Rafal Bartoszewski

A synonymous single nucleotide polymorphism in δF508 CFTR alters the secondary structure of the mRNA and the expression of the mutant protein

Recent advances in our understanding of translational dynamics indicate that codon usage and mRNA secondary structure influence translation and protein folding. The most frequent cause of cystic fibrosis (CF) is the deletion of three nucleotides (CTT) from the cystic fibrosis transmembrane conductance regulator (CFTR) gene that includes the last cytosine (C) of isoleucine 507 (Ile507ATC) and the two thymidines (T) of phenylalanine 508 (Phe508TTT) codons. The consequences of the deletion are the loss of phenylalanine at the 508 position of the CFTR protein (ΔF508), a synonymous codon change for isoleucine 507 (Ile507ATT), and protein misfolding. Here we demonstrate that the ΔF508 mutation alters the secondary structure of the CFTR mRNA. Molecular modeling predicts and RNase assays support the presence of two enlarged single stranded loops in the ΔF508 CFTR mRNA in the vicinity of the mutation. The consequence of ΔF508 CFTR mRNA “misfolding” is decreased translational rate. A synonymous single nucleotide variant of the ΔF508 CFTR (Ile507ATC), that could exist naturally if Phe-508 was encoded by TTC, has wild type-like mRNA structure, and enhanced expression levels when compared with native ΔF508 CFTR. Because CFTR folding is predominantly cotranslational, changes in translational dynamics may promote ΔF508 CFTR misfolding. Therefore, we propose that mRNA “misfolding” contributes to ΔF508 CFTR protein misfolding and consequently to the severity of the human ΔF508 phenotype. Our studies suggest that in addition to modifier genes, SNPs may also contribute to the differences observed in the symptoms of various ΔF508 homozygous CF patients.

Knockdown of ASIC1 and Epithelial Sodium Channel Subunits Inhibits Glioblastoma Whole Cell Current and Cell Migration

High grade gliomas such as glioblastoma multiforme express multiple members of the epithelial sodium channel (ENaC)/Degenerin family, characteristically displaying a basally active amiloride-sensitive cation current not seen in normal human astrocytes or lower grade gliomas. Using quantitative real time PCR, we have shown higher expression of ASIC1, αENaC, and γENaC in D54-MG human glioblastoma multiforme cells compared with primary human astrocytes. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test this hypothesis we made dominant negative cDNAs for ASIC1, αENaC, γENaC, and δENaC. D54-MG cells transfected with the dominant negative constructs for ASIC1, αENaC, or γENaC showed reduced protein expression and a significant reduction in the amiloride-sensitive whole cell current as compared with untransfected D54-MG cells. Knocking down αENaC or γENaC also abolished the high PK+/PNa+ of D54-MG cells. Knocking down δENaC in D54-MG cells reduced δENaC protein expression but had no effect on either the whole cell current or K+ permeability. Using co-immunoprecipitation we show interactions between ASIC1, αENaC, and γENaC, consistent with these subunits interacting with each other to form an ion channel in glioma cells. We also found a significant inhibition of D54-MG cell migration after ASIC1, αENaC, or γENaC knockdown, consistent with the hypothesis that ENaC/Degenerin subunits play an important role in glioma cell biology.

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.

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.

Regulation of angiogenesis by hypoxia: the role of microRNA

Understanding the cellular pathways that regulate angiogenesis during hypoxia is a necessary aspect in the development of novel treatments for cardiovascular disorders. Although the pathways of angiogenesis have been extensively studied, there is limited information on the role of miRNAs in this process. miRNAs or their antagomirs could be used in future therapeutic approaches to regulate hypoxia-induced angiogenesis, so it is critical to understand their role in governing angiogenesis during hypoxic conditions. Although hypoxia and ischemia change the expression profile of many miRNAs, a functional role for a limited number of so-called hypoxamiRs has been demonstrated in angiogenesis. Here, we discuss the best examples that illustrate the role of hypoxamiRs in angiogenesis.

The hypoxia-inducible miR-429 regulates hypoxia-inducible factor- 1α expression in human endothelial cells through a negative feedback loop

Hypoxia‐inducible factors (HIFs) 1 and 2 are dimeric α/β transcription factors that regulate cellular responses to low oxygen. HIF‐1 is induced first, whereas HIF‐2 is associated with chronic hypoxia. To determine how HIF1A mRNA, the inducible subunit of HIF‐1, is regulated during hypoxia, we followed HIF1A mRNA levels in primary HUVECs over 24 hours using quantitative PCR. HIF1A and VEGF A (VEGFA) mRNA, a transcriptional target of HIF‐1, increased ~2.5‐ and 8‐fold at 2‐4 hours, respectively. To determine how the mRNAs were regulated, we identified a microRNA (miRNA), miR‐429, that destabilized HIF1A message and decreased VEGFA mRNA by inhibiting HIF1A. Target protector analysis, which interferes with miRNA‐mRNA complex formation, confirmed that miR‐429 targeted HIF1A message. Desferoxamine treatment, which inhibits the hydroxylases that promote HIF‐1α protein degradation, stabilized HIF‐1 activity during normoxic conditions and elevated miR‐429 levels, demonstrating that HIF‐1 promotes miR‐429 expression. RNA‐sequencing‐based transcriptome analysis indicated that inhibition of miRNA‐429 in HUVECs up‐regulated 209 mRNAs, a number of which regulate angiogenesis. The results demonstrate that HIF‐1 is in a negative regulatory loop with miR‐429, that miR‐429 attenuates HIF‐1 activity by decreasing HIF1A message during the early stages of hypoxia before HIF‐2 is activated, and this regulatory network helps explain the HIF‐1 transition to HIF‐2 during chronic hypoxia in endothelial cells.—Bartoszewska, S., Kochan, K., Piotrowski, A., Kamysz, W., Ochocka, R. J., Collawn, J. F., Bartoszewski, R. The hypoxia‐inducible miR‐429 regulates hypoxia hypoxia‐inducible factor‐1α expression in human endothelial cells through a negative feedback loop. FASEB J. 29, 1467‐1479 (2015). www.fasebj.org

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

Role of epithelial sodium channels in the regulation of lung fluid homeostasis

In utero, fetal lung epithelial cells actively secrete Cl(-) ions into the lung air spaces while Na(+) ions follow passively to maintain electroneutrality. This process, driven by an electrochemical gradient generated by the Na(+)-K(+)-ATPase, is responsible for the secretion of fetal fluid that is essential for normal lung development. Shortly before birth, a significant upregulation of amiloride-sensitive epithelial channels (ENaCs) on the apical side of the lung epithelial cells results in upregulation of active Na(+) transport. This process is critical for the reabsorption of fetal lung fluid and the establishment of optimum gas exchange. In the adult lung, active Na(+) reabsorption across distal lung epithelial cells limits the degree of alveolar edema in patients with acute lung injury and cardiogenic edema. Cl(-) ions are transported either paracellularly or transcellularly to preserve electroneutrality. An increase in Cl(-) secretion across the distal lung epithelium has been reported following an acute increase in left atrial pressure and may result in pulmonary edema. In contrast, airway epithelial cells secrete Cl(-) through apical cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels and absorb Na(+). Thus the coordinated action of Cl(-) secretion and Na(+) absorption is essential for maintenance of the volume of epithelial lining fluid that, in turn, maximizes mucociliary clearance and facilitates clearance of bacteria and debris from the lungs. Any factor that interferes with Na(+) or Cl(-) transport or dramatically upregulates ENaC activity in airway epithelial cells has been associated with lung diseases such as cystic fibrosis or chronic obstructive lung disease. In this review we focus on the role of the ENaC, the mechanisms involved in ENaC regulation, and how ENaC dysregulation can lead to lung pathology.

Interaction of ASIC1 and ENaC subunits in human glioma cells and rat astrocytes

Glioblastoma multiforme (GBM) is the most common and aggressive of the primary brain tumors. These tumors express multiple members of the epithelial sodium channel (ENaC)/degenerin (Deg) family and are associated with a basally active amiloride-sensitive cation current. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of ENaC and acid-sensing ion channel (ASIC) subunits. To test the hypothesis that ASIC1 interacts with αENaC and γENaC at the cellular level, we have used total internal reflection fluorescence microscopy (TIRFM) in live rat astrocytes transiently cotransfected with cDNAs for ASIC1-DsRed plus αENaC-yellow fluorescent protein (YFP) or ASIC1-DsRed plus γENaC-YFP. TIRFM images show colocalization of ASIC1 with both αENaC and γENaC. Furthermore, using TIRFM in stably transfected D54-MG cells, we also found that ASIC1 and αENaC both localize to a submembrane region following exposure to pH 6.0, similar to the acidic conditions found in the core of a glioblastoma lesion. Using high-resolution clear native gel electrophoresis, we found that ASIC1 forms a complex with ENaC subunits which migrates at ≈480 kDa in D54-MG glioma cells. These data suggest that different ENaC/Deg subunits interact and could combine to form a hybrid channel that likely underlies the amiloride-sensitive current seen in human glioma cells.