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Shyam Ramachandran,
Philip H. Karp,
Peng Jiang,
Lynda S. Ostedgaard,
Amy E. Walz,
John T. Fisher, Shaf Keshavjee,
Kim A. Lennox,
Ashley M. Jacobi,
Scott D. Rose,
Mark A. Behlke,
Michael J. Welsh,
Yi Xing,
Jr. Paul B. McCray
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ABSTRACT: Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing.
MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane
conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We
discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an
miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl− permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered
the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common
CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138
regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl− transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to
the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously.
This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis
mutation.
Proceedings of the National Academy of Sciences 08/2012; 109(33):13362-13367. · 9.68 Impact Factor
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Shyam Ramachandran,
Philip H Karp,
Peng Jiang,
Lynda S Ostedgaard,
Amy E Walz,
John T Fisher, Shaf Keshavjee,
Kim A Lennox,
Ashley M Jacobi,
Scott D Rose,
Mark A Behlke,
Michael J Welsh,
Yi Xing,
Paul B McCray
[show abstract]
[hide abstract]
ABSTRACT: Production of functional proteins requires multiple steps, including gene transcription and posttranslational processing. MicroRNAs (miRNAs) can regulate individual stages of these processes. Despite the importance of the cystic fibrosis transmembrane conductance regulator (CFTR) channel for epithelial anion transport, how its expression is regulated remains uncertain. We discovered that miRNA-138 regulates CFTR expression through its interactions with the transcriptional regulatory protein SIN3A. Treating airway epithelia with an miR-138 mimic increased CFTR mRNA and also enhanced CFTR abundance and transepithelial Cl(-) permeability independent of elevated mRNA levels. An miR-138 anti-miR had the opposite effects. Importantly, miR-138 altered the expression of many genes encoding proteins that associate with CFTR and may influence its biosynthesis. The most common CFTR mutation, ΔF508, causes protein misfolding, protein degradation, and cystic fibrosis. Remarkably, manipulating the miR-138 regulatory network also improved biosynthesis of CFTR-ΔF508 and restored Cl(-) transport to cystic fibrosis airway epithelia. This miRNA-regulated network directs gene expression from the chromosome to the cell membrane, indicating that an individual miRNA can control a cellular process more broadly than recognized previously. This discovery also provides therapeutic avenues for restoring CFTR function to cells affected by the most common cystic fibrosis mutation.
Proceedings of the National Academy of Sciences 08/2012; 109(33):13362-7. · 9.68 Impact Factor
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ABSTRACT: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF) lung disease. CFTR is expressed in airway epithelia, but how CF alters electrolyte transport across airway epithelia has remained uncertain. Recent studies of a porcine model showed that in vivo, excised, and cultured CFTR(-/-) and CFTR(ΔF508/ΔF508) airway epithelia lacked anion conductance, and they did not hyperabsorb Na(+). Therefore, we asked whether Cl(-) and Na(+) conductances were altered in human CF airway epithelia. We studied differentiated primary cultures of tracheal/bronchial epithelia and found that transepithelial conductance (Gt) under basal conditions and the cAMP-stimulated increase in Gt were markedly attenuated in CF epithelia compared with non-CF epithelia. These data reflect loss of the CFTR anion conductance. In CF and non-CF epithelia, the Na(+) channel inhibitor amiloride produced similar reductions in Gt and Na(+) absorption, indicating that Na(+) conductance in CF epithelia did not exceed that in non-CF epithelia. Consistent with previous reports, adding amiloride caused greater reductions in transepithelial voltage and short-circuit current in CF epithelia than in non-CF epithelia; these changes are attributed to loss of a Cl(-) conductance. These results indicate that Na(+) conductance was not increased in these cultured CF tracheal/bronchial epithelia and point to loss of anion transport as key to airway epithelial dysfunction in CF.
Proceedings of the National Academy of Sciences 06/2011; 108(25):10260-5. · 9.68 Impact Factor
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ABSTRACT: The family of zinc- and calcium-dependent matrix metalloproteases (MMPs) play an important role in remodeling of the airways in disease. Transcriptional regulation by proinflammatory cytokines increases lymphocyte-derived MMP9 levels in the airway lumen of asthmatics. Moreover, the levels of the MMP9 inhibitor, tissue inhibitor of metalloprotease (TIMP1), are decreased leading to increased protease activity. The mechanism by which MMP9 activity leads to asthma pathogenesis and remodeling remains unclear. Using a model of well-differentiated human airway epithelia, we found that apical MMP9 significantly increases transepithelial conductance. Moreover, apical MMP9 treatment decreased immunostaining of tight junction proteins suggesting disruption of barrier function. Consistent with this, viruses gained access to the epithelial basolateral surface after MMP9 treatment, which increased infection efficiency. All of these effects were blocked by TIMP1. In addition, loss of epithelial integrity correlated with increased epithelial cell death. Thus we hypothesized that MMP9 exerts its effects on the epithelium by cleaving one or more components of cell-cell junctions and triggering anoikis. Taken together, these data suggest that a component of airway remodeling associated with asthma may be directly regulated by MMP9.
AJP Lung Cellular and Molecular Physiology 04/2009; 296(5):L751-62. · 3.66 Impact Factor
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ABSTRACT: Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl(-) transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.
Proceedings of the National Academy of Sciences 03/2009; 106(10):3865-70. · 9.68 Impact Factor
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ABSTRACT: Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel regulated by phosphorylation. Most of the disease-associated morbidity is the consequence of chronic lung infection with progressive tissue destruction. As an approach to investigate the cellular effects of CFTR mutations, we used large-scale microarray hybridization to contrast the gene expression profiles of well-differentiated primary cultures of human CF and non-CF airway epithelia grown under resting culture conditions. We surveyed the expression profiles for 10 non-CF and 10 DeltaF508 homozygote samples. Of the 22,283 genes represented on the Affymetrix U133A GeneChip, we found evidence of significant changes in expression in 24 genes by two-sample t-test (P < 0.00001). A second, three-filter method of comparative analysis found no significant differences between the groups. The levels of CFTR mRNA were comparable in both groups. There were no significant differences in the gene expression patterns between male and female CF specimens. There were 18 genes with significant increases and 6 genes with decreases in CF relative to non-CF samples. Although the function of many of the differentially expressed genes is unknown, one transcript that was elevated in CF, the KCl cotransporter (KCC4), is a candidate for further study. Overall, the results indicate that CFTR dysfunction has little direct impact on airway epithelial gene expression in samples grown under these conditions.
AJP Lung Cellular and Molecular Physiology 11/2005; 289(4):L545-53. · 3.66 Impact Factor
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ABSTRACT: Adenovirus-mediated gene transfer to airway epithelia is inefficient in part because its receptor is absent on the apical surface of the airways. Targeting adenovirus to other receptors, increasing the viral concentration, and even prolonging the incubation time with adenovirus vectors can partially overcome the lack of receptors and facilitate gene transfer. Unfortunately, mucociliary clearance would prevent prolonged incubation time in vivo. Thixotropic solutions (TS) are gels that upon a vigorous shearing force reversibly become liquid. We hypothesized that formulating recombinant adenoviruses in TS would decrease virus clearance and thus enhance gene transfer to the airway epithelia. We found that clearance of virus-sized fluorescent beads by human airway epithelia in vitro and by monkey trachea in vivo were markedly decreased when the beads were formulated in TS compared with phosphate-buffered saline (PBS). Adenovirus formulated in TS significantly increased adenovirus-mediated gene transfer of a reporter gene in human airway epithelia in vitro and in murine airway epithelia in vivo. Furthermore, an adenovirus encoding the cystic fibrosis transmembrane regulator (CFTR) gene (AdCFTR) formulated in TS was more efficient in correcting the chloride transport defect in cystic fibrosis airway epithelia than AdCFTR formulated in PBS. These data indicate a novel strategy to augment the efficiency of gene transfer to the airways that may be applicable to a number of different gene transfer vectors and could be of value in gene transfer to cystic fibrosis (CF) airway epithelia in vivo.
American Journal of Respiratory Cell and Molecular Biology 09/2002; 27(2):133-40. · 5.13 Impact Factor
