Andras Rab

The Unfolded Protein Response (UPR)-activated Transcription Factor X-box-binding Protein 1 (XBP1) Induces MicroRNA-346 Expression That Targets the Human Antigen Peptide Transporter 1 (TAP1) mRNA and Governs Immune Regulatory Genes

Background: The adaptive unfolded protein response (UPR) promotes endoplasmic reticulum (ER) expansion and reduces ER load. Results: UPR-activated XBP1 induces miR-346 expression that targets TAP1. Conclusion: We identify a novel function for XBP1 and an miRNA-mediated pathway for ER load reduction through TAP1. Significance: Novel interventions for protein folding disorders will require an understanding of how microRNAs regulate gene expression during ER stress. To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3′-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

Activation of the Unfolded Protein Response by ΔF508 CFTR

Environmental insults and misfolded proteins cause endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR). The UPR decreases endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency. Herein, we investigated the effects of the folding-deficient deltaF508 CFTR on ER stress induction and UPR activation. For these studies, we developed and characterized stable clones of Calu3deltaF cells that express different levels of endogenous wild-type (WT) and recombinant deltaF508 CFTR. We also present a novel RT-PCR-based assay for differential quantification of wild-type CFTR mRNA in the presence of deltaF508 CFTR message. The assay is based on a TaqMan minor groove binding (MGB) probe that recognizes a specific TTT sequence (encoding phenylalanine at position 508 in human CFTR). The MGB probe is extremely specific and sensitive to changes in WT CFTR message levels. In RNA samples that contain both WT and deltaF508 CFTR mRNAs, measurement of WT CFTR mRNA levels (using the MGB probe) and total CFTR mRNA (using commercial primers) allowed us to calculate deltaF508 CFTR mRNA levels. The results indicate that overexpression of deltaF508 CFTR causes ER stress and activates the UPR. UPR activation precedes a marked decrease in endogenous WT CFTR mRNA expression. Furthermore, polarized airway epithelial cell lines are important tools in cystic fibrosis research, and herein we provide an airway epithelial model to study the biogenesis and function of WT and deltaF508 CFTR expressed within the same cell.

Cigarette smoke and CFTR: implications in the pathogenesis of COPD

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disorder consisting of chronic bronchitis and/or emphysema. COPD patients suffer from chronic infections and display exaggerated inflammatory responses and a progressive decline in respiratory function. The respiratory symptoms of COPD are similar to those seen in cystic fibrosis (CF), although the molecular basis of the two disorders differs. CF is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene encoding a chloride and bicarbonate channel (CFTR), leading to CFTR dysfunction. The majority of COPD cases result from chronic oxidative insults such as cigarette smoke. Interestingly, environmental stresses including cigarette smoke, hypoxia, and chronic inflammation have also been implicated in reduced CFTR function, and this suggests a common mechanism that may contribute to both the CF and COPD. Therefore, improving CFTR function may offer an excellent opportunity for the development of a common treatment for CF and COPD. In this article, we review what is known about the CF respiratory phenotype and discuss how diminished CFTR expression-associated ion transport defects may contribute to some of the pathological changes seen in COPD.

The Mechanism of Cystic Fibrosis Transmembrane Conductance Regulator Transcriptional Repression during the Unfolded Protein Response

The unfolded protein response (UPR) aids cellular recovery by increasing the capacity and decreasing the protein load of the endoplasmic reticulum (ER). Although the main pathways of the UPR are known, the mechanisms of UPR-associated transcriptional repression have not been explored in mammalian cells. Previous studies indicate that endogenous cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels and protein maturation efficiency decrease when the UPR is activated. In the present study, we demonstrate that inhibition of CFTR expression under ER stress leads to reduced cAMP-activated chloride secretion in epithelial monolayers, an indication of diminished CFTR function. Moreover, ER stress and the UPR obliterate endogenous ΔF508 CFTR mRNA expression in CFPAC-1 cells without affecting recombinant ΔF508 CFTR mRNA levels or mRNA half-life. These results emphasize that transcriptional repression of CFTR under ER stress, in concert with decreased CFTR maturation efficiency, leads to diminished function. Using human CFTR promoter reporter constructs, we confined the ER stress-associated CFTR transcriptional repression to the minimal promoter. Chromatin immunoprecipitation assays established the binding of the UPR-activated ATF6 transcription factor to this region during ER stress, which links the repression to the UPR. Methylation-specific PCR (MSP) revealed hypermethylation of CpG sites inside and in the vicinity of the MAZ transcription factor binding region of CFTR, demonstrating methylation-dependent repression. Using pharmacological inhibitors, we show that both DNA methylation and histone deacetylation contribute to CFTR transcriptional inhibition. These studies provide novel insight into the mechanism of gene repression during the mammalian UPR.

The silent codon change I507-ATC→ATT contributes to the severity of the ΔF508 CFTR channel dysfunction

The most common disease‐causing mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is the out‐of‐frame deletion of 3 nucleotides (CTT). This mutation leads to the loss of phenylalanine‐508 (ΔF508) and a silent codon change (SCC) for isoleucine‐507 (I507‐ATC→ATT). ΔF508 CFTR is misfolded and degraded by endoplasmic reticulum‐associated degradation (ERAD). We have demonstrated that the I507‐ATC→ATT SCC alters ΔF508 CFTR mRNA structure and translation dynamics. By comparing the biochemical and functional properties of the I507‐ATT and I507‐ATC ΔF508 CFTR, we establish that the I507‐ATC→ATT SCC contributes to the cotranslational misfolding, ERAD, and to the functional defects associated with ΔF508 CFTR We demonstrate that the I507‐ATC ΔF508 CFTR is less susceptible to the ER quality‐control machinery during translation than the I507‐ATT, although 27°C correction is necessary for sufficient cell‐surface expression. Whole‐cell patch‐clamp recordings indicate sustained, thermally stable cAMP‐activated Cl– transport through I507‐ATC and unstable function of the I507‐ATT ΔF508 CFTR Single‐channel recordings reveal improved gating properties of the I507‐ATC compared to I507‐ATT ΔF508 CFTR (NPo=0.45±0.037 vs. NPo=0.09±0.002; P<0.001). Our results signify the role of the I507‐ATC→ATT SCC in the ΔF508 CFTR defects and support the importance of synonymous codon choices in determining the function of gene products.—Lazrak, A., Fu, L., Bali, V., Bartoszewski, R., Rab, A., Havasi, V., Keiles, S., Kappes, J., Kumar, R., Lefkowitz, E., Sorscher, E. J., Matalon, S., Collawn, J. F., Bebok, Z. The silent codon change I507‐ATC→ATT contributes to the severity of the ΔF508 CFTR FASEB J. 27, 4630–4645 (2013). www.fasebj.org

CFTR expression regulation by the unfolded protein response.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel and key regulator of epithelial functions. Mutations in the CFTR gene lead to reduced or dysfunctional CFTR protein and cause cystic fibrosis (CF), a generalized exocrinopathy affecting multiple organs. In the airways, loss of CFTR function leads to thickened mucus, reduced mucociliary clearance, chronic infections, and respiratory failure. Common airway disorders such as bronchitis and chronic obstructive pulmonary disease (COPD) also present CF-like symptoms such as mucus congestion and chronic inflammation without mutations in CFTR. The primary risk factors for COPD and chronic bronchitis include environmental stress insults such as pollutants and infections that often result in hypoxic conditions. Furthermore, environmental factors such as cigarette smoke and reactive oxygen species have been implicated in reduced CFTR function. Activation of cellular stress responses by these factors promotes differential, stress-associated gene expression regulation. During our investigations on the mechanisms of CFTR expression regulation, we have shown that the ER stress response, the unfolded protein response (UPR), decreases CFTR expression at the transcriptional, translational, and maturational levels. Here, we provide a detailed description of the methods we employ to study CFTR expression regulation by the UPR. Similar approaches are applicable in studies on other genes and how they are affected by the UPR.

Chymase Mediates Injury and Mitochondrial Damage in Cardiomyocytes during Acute Ischemia/Reperfusion in the Dog

Cardiac ischemia and reperfusion (I/R) injury occurs because the acute increase in oxidative/inflammatory stress during reperfusion culminates in the death of cardiomyocytes. Currently, there is no drug utilized clinically that attenuates I/R injury in patients. Previous studies have demonstrated degranulation of mast cell contents into the interstitium after I/R. Using a dog model of I/R, we tested the role of chymase, a mast cell protease, in cardiomyocyte injury using a specific oral chymase inhibitor (CI). 15 adult mongrel dogs had left anterior descending artery occlusion for 60 min and reperfusion for 100 minutes. 9 dogs received vehicle and 6 were pretreated with a specific CI. In vivo cardiac microdialysis demonstrated a 3-fold increase in interstitial fluid chymase activity in I/R region that was significantly decreased by CI. CI pretreatment significantly attenuated loss of laminin, focal adhesion complex disruption, and release of troponin I into the circulation. Microarray analysis identified an I/R induced 17-fold increase in nuclear receptor subfamily 4A1 (NR4A1) and significantly decreased by CI. NR4A1 normally resides in the nucleus but can induce cell death on migration to the cytoplasm. I/R caused significant increase in NR4A1 protein expression and cytoplasmic translocation, and mitochondrial degradation, which were decreased by CI. Immunohistochemistry also revealed a high concentration of chymase within cardiomyocytes after I/R. In vitro, chymase added to culture HL-1 cardiomyocytes entered the cytoplasm and nucleus in a dynamin-dependent fashion, and promoted cytoplasmic translocation of NR4A1 protein. shRNA knockdown of NR4A1 on pre-treatment of HL-1 cells with CI significantly decreased chymase-induced cell death and mitochondrial damage. These results suggest that the beneficial effects of an orally active CI during I/R are mediated in the cardiac interstitium as well as within the cardiomyocyte due to a heretofore-unrecognized chymase entry into cardiomyocytes.

ΔF508 CFTR Surface Stability Is Regulated by DAB2 and CHIP-Mediated Ubiquitination in Post-Endocytic Compartments

The ΔF508 mutant form of the cystic fibrosis transmembrane conductance regulator (ΔF508 CFTR) that is normally degraded by the ER-associated degradative pathway can be rescued to the cell surface through low-temperature (27°C) culture or small molecular corrector treatment. However, it is unstable on the cell surface, and rapidly internalized and targeted to the lysosomal compartment for degradation. To understand the mechanism of this rapid turnover, we examined the role of two adaptor complexes (AP-2 and Dab2) and three E3 ubiquitin ligases (c-Cbl, CHIP, and Nedd4-2) on low-temperature rescued ΔF508 CFTR endocytosis and degradation in human airway epithelial cells. Our results demonstrate that siRNA depletion of either AP-2 or Dab2 inhibits ΔF508 CFTR endocytosis by 69% and 83%, respectively. AP-2 or Dab2 depletion also increases the rescued protein half-life of ΔF508 CFTR by ~18% and ~91%, respectively. In contrast, the depletion of each of the E3 ligases had no effect on ΔF508 CFTR endocytosis, whereas CHIP depletion significantly increased the surface half-life of ΔF508 CFTR. To determine where and when the ubiquitination occurs during ΔF508 CFTR turnover, we monitored the ubiquitination of rescued ΔF508 CFTR during the time course of CFTR endocytosis. Our results indicate that ubiquitination of the surface pool of ΔF508 CFTR begins to increase 15 min after internalization, suggesting that CFTR is ubiquitinated in a post-endocytic compartment. This post-endocytic ubiquination of ΔF508 CFTR could be blocked by either inhibiting endocytosis, by siRNA knockdown of CHIP, or by treating cells with the CFTR corrector, VX-809. Our results indicate that the post-endocytic ubiquitination of CFTR by CHIP is a critical step in the peripheral quality control of cell surface ΔF508 CFTR.

Robust Stimulation of W1282X-CFTR Channel Activity by a Combination of Allosteric Modulators.

W1282X is a common nonsense mutation among cystic fibrosis patients that results in the production of a truncated Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) channel. Here we show that the channel activity of the W1282X-CFTR polypeptide is exceptionally low in excised membrane patches at normally saturating doses of ATP and PKA (single channel open probability (PO) < 0.01). However, W1282X-CFTR channels were stimulated by two CFTR modulators, the FDA-approved VX-770 and the dietary compound curcumin. Each of these compounds is an allosteric modulator of CFTR gating that promotes channel activity in the absence of the native ligand, ATP. Although W1282X-CFTR channels were stimulated by VX-770 in the absence of ATP their activities remained dependent on PKA phosphorylation. Thus, activated W1282X-CFTR channels should remain under physiologic control by cyclic nucleotide signaling pathways in vivo. VX-770 and curcumin exerted additive effects on W1282X-CFTR channel gating (opening/closing) in excised patches such that the Po of the truncated channel approached unity (> 0.9) when treated with both modulators. VX-770 and curcumin also additively stimulated W1282X-CFTR mediated currents in polarized FRT epithelial monolayers. In this setting, however, the stimulated W1282X-CFTR currents were smaller than those mediated by wild type CFTR (3–5%) due presumably to lower expression levels or cell surface targeting of the truncated protein. Combining allosteric modulators of different mechanistic classes is worth considering as a treatment option for W1282X CF patients perhaps when coupled with maneuvers to increase expression of the truncated protein.

In Vitro Longitudinal Relaxivity Profile of Gd(ABE-DTTA), an Investigational Magnetic Resonance Imaging Contrast Agent.

Purpose MRI contrast agents (CA) whose contrast enhancement remains relatively high even at the higher end of the magnetic field strength range would be desirable. The purpose of this work was to demonstrate such a desired magnetic field dependency of the longitudinal relaxivity for an experimental MRI CA, Gd(ABE-DTTA). Materials and Methods The relaxivity of 0.5mM and 1mM Gd(ABE-DTTA) was measured by Nuclear Magnetic Relaxation Dispersion (NMRD) in the range of 0.0002 to 1T. Two MRI and five NMR instruments were used to cover the range between 1.5 to 20T. Parallel measurement of a Gd-DTPA sample was performed throughout as reference. All measurements were carried out at 37°C and pH 7.4. Results The relaxivity values of 0.5mM and 1mM Gd(ABE-DTTA) measured at 1.5, 3, and 7T, within the presently clinically relevant magnetic field range, were 15.3, 11.8, 12.4 s-1mM-1 and 18.1, 16.7, and 13.5 s-1mM-1, respectively. The control 4 mM Gd-DTPA relaxivities at the same magnetic fields were 3.6, 3.3, and 3.0 s-1mM-1, respectively. Conclusions The longitudinal relaxivity of Gd(ABE-DTTA) measured within the presently clinically relevant field range is three to five times higher than that of most commercially available agents. Thus, Gd(ABE-DTTA) could be a practical choice at any field strength currently used in clinical imaging including those at the higher end.