Paul Kogut

Fine Mapping and Positional Candidate Studies Identify HLA-G as an Asthma Susceptibility Gene on Chromosome 6p21

Asthma affects nearly 14 million people worldwide and has been steadily increasing in frequency for the past 50 years. Although environmental factors clearly influence the onset, progression, and severity of this disease, family and twin studies indicate that genetic variation also influences susceptibility. Linkage of asthma and related phenotypes to chromosome 6p21 has been reported in seven genome screens, making it the most replicated region of the genome. However, because many genes with individually small effects are likely to contribute to risk, identification of asthma susceptibility loci has been challenging. In this study, we present evidence from four independent samples in support of HLA-G as a novel asthma and bronchial hyperresponsiveness susceptibility gene in the human leukocyte antigen region on chromosome 6p21, and we speculate that this gene might contribute to risk for other inflammatory diseases that show linkage to this region.

Physiological control of smooth muscle-specific gene expression through regulated nuclear translocation of serum response factor.

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcription-promoting activity.

Myosin, Transgelin, and Myosin Light Chain Kinase: Expression and Function in Asthma

RATIONALE Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. OBJECTIVES We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. METHODS We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. MEASUREMENTS AND MAIN RESULTS We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. CONCLUSIONS Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma.

Lysophosphatidic Acid Enhances Pulmonary Epithelial Barrier Integrity and Protects Endotoxin-Induced Epithelial Barrier Disruption and Lung Injury

Lysophosphatidic acid (LPA), a bioactive phospholipid, induces a wide range of cellular effects, including gene expression, cytoskeletal rearrangement, and cell survival. We have previously shown that LPA stimulates secretion of pro- and anti-inflammatory cytokines in bronchial epithelial cells. This study provides evidence that LPA enhances pulmonary epithelial barrier integrity through protein kinase C (PKC) δ- and ζ-mediated E-cadherin accumulation at cell-cell junctions. Treatment of human bronchial epithelial cells (HBEpCs) with LPA increased transepithelial electrical resistance (TER) by ∼2.0-fold and enhanced accumulation of E-cadherin to the cell-cell junctions through Gαi-coupled LPA receptors. Knockdown of E-cadherin with E-cadherin small interfering RNA or pretreatment with EGTA (0.1 mm) prior to LPA (1 μm) treatment attenuated LPA-induced increases in TER in HBEpCs. Furthermore, LPA induced tyrosine phosphorylation of focal adhesion kinase (FAK) and overexpression of the FAK inhibitor, and FAK-related non-kinase-attenuated LPA induced increases in TER and E-cadherin accumulation at cell-cell junctions. Overexpression of dominant negative protein kinase δ and ζ attenuated LPA-induced phosphorylation of FAK, accumulation of E-cadherin at cell-cell junctions, and an increase in TER. Additionally, lipopolysaccharide decreased TER and induced E-cadherin relocalization from cell-cell junctions to cytoplasm in a dose-dependent fashion, which was restored by LPA post-treatment in HBEpCs. Intratracheal post-treatment with LPA (5 μm) reduced LPS-induced neutrophil influx, protein leak, and E-cadherin shedding in bronchoalveolar lavage fluids in a murine model of acute lung injury. These data suggest a protective role of LPA in airway inflammation and remodeling.

What evidence implicates airway smooth muscle in the cause of BHR

Bronchial hyperresponsiveness (BHR), the occurrence of excessive bronchoconstriction in response to relatively small constrictor stimuli, is a cardinal feature of asthma. Here, we consider the role that airway smooth muscle might play in the generation of BHR. The weight of evidence suggests that smooth muscle isolated from asthmatic tissues exhibits normal sensitivity to constrictor agonists when studied during isometric contraction, but the increased muscle mass within asthmatic airways might generate more total force than the lesser amount of muscle found in normal bronchi. Another salient difference between asthmatic and normal individuals lies in the effect of deep inhalation (DI) on bronchoconstriction. DI often substantially reverses induced bronchoconstriction in normals, while it often has much less effect on spontaneous or induced bronchoconstriction in asthmatics. It has been proposed that abnormal dynamic aspects of airway smooth muscle contraction—velocity of contraction or plasticity-elasticity balance—might underlie the abnormal DI response in asthma. We suggest a speculative model in which abnormally long actin filaments might account for abnormally increased elasticity of contracted airway smooth muscle.

Inhibition of Transforming Growth Factor β-enhanced Serum Response Factor-dependent Transcription by SMAD7

Transforming growth factor (TGF)-β is present in large amounts in the airways of patients with asthma and with other diseases of the lung. We show here that TGFβ treatment increased transcriptional activation of SM22α, a smooth muscle-specific promoter, in airway smooth muscle cells, and we demonstrate that this effect stems in part from TGFβ-induced enhancement of serum response factor (SRF) DNA binding and transcription promoting activity. Overexpression of Smad7 inhibited TGFβ-induced stimulation of SRF-dependent promoter function, and chromatin immunoprecipitation as well as co-immunoprecipitation assays established that endogenous or recombinant SRF interacts with Smad7 within the nucleus. The SRF binding domain of Smad7 mapped to the C-terminal half of the Smad7 molecule. TGFβ treatment weakened Smad7 association with SRF, and conversely the Smad7-SRF interaction was increased by inhibition of the TGFβ pathway through overexpression of a dominant negative mutant of TGFβ receptor I or of Smad3 phosphorylation-deficient mutant. Our findings thus reveal that SRF-Smad7 interactions in part mediate TGFβ regulation of gene transcription in airway smooth muscle. This offers potential targets for interventions in treating lung inflammation and asthma.

Cyclooxygenase-2 and MicroRNA-155 Expression Are Elevated in Asthmatic Airway Smooth Muscle Cells

Cyclooxygenase-2 (COX-2) expression and PGE2 secretion from human airway smooth muscle cells (hASMCs) may contribute to β2-adrenoceptor hyporesponsiveness, a clinical feature observed in some patients with asthma. hASMCs from patients with asthma exhibit elevated expression of cytokine-responsive genes, and in some instances this is attributable to an altered histone code and/or microRNA expression. We hypothesized that COX-2 expression and PGE2 secretion might be elevated in asthmatic hASMCs in response to proinflammatory signals in part due to altered histone acetylation and/or microRNA expression. hASMCs obtained from nonasthmatic and asthmatic human subjects were treated with cytomix (IL-1β, TNF-α, and IFN-γ). A greater elevation of COX-2 mRNA, COX-2 protein, and PGE2 secretion was observed in the asthmatic cells. We investigated histone H3/H4-acetylation, transcription factor binding, mRNA stability, p38 mitogen-activated protein kinase signaling, and microRNA (miR)-155 expression as potential mechanisms responsible for the differential elevation of COX-2 expression. We found that histone H3/H4-acetylation and transcription factor binding to the COX-2 promoter were similar in both groups, and histone H3/H4-acetylation did not increase after cytomix treatment. Cytomix treatment elevated NF-κB and RNA polymerase II binding to similar levels in both groups. COX-2 mRNA stability was increased in asthmatic cells. MiR-155 expression was higher in cytomix-treated asthmatic cells, and we show it enhances COX-2 expression and PGE2 secretion in asthmatic and nonasthmatic hASMCs. Thus, miR-155 expression positively correlates with COX-2 expression in the asthmatic hASMCs and may contribute to the elevated expression observed in these cells. These findings may explain, at least in part, β2-adrenoceptor hyporesponsiveness in patients with asthma.

Steroids Augment Relengthening of Contracted Airway Smooth Muscle: Potential Additional Mechanism of Benefit in Asthma

Breathing (especially deep breathing) antagonises development and persistence of airflow obstruction during bronchoconstrictor stimulation. Force fluctuations imposed on contracted airway smooth muscle (ASM) in vitro result in its relengthening, a phenomenon called force fluctuation-induced relengthening (FFIR). Because breathing imposes similar force fluctuations on contracted ASM within intact lungs, FFIR represents a likely mechanism by which breathing antagonises bronchoconstriction. While this bronchoprotective effect appears to be impaired in asthma, corticosteroid treatment can restore the ability of deep breaths to reverse artificially induced bronchoconstriction in asthmatic subjects. It has previously been demonstrated that FFIR is physiologically regulated through the p38 mitogen-activated protein kinase (MAPK) signalling pathway. While the beneficial effects of corticosteroids have been attributed to suppression of airway inflammation, the current authors hypothesised that alternatively they might exert their action directly on ASM by augmenting FFIR as a result of inhibiting p38 MAPK signalling. This possibility was tested in the present study by measuring relengthening in contracted canine tracheal smooth muscle (TSM) strips. The results indicate that dexamethasone treatment significantly augmented FFIR of contracted canine TSM. Canine tracheal ASM cells treated with dexamethasone demonstrated increased MAPK phosphatase-1 expression and decreased p38 MAPK activity, as reflected in reduced phosphorylation of the p38 MAPK downstream target, heat shock protein 27. These results suggest that corticosteroids may exert part of their therapeutic effect through direct action on airway smooth muscle, by decreasing p38 mitogen-activated protein kinase activity and thus increasing force fluctuation-induced relengthening.

Akt activation induces hypertrophy without contractile phenotypic maturation in airway smooth muscle

Airway smooth muscle (ASM) hypertrophy is a cardinal feature of severe asthma, but the underlying molecular mechanisms remain uncertain. Forced protein kinase B/Akt 1 activation is known to induce myocyte hypertrophy in other muscle types, and, since a number of mediators present in asthmatic airways can activate Akt signaling, we hypothesized that Akt activation could contribute to ASM hypertrophy in asthma. To test this hypothesis, we evaluated whether Akt activation occurs naturally within airway myocytes in situ, whether Akt1 activation is sufficient to cause hypertrophy of normal airway myocytes, and whether such hypertrophy is accompanied by excessive accumulation of contractile apparatus proteins (contractile phenotype maturation). Immunostains of human airway sections revealed concordant activation of Akt (reflected in Ser(473) phosphorylation) and of its downstream effector p70(S6Kinase) (reflected in Thr(389) phosphorylation) within airway muscle bundles, but there was no phosphorylation of the alternative Akt downstream target glycogen synthase kinase (GSK) 3β. Artificial overexpression of constitutively active Akt1 (by plasmid transduction or lentiviral infection) caused a progressive increase in size and protein content of cultured canine tracheal myocytes and increased p70(S6Kinase) phosphorylation but not GSK3β phosphorylation; however, constitutively active Akt1 did not cause disproportionate overaccumulation of smooth muscle (sm) α-actin and SM22. Furthermore, mRNAs encoding sm-α-actin and SM22 were reduced. These results indicate that forced Akt1 signaling causes hypertrophy of cultured airway myocytes without inducing further contractile phenotypic maturation, possibly because of opposing effects on contractile protein gene transcription and translation, and suggest that natural activation of Akt1 plays a similar role in asthmatic ASM.

Nuclear Import of Serum Response Factor in Airway Smooth Muscle

We have previously shown that the transcription-promoting activity of serum response factor (SRF) is partially regulated by its extranuclear redistribution. In this study, we examined the cellular mechanisms that facilitate SRF nuclear entry in canine tracheal smooth muscle cells. We used in vitro pull-down assays to determine which karyopherin proteins bound SRF and found that SRF binds KPNA1 and KPNB1 through its nuclear localization sequence. Immunoprecipitation studies also demonstrated direct SRF-KPNA1 interaction in HEK293 cells. Import assays demonstrated that KPNA1 and KPNB1 together were sufficient to mediate rapid nuclear import of SRF-GFP. Our studies also suggest that SRF is able to gain nuclear entry through an auxiliary, nuclear localization sequence-independent mechanism.