Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A–induced cytotoxicity

Abstract

Progressive pulmonary disease and infections with Pseudomonas aeruginosa remain an intractable problem in cystic fibrosis (CF). At the cellular level, CF is characterized by organellar hyperacidification, which results in altered protein and lipid glycosylation. Altered pH of the trans-Golgi network (TGN) may further disrupt the protein processing and packaging that occurs in this organelle. Here we measured activity of the major TGN endoprotease furin and demonstrated a marked upregulation in human CF cells. Increased furin activity was linked to elevated production in CF of the immunosuppressive and tissue remodeling cytokine TGF-β and its downstream effects, including macrophage deactivation and augmented collagen secretion by epithelial cells. As furin is responsible for the proteolytic processing of a range of endogenous and exogenous substrates including growth factors and bacterial toxins, we determined that elevated furin-dependent activation of exotoxin A caused increased cell death in CF respiratory epithelial cells compared with genetically matched CF transmembrane conductance regulator-corrected cells. Thus elevated furin levels in CF respiratory epithelial cells contributes to bacterial toxin-induced cell death, fibrosis, and local immunosuppression. These data suggest that the use of furin inhibitors may represent a strategy for pharmacotherapy in CF.

Bcl-2 Suppresses Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression in Cystic Fibrosis Airways

“…Such effects of Bcl-2 may be site specific. In this context and in the context of the present study, it is noteworthy that others recently described a paradoxical proapoptotic effect of high-level Bcl-2 expression when Bcl-2 is localized to the nucleus (49 Although the literature is mixed on the ability of CF cells to undergo apoptosis, recent reports indicated that CF airway epithelial cells are more prone to apoptosis and/or necrosis than non-CF cells (54,55). Despite the antiapoptotic effects that Bcl-2 could have in the CF airway, we sought to define whether down-regulation of SERCA2 in airway epithelial cells, whether via Bcl-2 or other mechanisms, affects cell survival there.…”

Regulation of the epithelial Na+ channel and airway surface liquid volume by serine proteases

“…Extracellular proteases may be more influenced by changes in the ASL microenvironment, especially during chronic airway disease, since the activity of these proteases can be affected by changes in ASL pH and/or the presence of extracellular protease inhibitors. However, intracellular proteases may also be affected by such changes as the lack of CFTR [103]. …”

“…However, it is also possible that abnormal proteolytic cleavage of ENaC contributes to Na + hyper-absorption. Due to secondary defects, which may include abnormal local pH environments and/or chronic inflammation, furin, prostasin, and CAP2 have been shown to be upregulated in the absence of CFTR [99, 103, 146]. This upregulation may contribute to a failure of CF airways to affect suitable ASL volume regulation, leading to ASL volume collapse which is indicative of a failure of mucus clearance in vivo.…”

Polyarginine peptide IND-1 enhances recombinant human bone morphogenetic protein-2 yield in mammalian cells

“…Equal volumes of the substrate, prepared in reaction buffer [25 mM Tris, 1 mM CaCl 2 , 0.5% (w/v) Brij-35, pH 9.0], and cell supernatants were incubated at 37°C for 60 min. The fluorescent AMC liberated by PC cleavage was measured at 380 nm excitation and 460 nm emission (Molloy et al 1992;Ornatowski et al 2007) with a fluorescence microplate reader. A standard curve using 7-amino-4-methyl-coumarin (AMC) was constructed.…”