Cyclic AMP signaling pathways play crucial roles in photoreceptor cells and other retinal cell types. Previous studies demonstrated a circadian rhythm of cyclic AMP level in chick photoreceptor cell cultures that drives the rhythm of activity of the melatonin synthesizing enzyme arylalkylamine N-acetyltransferase (Ivanova and Iuvone, 2003a) and the rhythm of affinity of the cyclic nucleotide-gated channel for cyclic GMP (Ko et al., 2004). Here we report that the photoreceptor circadian clock generates a rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase activity, which accounts for the temporal changes in the cyclic AMP levels in the photoreceptors. The circadian rhythm of cyclic AMP in photoreceptor cell cultures is abolished by treatment with the L-type Ca2+ channel antagonist nitrendipine, while the Ca2+ channel agonist, Bay K 8644, increased cyclic AMP levels with continued circadian rhythmicity in constant darkness. These results indicate that the circadian rhythm of cyclic AMP is dependent, in part, on Ca2+ influx. Photoreceptor cell cultures exhibit a circadian rhythm in Ca2+/calmodulin-stimulated adenylyl cyclase enzyme activity with high levels at night and low levels during the day, correlating with the temporal changes of cyclic AMP in these cells. Both of the Ca2+/calmodulin-stimulated adenylyl cyclase genes, type 1 and type 8 (Adcy1 and Adcy8), displayed significant daily rhythms of mRNA expression under a light-dark cycle, but only the Adcy1 transcript rhythm persisted in constant darkness. Similar rhythms of Adcy1 mRNA level and Ca2+/calmodulin-stimulated adenylyl cyclase activity were observed in retinas of 2 week old chickens. These results indicate that a circadian clock controls the expression of Adcy1 mRNA and Ca2+/calmodulin-stimulated adenylyl cyclase activity; and calcium influx into these cells gates the circadian rhythm of cyclic AMP, a key component in the regulation of photoreceptor function.
Dopamine is a retinal neuromodulator that has been implicated in many aspects of retinal physiology. Photoreceptor cells express dopamine D4 receptors that regulate cAMP metabolism. To assess the effects of dopamine on photoreceptor physiology, we examined the morphology, electrophysiology, and regulation of cAMP metabolism in mice with targeted disruption of the dopamine D4 receptor gene. Photoreceptor morphology and outer segment disc shedding after light onset were normal in D4 knock-out (D4KO) mice. Quinpirole, a dopamine D2/ D3/D4 receptor agonist, decreased cAMP synthesis in retinas of wild-type (WT) mice but not in retinas of D4KO mice. In WT retinas, the photoreceptors of which were functionally isolated by incubation in the presence of exogenous glutamate, light also suppressed cAMP synthesis. Despite the similar inhibition of cAMP synthesis, the effect of light is directly on the photo-receptors and independent of dopamine modulation, because it was unaffected by application of the D4 receptor antagonist L-745,870. Nevertheless, compared with WT retinas, basal cAMP formation was reduced in the photoreceptors of D4KO retinas, and light had no additional inhibitory effect. The results suggest that dopamine, via D4 receptors, normally modulates the cascade that couples light responses to adenylyl cyclase activity in photoreceptor cells, and the absence of this modulation results in dysfunction of the cascade. Dark-adapted electroretinogram (ERG) responses were normal in D4KO mice. However, ERG b-wave responses were greatly suppressed during both light adaptation and early stages of dark adaptation. Thus, the absence of D4 receptors affects adaptation, altering transmission of light responses from photoreceptors to inner retinal neurons. These findings indicate that dopamine D4 receptors normally play a major role in regulating photoreceptor cAMP metabolism and adaptive retinal responses to changing environmental illumination.
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Nikita Pozdeyev;
Carla Taylor;
Rashidul Haque;
Shyam S. Chaurasia;
Amy Visser;
Aamera Thazyeen;
Yuhong Du;
Haian Fu;
Joan Weller;
David C. Klein;
P Michael Iuvone
14-3-3 proteins are a ubiquitous, highly conserved family of chaperone proteins involved in signal transduction, regulation of cell cycle, intracellular trafficking/targeting, cytoskeletal structure, and transcription. Although 14-3-3 proteins are among the most abundant proteins in the CNS, very little is known about their functional roles in the vertebrate retina. In the present study, we demonstrated that photoreceptors express 14-3-3 protein(s) and identified a 14-3-3 binding partner in photoreceptor cells, the melatonin-synthesizing enzyme arylalkylamine N-acetyltransferase (AANAT). Importantly, our data demonstrate that the binding of 14-3-3 to AANAT is regulated by light, with dramatic functional consequences. During the night in darkness, retinal AANAT is phosphorylated and forms a complex with 14-3-3 proteins with an apparent molecular weight of ∼90 kDa. Phosphorylation of AANAT facilitates the binding of enzyme to 14-3-3 proteins. Within the complex, AANAT is catalytically activated and protected from dephosphorylation and degradation. Light disrupts the AANAT/14-3-3 complex, leading to catalytic inactivation, dephosphorylation, and proteolytic degradation of the enzyme. In the presence of the proteasome inhibitor, lactacystin, light results in the formation of a high molecular weight complex (>150 kDa), which may represent an intermediate in the AANAT degradation process. These findings provide new insight into the roles of 14-3-3 proteins in photoreceptor cells and to the mechanisms controlling melatonin synthesis in the vertebrate retina.
Neonatal aphakia is associated with retardation of the axial elongation of the neonatal eye. In contrast, form deprivation increases axial elongation, an effect that has been associated with decreased retinal dopamine metabolism. The present investigation was conducted to test the hypothesis that neonatal aphakia induces an effect on the levels of retinal dopamine opposite to form deprivation. Lensectomy and vitrectomy were performed on the right eyes of rhesus monkeys at approximately 1 week of age; their left eyes were unmanipulated. Axial length was measured by A-scan ultrasonography. Prior to surgery, mean axial length of the right and left eyes was identical. Following lens removal, both eyes continued to elongate, however the aphakic eyes elongated at a slower rate resulting in a significant shorter axial length compared to that of the unmanipulated eye. Removal of the crystalline lens had no effect on steady-state dopamine levels in either central or peripheral retina. However, levels of the dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid were significantly elevated in central retina, but not in the peripheral retina of aphakic eyes. Our results support the hypothesis that dopamine is a component of the retinal signaling pathways that are involved in the regulation of eye growth and emmetropization.
Purpose: In recent years, microRNAs (miRNAs) have been reported to play important roles in a broad range of biologic processes, including oxidative stress-mediated ocular diseases. In addition, the polyphenolic compound curcumin has been shown to possess anti-inflammatory, antioxidant, anticancer, antiproliferative, and proapoptotic activities. The aim of this study was to investigate the impact of curcumin on the expression profiles of miRNAs in ARPE-19 cells exposed to oxidative stress.
Methods: MiRNA expression profiles were measured in ARPE-19 cells treated with 20 μΜ curcumin and 200 μΜ H2O2. PCR array analysis was performed using web-based software from SABiosciences. The cytotoxicity of ARPE-19 cells was determined with the CellTiter-Blue cell viability assay. The effects of curcumin on potential miRNA targets were analyzed with quantitative real-time PCR and western blotting.
Results: Curcumin treatment alone for 6 h had no effect on ARPE-19 cell viability. Incubation with H2O2 (200 µM) alone for 18 h decreased cell viability by 12.5%. Curcumin alone downregulated 20 miRNAs and upregulated nine miRNAs compared with controls. H2O2 downregulated 18 miRNAs and upregulated 29 miRNAs. Furthermore, curcumin pretreatment in cells exposed to H2O2 significantly reduced the H2O2-induced expression of 17 miRNAs. As determined with quantitative real-time PCR and western blotting, curcumin increased the expression of antioxidant genes and reduced angiotensin II type 1 receptor, nuclear factor-kappa B, and vascular endothelial growth factor expression at the messenger RNA and protein levels.
Conclusions: The results demonstrated that curcumin alters the expression of H2O2-modulated miRNAs that are putative regulators of antioxidant defense and renin-angiotensin systems, which have been reported to be linked to ocular diseases.
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Rashidul Haque;
Nelson W. Chong;
Fatima Ali;
Shyam S. Chaurasia;
Trisha Sengupta;
Eugene Chun;
Jennifer Christina Howell;
David C. Klein;
P Michael Iuvone
Arylalkylamine N-acetyltransferase (AANAT) is the key regulatory enzyme controlling the daily rhythm of melatonin biosynthesis. In chicken retinal photoreceptor cells, Aanat transcription and AANAT activity are regulated in part by cAMP-dependent mechanisms. The purpose of this study was to identify regulatory elements within the chicken Aanat promoter responsible for cAMP-dependent induction. Photoreceptor-enriched retinal cell cultures were transfected with a luciferase reporter construct containing up to 4kb of 5′-flanking region and the first exon of Aanat. Forskolin treatment stimulated luciferase activity driven by the ~4kb promoter construct and by all 5′-deletion constructs except the smallest, Aanat (−217 to +120)luc. Maximal basal and forskolin-stimulated expression levels were generated by the Aanat (−484 to +120)luc construct. This construct lacks a canonical cyclic AMP-response element (CRE), but contains two other potentially important elements in its sequence: an eight times TTATT repeat (TTATT8) and a CRE-like sequence (CLS). Electrophoretic mobility shift assays (EMSA), luciferase reporter assays, chromatin immunoprecipitation, and siRNA experiments provide evidence that these elements bind c-Fos, JunD, and CREB to enhance basal and forskolin-stimulated Aanat transcription. We propose that the CLS and TTATT8 elements in the 484 bp proximal promoter interact to mediate cAMP-dependent transcriptional regulation of Aanat.
Purpose: The (pro)renin receptor (PRR), a component of the renin-angiotensin system (RAS), plays an important role in the physiologic and pathophysiological regulation of blood pressure and fluid/electrolyte homeostasis. The RAS including the PRR has been identified in retinal endothelial cells and other ocular tissues. In this study, the potential involvement of miRNAs in the posttranscriptional regulation of PRR was investigated in human retinal endothelial cells (hRECs) under high glucose (HG) conditions. Methods: miRNA-152 (miR-152) was identified in silico as a potential regulator of PRR, and this was confirmed by quantitative real-time PCR (qRT-PCR) and PRR 3′-untranslated region (UTR) reporter assays. Using RNA interference, both AT1R and PRR were implicated in the HG-mediated induction of vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR-2), and transforming growth factor β1 (TGFβ1). Results: The downregulation of miR-152 was observed in hRECs and rat retinal tissues under HG conditions. In parallel, PRR (target of miR-152), VEGF, VEGFR-2, and TGFβ1 at mRNA levels were elevated. However, the transfection of hRECs with miR-152 mimics in HG conditions resulted in the suppression of the PRR expression, as well as reduced VEGF, VEGFR-2, and TGFβ1 production. This was reversed by transfecting cells with the antisense (antagomir) of miR-152, suggesting the glucose-induced upregulation of VEGF, VEGFR-2, and TGFβ1 is mediated through PRR, and this regulation is likely achieved through the HG-mediated modulation of miRNAs. Conclusions: We have demonstrated that miR-152 interacting with PRR regulates downstream VEGF, VRGFR-2, and TGFβ1 expressions in hRECs in HG conditions. These studies suggest miR-152 and PRR may play a role in the pathogenesis of diabetic retinopathy (DR).