Patrick G. Quinn

Diabetic Retinopathy Seeing Beyond Glucose-Induced Microvascular Disease

Diabetic retinopathy remains a frightening prospect to patients and frustrates physicians. Destruction of damaged retina by photocoagulation remains the primary treatment nearly 50 years after its introduction. The diabetes pandemic requires new approaches to understand the pathophysiology and improve the detection, prevention, and treatment of retinopathy. This perspective considers how the unique anatomy and physiology of the retina may predispose it to the metabolic stresses of diabetes. The roles of neural retinal alterations and impaired retinal insulin action in the pathogenesis of early retinopathy and the mechanisms of vision loss are emphasized. Potential means to overcome limitations of current animal models and diagnostic testing are also presented with the goal of accelerating therapies to manage retinopathy in the face of ongoing diabetes.

Tyrosine hydroxylase transcription depends primarily on cAMP response element activity, regardless of the type of inducing stimulus.

In neurons and neuroendocrine cells, tyrosine hydroxylase (TH) gene expression is induced by stimuli that elevate cAMP, by depolarization, and by hypoxia. Using these stimuli, we examined TH promoter mutants, cAMP response element binding protein (CREB) phosphorylation site mutants, and transcriptional interference with dominant negative transcription factors to assess the relative contributions of CREB/AP-1 family members to the regulation of basal and inducible TH transcription in PC12 cells. We found that basal transcription depends on transcription factor activity at the partial dyad (-17 bp), CRE (-45 bp), and AP1 (-205 bp) elements. Induced transcription is regulated primarily by activity at the CRE, with only small contributions from the AP1 or hypoxia response element 1 (HRE1; -225 bp) elements, regardless of inducing stimulus. CREB, ATF-1, and CREMtau all mediate CRE-dependent transcription, with CREB and CREMtau being more effective than ATF-1. Phosphorylation of CREB on Ser133, but not on Ser142 or Ser143, is required for induced transcription, regardless of inducing stimulus.

Assessment of the Roles of Mitogen-activated Protein Kinase, Phosphatidylinositol 3-Kinase, Protein Kinase B, and Protein Kinase C in Insulin Inhibition of cAMP-induced Phosphoenolpyruvate Carboxykinase Gene Transcription

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP and protein kinase A, and this induction is inhibited by insulin. Conflicting reports have suggested that insulin inhibits induction by cAMP by activating the Ras/mitogen-activated protein kinase (MAPK) pathway or by activating the phosphatidylinositol 3-kinase (PI3-kinase), but not MAPK, pathway. Insulin activated PI3-kinase phosphorylates lipids that activate protein kinase B (PKB) and Ca2+/diacylglycerol-insensitive forms of protein kinase C (PKC). We have assessed the roles of these pathways in insulin inhibition of cAMP/PKA-induced transcription of PEPCK by using dominant negative and dominant active forms of regulatory enzymes in the Ras/MAPK and PKB pathways and chemical inhibitors of PKC isoforms. Three independently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS, Ras, and MAPK, had no effect upon insulin inhibition. However, dominant active Ras prevented induction of PEPCK and also stimulated transcription mediated by Elk, a MAPK target. Insulin did not stimulate Elk-mediated transcription, indicating that insulin did not functionally activate the Ras/MAPK pathway. Inhibitors of PI3-kinase, LY294002 and wortmannin, abolished insulin inhibition of PEPCK gene transcription. However, inhibitors of PKC and mutated forms of PKB, both of which are known downstream targets of PI3-kinase, had no effect upon insulin inhibition. Dominant negative forms of PKB did not interfere with insulin inhibition and a dominant active form of PKB did not prevent induction by PKA. Phorbol ester-mediated inhibition of PEPCK transcription was blocked by bisindole maleimide and by staurosporine, but insulin-mediated inhibition was unaffected. Thus, insulin inhibition of PKA-induced PEPCK expression does not require MAPK activation but does require activation of PI3-kinase, although this signal is not transmitted through the PKB or PKC pathways.

The CREB Constitutive Activation Domain Interacts with TATA-binding Protein-associated Factor 110 (TAF110) through Specific Hydrophobic Residues in One of the Three Subdomains Required for Both Activation and TAF110 Binding

The cAMP response element-binding protein (CREB) mediates both basal and PKA-inducible transcription through two separate and independently active domains, the constitutive activation domain (CAD) and the kinase-inducible domain, respectively. The CREB CAD interacts with the general transcription factor TFIID through one or more of the TATA-binding protein-associated factors (TAFs), one of which is TAF110. The CAD is composed of three subdomains, rich in either serine, hydrophobic amino acids, or glutamine. In the present study, analysis of deletion mutants of the CAD showed that all three CAD subdomains were required for effective interaction with TAF110 in a yeast two-hybrid assay. Therefore, a library of random point mutations within the CAD was analyzed in a reverse two-hybrid screen to identify amino acids that are essential for interaction with the TAF. Interaction defects resulted solely from mutations of hydrophobic amino acid residues within the hydrophobic cluster to charged amino acid residues. Together, the deletion and mutation analyses suggest that the entire CAD provides an environment for a specific hydrophobic interaction with TAF110 that is crucial for interaction. Our results provide further evidence for a model of basal activation by CREB involving interaction with TAF110 that promotes recruitment or stabilization of TFIID binding to the promoter, which facilitates pre-initiation complex assembly.

A tripartite array of transcription factor binding sites mediates cAMP induction of phosphoenolpyruvate carboxykinase gene transcription and its inhibition by insulin.

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) gene is induced upon activation of protein kinase A by cAMP and phosphorylation of Ser-133 in the transcription factor, cAMP-response element binding protein (CREB), and this induction is inhibited by insulin. We show here that insulin does not act by dephosphorylating CREB or by affecting heterologous kinases that phosphorylate Ser-129 or Ser-142 in CREB. In addition, insulin inhibition of minimal PEPCK promoter activity induced by CREB-GAL4 + protein kinase A was equivalent to inhibition of basal transcription, and thus cAMP-independent. On the other hand, nearly complete insulin inhibition is observed with the full PEPCK promoter (−600/+69), indicating that other factors are involved. The additional promoter elements required for induction by protein kinase A lie within −271 nucleotides of the start site and correspond to putative binding sites for activator protein-1 and CAAT/enhancer-binding protein (C/EBP), first identified by Roesler et al. (Roesler, W. J., McFie, P. J., and Puttick, D. M., (1993) J. Biol. Chem. 268, 3791–3796). This tripartite array of binding sites for CREB, C/EBP, and activator protein-1 (AP-1) factors forms a cAMP response unit that, together with the minimal promoter, can mediate both induction by cAMP and inhibition by insulin. Thus, for the PEPCK gene with a single CREB site, the CREB·CBP·RNA polymerase II complex cannot mediate either induction by cAMP or inhibition by insulin.

Recruitment of an RNA Polymerase II Complex Is Mediated by the Constitutive Activation Domain in CREB, Independently of CREB Phosphorylation

ABSTRACT The cAMP response element binding protein (CREB) is a bifunctional transcription activator, exerting its effects through a constitutive activation domain (CAD) and a distinct kinase inducible domain (KID), which requires phosphorylation of Ser-133 for activity. Both CAD and phospho-KID have been proposed to recruit polymerase complexes, but this has not been directly tested. Here, we show that the entire CREB activation domain or the CAD enhanced recruitment of a complex containing TFIID, TFIIB, and RNA polymerase II to a linked promoter. The nuclear extracts used mediated protein kinase A (PKA)-inducible transcription, but phosphorylation of CRG (both of the CREB activation domains fused to the Gal4 DNA binding domain) or KID-G4 did not mediate recruitment of a complex, and mutation of the PKA site in CRG abolished transcription induction by PKA but had no effect upon recruitment. The CREB-binding protein (CBP) was not detected in the recruited complex. Our results support a model for transcription activation in which the interaction between the CREB CAD and hTAFII130 of TFIID promotes the recruitment of a polymerase complex to the promoter.

Effect of IL-1β on Survival and Energy Metabolism of R28 and RGC-5 Retinal Neurons

PURPOSE Interleukin-(IL)1beta expression is increased in the retina during a variety of diseases involving the death of retinal neurons and contributes to neurodegenerative processes through an unknown mechanism. This study was conducted to examine the effects of IL-1beta on the metabolism and viability of RGC-5 and R28 retinal neuronal cells. METHODS Cellular reductive capacity was evaluated using WST-1 tetrazolium salt. Mitochondrial transmembrane potential was determined by JC-1 fluorescence. Cellular ATP levels were measured with a luciferase assay. Caspase-3/7 activation was detected with a DEVDase activity assay. Cell death and lysis was evaluated by measuring release of lactate dehydrogenase (LDH). Glycolysis was assessed by measuring glucose disappearance and lactate appearance in cell culture medium. Cellular respiration was followed polarographically. RESULTS IL-1beta treatment caused a pronounced decrease in cellular reductive potential. IL-1beta caused depletion of intracellular ATP, loss of mitochondrial transmembrane potential, caspase-3/7 activation, and LDH release. IL-1beta treatment increased rates of glucose utilization and lactate production. The cells were partially protected from IL-1beta toxicity by ample ambient glucose. However, glucose did not block the ability of IL-1beta to cause a decline in mitochondrial transmembrane potential or ATP depletion. IL-1beta decreased oxygen consumption of the R28 cells by nearly half, but did not lower cytochrome c oxidase activity. CONCLUSIONS The present results suggest that IL-1beta inhibits mitochondrial energy metabolism of these retinal neuronlike cells.

ATF/CREB elements in the herpes simplex virus type 1 latency-associated transcript promoter interact with members of the ATF/CREB and AP-1 transcription factor families

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) promoter 1 (LP1) is an inducible and cell type-specific promoter involved in regulating the production of an 8.3-kb primary LAT transcript during acute and latent infection of peripheral sensory neurons and during subsequent virus reactivation. A number of cis-acting regulatory elements have been identified in LP1, including two cyclic-AMP (cAMP) response element (CRE)-like sequences, designated CRE-1 and CRE-2. CRE-1 has previously been shown to confer cAMP responsiveness to LP1 and to regulate reactivation of HSV-1 from latency in vivo. A role for CRE-2 in modulating inducible activity is not yet as clear; however, it has been shown to support basal expression in neuronal cells in vitro. Electrophoretic mobility shift (EMS) analyses demonstrate that the LP1 CRE-like elements interact with distinct subsets of neuronal ATF/CREB and Jun/Fos proteins including CREB-1, CREB-2, ATF-1, and JunD. The factor-binding properties of each LP1 CRE element distinguish them from each other and from a highly related canonical CRE binding site and the TPA response element (TRE). LP1 CRE-1 shares binding characteristics of both a canonical CRE and a TRE. LP1 CRE-2 is more unusual in that it shares more features of a canonical CRE site than a TRE with two notable exceptions: it does not bind CREB-1 very well and it binds CREB-2 better than the canonical CRE. Interestingly, a substantial proportion of the C1300 neuroblastoma factors that bind to CRE-1 and CRE-2 have been shown to be immunologically related to JunD, suggesting that the AP-1 family of transcription factors may be important in regulating CRE-dependent LP1 transcriptional activity. In addition, we have demonstrated the two HSV-1 LP1 CRE sites to be unique with respect to their ability to bind neuronal AP1-related factors that are regulated by cAMP. These studies suggest that both factor binding and activation of bound factors may be involved in cAMP regulation of HSV-1 LP1 through the CRE elements, and indicate the necessity of investigating the expression and posttranslational modification of a variety of ATF/CREB and AP-1 factors during latency and reactivation.

Characterization of elements mediating regulation of phosphoenolpyruvate carboxykinase gene transcription by protein kinase A and insulin. Identification of a distinct complex formed in cells that mediate insulin inhibition.

The in vivopattern of induction of phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by cAMP and its inhibition by insulin is reproduced in H4IIe cells and is mediated by a bipartite cAMP/insulin response unit (C/IRU) consisting of a cAMP response element (−95/−87) and an upstream enhancer, AC (−271/−225). Studies in HepG2 cells showed that binding of AP-1 and CAAT/enhancer-binding protein (C/EBP) to AC is required for induction by cAMP, but insulin did not inhibit cAMP-induced PEPCK expression in HepG2 cells. Binding of H4IIe nuclear proteins to an AC element probe was inhibited by antibodies or a consensus site for C/EBP, but not AP-1. Transfection with dominant negative bZIP factors, which prevent endogenous factors from binding to DNA, showed that elimination of cAMP regulatory element-binding protein CREB or C/EBP activity blocked induction by protein kinase A (PKA), whereas elimination of AP-1 activity had no effect. In addition, promoters with multiple CREB sites, or a single CREB site and multiple C/EBP sites, mediated PKA induction, but this was inhibited to no greater extent than basal activity was by insulin. These results indicate that an AC factor other than C/EBP must mediate insulin inhibition. An A-site probe (−265/−247) or a probe across the middle of the AC element (−256/−237) competed for complexes formed by factors other than AP-1 or C/EBP. However, analysis of competitor oligonucleotides and antibodies for candidate factors failed to identify other factors. Scanning mutations throughout the AC element interfered with induction but allowed us to define five overlapping sites for regulatory factors in AC and to design probes binding just one or two factors. Comparison of the protein-DNA complexes formed on these smaller probes revealed that a specific complex present in rat liver and H4IIe cell nuclear extracts differed from those formed by HepG2 cell nuclear extracts. Our results suggest that multiple factors binding the AC element of the C/IRU interact with each other and CREB to regulate PEPCK induction by cAMP and inhibition by insulin and that the unique factor expressed in H4IIe cells is a candidate for involvement in insulin regulation of PKA-induced PEPCK gene transcription.

The coactivator dTAFII110/hTAFII135 is sufficient to recruit a polymerase complex and activate basal transcription mediated by CREB

A specific TATA binding protein-associated factor (TAF), dTAFII110/hTAFII135, interacts with cAMP response element binding protein (CREB) through its constitutive activation domain (CAD), which recruits a polymerase complex and activates transcription. The simplest explanation is that the TAF is a coactivator, but several studies have questioned this role of TAFs. Using a reverse two-hybrid analysis in yeast, we previously mapped the interaction between dTAFII110 (amino acid 1–308) and CREB to conserved hydrophobic amino acid residues in the CAD. That mapping was possible only because CREB fails to activate transcription in yeast, where all TAFs are conserved, except for the TAF recognizing CREB. To test whether CREB fails to activate transcription in yeast because it lacks a coactivator, we fused dTAFII110 (amino acid 1–308) to the TATA binding protein domain of the yeast scaffolding TAF, yTAFII130. Transformation of yeast with this hybrid TAF conferred activation by the CAD, indicating that interaction with yTFIID is sufficient to recruit a polymerase complex and activate transcription. The hybrid TAF did not mediate activation by VP16 or vitamin D receptor, each of which interacts with TFIIB, but not with dTAFII110 (amino acid 1–308). Enhancement of transcription activation by dTAFII110 in mammalian cells required interaction with both the CAD and TFIID and was inhibited by mutation of core hydrophobic residues in the CAD. These data demonstrate that dTAFII110/hTAFII135 acts as a coactivator to recruit TFIID and polymerase and that this mechanism of activation is conserved in eukaryotes.