A-Kinase Anchoring Proteins Diminish TGF-β1/Cigarette Smoke-Induced Epithelial-To-Mesenchymal Transition

Epithelial-to-mesenchymal transition (EMT) plays a role in chronic obstructive pulmonary diseases (COPD). Cyclic adenosine monophosphate (cAMP) can inhibit transforming growth factor-β1 (TGF-β1) mediated EMT. Although compartmentalization via A-kinase anchoring proteins (AKAPs) is central to cAMP signaling, functional studies regarding their therapeutic value in the lung EMT process are lacking. The human bronchial epithelial cell line (BEAS-2B) and primary human airway epithelial (pHAE) cells were exposed to TGF-β1. Epithelial (E-cadherin, ZO-1) and mesenchymal markers (collagen Ӏ, α-SMA, fibronectin) were analyzed (mRNA, protein). ELISA measured TGF-β1 release. TGF-β1-sensitive AKAPs Ezrin, AKAP95 and Yotiao were silenced while using siRNA. Cell migration was analyzed by wound healing assay, xCELLigence, Incucyte. Prior to TGF-β1, dibutyryl-cAMP (dbcAMP), fenoterol, rolipram, cilostamide, and forskolin were used to elevate intracellular cAMP. TGF-β1 induced morphological changes, decreased E-cadherin, but increased collagen Ӏ and cell migration, a process that was reversed by the inhibitor of δ/epsilon casein kinase I, PF-670462. TGF-β1 altered (mRNA, protein) expression of Ezrin, AKAP95, and Yotiao. St-Ht31, the AKAP antagonist, decreased E-cadherin (mRNA, protein), but counteracted TGF-β1-induced collagen Ӏ upregulation. Cigarette smoke (CS) increased TGF-β1 release, activated TGF signaling, augmented cell migration, and reduced E-cadherin expression, a process that was blocked by TGF-β1 neutralizing antibody. The silencing of Ezrin, AKAP95, and Yotiao diminished TGF-β1-induced collagen Ӏ expression, as well as TGF-β1-induced cell migration. Fenoterol, rolipram, and cilostamide, in AKAP silenced cells, pointed to distinct cAMP compartments. We conclude that Ezrin, AKAP95, and Yotiao promote TGF-β1-mediated EMT, linked to a TGF-β1 release by CS. AKAP members might define the ability of fenoterol, rolipram, and cilostamide to modulate the EMT process, and they might represent potential relevant targets in the treatment of COPD

Cigarette Smoke Up-regulates PDE3 and PDE4 to Decrease cAMP in Airway Cells

BACKGROUND AND PURPOSE: 3', 5'-cyclic adenosine monophosphate (cAMP) is a central second messenger that broadly regulates cell function and can underpin pathophysiology. In chronic obstructive pulmonary disease (COPD), a lung disease primarily provoked by cigarette smoke (CS), the induction of cAMP-dependent pathways, via inhibition of hydrolyzing phosphodiesterases (PDEs), is a prime therapeutic strategy. Mechanisms that disrupt cAMP signaling in airway cells, in particular regulation of endogenous PDEs are poorly understood. EXPERIMENTAL APPROACH: We used a novel Förster resonance energy transfer (FRET) based cAMP biosensor in mouse in vivo, ex vivo precision cut lung slices (PCLS), and in human in vitro cell models to track the effects of CS exposure. KEY RESULTS: Under fenoterol stimulated conditions, FRET responses to cilostamide were significantly increased in in vivo, ex vivo PCLS exposed to CS and in human airway smooth muscle cells exposed to CS extract. FRET signals to rolipram were only increased in the in vivo CS model. Under basal conditions, FRET responses to cilostamide and rolipram were significantly increased in in vivo, ex vivo PCLS exposed to CS. Elevated FRET signals to rolipram correlated with a protein upregulation of PDE4 subtypes. In ex vivo PCLS exposed to CS extract, rolipram reversed downregulation of ciliary beating frequency, whereas only cilostamide significantly increased airway relaxation of methacholine pre-contracted airways. CONCLUSION AND IMPLICATIONS: We show that CS upregulates expression and activity of both PDE3 and PDE4, which regulate real-time cAMP dynamics. These mechanisms determine the availability of cAMP and can contribute to CS-induced pulmonary pathophysiology

A taxonomic backbone for the global synthesis of species diversity in the angiosperm order Caryophyllales

The Caryophyllales constitute a major lineage of flowering plants with approximately 12500 species in 39 families. A taxonomic backbone at the genus level is provided that reflects the current state of knowledge and accepts 749 genera for the order. A detailed review of the literature of the past two decades shows that enormous progress has been made in understanding overall phylogenetic relationships in Caryophyllales. The process of re-circumscribing families in order to be monophyletic appears to be largely complete and has led to the recognition of eight new families (Anacampserotaceae, Kewaceae, Limeaceae, Lophiocarpaceae, Macarthuriaceae, Microteaceae, Montiaceae and Talinaceae), while the phylogenetic evaluation of generic concepts is still well underway. As a result of this, the number of genera has increased by more than ten percent in comparison to the last complete treatments in the Families and genera of vascular plants” series. A checklist with all currently accepted genus names in Caryophyllales, as well as nomenclatural references, type names and synonymy is presented. Notes indicate how extensively the respective genera have been studied in a phylogenetic context. The most diverse families at the generic level are Cactaceae and Aizoaceae, but 28 families comprise only one to six genera. This synopsis represents a first step towards the aim of creating a global synthesis of the species diversity in the angiosperm order Caryophyllales integrating the work of numerous specialists around the world

Monitoring local pulmonary cAMP levels:combining precision cut lung slice (PCLS) and fluorescence resonance energy transfer (FRET) technologies in mice

RATIONALE Cyclic AMP (cAMP) is one of the most important second messengers and is involved as a target for the therapy of chronic obstructive pulmonary disease (COPD), an airway disease primarily provoked by cigarette smoke . Cyclic nucleotide hydrolyzing phosphodiesterases (PDEs) are able to degrade cAMP or cGMP within subcellular compartments, thereby potentially altering pulmonary responses including airway contractility and inflammation. In the present study, we combine the precision cut lung slice (PCLS) technique in mice with fluorescence resonance energy transfer (FRET) to monitor cAMP in real time. METHODS To monitor the cAMP levels in lung tissues, transgenic mice (CAG-Epac1-camps) that express the FRET-based cAMP sensor Epac1-camps were used for the preparation of PCLS. The β2-adrenergic receptor agonist fenoterol was applied to elevate intracellular cAMP. To achieve PDE subtype specific inhibition, the PDE4 inhibitor rolipram, the PDE3 inhibitor cilostamide and the PDE2 inhibitor BAY60-7550 were used. The nonselective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) served as a control. Moreover, lung slices were exposed to 2.5 % cigarette smoke extract (CSE) for 24 hours used as a COPD model in vitro. RESULTS and CONCLUSIONS We provide evidence that the FRET and PCLS technologies can be combined in CAG-Epac1-camps mice to measure global cAMP. Moreover, we found in fenoterol-stimulated PCLS that PDE4 accounts for more than 80% of the total cAMP-PDE activity. Besides PDE4, PDE3 known as a cGMP-inhibited PDE also contributes to cAMP hydrolysis, indicating that cGMP may modulate the maintenance of local cAMP in PCLS. In contrast, the cGMP-activated PDE2 plays a limited role in cAMP hydrolysis. Exposure to CSE did not alter the FRET signal in the presence of the PDE4 inhibitor under fenoterol stimulated conditions. In contrast, we found that a significant increase could be observed in PDE3-dependent FRET responses (p<0.05). Under basal conditions, CSE treatment altered local cAMP levels by significantly increasing both PDE4 and PDE3 inhibitor effects. Therefore, as the major lung cyclic nucleotide hydrolyzing enzymes PDE3 and PDE4 are both involved in the local regulation of the cAMP levels in the β2-AR microdomain, our findings suggest that exposure to CSE induced alterations in the PDEs activity profile both under basal conditions and in the presence of the β2-agonist fenoterol