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Introduction: Air pollution from diesel combustion is linked in part to the generation of diesel exhaust particles (DEP). DEP exposure induces various processes, including inflammation and oxidative stress, which ultimately contribute to a decline in lung function. Cyclic AMP (cAMP) signaling is critical for lung homeostasis. The impact of DEP on cAMP signaling is largely unknown. Methods: We exposed human bronchial epithelial (BEAS-2B) cells to DEP for 24-72 h and evaluated mitochondrial bioenergetics, markers of oxidative stress and inflammation and the components of cAMP signaling. Mitochondrial bioenergetics was measured at 72 h to capture the potential and accumulative effects of prolonged DEP exposure on mitochondrial function. Results: DEP profoundly altered mitochondrial morphology and network integrity, reduced both basal and ATP-linked respiration as well as the glycolytic capacity of mitochondria. DEP exposure increased gene expression of oxidative stress and inflammation markers such as interleukin-8 and interleukin-6. DEP significantly affected mRNA levels of exchange protein directly activated by cAMP-1 and -2 (Epac1, Epac2), appeared to increase Epac1 protein, but left phospho-PKA levels unhanged. DEP exposure increased A-kinase anchoring protein 1, β2-adrenoceptor and prostanoid E receptor subtype 4 mRNA levels. Interestingly, DEP decreased mRNA levels of adenylyl cyclase 9 and reduced cAMP levels stimulated by forskolin (AC activator), fenoterol (β2-AR agonist) or PGE2 (EPR agonist). Discussion: Our findings suggest that DEP induces mitochondrial dysfunction, a process accompanied by oxidative stress and inflammation, and broadly dampens cAMP signaling. These epithelial responses may contribute to lung dysfunction induced by air pollution exposure. [ABSTRACT FROM AUTHOR]

Catenin is an 88-kDa member of the armadillo family of proteins that is associated with the cadherin-catenin complex in the plasma membrane. This complex interacts dynamically with the actin cytoskeleton to stabilize adherens junctions, which play a central role in force transmission by smooth muscle cells. Therefore, in the present study, we hypothesized a role for [beta]-catenin in the regulation of smooth muscle force production. [beta]-Catenin colocalized with smooth muscle [alpha]-actin (sm-[alpha]-actin) and N-cadherin in plasma membrane fractions and coimmunoprecipitated with sm-[alpha]-actin and N-cadherin in lysates of bovine tracheal smooth muscle (BTSM) strips. Moreover, immunocytochemistry of cultured BTSM cells revealed clear and specific colocalization of sm-[alpha]-actin and [beta]-catenin at the sites of cell-cell contact. Treatment of BTSM strips with the pharmacological [beta]-catenin/T cell factor-4 (TCF4) inhibitor PKFl15-584 (100 nM) reduced [beta]-catenin expression in BTSM whole tissue lysates and in plasma membrane fractions and reduced maximal KC1- and methacholine-induced force production. These changes in force production were not accompanied by changes in the expression of sm-[alpha]-actin or sin-myosin heavy chain (MHC). Likewise, small interfering RNA (siRNA) knockdown of [beta]-catenin in BTSM strips reduced [beta]-catenin expression and attenuated maximal KCl- and methacholine-induced contractions without affecting sm-[alpha]actin or sm-MHC expression. Conversely, pharmacological (SB216763, LiC1) or insulin-induced inhibition of glycogen synthase kinase-3 (GSK-3) enhanced the expression of [beta]-catenin and augmented maximal KC1- and methacholine-induced contractions. We conclude that [beta]-catenin is a plasma membrane-associated protein in airway smooth muscle that regulates active tension development, presumably by stabilizing cell-cell contacts and thereby supporting force transmission between neighboring cells. asthma; cadherin; GSK-3 doi: 10.1152/ajplung.00020.2010.

[beta]-Catenin plays a dual role in cellular signaling by stabilizing cadherin-mediated cell-cell contact and by regulating gene transcription associated with cell cycle progression. Nonetheless, its presence and function in airway smooth muscle have not been determined. We hypothesized a central role for [beta]-catenin in mitogenic signaling in airway smooth muscle in response to growth factor stimulation. Immunocytochemical and biochemical analysis revealed that human airway smooth muscle cells indeed express abundant [beta]-catenin, which was localized primarily to the plasma membrane in quiescent cells. Treatment of airway smooth muscle cells with PDGF or FBS induced sustained phosphorylation of glycogen synthase kinase-3 (GSK-3), a negative regulator in its unphosphorylated form that promotes [beta]-catenin degradation. GSK-3 phosphorylation was also increased in airway smooth muscle cells with a proliferative phenotype compared with quiescent airway smooth muscle cells with a mature phenotype. Parallel with the increase in GSK-3 phosphorylation, growth factor treatment induced an increased expression and nuclear presence of [beta]-catenin and activated promitogenic signaling in airway smooth muscle, including the phosphorylation of retinoblastoma protein, DNA synthesis ([[sup.3]H]thymidine incorporation), and cell proliferation. Importantly, small interfering RNA knockdown of [beta]-catenin strongly reduced retinoblastoma protein phosphorylation, [[sup.3]H]thymidine incorporation, and cell proliferation induced by PDGF and FBS. Collectively, these data reveal the existence of a GSK-3/[beta]-catenin signaling axis in airway smooth muscle that is regulated by growth factors and of central importance to mitogenic signaling. airway remodeling; asthma; proliferation; growth factor

We have previously demonstrated that long-term exposure of bovine tracheal smooth muscle (BTSM) strips to insulin induces a functional hypercontractile phenotype. To elucidate molecular mechanisms by which insulin might induce maturation of contractile phenotype airway smooth muscle (ASM) cells, we investigated effects of insulin stimulation in serum-free primary BTSM cell cultures on protein accumulation of specific contractile phenotypic markers and on the abundance and stability of mRNA encoding these markers. In addition, we used microscopy to assess insulin effects on ASM cell morphology, phenotype, and induction of phosphatidylinositol (PI) 3-kinase signaling. It was demonstrated that protein and mRNA levels of smooth muscle-specific contractile phenotypic markers, including sm-myosin, are significantly increased after stimulation of cultured BTSM cells with insulin (1 [micro]M) for 8 days compared with cells treated with serum-free media, whereas mRNA stability was unaffected. In addition, insulin treatment promoted the formation of large, elongate ASM cells, characterized by dramatic accumulation of contractile phenotype marker proteins and phosphorylated [p70.sup.S6K] (downstream target of PI 3-kinase associated with ASM maturation). Insulin effects on protein accumulation and cell morphology were abrogated by combined pretreatment with the Rho kinase inhibitor Y-27632 (1 [micro]M) or the PI 3-kinase inhibitor LY-294002 (10 [micro]M), indicating that insulin increases the expression of contractile phenotypic markers in BTSM in a Rho kinase- and PI 3-kinase-dependent fashion. In conclusion, insulin increases transcription and protein expression of contractile phenotypic markers in ASM. This could have important implications for the use of recently approved aerosolized insulin formulations in diabetes mellitus. airway smooth muscle; hypercontractile phenotype; insulin; phosphatidylinositol 3-kinase; Rho kinase doi:10.1152.ajpcell.00502.2006

Background Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix ( ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 ( GSK-3) regulates various intracellular signalling pathways; its role in TGF-β1-induced myofibroblast differentiation is currently largely unknown. Purpose To determine the contribution of GSK-3 signalling in TGF-β1-induced myofibroblast differentiation. Experimental Approach We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and m RNA expression were determined by immunoblotting and RT-PCR analysis respectively. Results Stimulation of MRC5 and primary human lung fibroblasts with TGF-β1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by si RNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling. Conclusion and Implication We demonstrate that GSK-3 signalling regulates TGF-β1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases. [ABSTRACT FROM AUTHOR]