Membrane-anchored Serine Protease Matriptase Is a Trigger of Pulmonary Fibrogenesis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a devastating disease that remains refractory to current therapies. OBJECTIVES: To characterize the expression and activity of the membrane-anchored serine protease matriptase in IPF in humans and unravel its potential role in human and experimental pulmonary fibrogenesis. METHODS: Matriptase expression was assessed in tissue specimens from patients with IPF versus control subjects using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blotting, while matriptase activity was monitored by fluorogenic substrate cleavage. Matriptase-induced fibroproliferative responses and the receptor involved were characterized in human primary pulmonary fibroblasts by Western blot, viability, and migration assays. In the murine model of bleomycin-induced pulmonary fibrosis, the consequences of matriptase depletion, either by using the pharmacological inhibitor camostat mesilate (CM), or by genetic down-regulation using matriptase hypomorphic mice, were characterized by quantification of secreted collagen and immunostainings. MEASUREMENTS AND MAIN RESULTS: Matriptase expression and activity were up-regulated in IPF and bleomycin-induced pulmonary fibrosis. In cultured human pulmonary fibroblasts, matriptase expression was significantly induced by transforming growth factor-ß. Furthermore, matriptase elicited signaling via protease-activated receptor-2 (PAR-2), and promoted fibroblast activation, proliferation, and migration. In the experimental bleomycin model, matriptase depletion, by the pharmacological inhibitor CM or by genetic down-regulation, diminished lung injury, collagen production, and transforming growth factor-ß expression and signaling. CONCLUSIONS: These results implicate increased matriptase expression and activity in the pathogenesis of pulmonary fibrosis in human IPF and in an experimental mouse model. Overall, targeting matriptase, or treatment by CM, which is already in clinical use for other diseases, may represent potential therapies for IPF.

Deleterious impact of hyperglycemia on cystic fibrosis airway ion transport and epithelial repair.

BACKGROUND: Cystic fibrosis (CF)-related diabetes (CFRD) is associated with faster pulmonary function decline. Thus, we evaluated the impact of hyperglycemia on airway epithelial repair and transepithelial ion transport, which are critical in maintaining lung integrity and function. METHODS: Non-CF and CF airway epithelial cells were exposed to low (LG) or high (HG) glucose before ion current and wound repair rate measurements. RESULTS: CFTR and K+ currents decreased after HG treatments. HG also reduced the wound healing rates of non-CF and CF cell monolayers. Although CFTR correction with VRT-325 accelerated the healing rates of CF cells monolayers under LG conditions, this improvement was significantly abrogated under HG conditions. CONCLUSIONS: Our data highlights a deleterious impact of hyperglycemia on ion transport and epithelial repair functions, which could contribute to the deterioration in lung function in CFRD patients. HG may also interfere with the beneficial effects of CFTR rescue on airway epithelial repair.

Identification of KvLQT1 K+ channels as new regulators of non-small cell lung cancer cell proliferation and migration.

K+ channels, which are overexpressed in several cancers, have been identified as regulators of cell proliferation and migration, key processes in cancer development/propagation. Their role in lung cancer has not been studied extensively; but we showed previously that KvLQT1 channels are involved in cell migration, proliferation and repair of normal lung epithelial cells. We now investigated the role of these channels in lung cancer cell lines and their expression levels in human lung adenocarcinoma (AD) tissues. First, we observed that the wound-healing rates of A549 lung adenocarcinoma cell monolayers were reduced by clofilium and chromanol or after silencing with KvLQT1-specific siRNA. Dose-dependent decrease of A549 cell growth and cell accumulation in G0/G1 phase were seen after KvLQT1 inhibition. Clofilium also affected 2D and 3D migration of A549 cells. Similarly, H460 cell growth, migration and wound healing were diminished by this drug. Because K+ channel overexpression has been encountered in some cancers, we assessed KvLQT1 expression levels in tumor tissues from patients with lung AD. KvLQT1 protein expression in tumor samples was increased by 1.5- to 7-fold, compared to paired non-neoplastic tissues, in 17 of 26 patients. In summary, our data reveal that KvLQT1 channel blockade efficiently reduces A549 and H460 cell proliferation and migration. Moreover, KvLQT1 overexpression in AD samples suggests that it could be a potential therapeutic target in lung cancer.

Improvement of defective cystic fibrosis airway epithelial wound repair after CFTR rescue.

Airway damage and remodelling are important components of lung pathology progression in cystic fibrosis (CF). Although repair mechanisms are engaged to restore the epithelial integrity, these processes are obviously insufficient to maintain lung function in CF airways. Our aims were therefore to study how the basic cystic fibrosis transmembrane conductance regulator (CFTR) defect could impact epithelial wound healing and to determine if CFTR correction could improve it. Wound-healing experiments, as well as cell migration and proliferation assays, were performed to study the early phases of epithelial repair in human CF and non-CF airway cells. CFTR function was evaluated using CFTR small interferring (si)RNA and inhibitor GlyH101 in non-CF cells, and conversely after CFTR rescue with the CFTR corrector VRT-325 in CF cells. Wound-healing experiments first showed that airway cells from CF patients repaired slower than non-CF cells. CFTR inhibition or silencing in non-CF primary airway cells significantly inhibited wound closure. GlyH101 also decreased cell migration and proliferation. Interestingly, wild-type CFTR transduction in CF airway cell lines or CFTR correction with VRT-325 in CFBE-ΔF508 and primary CF bronchial monolayers significantly improved wound healing. Altogether our results demonstrated that functional CFTR plays a critical role in wound repair, and CFTR correction may represent a novel strategy to promote the airway repair processes in CF.

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells.

Active Na+ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and KATP K+ channel activities exerts sustained control in Na+ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the alpha-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K+ channels, and 2) to determine the physiological impact of K+ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and KATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased alpha-ENaC expression, similarly to KATP activation by pinacidil. Conversely, pharmacological KvLQT1 and KATP inhibition or silencing with siRNAs down-regulated alpha-ENaC expression. Furthermore, K+ channel blockers significantly decreased alpha-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and KATP activation dose-dependently enhanced alpha-ENaC promoter activity. Finally, we noted a physiological impact of changes in K+ channel functions on ERK activity, alpha-, beta-, gamma-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K+ channels regulate alpha-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.

[K+ channels and lung epithelial physiology]. / Canaux potassiques et physiologie de l'épithélium respiratoire.

Transcripts of more than 30 different K(+) channels have been detected in the respiratory epithelium lining airways and alveoli. These channels belong to the 3 main classes of K(+) channels, i.e. i) voltage-dependent or calcium-activated, 6 transmembrane segments (TM), ii) 2-pores 4-TM and iii) inward-rectified 2-TM channels. The physiological and functional significance of this high molecular diversity of lung epithelial K(+) channels is not well understood. Surprisingly, relatively few studies are focused on K(+) channel function in lung epithelial physiology. Nevertheless, several studies have shown that KvLQT1, KCa and K(ATP) K(+) channels play a crucial role in ion and fluid transport, contributing to the control of airway and alveolar surface liquid composition and volume. K(+) channels are involved in other key functions, such as O(2) sensing or the capacity of the respiratory epithelia to repair after injury. This mini-review aims to discuss potential functions of lung K(+) channels.

Molecular diversity and function of K+ channels in airway and alveolar epithelial cells.

Multiple K(+) channels are expressed in the respiratory epithelium lining airways and alveoli. Of the three main classes [1) voltage-dependent or Ca(2+)-activated, 6-transmembrane domains (TMD), 2) 2-pores 4-TMD, and 3) inward-rectified 2-TMD K(+) channels], almost 40 different transcripts have already been detected in the lung. The physiological and functional significance of this high molecular diversity of lung epithelial K(+) channels is intriguing. As detailed in the present review, K(+) channels are located at both the apical and basolateral membranes in the respiratory epithelium, where they mediate K(+) currents of diverse electrophysiological and regulatory properties. The main recognized function of K(+) channels is to control membrane potential and to maintain the driving force for transepithelial ion and liquid transport. In this manner, KvLQT1, KCa and K(ATP) channels, for example, contribute to the control of airway and alveolar surface liquid composition and volume. Thus, K(+) channel activation has been identified as a potential therapeutic strategy for the resolution of pathologies characterized by ion transport dysfunction. K(+) channels are also involved in other key functions in lung physiology, such as oxygen-sensing, inflammatory responses and respiratory epithelia repair after injury. The purpose of this review is to summarize and discuss what is presently known about the molecular identity of lung K(+) channels with emphasis on their role in lung epithelial physiology.