Epithelial nuclear factor-κB signaling promotes lung carcinogenesis via recruitment of regulatory T lymphocytes.

The mechanisms by which chronic inflammatory lung diseases, particularly chronic obstructive pulmonary disease, confer enhanced risk for lung cancer are not well-defined. To investigate whether nuclear factor (NF)-κB, a key mediator of immune and inflammatory responses, provides an interface between persistent lung inflammation and carcinogenesis, we utilized tetracycline-inducible transgenic mice expressing constitutively active IκB kinase ß in airway epithelium (IKTA (IKKß trans-activated) mice). Intraperitoneal injection of ethyl carbamate (urethane), or 3-methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) was used to induce lung tumorigenesis. Doxycycline-treated IKTA mice developed chronic airway inflammation and markedly increased numbers of lung tumors in response to urethane, even when transgene expression (and therefore epithelial NF-κB activation) was begun after exposure to carcinogen. Studies using a separate tumor initiator/promoter model (MCA+BHT) indicated that NF-κB functions as an independent tumor promoter. Enhanced tumor formation in IKTA mice was preceded by increased proliferation and reduced apoptosis of alveolar epithelium, resulting in increased formation of premalignant lesions. Investigation of inflammatory cells in lungs of IKTA mice revealed a substantial increase in macrophages and lymphocytes, including functional CD4+/CD25+/FoxP3+ regulatory T lymphocytes (Tregs). Importantly, Treg depletion using repetitive injections of anti-CD25 antibodies limited excessive tumor formation in IKTA mice. At 6 weeks following urethane injection, antibody-mediated Treg depletion in IKTA mice reduced the number of premalignant lesions in the lungs in association with an increase in CD8 lymphocytes. Thus, persistent NF-κB signaling in airway epithelium facilitates carcinogenesis by sculpting the immune/inflammatory environment in the lungs.

Care of very low birth weight infants with respiratory distress syndrome: an evidence-based review.

Since 1959, when it was reported that many preterm infants had surfactant deficiency, there has been a remarkable improvement in the prevention of respiratory distress syndrome (RDS) and in the care of infants who develop RDS. Antenatal corticosteroids and surfactant replacement have improved the care of very low birth weight infants.

KGF alters gene expression in human airway epithelia: potential regulation of the inflammatory response.

Keratinocyte growth factor (KGF) regulates several functions in adult and developing lung epithelia; it causes proliferation, stimulates secretion of fluid and electrolytes, enhances repair, and may minimize injury. To gain insight into the molecular processes influenced by KGF, we applied KGF to primary cultures of well-differentiated human airway epithelia and used microarray hybridization to assess the abundance of gene transcripts. Of 7,069 genes tested, KGF changed expression levels of 910. Earlier studies showed that KGF causes epithelial proliferation, and as expected, treatment altered expression of numerous genes involved in cell proliferation. We found that KGF stimulated transepithelial Cl(-) transport, but the number of cystic fibrosis (CF) transmembrane conductance regulator (CFTR) transcripts fell. Although transcripts for ClC-1 and ClC-7 Cl(-) channels increased, KGF failed to augment transepithelial Cl(-) transport in CF epithelia, suggesting that KGF-stimulated Cl(-) transport in differentiated airway epithelia depends on the CFTR Cl(-) channel. Interestingly, KGF decreased transcripts for many interferon (IFN)-induced genes. IFN causes trafficking of Stat dimers to the nucleus, where they activate transcription of IFN-induced genes. We found that KGF prevented the IFN-stimulated trafficking of Stat1 from the cytosol to the nucleus, suggesting a molecular mechanism for KGF-mediated suppression of the IFN-signaling pathway. These results suggest that in addition to stimulating proliferation and repair of damaged airway epithelia, KGF stimulates Cl(-) transport and may dampen the response of epithelial cells to inflammatory mediators.

Absence of amiloride-sensitive sodium absorption in the airway of an infant with pseudohypoaldosteronism.

We report the measurement of transepithelial voltage across the nasal epithelium in a neonate with pseudohypoaldosteronism (PHA) type 1. A 5-day-old infant was seen with hyponatremia, hyperkalemia, and elevated plasma renin and aldosterone levels. Sweat Cl(-) concentration was also increased. Measurements of voltage showed a basal value of zero and the absence of an amiloride-sensitive voltage. However, voltage changed as expected for normal cyclic adenosine monophosphate-stimulated Cl(-) transport. These data demonstrate the absence of amiloride-sensitive Na(+) transport across airway epithelia in a neonate with PHA. The findings suggest that measurements of voltage could be of value in the diagnosis of PHA.

Effect of subunit composition and Liddle's syndrome mutations on biosynthesis of ENaC.

The epithelial Na+ channel (ENaC) is comprised of three homologous subunits: alpha, beta, and gamma, all of which are required for formation of the fully functional channel. This channel is responsible for salt reabsorption in the kidney, the airway, and the large bowel. Mutations in ENaC can cause human disease by increasing channel function in Liddle's syndrome, a form of hereditary hypertension, or by decreasing channel function in pseudohypoaldosteronism type I, a salt-wasting disease of infancy. We previously showed that ENaC is expressed on the cell surface as a minimally glycosylated, Triton-insoluble protein. In the present study we found that ENaC existed initially as a Triton-soluble protein that contained high-mannose glycosylation, presumably in the endoplasmic reticulum. This form of the protein disappeared as the Triton-insoluble, minimally glycosylated form became the more prevalent species. In pulse-chase studies of individually expressed subunits, we found that the Triton-soluble form of beta-ENaC accumulated initially, whereas the Triton-soluble form of alpha-ENaC decreased throughout the time course. However, when all three subunits were coexpressed, the alpha- and beta-subunits showed a similar pattern. The complex became Triton insoluble at some point after the endoplasmic reticulum, as incubation at 15 degrees C blocked the conversion to the insoluble form. Deletion of the carboxy-terminal tail of beta-ENaC causes Liddle's syndrome. This mutation increased the amount of newly synthesized Triton-insoluble ENaC heteromultimers but did not affect the half-life of insoluble protein. Therefore, subunit composition and mutations in individual subunits can influence biosynthesis of the ENaC complex.

Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal.

We previously demonstrated that the cystic fibrosis transmembrane conductance regulator (CFTR) is rapidly endocytosed in epithelial cells (Prince, L. S., Workman, R. B., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 5192-5196). To determine the structural features of CFTR required for endocytosis, we prepared chimeric molecules consisting of the amino-terminal (residues 2-78) and carboxyl-terminal tail regions (residues 1391-1476) of CFTR, each fused to the transmembrane and extracellular domains of the transferrin receptor. Functional analysis of the CFTR-(2-78) and CFTR-(1391-1476) indicated that both chimeras were rapidly internalized. Deletion of residues 1440-1476 had no effect on chimera internalization. Mutations of potential internalization signals in both cytoplasmic domains reveal that only one mutation inhibits internalization, Y1424A. Using a surface biotinylation reaction, we also examined internalization rates of wild type and mutant CFTRs expressed in COS-7 cells. We found that both wild type and A1440X CFTR were rapidly internalized, whereas the Y1424A CFTR mutant, like the chimeric protein, had approximately 40% reduced internalization activity. Deletions in the amino-terminal tail region of CFTR resulted in defective trafficking of CFTR out of the endoplasmic reticulum to the cell surface, suggesting that an intact amino terminus is critical for biosynthesis. In summary, our results suggest that both tail regions of CFTR are sufficient to promote rapid internalization of a reporter molecule and that tyrosine 1424 is required for efficient CFTR endocytosis.

Cell surface expression and biosynthesis of epithelial Na+ channels.

The epithelial Na+ channel (ENaC) complex is composed of three homologous subunits: alpha, beta and gamma. Mutations in ENaC subunits can increase the number of channels on the cell surface, causing a hereditary form of hypertension called Liddle's syndrome, or can decrease channel activity, causing pseudohypoaldosteronism type I, a salt-wasting disease of infancy. To investigate surface expression, we studied ENaC subunits expressed in COS-7 and HEK293 cells. Using surface biotinylation and protease sensitivity, we found that when individual ENaC subunits are expressed alone, they traffic to the cell surface. The subunits are glycosylated with high-mannose oligosaccharides, but seem to have the carbohydrate removed before they reach the cell surface. Moreover, subunits form a complex that cannot be disrupted by several non-ionic detergents. The pattern of glycosylation and detergent solubility/insolubility persists when the N-teminal and C-terminal cytoplasmic regions of ENaC are removed. With co-expression of all three ENaC subunits, the insoluble complex is the predominant species. These results show that ENaC and its family members are unique in their trafficking, biochemical characteristics and post-translational modifications.

Inhibition of the epithelial Na+ channel by interaction of Nedd4 with a PY motif deleted in Liddle's syndrome.

The epithelial Na+ channel (ENaC) plays a critical role in Na+ absorption in the kidney and other epithelia. Mutations in the C terminus of the beta or gammaENaC subunits increase renal Na+ absorption, causing Liddle's syndrome, an inherited form of hypertension. These mutations delete or disrupt a PY motif that was recently shown to interact with Nedd4, a ubiquitin-protein ligase expressed in epithelia. We found that Nedd4 inhibited ENaC when they were coexpressed in Xenopus oocytes. Liddle's syndrome-associated mutations that prevent the interaction between Nedd4 and ENaC abolished inhibition, suggesting that a direct interaction is required for inhibition by Nedd4. Inhibition also required activity of a ubiquitin ligase domain within the C terminus of Nedd4. Nedd4 had no detectable effect on the single channel properties of ENaC. Rather, Nedd4 decreased cell surface expression of both ENaC and a chimeric protein containing the C terminus of the beta subunit. Decreased surface expression resulted from an increase in the rate of degradation of the channel complex. Thus, interaction of Nedd4 with the C terminus of ENaC inhibits Na+ absorption, and loss of this interaction may play a role in the pathogenesis of Liddle's syndrome and other forms of hypertension.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Electrophysiological and biochemical evidence that DEG/ENaC cation channels are composed of nine subunits.

Members of the DEG/ENaC protein family form ion channels with diverse functions. DEG/ENaC subunits associate as hetero- and homomultimers to generate channels; however the stoichiometry of these complexes is unknown. To determine the subunit stoichiometry of the human epithelial Na+ channel (hENaC), we expressed the three wild-type hENaC subunits (alpha, beta, and gamma) with subunits containing mutations that alter channel inhibition by methanethiosulfonates. The data indicate that hENaC contains three alpha, three beta, and three gamma subunits. Sucrose gradient sedimentation of alphahENaC translated in vitro, as well as alpha-, beta-, and gammahENaC coexpressed in cells, was consistent with complexes containing nine subunits. FaNaCh and BNC1, two related DEG/ENaC channels, produced complexes of similar mass. Our results suggest a novel nine-subunit stoichiometry for the DEG/ENaC family of ion channels.

Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel.

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is found at the apical region of exocrine epithelial cells, both at the cell surface and in an apically localized intracellular compartment. To determine if this internal pool was due to endocytosis, a technique was developed that allows the rate of CFTR internalization from the cell surface to be monitored. A two-step periodate/hydrazide biotinylation procedure was used to derivatize cell surface glycoconjugates. Because both of these steps are required for derivatization and are conducted at 4 degrees C, the inclusion of a 37 degrees C incubation between the treatments resulted in an assay for the internalization of cell surface glycoconjugates. CFTR was found to be targeted to a rapidly recycling endocytic pathway, as approximately 50% of cell surface CFTR was internalized within minutes and unavailable for biotinylation. In contrast, the major glycoproteins of the apical surface were not significantly endocytosed during even longer incubations at 37 degrees C. Elevating cAMP levels either by forskolin or cAMP analogs, which has been shown to activate CFTR chloride channel activity, inhibited CFTR internalization. However, cAMP did not affect the internalization of G551D CFTR, a naturally occurring Gly-551-->Asp mutant that is expressed at the cell surface but lacks normal ion-channel function. In addition, the inhibition by cAMP of CFTR was not observed when cells were depleted of cellular chloride. The presence of CFTR in epithelial cells had previously been shown to confer a cAMP-mediated inhibition on the rate of fluid-phase endocytosis. This effect was not seen in chloride-depleted cells, suggesting that CFTR's ion-channel function and localization to incipient endosomes may be responsible for the observed inhibition. The finding that CFTR is targeted to the endocytic pathway may provide insight into the role of CFTR in normal exocrine function. In addition, these findings suggest that the expression of a regulated ion channel in a membranous subcellular compartment provides a mechanism by which a cell can regulate vesicular trafficking through that compartment.

Cell surface labeling of CFTR in T84 cells.

To distinguish cystic fibrosis transmembrane conductance regulator (CFTR) at the surface of epithelial cells from that present in intracellular membranes, intact T84 cells were treated with periodate and biotin-LC-hydrazide to derivatize exposed glycoconjugates. Cell lysates were then passed over a monomeric avidin column, which allows reversible avidin-biotin binding. After washing, biotinylated molecules were eluted with 2 mM biotin. CFTR was then immunoprecipitated with a mouse monoclonal antibody from both the unbound and biotin eluent fractions, radioactively labeled by in vitro phosphorylation, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Nonimmune mouse immunoglobulin G failed to precipitate any CFTR, and CFTR was detectable only in the wash fractions when cells were periodate treated but not labeled with biotin hydrazide. In biotinylated cells, CFTR levels were approximately equal in the unbound fraction and the biotin eluent. The proportion of biotinylated CFTR did not significantly increase when cells were labeled after treatment with 10 microM forskolin. These data demonstrate that in T84 cells CFTR is constitutively expressed on the cell surface and that activation of CFTR does not primarily depend on the cAMP-dependent trafficking of CFTR to the plasma membrane. The large unbiotinylated pool of maturely glycosylated CFTR suggests that CFTR resides in an intracellular compartment as well as being present at the cell surface.

The longitudinal relaxation time (T1) of the intracellular 23Na NMR signal in the isolated perfused rat heart during hypoxia and reoxygenation.

Each of six perfused rat hearts was subjected to 30 min of hypoxia followed by 60 min of reoxygenation. Inversion-recovery data on the intracellular Na NMR signal, differentiated by a shift reagent, 6 mM Dy(PPP)2, were obtained every 5 min, and T1 values were calculated. The T1 of the intracellular Na signal did not show any significant change either during hypoxia or upon reoxygenation, although the level of Nai increased about 50%. Such an increase of total Nai is expected to reduce the observed relaxation rate by diluting the fraction of Nai ions that interact with intracellular polyelectrolytes. The observed constancy of T1 in our study is explained on the basis of the typical values of the dissociation constants of sodium ions, in aqueous solutions, in interaction with polyelectrolytes. Although the constancy of intracellular sodium T1 during hypoxia may preclude the utilization of T1 weighting for the monitoring of pathology, its determination could be important for setting optimal acquisition times in high time-resolution experiments.