Liza Barki-Harrington

β-Arrestin–mediated β1-adrenergic receptor transactivation of the EGFR confers cardioprotection

Deleterious effects on the heart from chronic stimulation of β-adrenergic receptors (βARs), members of the 7 transmembrane receptor family, have classically been shown to result from Gs-dependent adenylyl cyclase activation. Here, we identify a new signaling mechanism using both in vitro and in vivo systems whereby β-arrestins mediate β1AR signaling to the EGFR. This β-arrestin–dependent transactivation of the EGFR, which is independent of G protein activation, requires the G protein–coupled receptor kinases 5 and 6. In mice undergoing chronic sympathetic stimulation, this novel signaling pathway is shown to promote activation of cardioprotective pathways that counteract the effects of catecholamine toxicity. These findings suggest that drugs that act as classical antagonists for G protein signaling, but also stimulate signaling via β-arrestin–mediated cytoprotective pathways, would represent a novel class of agents that could be developed for multiple members of the 7 transmembrane receptor family.

ERK-dependent PSD-95 induction in the gustatory cortex is necessary for taste learning, but not retrieval

The processes underlying long-term memory formation in the neocortex are poorly understood. Using taste learning, we found learning-related induction of PSD-95 in the gustatory cortex, which was temporally restricted, coupled to the learning of a novel, but not familiar, taste and controlled by ERK. Using temporally and spatially restricted RNA interference knockdown of PSD-95 in vivo, we found that PSD-95 induction is necessary for learning novel tastes, but not for the recollection of familiar ones.

DNA Methylation of Specific CpG Sites in the Promoter Region Regulates the Transcription of the Mouse Oxytocin Receptor

Oxytocin is a peptide hormone, well known for its role in labor and suckling, and most recently for its involvement in mammalian social behavior. All central and peripheral actions of oxytocin are mediated through the oxytocin receptor, which is the product of a single gene. Transcription of the oxytocin receptor is subject to regulation by gonadal steroid hormones, and is profoundly elevated in the uterus and mammary glands during parturition. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression of the oxytocin receptor in individuals with autism. Here, we hypothesized that transcription of the mouse oxytocin receptor is regulated by DNA methylation of specific sites in its promoter, in a tissue-specific manner. Hypothalamus-derived GT1-7, and mammary-derived 4T1 murine cell lines displayed negative correlations between oxytocin receptor transcription and methylation of the gene promoter, and demethylation caused a significant enhancement of oxytocin receptor transcription in 4T1 cells. Using a reporter gene assay, we showed that methylation of specific sites in the gene promoter, including an estrogen response element, significantly inhibits transcription. Furthermore, methylation of the oxytocin receptor promoter was found to be differentially correlated with oxytocin receptor expression in mammary glands and the uterus of virgin and post-partum mice, suggesting that it plays a distinct role in oxytocin receptor transcription among tissues and under different physiological conditions. Together, these results support the hypothesis that the expression of the mouse oxytocin receptor gene is epigenetically regulated by DNA methylation of its promoter.

Tyrosine Phosphorylation of the 2B Subunit of the NMDA Receptor Is Necessary for Taste Memory Formation

We aimed to test whether tyrosine phosphorylation of the NMDA receptor (NMDAR) in the insular cortex is necessary for novel taste learning. We found that in rats, novel taste learning leads to elevated phosphorylation of tyrosine 1472 of the NR2B subunit of the NMDAR and increases the interaction of phosphorylated NR2B with the major postsynaptic scaffold protein PSD-95. Injection of the tyrosine kinase inhibitor genistein directly into the insular cortex of rats before novel taste exposure prevented the increase in NR2B tyrosine phosphorylation and behaviorally attenuated taste-memory formation. Functionally, tyrosine phosphorylation of NR2B after learning was found to determine the synaptic distribution of the NMDAR, since microinjection of genistein to the insular cortex altered the distribution pattern of NMDAR caused by novel taste learning.

β-Arrestins: Multifunctional Cellular Mediators

Initially thought to play a role only in G-protein-coupled receptor desensitization, beta-arrestins are ascribed with new roles such as scaffolding and signaling proteins by their own right. This review explores the many functions of beta-arrestins, with an emphasis on their recently identified role as regulators of receptor signaling.

Brain region-specific methylation in the promoter of the murine oxytocin receptor gene is involved in its expression regulation

Oxytocin is a nine amino acid neuropeptide that is known to play a critical role in fetal expulsion and breast-feeding, and has been recently implicated in mammalian social behavior. The actions of both central and peripheral oxytocin are mediated through the oxytocin receptor (Oxtr), which is encoded by a single gene. In contrast to the highly conserved expression of oxytocin in specific hypothalamic nuclei, the expression of its receptor in the brain is highly diverse among different mammalian species or even within individuals of the same species. The diversity in the pattern of brain Oxtr expression among mammals is thought to contribute to the broad range of social systems and organizations. Yet, the mechanisms underlying this diversity are poorly understood. DNA methylation is a major epigenetic mechanism that regulates gene transcription, and has been linked to reduced expression levels of the Oxtr in individuals with autism. Here we hypothesize that DNA methylation is involved in the expression regulation of Oxtr in the mouse brain. By combining bisulfite DNA conversion and Next-Generation Sequencing we found that specific CpG sites are differentially methylated between distinct brain regions expressing different levels of Oxtr mRNA. Some of these CpG sites are located within putative binding sites of transcription factors known to regulate Oxtr expression, including estrogen receptor α (ERα) and SP1. Specifically, methylation of the SP1 site was found to positively correlate with Oxtr expression. Furthermore, we revealed that the methylation levels of these sites in the various brain regions predict the relationship between ERα and Oxtr mRNA levels. Collectively, our results suggest that brain region-specific expression of the mouse Oxtr gene is epigenetically regulated by DNA methylation of its promoter.

Inhibition of exocytosis or endocytosis blocks activity‐dependent redistribution of synapsin

J. Neurochem. (2012) 120, 248–258.

Underwater trauma causes a long-term specific increase in the expression of cyclooxygenase-2 in the ventral CA1 of the hippocampus

The pro-inflammatory enzyme cyclooxygenase-2 (COX-2) is regularly expressed in the hippocampal neurons, but its role in emotional trauma is not known. Here we show that a single acute stress caused by a near-drowning experience results in heightened anxiety-like behavior one month after the trauma. Biochemical analyses of dorsal and ventral hippocampal CA1, CA3 and dentate gyrus revealed decreased ubiquitination and elevated levels of COX-2 in the traumatized animals only in the ventral CA1. To reveal the identity of the ubiquitin E3 ligase that targets COX-2, we tested the effect of several representative E3 ligases on COX-2 expression in vitro. We found that while AIP4 and Nedd4 had no effect, Mdm2 lowered COX-2 expression by nearly 50%, an effect that was not observed by its dominant negative form. To test whether this also occurs in the hippocampus, we immunoprecipitated Mdm2 from dorsal and ventral CA1 of traumatized and control animals and probed for the presence of COX-2. Our results showed that the levels of Mdm2 were not affected by the trauma but there was significantly less COX-2 associated with Mdm2 in the ventral but not dorsal CA1 of the traumatized animals. Together these data propose that an increase in COX-2 expression in ventral CA1 following trauma is likely due to its attenuated degradation. Unraveling the pathways and mechanisms that control hippocampal COX-2 degradation is important to boost the development of novel therapeutic approaches designed to treat stress-related pathologies.

The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression

Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/− mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.

Down-regulation of Cyclooxygenase-2 by the Carboxyl Tail of the Angiotensin II Type 1 Receptor

Background: The levels of the pro-inflammatory enzyme COX-2 require tight regulation. Results: The carboxyl tail of Angiotensin II type 1 receptor (AT1) enhances COX-2 degradation. Conclusion: We identified a novel mechanism for COX-2 regulation that is independent of receptor activation. Significance: The tail sequence of AT1 may serve as a basis for design of novel therapeutic agents that degrade COX-2. The enzyme cyclooxygenase-2 (COX-2) plays an important role in the kidney by up-regulating the production of the vasoconstrictor hormone angiotensin II (AngII), which in turn down-regulates COX-2 expression via activation of the angiotensin II type 1 receptor (AT1) receptor. Chemical inhibition of the catalytic activity of COX-2 is a well-established strategy for treating inflammation but little is known of cellular mechanisms that dispose of the protein itself. Here we show that in addition to its indirect negative feedback on COX-2, AT1 also down-regulates the expression of the COX-2 protein via a pathway that does not involve G-protein or β-arrestin-dependent signaling. Instead, AT1 enhances the ubiquitination and subsequent degradation of the enzyme in the proteasome through elements in its cytosolic carboxyl tail (CT). We find that a mutant receptor that lacks the last 35 amino acids of its CT (Δ324) is devoid of its ability to reduce COX-2, and that expression of the CT sequence alone is sufficient to down-regulate COX-2. Collectively these results propose a new role for AT1 in regulating COX-2 expression in a mechanism that deviates from its canonical signaling pathways. Down-regulation of COX-2 by a short peptide that originates from AT1 may present as a basis for novel therapeutic means of eliminating excess COX-2 protein.