Mu opioid receptors (MOPs) in the pain pathway contribute to morphine analgesia. Morphine also stimulates reward/reinforcement through disinhibition of dopaminergic (DA) neurones in the ventral tegmental area (VTA), an effect implicated in its abuse and dependence. We hope to develop approaches to achieve sustained analgesia without affecting reward by exploiting differential MOP signalling mechanisms in the pain and reward pathways. MOPs, delta opioid receptors (DOPs) and β-arrestin2 (BAR2) are all necessary components of the signalling complex in nociceptive neurones for morphine analgesic tolerance; c-Src (a tyrosine kinase), thought to couple to MOP receptors through BAR2 has also been implicated. To investigate opioid receptor signalling in response to morphine we used a variety of different techniques that included behavioural measures of nociception, reinforcement and locomotion and electrophysiological methods to study DRG neurones from the pain pathway and brain slices containing VTA neurones. This study in mice confirms that morphine administered subcutaneously (SC) causes analgesia, analgesic tolerance, and has psychomotor effects leading to enhanced locomotion and reinforcement. In VTA neurones morphine and the selective MOP receptor agonist DAMGO caused concentration-dependent inhibition of the frequency of IPSCs. All these actions of morphine were absent from MOP-/- mice. Morphine exhibited reduced potency as 1) an analgesic, 2) stimulator of locomotion, 3) a reinforcer in CPP and 4) an inhibitor of sIPSC frequency, when applied to MOP+/- mice or their VTA neurones. Morphine analgesic tolerance developed faster and to a greater extent in MOP+/- mice than in WT mice. DOP-/- mice exhibited morphine analgesia with less tolerance, as did BAR2-/- mice. BAR2-/- mice also exhibited reduced morphine locomotion and an increased sensitivity to morphine reinforcement. Morphine tolerance was absent from BAR2-/-//DOP-/- mice. The inhibition of sIPSC frequency by morphine was reduced in BAR2+/- and BAR2-/- VTA neurones. Dasatinib and PP2 (c-Src tyrosine kinase inhibitors) prevented the development of morphine tolerance in WT and MOP+/- mice and dasatinib caused its reversal in the latter. The drugs had no significant analgesic effect alone. Dasatinib did not affect morphine preference or locomotor activation. PP2 reduced morphine’s inhibition of sIPSC frequency. As c-Src inhibition does not appear to alter the psychomotor effects produced by morphine and it acts to reduce morphine analgesic tolerance. We believe that cSrc is an attractive target to prevent the development of morphine analgesic tolerance without affecting hedonic homeostasis.

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Palmitic acid (PA), a type of saturated fatty acids, induces cardiovascular diseases by causing cardiomyocyte apoptosis with unclear mechanisms. Akt participates in PA-induced cardiomyocyte apoptosis. GSK-3β is a substrate of Akt, we investigated its role in PA-induced apoptosis. We reveal that PA inhibits GSK-3β phosphorylation accompanied by inactivation of Akt in H9c2 cardiomyocytes. We also reveal that inhibition the activity of GSK-3β by its inhibitor LiCl or knockdown by siRNA significantly attenuates PA-induced cardiomyocyte apoptosis, this suggesting that GSK-3β plays a pro-apoptotic role. To detect its downstream factors, we analyzed the roles of JNK, p38 MAPK and β-arrestin 2 (β-Arr2). Here, we report that GSK-3β regulate PA-induced cardiomyocyte apoptosis by affecting the distribution of β-Arr2. PA diminishes the protein level of β-Arr2 and changes its distribution from nucleus to cytoplasm. Either inhibition of β-Arr2 by its siRNA or overexpression of its protein level by transfection of β-Arr2 full-length plasmid promotes PA-induced cardiomyocyte apoptosis, which remind us to focus on the changes of its location. β-Arr2 siRNA decreased the background level of β-Arr2 in nucleus in normal H9c2 cells. Overexpression of β-Arr2 increased cytoplasm level of β-Arr2 as PA did. While LiCl, the inhibitor of GSK-3β decreased PA-induced apoptosis, accompany with increased nucleus level of β-Arr2. Then we concluded that GSK-3β is closely associated with cardiomyocyte apoptosis induced by PA, it performs its pro-apoptotic function by affecting the location of β-Arr2. LiCl inhibits PA-induced cardiomyocyte apoptosis, which might provide novel therapeutic for cardiovascular diseases induced by metabolic syndrome.

Stem-cell antigen 1-positive (Sca-1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5'-azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. b-arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of b-arrestin2 in Sca-1+ CSC differentiation, we used b-arrestin2-knockout mice and overexpression strategies. Real-time PCR revealed that b-arrestin2 promoted 5'-azacytizine-induced Sca-1+ CSC differentiation in vitro. Because the microRNA 155 (miR-155) may regulate b-arrestin2 expression, we detected its role and relationship with b-arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR-155. Real-time PCR revealed that miR-155, inhibited by b-arrestin2, impaired 5'-azacytizine-induced Sca-1+ CSC differentiation. On luciferase report assay, miR-155 could inhibit the activity of b-arrestin2 and GSK3β, which suggests a loop pathway between miR-155 and b-arrestin2. Furthermore, b-arrestin2-knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in b-arrestin2-Knockout mice, so the activity of GSK3β was regulated by b-arrestin2 not Akt. We transplanted Sca-1+ CSCs from b-arrestin2-knockout mice to mice with myocardial infarction and found similar protective functions as in wild-type mice but impaired arterial elastance. Furthermore, low level of b-arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β-arrestin2/miR-155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.

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Les β-arrestines (Arrbs) régulent diverses voies de signalisation dont la voie Wnt/β-caténine (Wnt), un acteur clé dans le cancer colorectal. Le but de mon projet était d'étudier l'implication et les mécanismes régulés par les Arrbs dans la tumorigenèse intestinale dépendante de la voie Wnt. L'inhibition de l'expression des Arrbs partielle ou totale dans des souris ApcΔ14/+ montre que seules les souris invalidées pour l'Arrb2 développent 33% des tumeurs détectées chez les souris ApcΔ14/+ ; Arrb2+/+. Ces tumeurs ont une croissance normale. Cependant, l'analyse de leur transcriptome montre qu'elles expriment notamment certains gènes liés au système immunitaire, alors que les tumeurs dépendantes de l'Arrb2 expriment des gènes différents impliqués entre autres dans la voie Wnt. L'invalidation de l'Arrb2 réduit l'expression de gènes cibles de la voie Wnt dans les cellules isolées de 12 sur 18 tumeurs de souris ApcΔ14/+, et inhibe l'augmentation d'activité Wnt et la formation de colonies en agar mou induite par l'invalidation d'Apc dans des cellules murines ApcMin/+. L'Arrb2 est donc essentielle pour l'initiation et la croissance des tumeurs intestinales présentant une activité Wnt élevée. Pour comprendre les mécanismes régulés par l'Arrb2 dans ce contexte, les complexes protéiques associés à l'Arrb2 ont été analysés par protéomique dans des cellules humaines de cancer colorectal SW480 exprimant ou non un dominant négatif de Tcf4. 132 partenaires de l'Arrb2 potentiellement imbriqués dans un réseau de 917 protéines, ont été identifiés dans les cellules où la voie Wnt est active. Une baisse de 80% de l'activité Wnt entraine la disparition de 41 protéines avec 256 interactions potentielles alors que 42 protéines apparaissent avec 244 interactions potentielles. Le rôle clé d'Arrb2 dans le cancer colorectal s'expliquerait par la connexion d'au moins une quarantaine de protéines dépendantes de l'activité Wnt à un réseau de signalisation complexe dont l'analyse est en cours. / Β-arrestins (Arrbs) participate in the regulation of multiple signaling pathways, including Wnt/β-catenin (Wnt), the major actor in human colorectal cancer. The aim of my project was to study the involvement of Arrbs and the mechanisms they regulate in Wnt-dependent intestinal tumorigenesis. The partial or total inhibition of Arrbs in ApcΔ14/+ mice showed that only mice with Arrb2 depletion developed only 33% of the tumors detected in their Arrb2-WT littermates. These remaining tumors grow normally and are Arrb2–independent. Transcriptomic analysis showed that they overexpressed genes that reflect a high interaction with the immune system, whereas those overexpressed in Arrb2–dependent tumors are predominantly involved in Wnt signaling. Moreover, Arrb2 siRNAs decreased the expression of Wnt target genes in cells isolated from 12 of 18 tumors from ApcΔ14/+ mice, completely reversed the increased Wnt activity and colony formation in soft agar induced by Apc siRNA treatment in ApcMin/+ cells. Therefore, Arrb2 is essential for the initiation and growth of intestinal tumors displaying elevated Wnt pathway activity. To better understand the mechanisms involved in this context, Arrb2 protein complexes were analyzed by a differential systematic proteomic approach in SW480 human colorectal carcinoma cells expressing or not a Tcf4 dominant negative. 132 Arrb2 partners potentially involved in a signaling network of 917 proteins were identified in cells with a high Wnt activity. Upon a 80% decrease of this activity 41 partners disappeared with their 256 potential interactions whereas 42 partners appeared with 244 new possible interactions. Arrb2 key role in colorectal cancer could be explained by the cross-talk of about 40 proteins dependent of Wnt activity with a highly complex signaling network that is currently analyzed.