Anti-apoptotic effects of Z alpha1-antitrypsin in human bronchial epithelial cells.

alpha(1)-antitrypsin (alpha(1)-AT) deficiency is a genetic disease which manifests as early-onset emphysema or liver disease. Although the majority of alpha(1)-AT is produced by the liver, it is also produced by bronchial epithelial cells, amongst others, in the lung. Herein, we investigate the effects of mutant Z alpha(1)-AT (ZAAT) expression on apoptosis in a human bronchial epithelial cell line (16HBE14o-) and delineate the mechanisms involved. Control, M variant alpha(1)-AT (MAAT)- or ZAAT-expressing cells were assessed for apoptosis, caspase-3 activity, cell viability, phosphorylation of Bad, nuclear factor (NF)-kappaB activation and induced expression of a selection of pro- and anti-apoptotic genes. Expression of ZAAT in 16HBE14o- cells, like MAAT, inhibited basal and agonist-induced apoptosis. ZAAT expression also inhibited caspase-3 activity by 57% compared with control cells (p = 0.05) and was a more potent inhibitor than MAAT. Whilst ZAAT had no effect on the activity of Bad, its expression activated NF-kappaB-dependent gene expression above control or MAAT-expressing cells. In 16HBE14o- cells but not HEK293 cells, ZAAT upregulated expression of cIAP-1, an upstream regulator of NF-kappaB. cIAP1 expression was increased in ZAAT versus MAAT bronchial biopsies. The data suggest a novel mechanism by which ZAAT may promote human bronchial epithelial cell survival.

Interleukin-8 up-regulation by neutrophil elastase is mediated by MyD88/IRAK/TRAF-6 in human bronchial epithelium.

Cystic fibrosis is characterized in the lungs by neutrophil-dominated inflammation mediated significantly by neutrophil elastase (NE). Previous work has shown that NE induces interleukin-8 (IL-8) gene expression and protein secretion in bronchial epithelial cells. We sought to determine the intracellular mechanisms by which NE up-regulates IL-8 in bronchial epithelial cells. The data show that stimulation of 16HBE14o(-) cells with NE induced IL-8 protein production and gene expression. Both responses were abrogated by actinomycin D, indicating that regulation is at the transcriptional level. Electrophoretic mobility shift assays demonstrated that nuclear factor kappaB (NFkappaB) was activated in 16HBE14o(-) cells stimulated with NE. Western blot analysis demonstrated that activation of NFkappaB by NE was preceded by phosphorylation and degradation of IkappaB proteins, principally IkappaBbeta. In addition, we observed that interleukin-1 receptor-associated kinase (IRAK) was degraded in 16HBE14o(-) cells stimulated with NE. Quantification of IL-8 reporter gene activity by luminometry demonstrated that dominant negative MyD88 (MyD88Delta) or TRAF-6 (TRAF-6Delta) inhibited IL-8 reporter gene expression in response to NE. Furthermore, MyD88Delta inhibited NE-induced IRAK degradation. These results show that NE induces IL-8 gene up-regulation in bronchial epithelial cells through an IRAK signaling pathway involving both MyD88 and TRAF-6, resulting in degradation of IkappaBbeta and nuclear translocation of NFkappaB. These findings may have implications for therapeutic treatments in the cystic fibrosis condition.

Both the wild type and a functional isoform of CFTR are expressed in kidney.

The cystic fibrosis transmembrane conductance regulator (CFTR) consists of five domains, two transmembrane-spanning domains, each composed of six transmembrane segments, a regulatory domain, and two nucleotide-binding domains (NBDs). CFTR is expressed in kidney, but its role in overall renal function is not well understood, because mutations in CFTR found in patients with cystic fibrosis are not associated with renal dysfunction. To learn more about the distribution and functional forms of CFTR in kidney, we used a combination of molecular, cell biological, and electrophysiological approaches. These include an evaluation of CFTR mRNA and protein expression, as well as both two-electrode and patch clamping of CFTR expressed either in Xenopus oocytes or mammalian cells. In addition to wild-type CFTR mRNA, an alternate form containing only the first transmembrane domain (TMD), the first NBD, and the regulatory domain (TNR-CFTR) is expressed in kidney. Although missing the second set of TMDs and the second NBD, when expressed in Xenopus oocytes, TNR-CFTR has cAMP-dependent protein kinase A (PKA)-stimulated single Cl- channel characteristics and regulation of PKA activation of outwardly rectifying Cl- channels that are very similar to those of wild-type CFTR. TNR-CFTR mRNA is produced by an unusual mRNA processing mechanism and is expressed in a tissue-specific manner primarily in renal medulla.

Alternate translation initiation codons can create functional forms of cystic fibrosis transmembrane conductance regulator.

To evaluate the function of transmembrane domain 1 (TMD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) and the methionines that function in translation initiation, a series of progressive 5' truncations in TMD1 were created to coincide with residues that might serve as translation initiation codons. Expression of the mutants in Xenopus oocytes demonstrated that internal sites in TMD1 can function as initiation codons. In addition, all of the mutants that progressively removed the first four transmembrane segments (M1-M4) of TMD1 expressed functional cAMP-regulated Cl- channels with ion selectivity identical to wild-type CFTR but with reduced open probability and single channel conductance. Further removal of transmembrane segments did not produce functional Cl- channels. These data suggest that segments M1-M4 are not essential components of the conduction pore or the selectivity filter of CFTR.

Mechano- and chemoreceptor modulation of respiratory muscles in response to upper airway negative pressure.

To investigate the influence of phasic pulmonary stretch receptors (n = 6) and chemoreceptors (n = 7) on the reflex response of the genioglossus (GG) muscle and diaphragm (DIA) to upper airway (UAW) negative pressure, we measured the response of the GG and DIA electromyogram (EMG) to three challenges: 1) negative pressure applied to the UAW during normoxia and hypercapnia, 2) end-expiratory tracheal occlusion, and 3) application of UAW negative pressure simultaneous with tracheal occlusion in spontaneously breathing tracheotomized anesthetized cats. Peak GG EMG was greatest when UAW negative pressure and end-expiratory tracheal occlusion were combined. No GG EMG activity was seen when UAW negative pressure was applied alone unless the animal was vagotomized or hypercapnic. DIA EMG increased in response to UAW negative pressure combined with occlusion. However, the increase in peak GG EMG was significantly greater than for the DIA with the same challenge. DIA EMG amplitude increased in response to occlusion alone but did not change when UAW negative pressure was applied alone. In the cat, phasic feedback from phasic pulmonary stretch receptors is a potent inhibitor of reflex activation of the GG in response to negative pressure applied to the UAW, which can be overridden by an increase in chemoreceptor drive.

Appearance of water channels in Xenopus oocytes expressing red cell CHIP28 protein.

Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions. Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane water channels.