Modern pulmonary disease and infections with remain an intractable problem in cystic fibrosis (CF). Toll-like receptor account activation (14), decreased amounts of iNOS (15), and interrupted cholesterol transportation (16). The wide range of deficiencies in CF is a reflection of the multifactorial nature of problems in CF, and only seldom can multiple manifestations of lung disease be ordered in a sequence of causal relationships. One exception is the following series of linked abnormalities (5): (a) CFTR lossCassociated aberrant sodium transport; (b) organellar hyperacidification due to uninhibited sodium transport out of the organellar lumen, thus permitting higher proton accumulation; (c) altered protein and lipid glycosylation due to TGN hyperacidification; (d) increased bacterial adhesion due to altered glycosylation of cell surfaceCdestined macromolecules; and (e) elevated inflammation in response to bacterial products due to hyperacidified endosomes in which many Toll-like receptors function. The reports on altered products of glycosylase action in the TGN 71386-38-4 supplier of CF respiratory epithelial cells (6, 13) indicate that the hyperacidified lumen of this organelle 71386-38-4 supplier (6) may have other consequences on the properties of CF cells. In addition to being the organelle carrying out terminal glycosylation modifications of proteins destined for secretion or for sorting to the plasma membrane, TGN is a biosynthetic station in which a number of proteins are processed from their pro forms to mature proteins, with the endoprotease furin being a main proprotein convertase in this compartment (17). Furin is primarily located in the TGN (17), but it also readily traffics to the plasma membrane and recycles via the endosomal organelles. The dynamic distribution of furin enables it to cleave and activate numerous intracellular and extracellular proproteins in both the biosynthetic and endocytic pathways (17). Furin is involved in the processing of the substrates, containing the minimal basic amino acid RXXR recognition motif, such as coagulation factors, hormones and growth factors, cell-surface receptors, and extracellular matrix proteins (17). However, furin is not limited to processing of endogenous cellular proteins; it can be co-opted by bacterial toxins and viral coat proteins for maturation and activation in the host (17). We wondered, given the TGN dysfunction, as evidenced by defective sialylation of CF proteins and glycolipids (6, 13), whether furin activity was perturbed in CF respiratory epithelial cells and what would be the physiological consequences of potentially altered furin action. We report here that CF cells show increased furin activity, which explains the abnormally high TGF- levels in CF cells (18), since proCTGF- is one of the furin substrates. Moreover, we demonstrate that elevated furin levels in CF cells renders them more sensitive to the main mutant genotype (20), displayed a higher furin activity than the CFTR-corrected, genetically matched S9 cells (20) (Figure ?(Figure1A).1A). Increased furin activity was also found when other CF and normal cells were compared, including pCEP-R cells, in which the CF phenotype is induced using a dominant-negative construct expressing the R domain of CFTR 71386-38-4 supplier (Figure ?(Figure1A).1A). Furin activity was also examined in primary human lung epithelial cells (Figure ?(Figure1B).1B). The assay for furin was controlled by including the furin inhibitor decanoyl-RVKR-chloromethylketone (CMK) in the reaction mixture (Figure ?(Figure1B).1B). Higher levels of furin in CF cells were detected by western blots (Figure ?(Figure1C).1C). Thus CF cells have elevated levels of furin, and this translates into a higher furin activity in CF cells compared with normal cells. Figure 1 Furin levels are elevated in CF cells. Increased furin activity is responsible for elevated TGF- production by CF cells. CF cells produce more TGF- than do normal cells (18) (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31499DS1). We tested whether elevated levels Rabbit polyclonal to ZFYVE16 of TGF- are due to increased activity of furin in CF cells. ProCTGF- is 71386-38-4 supplier processed by furin in the TGN, an organelle that is hyperacidified in CF (6, 9), releasing the mature TGF-. We tested whether excess TGF- is due to altered furin activity in CF cells by examining the effects of furin inhibitors CMK and Portland 1Cantitrypsin (1-PDX). IB3-1 cells were incubated in media with 50 M CMK (directly tested for inhibitory action on furin; Figure ?Figure1D)1D) or 5 M 1-PDX for 24 hours, and bioactive TGF- was determined using PAI/L assay detecting.