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.

3',5'-cyclic adenosine monophosphate response element-binding protein and CCAAT enhancer-binding protein are dispensable for insulin inhibition of phosphoenolpyruvate carboxykinase transcription and for its synergistic induction by protein kinase A and glucocorticoids.

Phosphoenolpyruvate carboxykinase (PEPCK) transcription is induced by cAMP/protein kinase A (PKA) and glucocorticoids [dexamethasone (Dex)] and is inhibited by insulin to regulate blood glucose. Recent reports suggested that CCAAT enhancer binding protein (C/EBP) binding to the PEPCK cAMP response element (CRE) plays a role in Dex induction and that insulin-induces inhibitory forms of C/EBPbeta to inhibit transcription. Here, we assessed the roles of CRE-binding protein (CREB) and C/EBP factors in mediating hormone-regulated transcription. Neither cAMP nor insulin regulated the phosphorylation of C/EBP. Cycloheximide did not block insulin inhibition, indicating that alternate translation of C/EBPbeta is not required. Dominant-negative CREB or C/EBP blocked induction by PKA, but neither affected regulation by Dex or insulin. Tethering the activation domains of CREB or C/EBP to a CRE-->Gal4 (G4) site mediated varying extents of basal and PKA-inducible activity, but neither activation domain affected induction by Dex or inhibition by insulin. Surprisingly, synergistic induction by PKA and Dex did not require the CRE and was unaffected by dominant-negative CREB or C/EBP. PKA and Dex also synergistically induced a minimal 3 x glucocorticoid response element promoter, but inhibited Dex induction of the mouse mammary tumor virus and IGF-binding protein 1 promoters, even though PKA alone did not regulate these promoters. These results suggest that PKA modifies the activity of other factors involved in Dex induction to mediate synergistic induction or inhibition in a promoter-specific manner. Our data indicate that the roles of CREB and C/EBP are restricted to mediating PEPCK induction by PKA, and that other factors mediate PEPCK induction by Dex, synergism between PKA and Dex, and inhibition by insulin.

Mechanisms of basal and kinase-inducible transcription activation by CREB.

The cAMP response element (CRE)-binding protein (CREB) stimulates basal transcription of CRE-containing genes and mediates induction of transcription upon phosphorylation by protein kinases. The basal activity of CREB maps to a carboxy-terminal constitutive activation domain (CAD), whereas phosphorylation and inducibility map to a central, kinase-inducible domain (KID). The CAD interacts with and recruits the promoter recognition factor TFIID through an interaction with a specific TATA-binding-protein-associated factor (TAF), dTAFII110/ hTAFII135. Interaction between the TAF and the CAD is mediated by a central cluster of hydrophobic amino acids, mutation of which disrupts TAF binding, polymerase recruitment, and transcription activation. Assessment of the contributions of the CAD and KID to recruitment of the polymerase complex versus enhancement of subsequent reaction steps (isomerization, promoter clearance, and reinitiation) showed that the CAD and P-KID act in a concerted mechanism to stimulate transcription. The CAD, but not the KID, mediated recruitment of a complex containing components of a transcription initiation complex, including pol II, IIB, and IID. However, the CAD was relatively ineffective in stimulating subsequent steps in the reaction mechanism. In contrast, phosphorylation of the KID in CREB effectively stimulated isomerization of the recruited polymerase complex and multiple-round transcription. A model for the activation of transcription by phosphorylated CREB is proposed, in which the polymerase is recruited by interaction of the CAD with TFIID and the recruited polymerase is activated further by phosphorylation of the KID in CREB.

Increased cytochrome P450 17alpha-hydroxylase promoter function in theca cells isolated from patients with polycystic ovary syndrome involves nuclear factor-1.

Cytochrome P450 17alpha-hydroxylase (CYP17) gene expression and androgen biosynthesis are persistently elevated in theca cells isolated from ovaries of women with polycystic ovary syndrome (PCOS). We previously reported that -235 to -109 bp of the CYP17 promoter confers increased CYP17 promoter function in PCOS theca cells. In this report, additional deletion and mutational analyses of the CYP17 promoter were performed to identify the sequences that contribute to increased CYP17 promoter function in PCOS theca cells. Results of these analyses established that augmented promoter function in PCOS theca cells results from preferentially increased basal regulation conferred by sequences between -188 and -147 bp of the CYP17 promoter. Scanning mutant analysis demonstrated that mutations within a 16-bp sequence, spanning -174 to -158 bp of the promoter, ablated increased basal CYP17 promoter function in PCOS theca cells. EMSA analysis demonstrated that the NF-1 family member, NF-1C, bound this sequence. Cotransfection of several NF-1C isoforms expressed in normal and PCOS cells repressed CYP17 promoter function. NF-1C protein and DNA binding were reduced in PCOS theca cell nuclear extracts, as compared with normal. Another NF-1C site between -102 and -90 bp of the promoter was also identified. However, mutation of this site had no effect on differential promoter function in PCOS theca cells. These studies demonstrate that 1) augmented CYP17 promoter function in PCOS theca cells results from increased basal regulation, and 2) diminished NF-1C-dependent repression may be one mechanism underlying increased basal CYP17 promoter activity and altered gene expression in PCOS theca cells.

Effect of IL-1beta 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.

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.