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Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by progressive scarring of the lungs and resulting in deterioration in lung function. Transforming growth factor-b (TGF-b) is one of the most established drivers of fibrotic processes. TGF-b promotes the transformation of tissue fibroblasts to myofibroblasts, a key finding in the pathogenesis of pulmonary fibrosis. We report here that TGF-b robustly upregulates the expression of the calcium-activated chloride channel anoctamin-1 (ANO1) in human lung fibroblasts (HLFs) at mRNA and protein levels. ANO1 is readily detected in fibrotic areas of IPF lungs in the same area with smooth muscle a-actin (SMA)-positive myofibroblasts. TGF-b-induced myofibroblast differentiation (determined by the expression of SMA, collagen-1, and fibronectin) is significantly inhibited by a specific ANO1 inhibitor, T16Ainh-A01, or by siRNA mediated ANO1 knockdown. T16Ainh-A01 and ANO1 siRNA attenuate profibrotic TGF-b signaling, including activation of RhoA pathway and AKT, without affecting initial Smad2 phosphorylation. Mechanistically, TGF-b treatment of HLFs results in a significant increase in intracellular chloride levels, which is prevented by T16Ainh-A01 or by ANO1 knockdown. The downstream mechanism involves the chloride-sensing “with-no-lysine (K)” kinase (WNK1). WNK1 siRNA significantly attenuates TGF-b-induced myofibroblast differentiation and signaling (RhoA pathway and AKT), whereas the WNK1 kinase inhibitor WNK463 is largely ineffective. Together, these data demonstrate that 1) ANO1 is a TGF-b-inducible chloride channel that contributes to increased intracellular chloride concentration in response to TGF-b; and 2) ANO1 mediates TGF-b-induced myofibroblast differentiation and fibrotic signaling in a manner dependent on WNK1 protein but independent of WNK1 kinase activity. NEW & NOTEWORTHY This study describes a novel mechanism of differentiation of human lung fibroblasts (HLFs) to myofibroblasts: the key process in the pathogenesis of pulmonary fibrosis. Transforming growth factor-b (TGF-b) drives the expression of calcium-activated chloride channel anoctmin-1 (ANO1) leading to an increase in intracellular levels of chloride. The latter recruits chloride-sensitive with-no-lysine (K) kinase (WNK1) to activate profibrotic RhoA and AKT signaling pathways, possibly through activation of mammalian target of rapamycin complex-2 (mTORC2), altogether promoting myofibroblast differentiation. [ABSTRACT FROM AUTHOR]

The ADP-ribosyl cyclase activity of CD38, a membrane protein expressed in human airway smooth muscle (ASM) cells, generates cyclic ADP-ribose (cADPR), a [Ca.sup.2+]-mobilizing agent, cADPR-mediated [Ca.sup.2+] responses to agonists are augmented in human ASM cells by TNF-[alpha]. CD38-deficient mice fail to develop airway hyperresponsiveness following intranasal TNF-[alpha] or IL-13 challenge, suggesting a role in asthma. The role of CD38 in human asthma remains unknown. We hypothesized that CD38 expression will be elevated in ASM cells from asthmatic donors (ASMA cells). CD38 mRNA and ADP-ribosyl cyclase activity were measured in ceils maintained in growth-arrested conditions and exposed to vehicle or TNF-[alpha] (10-40 ng/ml). TNF-[alpha]-induced induction of CD38 expression was greater in ASMA than in ASM cells from nonasthmatic donors (ASMNA). In four of the six donors, basal and TNF-[alpha]-induced ERK and p38 MAPK activation were higher in ASMA than ASMNA cells. JNK MAPK activation was lower in ASMA than ASMNA cells. Nuclear NF-[kappa]B (p50 subunit) and phosphorylated c-Jun were comparable in cells from both groups, although nuclear c-Fos (part of the AP-1 complex) levels were lower in ASMA than ASMNA cells. NF-[kappa]B or AP-1 binding to their consensus sequences was comparable in ASMNA and ASMA cells, as are the decay kinetics of CD38 mRNA. The findings suggest that the differential induction of CD38 by TNF-[alpha] in ASMA cells is due to increased transcriptional regulation involving ERK and p38 MAPK activation and is independent of changes in NF-[kappa]B or AP-1 activation. The findings suggest a potential role for CD38 in the pathophysiology of asthma. ADP-ribosyl cyclase; MAP kinases; nuclear factor-[kappa]B; smooth muscle doi: 10.1152/ajplung.00021.2010.

Chen B, Yates E, Huang Y, Kogut P, Ma L, Turner JR, Tao Y, Camoretti-Mercado B, Lang D, Svensson EC, Garcia JG, Gruber PJ, Morrisey EE, Solway J. Alternative promoter and GATA5 transcripts in mouse. Am J Physiol Gastrointest Liver Physiol 297: G1214-G1222, 2009. First published September 24, 2009; doi: 10.1152/ajpgi.00165.2009.--GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an [NH.sub.2]-terminally truncated 'short GATA5' comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated. differentiation; airway; gut; epithelium doi: 10.1152/ajpgi.00165.2009

The effects of activated eosinophils or their secretory products on sensory neuropeptides in dorsal root ganglion neurons were investigated. Sprague-Dawley rats were used as experimental models to examine the mechanisms behind the ability of sensory C fiber stimulants to induce substance P release. Results showed that inflammatory substances released from activated eosinophils directly stimulated dorsal root ganglion neurons. This effect was also demonstrated by major basic protein alone.

Guinea pig tracheal smooth muscle strips were treated with capsaicin, an inducer of tachykinin release, to determine the role of tachykinins in tracheal smooth muscle contraction induced by potassium cloride (KCl). The results show that KCL-induced tracheal smooth muscle contraction is mediated not only by voltage-dependent calcium channels and acetylcholine secretion but also by the release of tachykinins from sensory C fibers in the tracheal smooth muscle.

Rat dorsal root ganglion (DRG) cells secrete neuropeptides that appear to be mitogenic for guinea pig tracheal epithelial (GPTE) cells in primary culture. Proliferation is stimulated in GPTE cells by treatment with neurokinin (NK1), NK2, and CGRP1 receptor agonists in primary culture. These results suggest that airway sensory nerves modulates epithelial cell growth.

A study on mitogen-activated protein kinase (MAP) activation in bovine tracheal myocytes after treatment with four potential mitogens, PDGF, EGF, insulin like growth factor-1 (IGF-1) and 5-HT, shows that kinase activity may be trigged by G-protein receptors not thought to be tyrosine kinase activators, while the kinase activating potential of PDGF and IFG-1 is high. Tracheal myocyte proliferation appears to be heavily reliant on sustained MAP kinase activity.

Chronic immune sensitization with ovalbumin augments spontaneous contractile response of tracheal smooth muscle (TSH) and antagonizes relaxation of airway smooth muscles caused by beta-adrenoreceptor stimulation in guinea pigs. A contractile factor such as cyclooxygenase derivative is produced in the epithelial cells and its formation is potentiated by immune sensitization.

The possibility of employing a cultured human cell line of promyelocytic HL-60 cells to derive inflammatory hyperresponsiveness in a guinea pig synthesis is examined. The biological activity seen in these cells and in human polymorphonuclear leukocytes is compared by using a living explant of tracheal smooth muscle. In vitro examination reveals the excising of airways which hinder the in situ anatomic and circulatory associations conserved by this synthesis.

Selective restoration of calcium coupling to muscarinic [M.sub.3] receptors in contractile cultured airway myocytes. Am J Physiol Lung Cell Mol Physiol 278: L1091-L1100, 2000.--We previously demonstrated that after several days of serum deprivation about one-sixth of confluent cultured canine tracheal myocytes acquire an elongated, structurally and functionally contractile phenotype. These myocytes demonstrated significant shortening on ACh exposure. To evaluate the mechanism by which these myocytes acquire responsiveness to ACh, we assessed receptor-[Ca.sup.2+] coupling using fura 2-AM fluorescence imaging and muscarinic receptor expression using Western analysis. Cells were grown to confluence in 10% fetal bovine serum and then maintained for 7-13 days in serum-free medium. A fraction of serum-deprived cells exhibited reproducible intracellular [Ca.sup.2+] mobilization in response to ACh that was uniformly absent from airway myocytes before serum deprivation. The [Ca.sup.2+] response to [10.sup.-4] M ACh was ablated by inositol 1,4,5-trisphosphate ([IP.sub.3]) receptor blockade using [10.sup.-6] M xestospongin C but not by removal of extracellular [Ca.sup.2+]. Also, [10.sup.-7] M atropine or [10.sup.-7] M 4-diphenylacetoxy-N-methylpiperidine completely blocked the response to ACh, but intracellular [Ca.sup.2+] mobilization was not ablated by [10.sup.-6] M pirenzepine or [10.sup.-6] M methoctramine. In contrast, [10.sup.-5] M bradykinin (BK) was without effect in these ACh-responsive myocytes. Interestingly, myocytes that did not respond to ACh demonstrated robust increases in intracellular [Ca.sup.2+] on exposure to [10.sup.-5] M BK that were blocked by removal of extracellular [Ca.sup.2+] and were only modestly affected by [IP.sub.3] receptor blockade. Serum deprivation increased the abundance of [M.sub.3] receptor protein and of [BK.sub.2] receptor protein by two- to threefold in whole cell lysates within 2 days of serum deprivation, whereas [M.sub.2] receptor protein fell by [is greater than] 75%. An increase in [M.sub.3] receptor abundance and restoration of [M.sub.3] receptor-mediated [Ca.sup.2+] mobilization occur concomitant with reacquisition of a contractile phenotype during prolonged serum deprivation. These data demonstrate plasticity in muscarinic surface receptor expression and function in a subpopulation of airway myocytes that show mutually exclusive physiological and pharmacological diversity with other cells in the same culture.

Hamann, Kimm J., Joaquim E. Vieira, Andrew J. Halayko, Delbert Dorscheid, Steven R. White, Sean M. Forsythe, Blanca Camoretti-Mercado, Klaus F. Rabe, and Julian Solway. Fas cross-linking induces apoptosis in human airway smooth muscle cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 278: L618-L624, 2000.--Hypertrophy and hyperplasia lead to excess accumulation of smooth muscle in the airways of human asthmatic subjects. However, little is known about mechanisms that might counterbalance these processes, thereby limiting the quantity of smooth muscle in airways. Ligation of Fas on the surface of vascular smooth muscle cells and nonmuscle airway cells can lead to apoptotic cell death. We therefore tested the hypotheses that 1) human airway smooth muscle (HASM) expresses Fas, 2) Fas cross-linking induces apoptosis in these cells, and 3) tumor necrosis factor (TNF)-[Alpha] potentiates Fas-mediated airway myocyte killing. Immunohistochemistry using CH-11 anti-Fas monoclonal IgM antibody revealed Fas expression in normal human bronchial smooth muscle in vivo. Flow cytometry using DX2 anti-Fas monoclonal IgG antibody revealed that passage 4 cultured HASM cells express surface Fas. Surface Fas decreased partially during prolonged serum deprivation of cultured HASM cells and was upregulated by TNF-[Alpha] stimulation. Fas cross-linking with CH-11 antibody induced apoptosis in cultured HASM cells, and this effect was reduced by long-term serum deprivation and synergistically potentiated by concomitant TNF-[Alpha] exposure. TNF-[Alpha] did not induce substantial apoptosis in the absence of Fas cross-linking. These data represent the first demonstration that Fas is expressed on HASM and suggest a mechanism by which Fas-mediated apoptosis could act to oppose excess smooth muscle accumulation during airway remodeling in asthma. asthma; airway remodeling; cytokines

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 Ser473 phosphorylation) and of its downstream effector p70S6Kinase (reflected in Thr389 phosphorylation) within airway muscle bundles, but there was no phosphorylation of the alternative Akt downstream target glycogen synthase kinase (GSK) 3ß. 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 p70S6Kinase phosphorylation but not GSK3ß 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. [ABSTRACT FROM AUTHOR]

There is abundant evidence that tidal breathing, and especially tidal breathing at elevated minute ventilation, antagonizes the development and persistence of airflow obstruction during bronchoconstrictor stimulation in normal animals and people. Here, we studied the antiobstructive effect of different tidal breathing patterns in C57B1/6J and A/J mice during bronchoconstriction induced by continuous or bolus infusion of methacholine. Anesthetized, paralyzed mice were mechanically ventilated at 1,500 ml·kg-1·min-1, using each of three breathing patterns: 5 ml/kg, 300 breath/min; 10 ml/kg, 150 breath/min; or 20 ml/kg, 75 breath/min. Changing from 10 ml/kg, 150 breath/min to 20 ml/kg, 75 breath/min, breathing functionally antagonized bronchoconstriction, reducing the level of airflow obstruction induced by methacholine infusion or boluses equivalently in both strains. In marked contrast, changing from 10 ml/kg, 150 breath/min to 5 ml/kg, 300 breath/min, breathing substantially exacerbated methacholine-induced airflow obstruction in A/J mice, whereas it had no significant effect in C57B1/6J mice. Our results therefore demonstrate that 1) even at moderate, fixed minute ventilation, the precise breathing pattern can influence the degree of airflow obstruction substantially, and 2) the influence of breathing pattern on bronchoconstriction differs considerably between genetically diverse inbred mouse strains. These findings imply that differences in antiobstructive effects of breathing can contribute to differences in apparent airway constrictor responsiveness. Much attention has been placed on dysregulation of contractile function of airway smooth muscle in human disease. We suggest that important pathophysiology might also be found in impairment of the functional antagonist effect of tidal breathing on airflow obstruction. [ABSTRACT FROM AUTHOR]

Studies the effects of inflammatory mediators on airway smooth muscle contraction to airway hyperresponsiveness in asthma. Smooth muscle contractile apparatus dysfunction in asthma; Mediators involved in asthma inflammation.