Furthermore, recombinant beta-ST3 produced in Escherichia coli exhibits a proteolytic activity against alpha1-proteinase inhibitor, a substrate previously shown to be inactivated by the alpha-ST3. Therefore, although it was thought that all MMPs were synthesized as inactive zymogens and functioned extracellularly, this is the first MMP isoform reported that is generated by alternative promoter usage and directly translated as an active enzyme. Although the intracellular function of the beta-ST3 remains to be investigated, these data support the idea that the functions of MMPs are not restricted to the extracellular space.

Rat liver injury after normothermic ischemia is prevented by a phosphinic matrix metalloproteinase inhibitor.

Hepatic ischemia occurs in liver transplantation, hemodynamic or cardiogenic shock, and liver resection associated with trauma or tumor. Ischemia/reperfusion (I/R) injury results in microcirculation failure followed by apoptosis and necrosis. Matrix metalloproteinases (MMPs) are involved in many physiological and pathological processes, but their expression and function during liver I/R remains poorly documented. In this study, we evaluated the expression of nine MMPs and their natural inhibitors, tissue inhibitors of MMPs (TIMPs), in a rat model of liver I/R. Analysis of MMP and TIMP expression show that although most of these genes are not constitutively expressed in the normal liver, they are induced in a specific time-dependent manner following I/R. Stromelysin-1, gelatinase B, and collagenase-3 are induced during the early phase of acute liver injury associated with inflammation and increased necrosis/apoptosis, whereas gelatinase A, membrane type-MMP, stromelysin-3, metalloelastase, TIMP-1, and TIMP-2 are essentially detectable during the recovery phase of liver injury corresponding to hepatocyte regeneration. This observation suggested that MMPs and TIMPs could play both deleterious and beneficial roles following I/R. We thus tested the effect of a specific phosphinic MMP inhibitor on acute liver I/R injury. Inhibition of MMP activity was shown to significantly decrease liver injury in ischemic/reperfused liver tissue as assessed by histological studies and serum hepatic enzyme levels. We therefore propose that MMP inhibitors may be of clinical relevance in liver-associated ischemic diseases or after liver transplantation.