Calvin U. Cotton

Intronic enhancers coordinate epithelial-specific looping of the active CFTR locus

The regulated expression of large human genes can depend on long-range interactions to establish appropriate three-dimensional structures across the locus. The cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encompasses 189 kb of genomic DNA, shows a complex pattern of expression with both spatial and temporal regulation. The flanking loci, ASZ1 and CTTNBP2, show very different tissue-specific expression. The mechanisms governing control of CFTR expression remain poorly understood, although they are known to involve intronic regulatory elements. Here, we show a complex looped structure of the CFTR locus in cells that express the gene, which is absent from cells in which the gene is inactive. By using chromatin conformation capture (3C) with a bait probe at the CFTR promoter, we demonstrate close interaction of this region with sequences in the middle of the gene about 100 kb from the promoter and with regions 3′ to the locus that are about 200 kb away. We show that these interacting regions correspond to prominent DNase I hypersensitive sites within the locus. Moreover, these sequences act cooperatively in reporter gene constructs and recruit proteins that modify chromatin structure. The model for CFTR gene expression that is revealed by our data provides a paradigm for other large genes with multiple regulatory elements lying within both introns and intergenic regions. We anticipate that these observations will enable original approaches to designing regulated transgenes for tissue-specific gene therapy protocols.

Absence of the cystic fibrosis transmembrane regulator (Cftr) from myeloid-derived cells slows resolution of inflammation and infection

The absence or reduction of CFTR function causes CF and results in a pulmonary milieu characterized by bacterial colonization and unresolved inflammation. The ineffectiveness at controlling infection by species such as Pseudomonas aeruginosa suggests defects in innate immunity. Macrophages, neutrophils, and DCs have all been shown to express CFTR mRNA but at low levels, raising the question of whether CFTR has a functional role in these cells. Bone marrow transplants between CF and non‐CF mice suggest that these cells are inherently different; we confirm this observation using conditional inactivation of Cftr in myeloid‐derived cells. Mice lacking Cftr in myeloid cells overtly appear indistinguishable from non‐CF mice until challenged with bacteria instilled into the lungs and airways, at which point, they display survival and inflammatory profiles intermediate in severity as compared with CF mice. These studies demonstrate that Cftr is involved directly in myeloid cell function and imply that these cells contribute to the pathophysiological phenotype of the CF lung.

Dysfunction of Nrf-2 in CF Epithelia Leads to Excess Intracellular H2O2 and Inflammatory Cytokine Production

Cystic fibrosis is characterized by recurring pulmonary exacerbations that lead to the deterioration of lung function and eventual lung failure. Excessive inflammatory responses by airway epithelia have been linked to the overproduction of the inflammatory cytokine IL-6 and IL-8. The mechanism by which this occurs is not fully understood, but normal IL-1β mediated activation of the production of these cytokines occurs via H2O2 dependent signaling. Therefore, we speculated that CFTR dysfunction causes alterations in the regulation of steady state H2O2. We found significantly elevated levels of H2O2 in three cultured epithelial cell models of CF, one primary and two immortalized. Increases in H2O2 heavily contributed to the excessive IL-6 and IL-8 production in CF epithelia. Proteomic analysis of three in vitro and two in vivo models revealed a decrease in antioxidant proteins that regulate H2O2 processing, by ≥2 fold in CF vs. matched normal controls. When cells are stimulated, differential expression in CF versus normal is enhanced; corresponding to an increase in H2O2 mediated production of IL-6 and IL-8. The cause of this redox imbalance is a decrease by ∼70% in CF cells versus normal in the expression and activity of the transcription factor Nrf-2. Inhibition of CFTR function in normal cells produced this phenotype, while N-acetyl cysteine, selenium, an activator of Nrf-2, and the overexpression of Nrf-2 all normalized H2O2 processing and decreased IL-6 and IL-8 to normal levels, in CF cells. We conclude that a paradoxical decrease in Nrf-2 driven antioxidant responses in CF epithelia results in an increase in steady state H2O2, which in turn contributes to the overproduction of the pro-inflammatory cytokines IL-6 and IL-8. Treatment with antioxidants can ameliorate exaggerated cytokine production without affecting normal responses.

Activation of endogenous deltaF508 cystic fibrosis transmembrane conductance regulator by phosphodiesterase inhibition.

Many heterologously expressed mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) exhibit residual chloride channel activity that can be stimulated by agonists of the adenylate cyclase/protein kinase A pathway. Because of clinical implications for cystic fibrosis of activating mutants in vivo, we are investigating whether deltaF508, the most common disease-associated CFTR mutation, can be activated in airway epithelial cells. We have found that, 36Cl- efflux can be stimulated 19-61% above baseline by beta-adrenoreceptor agonists and cGI-phosphodiesterase inhibitors in transformed nasal polyp (CF-T43) cells homozygous for the deltaF508 mutation. The increase in 36Cl- permeability is diminished by protein kinase A inhibitors and is not mediated by an increase in intracellular calcium concentrations. Preincubation of CF-T43 cells with CFTR anti-sense oligonucleotides prevented an increase in 36Cl- efflux in response to beta-agonist and phosphodiesterase inhibitor. Primary cells isolated from CF nasal polyps gave similar results. These data indicate that endogenous levels of deltaF508 protein can be stimulated to increase 36Cl- permeability in airway epithelial cells.

Generation of a conditional null allele for Cftr in mice

The cystic fibrosis transmembrane conductance regulator (CFTR) gene encodes a cAMP‐regulated chloride channel that is important in controlling the exchange of fluid and electrolytes across epithelial cells. Mutation of CFTR can lead to cystic fibrosis (CF), the most common lethal genetic disease in Caucasians. CF is a systemic illness with multiple organ systems affected including pulmonary, gastrointestinal, pancreatic, immune, endocrine, and reproductive systems. To understand the role of CFTR in the various tissues in which it is expressed, we generated a murine conditional null allele of Cftr (Cftrfl10) in which loxP sites were inserted around exon 10 of the Cftr gene. The Cftrfl10 allele was validated by generating constitutive Cftr null (CftrΔ10) mice using the protamine‐cre system. The CftrΔ10/Δ10 mice displayed almost identical phenotypes to previously published CF mouse models, including poor growth, decreased survival, intestinal obstruction, and loss of Cftr function as assessed by electrophysiology measurements on gut and nasal epithelium. Mice containing the conditional null Cftr allele will be useful in future studies to understand the role of Cftr in specific tissues and developmental time points and lead to a better understanding of CF disease. genesis 46:546–552, 2008.

CX3CR1 is an important surface molecule for respiratory syncytial virus infection in human airway epithelial cells

Respiratory syncytial virus (RSV) is a major cause of severe pneumonia and bronchiolitis in infants and young children, and causes disease throughout life. Understanding the biology of infection, including virus binding to the cell surface, should help develop antiviral drugs or vaccines. The RSV F and G glycoproteins bind cell surface heparin sulfate proteoglycans (HSPGs) through heparin-binding domains. The G protein also has a CX3C chemokine motif which binds to the fractalkine receptor CX3CR1. G protein binding to CX3CR1 is not important for infection of immortalized cell lines, but reportedly is so for primary human airway epithelial cells (HAECs), the primary site for human infection. We studied the role of CX3CR1 in RSV infection with CX3CR1-transfected cell lines and HAECs with variable percentages of CX3CR1-expressing cells, and the effect of anti-CX3CR1 antibodies or a mutation in the RSV CX3C motif. Immortalized cells lacking HSPGs had low RSV binding and infection, which was increased markedly by CX3CR1 transfection. CX3CR1 was expressed primarily on ciliated cells, and ∼50 % of RSV-infected cells in HAECs were CX3CR1+. HAECs with more CX3CR1-expressing cells had a proportional increase in RSV infection. Blocking G binding to CX3CR1 with anti-CX3CR1 antibody or a mutation in the CX3C motif significantly decreased RSV infection in HAECs. The kinetics of cytokine production suggested that the RSV/CX3CR1 interaction induced RANTES (regulated on activation normal T-cell expressed and secreted protein), IL-8 and fractalkine production, whilst it downregulated IL-15, IL1-RA and monocyte chemotactic protein-1. Thus, the RSV G protein/CX3CR1 interaction is likely important in infection and infection-induced responses of the airway epithelium, the primary site of human infection.

Acute downregulation of ENaC by EGF involves the PY motif and putative ERK phosphorylation site.

The epithelial sodium channel (ENaC) is expressed in a variety of tissues, including the renal collecting duct, where it constitutes the rate-limiting step for sodium reabsorption. Liddles syndrome is caused by gain-of-function mutations in the β and γ subunits of ENaC, resulting in enhanced Na reabsorption and hypertension. Epidermal growth factor (EGF) causes acute inhibition of Na absorption in collecting duct principal cells via an extracellular signal–regulated kinase (ERK)–dependent mechanism. In experiments with primary cultures of collecting duct cells derived from a mouse model of Liddles disease (β-ENaC truncation), it was found that EGF inhibited short-circuit current (Isc) by 24 ± 5% in wild-type cells but only by 6 ± 3% in homozygous mutant cells. In order to elucidate the role of specific regions of the β-ENaC C terminus, Madin-Darby canine kidney (MDCK) cell lines that express β-ENaC with mutation of the PY motif (P616L), the ERK phosphorylation site (T613A), and C terminus truncation (R564stop) were created using the Phoenix retroviral system. All three mutants exhibited significant attenuation of the EGF-induced inhibition of sodium current. In MDCK cells with wild-type β-ENaC, EGF-induced inhibition of Isc (<30 min) was fully reversed by exposure to an ERK kinase inhibitor and occurred with no change in ENaC surface expression, indicative of an effect on channel open probability (Po). At later times (>30 min), EGF-induced inhibition of Isc was not reversed by an ERK kinase inhibitor and was accompanied by a decrease in ENaC surface expression. Our results are consistent with an ERK-mediated decrease in ENaC open probability and enhanced retrieval of sodium channels from the apical membrane.

Cystic fibrosis growth retardation is not correlated with loss of Cftr in the intestinal epithelium

Maldigestion due to exocrine pancreatic insufficiency leads to intestinal malabsorption and consequent malnutrition, a mechanism proposed to cause growth retardation associated with cystic fibrosis (CF). However, although enzyme replacement therapy combined with increased caloric intake improves weight gain, the effect on stature is not significant, suggesting that growth retardation has a more complex etiology. Mouse models of CF support this, since these animals do not experience exocrine pancreatic insufficiency yet are growth impaired. Cftr absence from the intestinal epithelium has been suggested as a primary source of growth retardation in CF mice, a concept we directly tested by generating mouse models with Cftr selectively inactivated or restored in intestinal epithelium. The relationship between growth and functional characteristics of the intestines, including transepithelial electrophysiology, incidence of intestinal obstruction, and histopathology, were assessed. Absence of Cftr exclusively from intestinal epithelium resulted in loss of cAMP-stimulated short-circuit current, goblet cell hyperplasia, and occurrence of intestinal obstructions but only slight and transient impaired growth. In contrast, specifically restoring Cftr to the intestinal epithelium resulted in restoration of ion transport and completely protected against obstruction and histopathological anomalies, but growth was indistinguishable from CF mice. These results indicate that absence of Cftr in the intestinal epithelium is an important contributor to the intestinal obstruction phenotype in CF but does not correlate with the observed growth reduction in CF.

Morphological and functional characterization of a conditionally immortalized collecting tubule cell line

A conditionally immortalized collecting tubule cell line, mCT1, was derived from the H-2Kb-ts A58 transgenic mouse (ImmortoMouse), which harbors a temperature-sensitive mutant of the SV40 large T antigen oncogene. Cells maintained under permissive conditions [33 degreesC with interferon-gamma (IFN-gamma)] form epithelial monolayers, express large T antigen, and proliferate (>50 passages). The cells retain properties characteristic of the renal collecting tubule (CT) including: vasopressin (VP)-stimulated cAMP accumulation, aquaporin-2 expression, high transepithelial electrical resistance, VP-stimulated ion transport, and amiloride-sensitive sodium absorption. When the cells are transferred to nonpermissive conditions (39 degreesC without IFN-gamma), the steady-state level of large T antigen protein declines (>95% decrease) and cell proliferation is arrested. This conditionally immortalized, murine renal cell line should prove useful for studies of CT physiology and large T antigen biology.A conditionally immortalized collecting tubule cell line, mCT1, was derived from the H-2Kb-ts A58 transgenic mouse (ImmortoMouse), which harbors a temperature-sensitive mutant of the SV40 large T antigen oncogene. Cells maintained under permissive conditions [33°C with interferon-γ (IFN-γ)] form epithelial monolayers, express large T antigen, and proliferate (>50 passages). The cells retain properties characteristic of the renal collecting tubule (CT) including: vasopressin (VP)-stimulated cAMP accumulation, aquaporin-2 expression, high transepithelial electrical resistance, VP-stimulated ion transport, and amiloride-sensitive sodium absorption. When the cells are transferred to nonpermissive conditions (39°C without IFN-γ), the steady-state level of large T antigen protein declines (>95% decrease) and cell proliferation is arrested. This conditionally immortalized, murine renal cell line should prove useful for studies of CT physiology and large T antigen biology.

Autosomal Recessive Polycystic Kidney Disease Epithelial Cell Model Reveals Multiple Basolateral Epidermal Growth Factor Receptor Sorting Pathways

We have discovered that multiple basolateral pathways mediate EGF receptor sorting in renal epithelial cells. The polycystic kidney disease allele in the BPK mouse model, Bicc1, interferes with one specific EGF receptor pathway, causing nonpolar delivery of the receptor without affecting overall cell polarity.