The N-Terminal Amphipathic Helix of Endophilin Does Not Contribute to Its Molecular Curvature Generation Capacity.

N-BAR proteins such as endophilin are thought to bend lipid membranes via scaffolding (the molding of membranes through the crescent protein shape) and membrane insertion (also called wedging) of amphipathic helices. However, the contributions from these distinct mechanisms to membrane curvature generation and sensing have remained controversial. Here we quantitatively demonstrate that the amphipathic N-terminal H0 helix of endophilin is important for recruiting this protein to the membrane, but does not contribute significantly to its intrinsic membrane curvature generation capacity. These observations elevate the importance of the scaffolding mechanism, rather than H0 insertion, for the membrane curvature generation by N-BAR domains. Furthermore, consistent with the thermodynamically required coupling between curvature generation and sensing, we observed that the H0-truncated N-BAR domain is capable of sensing membrane curvature. Overall, our contribution clarifies an important mechanistic controversy in the function of N-BAR domain proteins.

Rhinovirus infection induces extracellular matrix protein deposition in asthmatic and nonasthmatic airway smooth muscle cells.

Airway remodeling, which includes increases in the extracellular matrix (ECM), is a characteristic feature of asthma and is correlated to disease severity. Rhinovirus (RV) infections are associated with increased risk of asthma development in young children and are the most common cause of asthma exacerbations. We examined whether viral infections can increase ECM deposition and whether this increased ECM modulates cell proliferation and migration. RV infection of nonasthmatic airway smooth muscle (ASM) cells significantly increased the deposition of fibronectin (40% increase, n = 12) and perlecan (80% increase, n = 14), while infection of asthmatic ASM cells significantly increased fibronectin (75% increase, n = 9) and collagen IV (15% increase, n = 9). We then treated the ASM cells with the Toll-like receptor (TLR) agonists polyinosinic:polycytidylic acid, imiquimod, and pure RV RNA and were able to show that the mechanism through which RV induced ECM deposition was via the activation of TLR3 and TLR7/8. Finally, we assessed whether the virus-induced ECM was bioactive by measuring the amount of migration and proliferation of virus-naive cells that seeded onto the ECM. Basically, ECM from asthmatic ASM cells induced twofold greater migration of virus-naive ASM cells than ECM from nonasthmatic ASM cells, and these rates of migration were further increased on RV-modulated ECM. Increased migration on the RV-modulated ECM was not due to increased cell proliferation, as RV-modulated ECM decreased the proliferation of virus-naive cells. Our results suggest that viruses may contribute to airway remodeling through increased ECM deposition, which in turn may contribute to increased ASM mass via increased cell migration.

Rhinovirus infection induces expression of airway remodelling factors in vitro and in vivo.

BACKGROUND AND OBJECTIVE: A hallmark of asthma is airway remodelling, which includes increased deposition of extracellular matrix (ECM) protein. Viral infections may promote the development of asthma and are the most common causes of asthma exacerbations. We evaluated whether rhinovirus (RV) infection induces airway remodelling, as assessed by ECM deposition. METHODS: Primary human bronchial epithelial cells and lung parenchymal fibroblasts were infected with RV-2 or RV-16, or treated with RV-16 RNA, imiquimod (Toll-like receptor (TLR) 7/8 agonist) or polyinosinic : polycytidylic acid (poly I : C) (activator of TLR 3, retinoic-acid-inducible protein I and melanoma-differentiated-associated gene 5). Changes in ECM proteins and their transcription were measured by ELISA and quantitative real-time PCR. In addition, gene expression for ECM proteins was assessed in a mouse model of RV infection. RESULTS: RV infection increased deposition of the ECM protein, perlecan, by human bronchial epithelial cells, and collagen V and matrix-bound vascular endothelial growth factor were increased in both human bronchial epithelial cell and fibroblast cultures. Purified RV-16 RNA, poly I : C and imiquimod induced similar increases in ECM deposition to those observed with RV-infected fibroblasts. However, only poly I : C induced ECM deposition by bronchial epithelial cells, suggesting that RV-induced ECM deposition is mediated through TLR. Furthermore, gene expression for fibronectin and collagen I was increased in lung homogenates of mice infected with RV-1b. CONCLUSIONS: RV infection and TLR ligands promote ECM deposition in isolated cell systems and RV induces ECM gene expression in vivo, thus demonstrating that RV has the potential to contribute to remodelling of the airways through induction of ECM deposition.

Rhinovirus-induced exacerbations of asthma: How is the {beta}2-adrenoceptor implicated?

Rhinovirus (RV) infections are the major cause of asthma exacerbations in children and adults. Under normal circumstances, asthmatic airway obstruction improves spontaneously or characteristically briskly in response to inhaled beta(2)-adrenergic receptor (beta(2)AR) agonists. During virus-associated exacerbations, an impaired response to beta(2)AR agonists is observed; the reason for this is not known. The objective of this study was to determine the effect of RV infection on airway smooth muscle beta(2)AR function. The human cell line Beas-2B and primary human bronchial epithelial cells (HBECs) were infected with RV (multiplicity of infection = 1). After 1 or 5 days for primary and Beas-2B cells, respectively, cell culture supernatants were harvested, UV-irradiated to inactivate RV, and applied to human airway smooth muscle cells for 3 days to assess modifications of beta(2)AR function. RV conditioned medium from Beas-2B and HBECs decreased beta(2)AR agonist-induced cAMP by 50 and 65%, respectively (n = 5; P < 0.05). When cAMP was induced independently of the beta(2)AR using forskolin, no impairment was found. Using flow cytometry, we demonstrated that this decrease was likely the result of beta(2)AR desensitization because membrane but not total cell receptor beta(2)AR was decreased. Pretreatment of HBECs and Beas-2B cells but not human airway smooth muscle cells with the corticosteroids dexamethasone or fluticasone abolished virus-mediated beta(2)AR loss of function. This study shows that epithelial infection with RV induces a decrease of beta(2)AR function on airway smooth muscle cells, potentially explaining the clinical observation of loss of beta(2)AR agonist function during RV-induced asthma exacerbations.