Rationale: In experimental models, lung fibrosis is dependent on transforming growth factor (TGF)-b signaling. TGF-b is secreted in a latent complex with its propeptide, and TGF-b activators release TGF-b from this complex. Because the integrin avb6 is a major TGF-b activator in the lung, inhibition of avb6-mediated TGF-b activation is a logical strategy to treat lung fibrosis. Objectives: To determine, by genetic and pharmacologic approaches, whether murine radiation-induced lung fibrosis is dependent on avb6. Methods: Wild-type mice, avb6-deficient (Itgb6 2/2 ) mice, and mice heterozygous for a Tgfb1 mutation that eliminates integrin-mediated activation (Tgfb1 1/RGE ) were exposed to 14 Gy thoracic radiation. Some mice were treated with an anti-avb6 monoclonal antibody or a soluble TGF-b receptor fusion protein. avb6 expression was determined by immunohistochemistry. Fibrosis, inflammation, and gene expression patterns were assessed 20-32 weeks postirradiation. Measurements and Main Results: b6 Integrin expression increased within the alveolar epithelium 18 weeks postirradiation, just before onset of fibrosis. Itgb6 2/2 mice were completely protected from fibrosis, but not from late radiation-induced mortality. Anti-avb6 therapy (1-10 mg/kg/wk) prevented fibrosis, but only higher doses (6-10 mg/kg/wk) caused lung inflammation similar to that in Itgb6 2/2 mice. Tgfb1-haploinsufficient mice were also protected from fibrosis. Conclusions: avb6-Mediated TGF-b activation is required for radiationinduced lung fibrosis. Together with previous data, our results demonstrate a robust requirement for avb6 in distinct fibrosis models. Inhibition of avb6-mediated TGF-b activation is a promising new approach for antifibrosis therapy.

The human and shark Na-K-Cl cotransporters (NKCC), although 74% identical in amino acid sequence, exhibit marked differences in ion transport and bumetanide binding. We have utilized shark-human chimeras of NKCC1 to search for regions that confer the kinetic differences. Two chimeras (hs3.1 and its reverse sh3.1) with a junction point located at the beginning of the third transmembrane domain were examined after stable transfection in HEK-293 cells. Each carried out bumetanide-sensitive 86 Rb inf lux with cation affinities intermediate between shark and human cotransporters. In conjunction with the previous finding that the N and C termini are not responsible for differences in ion transport, the current observations identify the second transmembrane domain as playing an important role. Site-specific mutagenesis of two pairs of residues in this domain revealed that one pair is indeed involved in the difference in Na affinity, and a second pair is involved in the difference in Rb affinity. Substitution of the same residues with corresponding residues from NKCC2 or the Na-Cl cotransporter resulted in cation affinity changes, consistent with the hypothesis that alternative splicing of transmembrane domain 2 endows different versions of NKCC2 with unique kinetic behaviors. None of the changes in transmembrane domain 2 was found to substantially affect K m(Cl) , demonstrating that the affinity difference for Cl is specified by the region beyond predicted transmembrane domain 3. Finally, unlike Cl, bumetanide binding was strongly affected by shark-human replacement of transmembrane domain 2, indicating that the bumetanide-binding site is not the same as the Cl-binding site.

The human and shark Na-K-Cl cotransporters (NKCCs) are 74% identical in amino acid sequence yet they display marked differences in apparent affinities for the ions and bumetanide. In this study, we have used chimeras and point mutations to determine which transmembrane domains (tm's) are responsible for the differences in ion transport and in inhibitor binding kinetics. When expressed in HEK-293 cells, all the mutants carry out bumetanide-sensitive 86Rb influx. The kinetic behavior of these constructs demonstrates that the first seven tm's contain all of the residues conferring affinity differences. In conjunction with our previous finding that tm 2 plays an important role in cation transport, the present observations implicate the fourth and seventh tm helices in anion transport. Thus, it appears that tm's 2, 4, and 7 contain the essential affinity-modifying residues accounting for the human–shark differences with regard to cation and anion transport. Point mutations have narrowed the list of candidates to 13 residues within the three tm's. The affinity for bumetanide was found to be affected by residues in the same tm 2–7 region, and also by residues in tm's 11 and 12. Unlike for the ions, changes in bumetanide affinity were nonlinear and difficult to interpret: the K i(bumetanide) of a number of the constructs was outside the range of sNKCC1 and hNKCC1 K is.

The Na-K-Cl cotransporter (NKCC) mediates the coupled movement of ions into most animal cells, playing important roles in maintenance of cell volume and in epithelial Cl transport. Two forms of NKCC have been described: NKCC1, the "housekeeping" isoform that is also responsible for Cl accumulation in secretory epithelial cells, and NKCC2, which mediates apical Na؉K؉Cl entry into renal epithelial cells. Here we examine the kinetic properties of NKCC1, NKCC2, and the endogenous HEK-293 cell cotransporter. Stable expression of rabbit NKCC2A was obtained in HEK-293 cells utilizing a chimera (h 1 r 2A 0.7) in which the 5-untranslated region and cDNA encoding 104 amino acids of the N terminus are replaced by the corresponding sequence of NKCC1. h 1 r 2A 0.7 exhibits Na and Cl affinities near those of NKCC1, but it has a 4-fold lower Rb affinity, and a 3-fold higher affinity for the inhibitor bumetanide. The activity of h 1 r 2A 0.7 is increased on incubation in low [Cl] media as is NKCC1, but the resting level of activity is higher in h 1 r 2A 0.7 and activation is more rapid. h 1 r 2A 0.7 exhibits an appropriate volume response, unlike NKCC1 for which concomitant changes in [Cl] i appear to be the overriding factor. These results support a model in which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl] i . Reverse transcriptasepolymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers. Surprisingly, we found that the behavior of the endogenous HEK cell Na-K-Cl cotransporter is unlike either of the two forms which have been described: compared with NKCC1, HEK cell cotransporter has a 2.5-fold lower Na affinity, an 8-fold lower Rb affinity, and a 4-fold higher bumetanide affinity. These results suggest the presence of a novel isoform of NKCC in HEK-293 cells.The Na-K-Cl cotransporter (NKCC or BSC) 1 mediates the coupled movement of Na, K, and Cl ions across the plasma membrane of animal cells. The transporter plays an important role in electrolyte movement across polarized epithelia and is also thought to be involved in regulation of intracellular volume and intracellular [Cl] (1, 2). NKCC is a member of the Na-coupled group of cation-chloride cotransporters (CCCs) (1, 3), a family which also includes K-Cl cotransporters (KCC) (3,4). Three Na-coupled cation-chloride cotransporters have been described to date. 1) The "secretory" (or "housekeeping" or "basolateral") Na-K-Cl cotransporter, NKCC1 (or BSC2), is widely distributed in mammalian tissues (5, 6) and is especially prominent in the basolateral membranes of secretory epithelial cells; within the kidney, NKCC1 is found in epithelial cells in the collecting duct and in the glomerulus (7,8).2) The "renal" or "apical" Na-K-Cl cotransporter, NKCC2 (or BSC1) (9, 10), is found only in the apical membrane of epithelial cells in the thick ascending limb of the loop of Henle (TAL) (11)(12)(13)(14)(15). Three splice variants of NKCC2 (A, B, and F), differing in the sequence of the second pred...

Mercury alters the function of proteins by reacting with cysteinyl sulfhydryl (SH−) groups. The inorganic form (Hg2+) is toxic to epithelial tissues and interacts with various transport proteins including the Na+ pump and Cl− channels. In this study, we determined whether the Na+-K+-Cl−cotransporter type 1 (NKCC1), a major ion pathway in secretory tissues, is also affected by mercurial substrates. To characterize the interaction, we measured the effect of Hg2+ on ion transport by the secretory shark and human cotransporters expressed in HEK-293 cells. Our studies show that Hg2+inhibits Na+-K+-Cl−cotransport, with inhibitor constant ( K i) values of 25 μM for the shark carrier (sNKCC1) and 43 μM for the human carrier. In further studies, we took advantage of species differences in Hg2+ affinity to identify residues involved in the interaction. An analysis of human-shark chimeras and of an sNKCC1 mutant (Cys-697→Leu) reveals that transmembrane domain 11 plays an essential role in Hg2+binding. We also show that modification of additional SH− groups by thiol-reacting compounds brings about inhibition and that the binding sites are not exposed on the extracellular face of the membrane.