Sang-Nam Lee

Crosstalk between platelet-derived growth factor-induced Nox4 activation and MUC8 gene overexpression in human airway epithelial cells.

Reactive oxygen species (ROS) contribute to chronic airway inflammation, and NADPH oxidase (Nox) is an important source of ROS. However, little is known of the role that ROS play in chronic upper respiratory tract inflammation. We investigated the mechanism of ROS generation and its association with mucin gene overexpression in the nasal epithelium. The level of platelet-derived growth factor (PDGF) expression was increased in sinusitis mucosa, and high-level PDGF expression induced intracellular ROS, followed by MUC8 gene overexpression in normal human nasal epithelial cells. Knockdown of Nox4 expression with Nox4 siRNA decreased PDGF-induced intracellular ROS and MUC8 expression. Infection with an adenovirus containing Nox4 cDNA resulted in Nox4 overexpression and increased intracellular levels of ROS and MUC8 expression. PDGF and Nox4 overexpression are essential components of intracellular ROS generation and may contribute to chronic inflammation in the nasal epithelium through induction of MUC8 overexpression.

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins

Background: The neuroendocrine protein 7B2 blocks the aggregation of certain secreted proteins. Results: 7B2 co-localizes with protein aggregates in Parkinson and Alzheimer disease brains; blocks the fibrillation of Aβ1–40, Aβ1–42, and α-synuclein; and blocks Aβ1–42-induced Neuro-2A cell death. Conclusion: 7B2 inhibits the cytotoxicity of Aβ1–42 by modulation of oligomer formation. Significance: 7B2 is a novel anti-aggregation secretory chaperone associated with neurodegenerative disease. Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ1–42, Aβ1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.

Overexpressed proprotein convertase 1/3 induces an epithelial-mesenchymal transition in airway epithelium

The proprotein convertases (PCs) are serine proteases responsible for the proteolytic maturation of many precursor proteins involved in upper airway remodelling during nasal polyposis. We have previously found that PC1/3 is expressed in human nasal mucosa. However, whether PC1/3 is related to nasal polyp formation has not been investigated. To gain insight into the functional role of PC1/3 in nasal polyps, we determined PC1/3 expression in nasal polyps by immunostaining, Western blotting and enzyme assays and generated stable cells expressing PC1/3 using airway epithelial cell line NCI-H292. Nasal polyps exhibit increased PC1/3 expression compared to normal nasal mucosa. PC1/3 was expressed in neuroendocrine cells in normal nasal mucosa and it was also expressed in goblet and ciliated cells in nasal polyps. NCI-H292 cells stably expressing PC1/3 displayed morphological changes, enhanced cell proliferation and migration, downregulation of E-cadherin and cytokeratins and upregulation of N-cadherin, vimentin, matrix metalloproteinase-2, collagen-I, snail and twist. Importantly, PC1/3 expression was positively correlated with epithelial-mesenchymal transition in cultured human nasal epithelial cells and in nasal polyps. Taken together, our data suggest that PC1/3 overexpression induces morphological and phenotypic epithelial-mesenchymal transition changes of airway epithelial cells and these changes may contribute to the pathogenesis of nasal polyps. PC1/3 overexpression induces morphological and phenotypic epithelial-mesenchymal transition changes in airway epithelium http://ow.ly/on9hZ

α-Melanocyte–Stimulating Hormone Inhibits Tumor Necrosis Factor α–Stimulated MUC5AC Expression in Human Nasal Epithelial Cells

Mucin hypersecretion is an important clinical feature of several respiratory diseases, including asthma, cystic fibrosis, nasal allergy, rhinitis, and sinusitis. It has been shown that α-melanocyte-stimulating hormone (α-MSH), a proopiomelanocortin (POMC)-derived peptide, has immunomodulatory activities by inhibiting NF-κB activation induced by proinflammatory cytokines such as TNF-α. Because MUC5AC expression is known to be up-regulated by TNF-α via NF-κB activation, we evaluated the inhibitory effect of α-MSH on MUC5AC gene expression induced by TNF-α in normal human nasal epithelial (NHNE) cells. Melanocortin-1-receptor (MC-1R) was detected by RT-PCR, Western blotting, and immunofluorescent labeling in NHNE cells. α-MSH suppressed NF-κB/p65 phosphorylation induced by TNF-α as well as IkB-α degradation in a dose-dependent manner, as assessed by Western blotting. In addition, α-MSH inhibited TNF-α-induced nuclear translocation of NF-κB and NF-κB luciferase activity. Real-time quantitative PCR data showed that α-MSH inhibited TNF-α-induced expression of MUC5AC, and this effect of α-MSH was neutralized by knockdown of MC-1R using MC-1R shRNA lentivirus. Analyses using RT-PCR and Western blotting showed the expression of POMC and two key enzymes in the POMC processing, proprotein convertases (PC)1 and PC2, and 7B2, which is required for enzymatic activity of PC2, in normal human nasal mucosa. We conclude that α-MSH down-regulates MUC5AC expression by inhibiting TNF-α-induced NF-κB activity through MC-1R stimulation in NHNE cells and that normal human nasal mucosa possesses the POMC processing machinery. Therefore, α-MSH may be a promising candidate to decrease mucin overproduction initiated by NF-κB activation.

Proprotein convertase inhibition promotes ciliated cell differentiation – a potential mechanism for the inhibition of Notch1 signalling by decanoyl-RVKR-chloromethylketone

Chronic repetitive rounds of injury and repair in the airway lead to airway remodelling, including ciliated cell loss and mucous cell hyperplasia. Airway remodelling is mediated by many growth and differentiation factors including Notch1, which are proteolytically processed by proprotein convertases (PCs). The present study evaluated a novel approach for controlling basal cell‐type determination based on the inhibition of PCs. It was found that decanoyl‐RVKR‐chloromethylketone (CMK), a PC inhibitor, promotes ciliated cell differentiation and has no effect on the ciliary beat frequency in air–liquid interface (ALI) cultures of human nasal epithelial cells (HNECs). Comparative microarray analysis revealed that CMK considerably increases ciliogenesis‐related gene expression. Use of cell‐permeable and cell‐impermeable PC inhibitors suggests that intracellular PCs regulate basal cell‐type determination in ALI culture. Furthermore, CMK effect on ciliated cell differentiation was reversed by a Notch inhibitor N‐[N‐(3,5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT). CMK inhibited the processing of Notch1, a key regulator of basal cell differentiation toward secretory cell lineages in the airway epithelium, and down‐regulated the expression of Notch1 target genes together with furin, a PC. Specific lentiviral shRNA‐mediated knockdown of furin resulted in reduced Notch1 processing and increased numbers of ciliated cells in HNECs. Moreover, CMK inhibited Notch1 processing and promoted regeneration and ciliogenesis of the mouse nasal respiratory epithelium after ZnSO4 injury. These observations suggest that PC inhibition promotes airway ciliated cell differentiation, possibly through suppression of furin‐mediated Notch1 processing.

Proprotein convertase 5/6A is associated with bone morphogenetic protein-2-induced squamous cell differentiation

Results Using a retinoic acid deficiency-induced squamous metaplasia model of HNECs, we observed a significant increase in the expression of PC5/6A, a PC member, and BMP-2, a candidate substrate for PC5/6A. Specific lentiviral shRNA-mediated PC5/6A knockdown decreased BMP-2 expression and maturation, decreased expression of squamous cell markers, and increased expression of ciliated cell markers. Dec-RVKR-CMK, a PC inhibitor, and LDN-193189, a BMP receptor inhibitor, suppressed squamous differentiation, promoted mucociliary differentiation, and down-regulated the BMP-2/Smad1/5/8/p38 signalling pathways. Dec-RVKRCMK also decreased expression of PC5/6A, but not furin, another PC member, suggesting the involvement of PC5/6A in squamous differentiation of HNECs. Overexpression of PC5/6A and BMP-2 in the human nasal epithelial cell line RPMI-2650 demonstrated that PC5/6A can activate BMP-2. Under retinoic acid-sufficient culture conditions for mucociliary differentiation of HNECs, short-term expression of PC5/6A by the adenovirus system and addition of exogenous BMP-2 induced squamous differentiation. Furthermore, PC5/6A and BMP-2 were highly expressed in metaplastic squamous epithelium of human nasal polyps.