BACKGROUND The mutations that have been implicated in pulmonary fibrosis account for only a small proportion of the population risk. METHODS Using a genomewide linkage scan, we detected linkage between idiopathic interstitial pneumonia and a 3.4-Mb region of chromosome 11p15 in 82 families. We then evaluated genetic variation in this region in gel-forming mucin genes expressed in the lung among 83 subjects with familial interstitial pneumonia, 492 subjects with idiopathic pulmonary fibrosis, and 322 controls. MUC5B expression was assessed in lung tissue. RESULTS Linkage and fine mapping were used to identify a region of interest on the p-terminus of chromosome 11 that included gel-forming mucin genes. The minor-allele of the single-nucleotide polymorphism (SNP) rs35705950, located 3 kb upstream of the MUC5B transcription start site, was present at a frequency of 34% among subjects with familial interstitial pneumonia, 38% among subjects with idiopathic pulmonary fibrosis, and 9% among controls (allelic association with familial interstitial pneumonia, P = 1.2×10−15; allelic association with idiopathic pulmonary fibrosis, P = 2.5×10−37). The odds ratios for disease among subjects who were heterozygous and those who were homozygous for the minor allele of this SNP were 6.8 (95% confidence interval [CI], 3.9 to 12.0) and 20.8 (95% CI, 3.8 to 113.7), respectively, for familial interstitial pneumonia and 9.0 (95% CI, 6.2 to 13.1) and 21.8 (95% CI, 5.1 to 93.5), respectively, for idiopathic pulmonary fibrosis. MUC5B expression in the lung was 14.1 times as high in subjects who had idiopathic pulmonary fibrosis as in those who did not (P<0.001). The variant allele of rs35705950 was associated with up-regulation in MUC5B expression in the lung in unaffected subjects (expression was 37.4 times as high as in unaffected subjects homozygous for the wild-type allele, P<0.001). MUC5B protein was expressed in lesions of idiopathic pulmonary fibrosis. CONCLUSIONS A common polymorphism in the promoter of MUC5B is associated with familial interstitial pneumonia and idiopathic pulmonary fibrosis. Our findings suggest that dys-regulated MUC5B expression in the lung may be involved in the pathogenesis of pulmonary fibrosis. (Funded by the National Heart, Lung, and Blood Institute and others.)

Shear stress causes the vascular endothelium to release nitric oxide (NO), which is an important regulator of vascular tone. However, direct measurement of NO release after the imposition of laminar flow has not been previously accomplished because of chemical (oxidative degradation) and physical (diffusion, convection, and washout) complications. Consequently, the mechanism, time course, kinetics, and Ca 2+ dependence of NO release due to shear stress remain incompletely understood. In this study, we characterized these parameters by using fura 2 fluorescence and a polymeric porphyrin/Nafion-coated carbon fiber microsensor (detection limit, 5 nmol/L; response time, 1 millisecond) to directly measure changes in [Ca 2+ ] i and NO release due to shear stress or agonist (ATP or brominated Ca 2+ ionophore [Br-A23187]) from bovine aortic endothelial cells. The cells were grown to confluence on glass coverslips, loaded with fura 2-AM, and mounted in a parallel-plate flow chamber (volume, 25 μL). The microsensor was positioned ≈100 μm above the cells with its long axis parallel to the direction of flow. Laminar flow of perfusate was maintained from 0.04 to 1.90 mL/min, which produced shear stresses of 0.2 to 10 dyne/cm 2 . Shear stress caused transient NO release 3 to 5 seconds after the initiation of flow and 1 to 3 seconds after the rise in [Ca 2+ ] i , which reached a plateau after 35 to 70 seconds. Although the amount (peak rate) of NO release increased as a function of the shear stress (0.08 to 3.80 pmol/s), because of the concomitant increase in the flow rate, the peak NO concentration (133±9 nmol/L) remained constant. Maintenance of flow resulted in additional transient NO release, with peak-to-peak intervals of 15.5±2.5 minutes. During this 13- to 18-minute period, when the cells were unresponsive to shear stress, exogenous ATP (10 μmol/L) or Br-A23187 (10 μmol/L) evoked NO release. Prior incubation of the cells with exogenous NO or the removal and EGTA (100 μmol/L) chelation of extracellular Ca 2+ blocked shear stress but not ATP-dependent NO release. The kinetics of shear stress–induced NO release (2.23±0.07 nmol/L per second) closely resembled the kinetics of Ca 2+ flux but differed markedly from the kinetics of ATP-induced NO release (5.64±0.32 nmol/L per second). These data argue that shear stress causes a Ca 2+ -mediated ATP-independent transient release of NO, where the peak rate of release but not the peak concentration depends on the level of shear stress. The transient nature of this response may be due to NO-induced inhibition of Ca 2+ influx via a mechanism yet to be determined.

Voltage-gated potassium channel beta subunits are cytoplasmic proteins that co-purify with the pore-forming alpha subunits. One of these subunits, Kv beta 1 from rat brain, was previously demonstrated to increase the rate of inactivation of Kv1.1 and Kv1.4 when co-expressed in Xenopus oocytes. We have cloned and characterized a novel voltage-gated K+ channel beta subunit. The cDNA, designated Kv beta 3, has a 408-amino acid open reading frame. It possesses a unique 79-amino acid N-terminal leader, but is identical with rat Kv beta 1 over the 329 C-terminal amino acids. The Kv beta 3 transcript was found in many tissues, but was most abundant in aorta and left ventricle of the heart. Co-expression of Kv beta 3 with K+ channel alpha subunits shows that this beta subunit can increase the rate of inactivation from 4- to 7-fold in a Kv1.4 or Shaker B channel. Kv beta 3 had no effect on Kv1.1, unlike Kv beta 1 which can increase rate of inactivation of this alpha subunit more than 100-fold. Other kinetic parameters were unaffected. This study shows that voltage-gated K+ channel beta subunits are present outside the central nervous system, and that at least one member of this family selectively modulates inactivation of K+ channel alpha subunits.

As excess mucin expression can contribute to the exacerbation of asthma, the present authors hypothesised that Mycoplasma pneumoniae significantly induces MUC5AC (the major airway mucin) expression in airway epithelial cells isolated directly from asthmatic subjects.A total of 11 subjects with asthma and six normal controls underwent bronchoscopy with airway brushing. Epithelial cells were cultured at an air-liquid interface and incubated with and without M. pneumoniae for 48 h, and in the presence and absence of nuclear factor (NF)-kB and a toll-like receptor (TLR)2 inhibitor. Quantitative PCR was performed for MUC5AC and TLR2 mRNA. MUC5AC protein and total protein were determined by ELISA.M. pneumoniae exposure significantly increased MUC5AC mRNA and protein expression after 48 h in epithelial cells isolated from asthmatic, but not from normal control subjects, at all concentrations as compared to unexposed cells. TLR2 mRNA expression was significantly increased in asthmatic epithelial cells at 4 h compared with unexposed cells. NF-kB and TLR2 inhibition reduced MUC5AC expression to the level of the unexposed control in both groups.Mycoplasma pneumoniae exposure significantly increased MUC5AC mRNA and protein expression preferentially in airway epithelial cells isolated from asthmatic subjects. The toll-like receptor 2 pathway may be involved in this process.