Post-transcriptional regulation of mu-opioid receptor: Role of the RNA-binding proteins heterogeneous nuclear ribonucleoprotein H1 and F

Classical opioids have been historically used for the treatment of pain and are among the most widely used drugs for both acute severe pain and long-term pain. Morphine and endogenous mu-opioid peptides exert their pharmacological actions mainly through the mu-opioid receptor (MOR). However, the expression of opioid receptor (OR) proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 50-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. To identify proteins binding to the 50-UTR as potential regulators of the mouse MOR gene, affinity column chromatography using 50-UTR-specific RNA oligonucleotides was performed using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified two heterogeneous ribonucleoproteins (hnRNPs) that bound to RNA sequences of interest: hnRNP H1 and hnRNP F. Binding of these proteins to the RNA region was M4-region sequence-specific as confirmed by Western-blot analysis and RNA supershift assay. Furthermore, a cotransfection study showed that the presence of hnRNP H1 and F resulted in repressed expression of the mouse MOR. Our data suggest that hnRNP H1 and F can function as repressors of MOR translation dependent on the M4 (-75 to -71 bp upstream of ATG) sequences. We demonstrate for the first time a role of hnRNPs as posttranscriptional repressors in MOR gene regulation. © Springer Basel AG 2011

Vimentin interacts with the 5'-untranslated region of mouse mu opioid receptor (MOR) and is required for post-transcriptional regulation

The opioid receptors are among the most highly studied members of the superfamily of G-protein coupled receptors. Morphine and endogenous mu opioid peptides exert their pharmacological actions mainly through the mu opioid receptor (MOR). Expression of opioid receptor proteins is controlled by extensive transcriptional and post-transcriptional processing. Previously, the 5'-untranslated region (UTR) of the mouse MOR was found to be important for post-transcriptional regulation of the MOR gene in neuronal cells. Here, we demonstrate for the first time, the role of vimentin as a post-transcriptional repressor in MOR gene regulation. To identify potential regulators of the mouse MOR gene, we performed affinity column chromatography using 5'-UTR-specific RNA oligonucleotides using neuroblastoma NS20Y cells. Chromatography was followed by two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry. We identified an intermediate filament protein, vimentin, which bound specifically to the region between -175 and -150 (175-150) of the MOR 5'-UTR. Binding was confirmed by western blot analysis and RNA supershift assay. Furthermore, a co-transfection study demonstrated that the presence of vimentin resulted in reduced expression of the mouse MOR. Our data suggest that vimentin functions as a repressor of MOR translation, dependent on 175-150 of the MOR 5'-UTR. Copyright © 2013 Landes Bioscience

Novel function of the poly(c)-binding protein α-CP2 as a transcriptional activator that binds to single-stranded DNA sequences

α-complex protein 2; Electrophoretic mobility shift assays; Poly(C) sequence; Post-transcriptional regulation; Transcriptional activator; Transcriptional regulatio

Novel dual-binding function of a poly (C)-binding protein 3, transcriptional factor which binds the double-strand and single-stranded DNA sequence

Poly(C)-binding proteins (PCBPs) are generally known as RNA-binding proteins that interact in a sequence-specific manner with single-stranded poly(C) sequences. These proteins are mainly involved in various posttranscriptional regulations (e.g., mRNA stabilization or translational activation/silencing). This study reports a novel dual-binding function for PCBP3, a member of the PCBP family. Recombinant PCBP3 was purified using affinity column chromatography and its identity confirmed by MALDI-TOF mass spectrometry. The protein folding conditions of the purified and renatured PCBP3 were optimized. Electrophoretic mobility shift assays demonstrated that the recombinant PCBP3 is capable of binding to both double- and single-strand poly(C) sequences. Furthermore, plasmids expressing PCBP3 repressed the expression of luciferase reporters when cotransfected with single-strand (pGL-SS) and double-strand (pGL-DS) constructs containing poly(C) sequences in their promoters. This study demonstrates for the first time that PCBP3 can function as a repressor dependent on binding to single-strand and double-stranded poly(C) sequences. © 2012 Elsevier B.V.

Renoprotective effect of red ginseng in gentamicin-induced acute kidney injury

Aminoglycoside-induced nephrotoxicity is one of the prevalent causes of acute kidney injury (AKI). Oxidative stress-mediated apoptosis of renal tubular cells is known to be a major mechanism of renal injury. Red ginseng extract (RGE) has been reported to possess antioxidant and immune-modulatory activities. We investigated the effect of RGE on gentamicin (GM)-induced apoptosis and oxidative stress in cultured renal tubular cells and animal model of GM-induced AKI. GM induced the generation of reactive oxygen species (ROS) with an increase in NADPH oxidase (NOX) activity and mitochondrial oxidation in NRK-52E cells that were ameliorated with RGE. GM-induced apoptosis of NRK-52E cells, which was associated with an increased expression of mitochondrial Bax, cytosolic cytochrome c, and cleaved caspase-9 and -3, along with a decrease in bcl-2 expression, was also blocked by RGE. In an animal model of GM-induced AKI, RGE treatment significantly attenuated renal dysfunction, cell apoptosis, and tubular damage. RGE ameliorated ROS production in rats with GM-induced AKI, as demonstrated by an increase in the reduced form of glutathione in renal cortex and a decrease in urinary excretion of 8-hydroxy-2'-deoxyguanosine. Our results suggest that RGE protects the kidney from GM-induced AKI via the mechanism of modulation of oxidative stress

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acidinduced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of Ecadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin. © 2013 the American Physiological Society