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Research ArticleCell biology Free access | 10.1172/JCI31499
Elevated furin levels in human cystic fibrosis cells result in hypersusceptibility to exotoxin A–induced cytotoxicity
Wojciech Ornatowski,1 Jens F. Poschet,1 Elizabeth Perkett,2,3 Jennifer L. Taylor-Cousar,3,4 and Vojo Deretic1,2
1Department of Molecular Genetics and Microbiology, 2Department of Cell Biology and Physiology, 3Department of Pediatrics, and 4Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, New Mexico, USA.
Address correspondence to: Vojo Deretic, Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, MSC08-4660, 915 Camino de Salud, Albuquerque, New Mexico 87131, USA. Phone: (505) 272-0291; Fax: (505) 272-5309; E-mail: vderetic@salud.unm.edu.
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Published October 18, 2007 - More info
Published in Volume 117, Issue 11 on November 1, 2007
J Clin Invest. 2007;117(11):3489–3497. https://doi.org/10.1172/JCI31499.
Published October 18, 2007 - Version history
Received: January 12, 2007; Accepted: August 14, 2007
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.
Cystic fibrosis (CF), the most common lethal inheritable disease in Caucasians (1), is caused by mutations in the gene encoding a chloride channel termed CF transmembrane conductance regulator (CFTR), resulting in multi-organ dysfunction. The main life-shortening pathology in CF is associated with chronic Pseudomonas aeruginosa lung infections (1). Mutations in CFTR have pleiotropic effects on the function of other ion transporters (2, 3). Of particular importance is the CFTR-dependent regulation of the amiloride-sensitive epithelial sodium channel in respiratory epithelial cells (2, 3), causing electrolyte and fluid hyperabsorption in the CF lung (2, 4) and hyperacidification of intracellular organelles of CF lung epithelial cells including trans-Golgi network (TGN) and endosomal compartments (5–10). In addition, there is an abundance of proposed defects related to pathology in CF, including defective phagosomal acidification in macrophages (11), altered P. aeruginosa uptake by respiratory epithelial cells (12), impaired glycosylation (13), enhanced Toll-like receptor activation (14), reduced levels of iNOS (15), and disrupted cholesterol transport (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 loss–associated 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 surface–destined 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 of CF respiratory epithelial cells (6, 13) indicate that the hyperacidified lumen of this organelle (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-β levels in CF cells (18), since pro–TGF-β is one of the furin substrates. Moreover, we demonstrate that elevated furin levels in CF cells renders them more sensitive to the main P. aeruginosa toxin (19), exotoxin A (ExoA), which requires furin-dependent processing for activation.
Increased furin activity in CF cells. To determine furin activity, permeabilized cells were assayed with the fluorogenic peptide substrate boc-RVRR-amc, which is cleaved to liberate a fluorescent product, reflecting furin activity. A CF bronchial epithelial cell line, IB3-1, from a patient with the DF508/W1282X CFTR mutant genotype (20), displayed a higher furin activity than the CFTR-corrected, genetically matched S9 cells (20) (Figure 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 (Figure 1A). Furin activity was also examined in primary human lung epithelial cells (Figure 1B). The assay for furin was controlled by including the furin inhibitor decanoyl-RVKR-chloromethylketone (CMK) in the reaction mixture (Figure 1B). Higher levels of furin in CF cells were detected by western blots (Figure 1C). Thus CF cells have elevated levels of furin, and this translates into a higher furin activity in CF cells compared with normal cells.
Furin levels are elevated in CF cells. Furin activity was measured by monitoring cleavage of fluorogenic substrate boc-RVRR-amc in extracts from lung epithelial cells. One unit of activity was defined as the amount of enzyme required to liberate 1 pmol of AMC from boc-RVRR-amc. (A) IB3-1, pCEP-R, and CFBE (all 3 cell lines with a CF phenotype) and S9, pCEP, and 16HBE (all 3 cell lines with CFTR-corrected or non-CF phenotype). (B) Furin activity in primary human (CF and normal) lung epithelial cells measured in the presence or absence of the furin inhibitor CMK. (C) Furin western blot and densitometry analysis. (D) Human lung epithelial cells after treatment with furin inhibitors CMK and α1-PDX. Production of TGF-β was measured in IB3-1 (CF) and S9 (CFTR corrected) epithelial cells using TGF-β–responsive luciferase assay. CMK, 50 μM; α1-PDX, 5 μM. Data are presented as percentage of furin or TGF-β, for which 100% corresponds to protein levels in non-CF cells (n = 3 experiments). Mean ± SEM (*P < 0.05, **P < 0.01; †P ≥ 0.05).
Increased furin activity is responsible for elevated TGF-β production by CF cells. CF cells produce more TGF-β than do normal cells (18) (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI31499DS1). We tested whether elevated levels of TGF-β are due to increased activity of furin in CF cells. Pro–TGF-β is processed by furin in the TGN, an organelle that is hyperacidified in CF (6, 9), releasing the mature TGF-β. We tested whether excess TGF-β is due to altered furin activity in CF cells by examining the effects of furin inhibitors CMK and Portland α1–antitrypsin (α1-PDX). IB3-1 cells were incubated in media with 50 μM CMK (directly tested for inhibitory action on furin; Figure 1D) or 5 μM α1-PDX for 24 hours, and bioactive TGF-β was determined using PAI/L assay detecting biologically active TGF-β. The amounts of TGF-β released by IB3-1 cells were reduced by CMK and α1-PDX treatment (P < 0.01) (Figure 1D). Furin inhibitors brought the levels of TGF-β in IB3-1 cells to those in genetically matched, CFTR-corrected cells (S9) (P < 0.05). These results show that increased furin activity in CF epithelial cells governs the abnormally high TGF-β levels and that this can be corrected with furin inhibitors.
Normalization of furin activity in CF cells. Furin is proteolytically processed into its mature form within the TGN (17) as a step necessary for its convertase action. Chloroquine corrects the hyperacidification of a subset of intracellular organelles that are abnormally acidic in CF lung epithelial cells (5, 6). Chloroquine restored the pH of the TGN in IB3-1 mutant cells to levels matching those in CFTR-transfected S9 cells (Supplemental Figure 2). We tested whether furin processing is enhanced in CF lung epithelial cells due to the hyperacidification of the TGN. When CF cells were incubated with 0.1 mM chloroquine, total furin activity was significantly inhibited in all cells tested (Figure 2, A–C). The effect of hyperacidification was additionally confirmed by using primary human CF lung epithelial cells and applying another acidification antagonist, bafilomycin A1, an inhibitor of vacuolar H+ ATPase responsible for acidification of intracellular organelles including endosomes and TGN. Bafilomycin A1 brought furin activity levels in CF primary cells to levels seen in normal primary cells (Figure 2D). These results indicate that excessive furin processing/activation in CF cells can be normalized by correcting organellar pH with chloroquine.
Chloroquine and blocking antibodies against TGF-β normalize furin activity in CF cells. Cells were incubated in full medium with 10 ng/ml TGF-β1 or in the presence of 5 μg/ml TGF-β antibodies or were treated with 0.1 mM chloroquine (CQ). (A) IB3-1 (CF) and S9 (CFTR corrected) cells. (B) pCEP-R (overexpressing the CFTR R domain) and pCEP (mock-transfected) cells. (C) CFBE (CF) and 16HBE (normal) cell lines. (D) Additionally, furin levels were normalized with 100 nM bafilomycin A1 (BA) or 50 μM furin inhibitor (CMK) in primary (CF and normal) human lung epithelial cells. Data are presented as percentage of furin, where 100% corresponds to furin activity in non-CF cells (n = 3 experiments). Mean ± SEM (*P < 0.05, **P < 0.01).
Higher furin levels in CF cells and positive feedback loop with TGF-β. TGF-β is known to induce the expression of furin in a positive feedback loop (21). To test whether these relationships contribute to elevated furin levels in CF, we treated CF cells with blocking antibodies against TGF-β and measured furin (Figure 2, A–D). After treatment with blocking antibodies against TGF-β, furin activity decreased in CF cells. Irrelevant antibodies had no effect (data not shown). In a converse experiment, pretreatment of normal cells (S9, pCEP, 16HBE, and primary normal human lung epithelial cells) with TGF-β1 caused elevated furin activity (Figure 2, A–D). Thus TGF-β and furin are linked in a positive amplification loop in respiratory epithelial cells, which augments furin levels in CF.
Elevated furin and TGF-β levels increase collagen production. In pulmonary fibrosis, respiratory epithelial cells produce more collagen with TGF-β being the most direct stimulator of collagen production (22, 23). To examine whether increased TGF-β levels lead to altered collagen production by CF cells, collagen levels were measured using Sircol collagen assay (Figure 3A). Total soluble collagen was increased in all CF lines tested (IB3-1, pCEP-R, and CFBE) relative to normal or CFTR-corrected respiratory epithelial cells. Blocking TGF-β antibodies decreased the soluble collagen content in IB3-1 cells. A furin inhibitor, α1-PDX also downregulated the level of soluble collagen (Figure 3B). The total soluble collagen secretion was markedly decreased when IB3-1 cells were treated with chloroquine (Figure 3B).
TGF-β contributes to increased collagen production. (A) Total soluble collagen (%) was measured in the media from human airway epithelial cells after 24 hours. Media containing collagen from non-CF (open bars) and CF (filled bars) cell lines. (B) IB3-1 cells incubated in full medium with 5 μg/ml blocking TGF-β antibodies in the presence of furin inhibitor (α1-PDX) or treated with 0.1 mM chloroquine. 100% corresponds to collagen levels in non-CF cells. Mean ± SEM (n = 3 experiments; **P < 0.01).
TGF-β suppresses antipseudomonal action of macrophages and diminishes iNOS expression in CF epithelial cells. TGF-β contributes to the persistence of certain infections by downregulating the antimicrobial activity of macrophages (24–26). To determine whether the increased TGF-β affected P. aeruginosa killing by macrophages, we examined the effect of TGF-β on the ability of primary human peripheral blood monocyte-derived macrophages (MDM) to eliminate bacteria (Supplemental Figure 3). The TGF-β–treated MDM displayed a 2-fold reduction in their ability to kill P. aeruginosa (Supplemental Figure 3A). To test directly whether increased TGF-β produced by CF respiratory epithelial cells can affect the ability of human macrophages to kill P. aeruginosa, we conducted coculture experiments with CF primary cells and MDM. When cocultures consisting of CF primary lung epithelial cells and MDM were infected with P. aeruginosa, bacterial survival decreased when blocking TGF-β antibodies were added (Supplemental Figure 3B). A similar enhancement in bacterial killing was observed when a furin inhibitor (CMK) was used to pretreat respiratory epithelial cells (Supplemental Figure 3B). TGF-β blocking antibody and CMK enhanced the killing ability of macrophages cocultured with CF respiratory epithelial cells to equal that of MDM coculture with normal respiratory epithelial cells. These observations show that TGF-β inhibits P. aeruginosa elimination by human macrophages and that TGF-β production by respiratory epithelial cells affects the ability of macrophages to kill this bacterium. We also found that altered TGF-β contributes to lower than normal expression of iNOS (which too plays an antibacterial role) in CF lung epithelial cells (Supplemental Figure 3, C–E). These results link increased furin activity and elevated TGF-β levels to defective antipseudomonal capacity of the CF lung.
CF cells are more sensitive to P. aeruginosa ExoA. ExoA is detectable in sputum samples of patients infected with P. aeruginosa (27, 28). ExoA is processed by furin to produce a C-terminal 37-kDa fragment which translocates to the cytosol and inhibits protein synthesis (29–31). ExoA is cleaved by furin in acidic intracellular compartments to produce an active toxin that ADP-ribosylates elongation factor 2, leading to cessation of protein synthesis, blocking of Sec61 translocon, and cell death (32–35). The CF and normal cell pairs were treated with ExoA for 24 hours (Figure 4, A–C), and effects on cell viability determined by WST-1 cytotoxicity assay. In Figure 4, A–C, the effects of ExoA-induced cytotoxicity were significantly higher in CF cell lines. Inhibition of furin with CMK resulted in protection from the toxin and increase in cell viability (Figure 4, A–C). Results from an experiment in which pCEP-R cells and their non-CF counterpart pCEP cells were treated with increasing concentrations (0 to 3,000 ng/ml) of ExoA are displayed in Figure 4D and indicate that CF cells showed increased sensitivity to ExoA over a range of toxin concentrations.
CF cells are more sensitive to P. aeruginosa ExoA. CF and matched non-CF respiratory epithelial cells were cultured overnight in 96-well plates with media containing 10% serum. Cells were incubated with ExoA for 24 hours. Cell viability was measured by adding WST-1 as described in Methods. (A) AS1 normal human respiratory epithelial cells stably transfected with CFTR antisense construct (inducing CF phenotype) and their matching control S1 (cells stably transfected with the sense construct). (B) pCEP-R (overexpressing the dominant-negative CFTR R domain) and pCEP (mock-transfected). (C) IB3-1 and S9 (CFTR corrected). Human lung epithelial cells were incubated with furin inhibitor CMK (50 μM) for 24 hours at 37°C. (D) Dose-dependent cytotoxicity of ExoA. Lung epithelial cells were treated for 24 hours with increasing concentrations of P. aeruginosa ExoA (0 to 3,000 ng/ml). Data are presented as percentage of live cells, for which 100% corresponds to ExoA untreated cells (n = 3 experiments). Mean ± SEM (*P < 0.05, **P < 0.01, †P ≥ 0.05).
The hypersusceptibility of CF cells to ExoA was also tested using another, more sensitive assay, based on ExoA inhibition of protein synthesis. The concentration of ExoA had to be titrated down to 100 ng/ml to observe differences between IB3-1 and S9 cells after 24 hours of incubation with the toxin (Figure 5A). The primary cells showed even higher sensitivity to ExoA in the protein synthesis inhibition assay, and to observe the differences between primary CF and normal lung epithelial cells, the incubation with the toxin was shortened to 4 hours (Supplemental Figure 5A). The difference between CF (IB3-1) and CFTR-corrected (S9) cells was abrogated by the inclusion of bafilomycin A1 and was significantly diminished with the furin inhibitor CMK (Figure 5B). The primary CF human lung epithelial cells showed similar response to CMK or bafilomycin A1 inhibitors (Figure 5C and Supplemental Figure 5). These results demonstrate that CF cells are more sensitive to ExoA than are CFTR-corrected or non-CF cells.
CF cells show increased sensitivity to ExoA inhibition of protein synthesis. CF and non-CF cells were incubated in media containing either increasing concentrations of ExoA for 24 hours (A) or 100 ng ExoA/ml (B) with IB3-1 and S9 (CFTR-corrected) cells. (C) CF primary lung epithelial cells in the presence or absence of furin inhibitor CMK (50 μM) or treated with 100 nM bafilomycin A1 (BA). Protein synthesis levels were determined by measuring the incorporation of [3H] leucine into TCA-precipitable cellular proteins and are expressed as percentage of control cells that received no toxin. Mean ± SEM for 3 separate experiments; *P < 0.05, **P < 0.01, †P ≥ 0.05.
Increased furin activity causes ExoA-induced cytotoxicity in CF. ExoA was distributed in respiratory epithelial cells, in keeping with its retrograde trafficking route (Figure 6A). Only a limited direct overlap or juxtaposition could be seen between GFP-furin and ExoA (Figure 6A). Instead, the strongest localization of ExoA was with cellubrevin-pHluorin GFP (Figure 6A), which localizes to the recycling endosome, also hyperacidified in CF cells (8, 9). The bulk of cellular furin under steady-state conditions was, as expected, in the Golgi/TGN region, with no discernible differences between CF and CFTR-corrected cells (Supplemental Figure 4).
Furin-dependent processing of ExoA is increased in CF cells. (A) Localization of ExoA in CFTR mutant IB3-1 cells by fluorescence microscopy (insets, close apposition). Cells were transfected with GFP-furin or cellubrevin-pHluorin GFP and incubated with ExoA conjugated to secondary Alexa Fluor 568 (red). EEA1 was visualized using primary human anti-EEA1 antibody and Alexa Fluor 468–conjugated antibody (green). Scale bars: 5 μm; 2 μm (insets). (B) SDS-PAGE analysis of ExoA processing in IB3-1 (CFTR mutant) cells, IB3-1 cells incubated with CMK (50 μM), and S9 (CFTR-corrected IB3-1) cells. Proteins in cell extracts were separated by SDS-PAGE and immunoblotted with anti-ExoA. ExoA66, full size, inactive ExoA toxin precursor; ExoA37, proteolytically processed 37-kDa active ExoA fragment. (C) Densitometry analysis of ExoA66/ExoA37 ratio.
Nevertheless, it is known that ExoA and furin transit through the same intracellular organelles (29, 31, 36), and either increased furin quantity or its activity under more acidic conditions in CF cells may explain increased cytotoxicity of ExoA to CF cells. Furin processes ExoA to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2, thereby causing cell death (29, 31, 36). To test whether CF cells displayed increased ExoA processing capacity and conversion into its active 37-kDa form, IB3-1 (CF) or S9 (CFTR-corrected) cells were exposed to ExoA (1,000 ng/ml) for 24 hours at 37°C. Products were analyzed by SDS-PAGE followed by immunoblotting and probed with the ExoA polyclonal antibody, which reacts with full-size ExoA (66 kDa) and the active proteolytic fragment of 37-kDa ExoA. Cleavage of ExoA was enhanced in IB3-1 cells relative to S9 cells, generating more of the 37-kDa fragment (Figure 6, B and C). No 37-kDa product was seen when ExoA was incubated with CMK furin inhibitor (Figure 6, B and C), showing that generation of the active toxin depended on furin.
The protein convertase furin quantity and activity are elevated in CF respiratory epithelial cells and lead to a number of previously unappreciated physiological consequences stemming from what we believe to be a newly recognized aberration in CF. The downstream consequences include increased levels of immunosuppressant and tissue remodeling cytokine TGF-β (Figure 7), with attendant downregulation of macrophage bactericidal functions, lowered iNOS expression in CF epithelial cells, and elevated secretion of collagen; and increased capacity of CF respiratory epithelial cells to activate the main toxic product of P. aeruginosa, ExoA, rendering them more susceptible to the effects of this toxin as measured by cell death (Figure 7). Of note is that these newly uncovered deficiencies are linked to the previously reported organellar hyperacidification (TGN and endosome) in CF, thus affirming previous findings (6, 9, 18). Furthermore, our observations are germane to the known but, to our knowledge, hitherto unexplained pathology in CF and thus clarify issues in CF. The newly recognized spectrum of abnormalities in CF cells and tissues does come with a silver lining, since all newly described defects are interconnected and can be treated at their root by normalizing organellar pH or by inhibiting furin.
Model depicting pathological cascade in CF based on increased furin activity and elevated TGF-β levels. Low intra-organellar (TGN and recycling endosome) pH in CF cells increases furin activity in respiratory epithelial cells, which causes a cascade of downstream effects. Left: Increased TGF-β maturation due to its enhanced processing contributes to suppression of iNOS and increased secretion of fibrotic tissue components (collagen). Increased TGF-β further augments furin levels in a positive feedback loop. Increased TGF-β leads to tissue fibrosis contributing to bronchiectasis, and local immunosuppression. Right: Increased activation of P. aeruginosa ExoA by furin in the TGN leads to higher cytotoxicity. These defects underlie the majority of salient aspects of lung disease in CF.
Furin, a ubiquitously expressed proteolytic-processing convertase, has been previously implicated, although not in CF, in a number of pathological processes (17). The increased furin levels and activity in CF can explain the recently reported elevated TGF-β in CF cells (18). Pro–TGF-β is normally processed by furin (17) releasing the mature TGF-β. Although additional factors may contribute to the higher active TGF-β levels in CF, furin processing of TGF-β probably plays an important role, since it showed a rate-limiting effect even in non-CF cells, based on decreased TGF-β levels in the presence of furin inhibitors (Figure 1D). The hyperacidification of TGN in CF most likely initiates increased TGF-β processing by furin. A positive feedback loop between TGF-β and furin (21), also observed in our work, amplifies levels of both furin and TGF-β in CF, leading to increased furin amounts detectable by immunoblots. The bulk of cellular furin is located in TGN (17) but it traffics appreciably to and from the plasma membrane, passing through endosomal organelles that are also hyperacidified in CF respiratory epithelial cells (7–9). In addition to its intracellular action, furin is released from cells to process a number of extracellular substrates (17). Excess furin released from CF cells could play a role in the final activation of secreted TGF-β by liberating it from latent complexes where it is trapped by association with other proteins, through a previously described process distinct from the intracellular processing of the TGF-β pro form (37). TGF-β activation may also be exacted within the hyperacidified TGN in CF cells, since acidification is the most efficient way of dissociating TGF-β from other components within the latent TGF-β complexes (38).
Our measurements show that CF respiratory epithelial cells secrete more collagen than their CFTR-corrected counterparts. TGF-β is one of the most potent regulators of connective tissue synthesis and controls deposition of profibrotic extracellular matrix protein. It plays a role in lung tissue remodeling following injury (39), and despite its crucial role in tissue repair, a fine balance is required, as increased levels of TGF-β or its receptors have been shown to result in severe pulmonary fibrosis in rat models (40). A genetic comprehensive study has indicated that TGF-β gene modifies lung disease severity in CF (41). Thus elevated TGF-β activity downstream of furin upregulation may explain salient aspects of the failure of tissue to remodel appropriately following injury in CF, including bronchiectasis and fibrosis. As shown here, chloroquine, furin inhibitors, or TGF-β blocking antibodies normalize collagen production, confirming the cascade depicted in Figure 7. TGF-β has been detected in CF specimens, including histological preparations (42) and bronchoalveolar lavage fluid (BALF) (43). A recent direct measurement of TGF-β1 in BALF from 43 CF patients (43) found a correlation between TGF-β1 in BALF and reticular basement membrane thickness (RBMT) in endobronchial biopsies in CF. Since the same study reported an increase in RBMT in CF compared with normal subjects, at least 1 parameter of CF respiratory disease shows a positive correlation with TGF-β levels in BALF in CF. In further agreement with our observations, the study by Hilliard et al. (43) also showed a 5-fold increase in collagen levels in BALF from CF compared with normal subjects, which can be explained by elevated TGF-β1 reported here. Lastly, CF patients with the TGF-β1 high-producer genotype have more rapid deterioration in lung function (44, 45), in keeping with our findings and with the results of studies in human populations showing that TGF-β is a genetic modifier in CF (41).
TGF-β is a recognized modulator of immune function and can act as an immunosuppressant. TGF-β plays a central role in the pathogenesis of several chronic infectious diseases; a range of macrophage-deactivating properties that lead to inhibition of macrophage ability to kill several pathogens, including Trypanosoma cruzii, Mycobacterium tuberculosis, and Leishmania major, has been ascribed to TGF-β (24–26). Although macrophage function in CF has not been extensively studied, a recent report with CF transgenic mice has indicated a potential problem in the ability of macrophages from CF animals to kill P. aeruginosa (11). The evidence presented here supports the possibility that increased TGF-β production by respiratory epithelial cells affects the ability of phagocytes to control P. aeruginosa in CF. A local suppression of macrophage function by TGF-β derived from respiratory epithelial cells is compatible with the lung-restricted immunodeficiency in lieu of compromised systemic immunity in CF, a feature of CF that has previously eluded satisfactory explanation.
In addition to the above trans effects, an autocrine action of elevated TGF-β observed here, most likely along with the previously reported STAT1 mechanisms (46), may lead to the lower than normal iNOS expression in CF respiratory epithelia (15, 47). This is compatible with TGF-β acting as a known suppressor of iNOS expression (48). Since iNOS-generated nitric oxide has antibacterial actions in addition to its other physiologic functions, iNOS suppression by TGF-β may further compound the ability of the CF lung to combat bacterial pathogens.
The most surprising aspect of our study is the finding that P. aeruginosa toxin ExoA causes significantly higher cell death in CF cells relative to CFTR-corrected controls. This is caused by the cryptic ability of CF cells to activate ExoA more efficiently than normal cells. Furin-dependent cleavage of ExoA occurs at low pH and takes place in the TGN and endosomes, where the acidic environment induces a conformational change in ExoA that renders it furin susceptible (30, 49). The increased processing of ExoA in CF is most likely the result of a combination of elevated furin levels and lower than normal pH in the relevant organelles of the CF respiratory epithelial cells. In order for ExoA to exert its cytotoxic activity, it has to be proteolytically converted to produce the 37-kDa lethal fragment, which translocates to the cytosol and inhibits protein synthesis (30, 31, 49). As shown here, inhibition of furin can block proteolytic activation of ExoA and exotoxin-mediated death of human respiratory epithelial cells. ExoA is a P. aeruginosa product expressed in the majority of CF patients, and ExoA serology can be used, with various levels of sensitivity, to monitor onset of P. aeruginosa colonization, chronic infection, progress of antimicrobial therapy, or recrudescence (50). Thus it is likely that the hypersusceptibility of CF epithelial cells to this toxin produced by P. aeruginosa (Supplemental Figure 5B) plays a role in CF pathogenesis.
Our work demonstrates that the serine protease furin is generated in excess by CF respiratory epithelial cells. The following model emerges: increased furin action brings about elevated production of fibrotic tissue constituents via TGF-β (Figure 7). Furthermore, the cytotoxicity of ExoA produced by P. aeruginosa, the most prevalent CF pathogen, is increased due to enhanced ExoA processing via furin in CF cells. We propose that these 2 effects synergize, contributing to irreversible tissue damage, fibrosis, and loss of elasticity, thus playing a substantial role in bronchiectasis and resulting in irreversible pathology in the CF lung, which leads to respiratory failure and premature death. Based on the reports that the organellar pH defect in CF can be normalized using the FDA-approved drug chloroquine (6, 9, 18), we applied this compound here to show correction of the increased furin levels in CF. Chloroquine is widely used for treatment of chronic inflammatory diseases such as rheumatoid arthritis and is being tested in clinical studies for its effects in CF (http://www.cff.org/research/ClinicalResearch/Find/?IDS=1&SearchAll=1?). In addition to organellar hyperacidification targeted by chloroquine, the beneficial effects of furin inhibitors described here strongly suggest that furin is a suitable pharmacological target and that provides new opportunities for therapeutic intervention in correcting a set of linked abnormalities in CF.
Cells and constructs. IB3-1 is a human bronchial epithelial cell line derived from a CF patient with a ΔF508/W1282X CFTR mutant genotype (51). S9 is an IB3-1 derivative stably transfected with a functional CFTR and is corrected for chloride conductance (20). 9HTEo- cell derivatives stably overexpress the dominant-negative CFTR R domain (pCEP-R) or are mock-transfected (pCEP), acting as normal lung epithelial cells (52). NuLi-1 (normal lung) cell line was derived from human airway epithelium of normal CFTR genotype; CuFi-3 (CF) cell line was derived from a CF patient (53). The simian virus 40 large T antigen–transformed human bronchial epithelial cell line 16HBE14o-, which maintains tight junctions (54, 55), and CF human bronchial epithelial cells CFBE41o- (CFBE, homozygous for the ΔF508 mutation) were from D. Gruenert (California Pacific Medical Center Research Institute, San Francisco, California, USA). The 16HBE cells were transfected with plasmids expressing the first 131 nucleotides of cftr in either the sense (S1) or antisense (AS3) orientations (S6). CF primary cells CFB-6-04 were from J. Zabner (University of Iowa, Iowa City, Iowa, USA) and human bronchial epithelial cells (NHBE) from Lonza Inc. Mink lung epithelial cells transfected with the plasminogen activator inhibitor-1/luciferase (PAI/L) DNA construct were from D.B. Rifkin (New York University School of Medicine, New York, New York, USA). Human peripheral blood monocyte-derived macrophages were isolated from individual donors by countercurrent centrifugal elutriation, and adherent monocytes were cultured in RPMI 1640 medium supplemented with 10% FBS (HyClone). All cells were grown at 37°C in 5% CO2 under standard conditions. GFP-furin was from G. Thomas (Vollum Institute, Portland, Oregon, USA). Cellubrevin-pHluorin GFP was from J. Rothman (Memorial Sloan-Kettering Cancer Center, New York, New York, USA).
Furin enzyme activity assay. Adherent cells (1.5 × 104) were assayed in the presence of 0.25% Triton X-100 as a permeabilizing agent using boc-RVRR-amc (100 μM) (Bachem) as a furin substrate. Fluorescence was measured at 380-nm excitation and 460-nm emission wavelengths (37, 57). The data were normalized to cell density by parallel staining of another 96-well plate with crystal violet. For released furin activity, supernatants were cleared of cell debris by centrifugation and assayed as described above. Data were normalized to the protein concentration in the supernatant.
PAI/L bioassay for TGF-β. The PAI/L assay using mink lung epithelial cells (MLEC) stably transfected with a luciferase reporter construct under the control of a TGF-β–specific, truncated PAI-1 promoter was performed as described previously (58). Total TGF-β was determined following acid activation of latent TGF-β in conditioned medium from respiratory cell cultures by incubation of samples with 4 N HCL for 1 hour, followed by neutralization with 4 N NaOH prior to assaying. Luciferase activity in conditioned medium was determined using an Orion Microplate Luminometer (Berthold Detection Systems). The amounts of TGF-β were normalized per cell number.
Western blot analysis. Protein samples were prepared by homogenizing lung epithelial cells in lysis buffer on ice. Protein concentrations were determined using a micro-BCA protein assay reagent kit (Pierce Biotechnology). Separated proteins were transferred onto nitrocellulose membranes, blocked, and probed overnight with antibody against furin (MON-152; Alexis Biochemicals), rabbit anti-ExoA (Sigma-Aldrich), and rabbit anti-iNOS antibody (Transduction Laboratories). Immune complexes were detected with horseradish peroxidase-conjugated IgG (Pierce Biotechnology). Signal was visualized by incubating with Super Signal chemiluminescent substrate (Pierce Biotechnology). Densitometry analysis of western blots was performed using NIH ImageJ 1.62 software.
Collagen measurements. Total soluble collagen was measured in culture supernatants by the Sircol assay (Biocolor).
Cytotoxicity assay. Airway epithelial cells (105/ml) were seeded to 96-well plates 1 day before the assay. ExoA (Calbiochem) at 1,000 ng/ml concentration was incubated with the cells for 24 hours at 37κ C. Cell viability was monitored as previously described (59) using the compound WST-1 (4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) (Roche Applied Science). This dye reflected the activity of mitochondrial dehydrogenase present in living cells; the difference in the absorbencies at 450 and 630 nm was measured 2 hours after addition to cells. The inhibitory concentration of CMK (Calbiochem) for ExoA-mediated cell death was assessed, and 50 μM was employed in subsequent experiments.
Protein synthesis inhibition assay. Incorporation of [3H] leucine into newly synthesized proteins was used to assess the effect of ExoA on CF cells as previously described (60). Cells grown to 50%–80% confluency in 12-well plates were exposed to ExoA (Calbiochem) for 24 hours, bathed with leucine-free medium for 30 minutes, and incubated for 1 hour with 1 μCi [3H] leucine/ml in HEPES-buffered, leucine-free media. The medium and extracellular radiolabel were then removed and replaced with 10% trichloroacetic acid (TCA) in PBS for 1 hour at 4°C. After a second 10-minute wash with 10% TCA-PBS to remove TCA-soluble radiolabel, the cells were solubilized. The numbers of TCA-precipitable cpm were determined by scintillation counting. Data are presented as a percentage of protein synthesis compared with that in cells that were not challenged with toxin. Samples were run in triplicate, and the experiment was repeated at least twice for each cell line.
Fluorescence microscopy. All microscopy methods are described in Supplemental Methods.
Statistics. All experiments were performed 3 times. Data are expressed as mean ± SEM. All statistical analyses were carried out using Fisher's protected least significant difference post hoc test (ANOVA) (SUPERANOVA version 1.11; Abacus Concepts).
View Supplemental data
We thank P. Zeitlin for IB3-1 and S9, J. Zabner for primary human CF lung epithelial cells (CFB-6-04), NuLi and CuFi cells, P. Davis for 9HTEo- cells, D. Gruenert for 16HBEo- and CFBE41o- cells, D. Rifkin for MLEC, and R. Elmaoued, S. McGinn, and J. Chua for help with experiments. W. Ornatowski was a Cystic Fibrosis Foundation postdoctoral fellow. This work was supported by NIH grants AI 50825 and AI 31139.
Nonstandard abbreviations used: CF, cystic fibrosis; CFTR, CF transmembrane conductance regulator; CMK, decanoyl-RVKR-chloromethylketone; ExoA, exotoxin A; α1-PDX, Portland α1-antitrypsin; TGN, trans-Golgi network.
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Jens F. Poschet's present address is: Sandia National Laboratories, Albuquerque, New Mexico, USA.
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Home > The Tale of Nokdu > The Tale of Nokdu: Episode 6 (11 & 12) Live Recap
V October 21, 2019 Recaps
The Tale of Nokdu: Episode 6 (11 & 12) Live Recap
The Tale of Nokdu Recap Episode 6 (11&12), image KBS
We were left with a huge cliffhanger last week when Yool-moo walked in on Nokdu giving Dong-mu a half naked kiss. I mean, there is not really a good way to talk yourself out of that one. Of course they have to come clean right? I'm really looking forward to finding out!
Our recap starts at 22:00 KST!
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KOREAN DRAMA THE TALE OF NOKDU EPISODE 6 (11&12) RECAP
Yool-moo walks up to Dong-ju's house and pauses for a moment before going inside. he looks at the ladies shoes and then takes a step toward the door.
Inside, Nokdu is slowly holding Dongju's cheeks. he leans in for a kiss when Yool-moo pushes the doors open. Dongju and Nokdu look at each other quickly and then look at Yool-moo.
Dongju quickly double leg kicks Nokdu, he goes flying backwards. Dongju says it is a misunderstanding! Yool-moo yells, come out! They both start to frantically hurry to standing and we hear the lady ajummas walking around the corner.
Dongju quicky pulls Yool-moo inside and covers his mouth. The lady ajummas call for Nokdu to come out and talk to them so he has to quickly put on his clothing. Nokdu goes out and the ladies ask about Dongju, they give him something to wash their hair. They can use it.
They also say that men are nothing, you two should get back together well. Nokdu says, yes, true, you two should all be tired, go back and sleep. They are all like, huh? We are not tired at all. He pushes them away and turns back to the door.
Nokdu goes back inside. Yool-moo fixes his hat and asks if this is why he had to withhold his identity? Dongju says yes. He asks about what he just saw. Dongju says that it was an accident.
He says he can understand that it was an accident for Dongju but not for that guy! Dongju tries to say that he is not a pervert and all that. If people know that he is a guy then I will be in trouble too beacase I helped hide him. If you stay longer than you will also be in trouble.
Yool-moo says that he hates that he cannot refuse her. He goes over to Nokdu and starts to pull him with him. You cannot stay in the same room as Dongju! Dongju tells him to stop and says that she will sleep at the Gisaeng Jip tonight. They both leave.
Cut to Dongju laying in bed thinking that Nokdu does not have a widow that he likes. She thinks about the kiss. Her friend tells her to turn off her light so she can sleep.
So Dongju grabs the lantern and takes it outside to walk around. She runs ito Nokdu doing the same thing. So they go somewhere to sit and chat.
She ells him that if he talks about the kiss then she will kill him. Don't talk about who I like anymore either. And don't talk about BS. He says, let me talk at least! I don't have a widow or anything. The person we saw in Seoul…
She tells him that she does not care, it is not like he thinks, she is not interested in him. He says she is lying. She says she is not. he asks if she is angry that he kissed her? She says that it was nothing. He says that he felt something with the kiss. She asks if he ever dated a woman ever?
He stands up and says there were so many women who loved him in his hometown! She tells him that he shouldn't have no one, that is why you misunderstood it and pushed things your way. He asks, what? She tells him to stop coming on to her if you understand. I came here with you to tell you that.
She walks away, he thinks that she thinks he is coming on to her?
The Prince is being taken away from the palace. he cries for his mother as he is put inside the carriage to be taken away. All the politicians line up to watch him go.
Heo Yoon meets with the King in the throne room to ask if he cried a lot? Yoon says that he was okay once he left. The King says that he heard it was the same for his brother. he wonders if it would be the same for him.
Heo Yoon says that he would be the same. The King talks about how his father left a cruel letter. If Heo Yoon did not remove that letter, then he would not be there at all. This is all because of you. Heo Yoon bows.
Later on, Heo Yoon opens the box with the secret letter. We flashback to 6 years ago where heo Yoon is talking to someone who is sitting inf ront of him. He says that this is true. The man says that Gwanghae became the King without expectations and the grand Prince is too young. he needed to wait for the chance.
Heo Yoon told him that you cannot avoid the crime for starting a coup. The man says that he will regret it someday that he took this letter from him. This letter is the Kings letter.
Cut to this man dead on the ground next to his son or grandson. it looks like they killed his entire family. Heo Yoon was responsible for it. heo Yoon wals away but is stopped by Yoon-jeo.
He and Yoon-jeo go somewhere to talk. Yoon-jeo says that the proper son of the King grew up smart. Now Gwanghae is the King so we should put everything back to normal. Heo Yoon says no, we should not. Yoon does not understand why, he is the King now, he should not be insecure anymore.
Heo Yoon shows the previous Kings letter which says that the young prince should become the King, not Gwanghae. Heo Yoon says that he just killed the first Prime Minister, do you think the King's worry has ended? What if nothing changes when he becomes King? Me and you and the boy can die.
He takes the letter back and tells Yoon not to wish for useless things. If you come into the world again, I cannot give you any more breaks.
In the present, Heo Yoon takes out the letter and looks at it. But hen he is called out. A man tells him that Yoon-jeo disappeared and all the people watching him are dead. Heo Yoon tells him to find Yoon, no matter what.
It looks like they found a dead body in the widow town and they are bringing that dead body back. There was evidence of torture on the dead body so they might have found that guy. Heo Yoon says that he will kill the military minister and take the little back. he heads out.
He rides right by Yoon-jeo but does not see him. Yoon-jeo sees Heo Yoon though.
WIDOW VILLAGE
Nokdu talks to his military master near the gisaeng village and says that he found out some important information with someone. The master, Jang Koon, says that they need to get that guy and torture him. Nokdu says that this person is the King.
Jang-koon says that this does not make any sense. Nokdu says that he knows that it doesn't, but what if his father knows the King? What if that is why we were hiding in the island. jang-koon wonders if it is a coup? Nokdu thinks it shouldn't be like that? His mother said it was because of him.
Jang-koon says it should be a mistake, his father is a nice person so it should not be like that. Nokdu thinks that he shouldn move too hastily. He needs to find out more. jang-koon talkes a good look at Nokdu and asks what is wrong with his eyes? Did you cry in your pillow all day becasue of a woman? He says no and then asks about Aengdoo.
Cut to Aengdoo crying and saying that her brother is her sister! She says this to Yool-moo. Yool-moo sighs and says that he already knows. he picks her up and says that he will take her somewhere. So he puts her over his shoulder and casually walks away.
She is sad to be caried away, but then starts to smell food as Yool-moo took her to the kitchen. She asks if he is not going to roll her in carpet and hit her? He tells her, if he did that then who would eat my food? he opens up a dish and tells him that they can talk more after she eats a lot. Aengmoo looks like she falls in love.
Elsewhere, Dongju tells Nokdu that she is going to the kitchen and heads out. Nokdu runs after her and says that yool-moo is calling her to the kitchen, do you know why? She casually tells him to separate her from him. Nokdu says, ah, so you know. She keeps walking away. he keeps stammering to keep her with him.
She turns and tells him to stop coming on to her. They pause for a moment and look at each other. He tells her no, I will make you like me. She asks how? So he grabs her and pulls her away over his shoulder. But then we have a lot of hitting sounds off camera and see Dongju walking away again. Nokdu is all bloody. But he still follows her.
She sighs and tells him, are you really going to do this? Go away. So he sadly turns and walks away with his tail between his legs.
Dongju goes to talk to Yool-moo. he tells her that he will ignore everything but asks if she is still going to stay with him in the same room? She asks, don't you trust me? Don't worry, nothing you worry about will happen.
He says okay, but he has a question. You said that you like…well I heard…tell me, was that to him?
She looks a bit nervous and says, of course not. I have my own criteria. Yool-moo agrees and says that she should not like that kind of guy.
Then he asks, so, was it m-m-m-m-me?
She quickly asserts no, I was just drunk!
He tells her that she did not have to deny it like that.
Nokdu gets to the kitchen building with a napkin in his bloody nose and speaks with aengdoo. She asks if he insulted her Yool-moo. He is all like, your Yool-moo? She says that she thought about marriage and thinks that Yool-moo is much better than Nokdu. He is so handsome and kind and nice to me. He is a really nice guy! She peers over the fence to look at Yool-moo. Nokdu looks jealous.
GISAENG HOUSE
Meanwhile, Yeon geun and Jang-koon talk and drink together. Yeon Geun asks if he is like a father to the young widow, Nokdu? Jang-koon says he is, he raised her. So Yeon Geun tells him to help him! jang-koon says, ah, I know women's hearts well, the way to seduce women is…
Cut to Nokdu's face coming is as he also wants to listen to this closely.
Jang-koon said that you pretend to be mean to her but then you are nice. For example, WHY CAN'T YOU DO THI, then do whatever that is as if you can't help yourself.
Nokdu takes this advice to the river where Dongju is washing the dishes. he takes the dishes away from her. YOU CAN'T DO THIS, YOU GO DIE. She is all like, what in the world?
So he takes the dishes to the water and starts to wash them. She is all like, what are you doing? I can do this. He pushes her away and says that she has to do this for her!
She sighs and his him in the head with one of the bowls. he tells her that he is doing this for her so she will think about how he is not kind to her and does nice things for her! Then he starts washing the dishes again.
She sits next to him and watches him. he says that she does not care about him but she looks at him like that. She asks how she is looking at him. He corrects her face and says, like this.
She pulls her face away and walks off. he asks where she is going. She tells him none of your business. he tells her not to be late and come home! Then he sighs and thinks that she does not respect what her mother says.
The King talks to one of his people and asks if they are using one of the governor? The man says yes, a lot of people will want the little prince. It is actually good for us if they move. The King thinks this is a good chance to see who will betray him.
In her cave, Dongju pulls out a wooden box, but we do not see what is inside it.
Cut to the assassin ladies talking in the kitchen. Ssook tells the other two that the military minister is at the center of the little prince issue. He has his fathers 60th birthday tomorrow. There are only family members invited. They might worry about assassins. But they will never guess that the kitchen ajummas are assassins.
Ssook says that she will go to hanyang with the other female assassins and will see what happened to Deul-rae (the one that died). Yeon-boon says that she will go to the island with the others. She stretches and says that no assassins will be in the town.
Cut to Nokdu walking to the kitchen and mubling abotu Yool-moo, that guy. Then he sees the three assassins leaving and asks if they are going far away? They say that they are going to a 60th birthday party, they will cook there. Nokdu asks if she can go?
One of the women says that hanyang is in big chaos becuase the King sent someone to the island~. But she is stopped from saying anymore. The tell Nokdu that she does not need to know and they pat her on the back as they leave.
Nokdu says the islands name and wonders about it.
HEO YOON
Heo Yoon speaks with his men about their plan as they walk inside a building. Everything is ready and they have the assassins. But then Yoon-jeo comes in and puts a knife to Heo Yoon's neck.
He asks him what he is up to again. Heo Yoon slowly turns around. Yoon tells him, it is nice to see you again.
In one of the huge noble houses, the King comes by. The man who opens the gate bows immediately. The King asks if he is inside? The man says yes, he is taking a break now. I think they are at Heo Yoon's house.
The King says that he will surprise Heo Yoon today.
Inside, Yoon still has the sword to Heo Yoon's neck. He asks why he did that to his son. Heo Yoon asks where his son, no, the Kings son is. he is able to break way and pull his sword out.
But then they see a shadow.
The King bursts into the room. Only Heo Yoon is there. But the King knows that something must have been going on. He opens the door to where Yoon was hiding, but he is not there anymore.
The King asks Heo Yoon, why don't we play chess now? Heo Yoon says yes, King.
Yoon is able to run out, he is stopped by Heo Yoon's right hand man who tells him to follow him, there are soldiers everywhere.
Cut to the island where the little prince is. The assassins are watching and waiting for their moment.
Meanwhile, Dongju thinks that about her plan, her plan was to leave after she completed her revenge.
Inside her home, Aengdoo tells Nokdu that Dongju will not be hungry because Yool-moo made her so much food. He is a much better guy than you. Nokdu yells, that jashik! And starts to run out but then Dongju comes in so he quickly sits to eat dinner and asks Nokdu if she ate?
Dongju says that she ate which makes Aengdoo tells Nokdu that she told him so. Dongju goes to bed right away. Nokdu eats angrily behind her.
The next day, Yool-moo meets with The King and brings him some food. The King asks him why he makes these things. What do you think about it? he says that he was not thinking about it because he was with the woman he liked. The King smiles and asks if it was a gisaeng?
Yool-moo says yes, your highness. The King smiles and tells him that he envies that he is free and has a lot of time, nephew. Yool-moo smiles and tells him not to be too jealous of it. he then starts to eat the food as well. It looks like they have a good relationship.
In the village, a man tells a guard to search the house for anything suspicious. The assassins are already in the house preparing the meal.
Elsewhere, Nokdu remembers what a horse looked like and goes to a stable to see if he can find that particular horse. The horse has black and white on his tail.
But he does not see it. he turns and sees a noble come in who announces himself as the governor of Gongwa island. he heard that this place was good. He is stopping by on his way to Gongwa.
The gisaengs show him to a certain room. Nokdu follows along.
GISAENG ROOM
inside one of the rooms, the governor and the gisaengs talk about the little King on the island. he says that he has a chance to get promoted because of this little King. One of the gisanegs asks what the relationship is with his promotion and the little King.
The governor is about to tell her but says nothing, just know that a good think is about to happen. Nokdu eavesdrops on this conversation.
Later on, the governor stumbles out of the room. Nokdu follows him. Cheon follows along as well.
Elsewhere, Dongju talks to a merchant who asks if she is making her own business? She says no, someon wanted me to ask about it, that is all. But then she sees Nokdu following the drunk guy.
Nokdu ends up putting a knife to the mans neck and tells him to tell her about the party! Dongju comes out right then so nokdu puts his knife away which allows for the man to run away. She tells Dongju to call for help and then chases after the governor.
Nokdu stops the governor but Cheon is there and sees this. The governor says that Nokdu was about to kill him! Cheon takes the knife and puts it to the governors throat! She tells him to tell her everything. Then she slits his throat!
He must have told her everything though, because she tells Nokdu that it is true that they are going to even kill the little prince. They sent him to the island already. She tells Nokdu that this is in a hurry. When you find Ssook, retract everyone from the party and go to the temple where the military minister is hiding now. it is a trap.
Nokdu says that she will do it and leaves.
But then another set of footprints shows up. Cheon is surprised to see them.
Nokdu rides off on a horse in a hurry to the governors mansion.
The assassins think that something went wrong, why is it so quiet here? But then one of the ladies gets bumped and a knife falls. All the mens heads turn to the sound of metal. then they all stand and say, get them! The assassins are women!
All the men at the party are trained swords men that alla start to fight. The assassins tell each other that they have to kill everyone that saw them!
They start to fight them off when Nokdu shows up and slides into action! She tells Ssook that their boss sent her. She knew that this would happen so she wanted to warn you.
Nokdu has a pot that she is carrying around and hits one of the men with it. She tells Ssook that the military minister is hiding at a mountain temple. Ssook asks if she thinks she can go find him?
Ssook kills a few more people while asking this. Nokdu asks how she can leave now? Ssook says it is too dangerous here for you. Buy us some time and go!
Nokdu takes off and secretly hops the gate like it aint no thang.He thinks to himself that buying time is no fun at all.
Nokdu walks to the temple bummed. he gets to the guards at the temple who ask why he is there. So Nokdu punches them both in the neck and takes them out.
In the party, the assassins have bested all the men. They head out to the temple, but we see that one of the men is not dead yet.
When they get there, Nokdu is sitting next to the all bound up minister of the military looking very innocent. Ssook is amazed.
All the assassin head back, Ssook walks with Nokdu who asks why Ssook does this dangerous work. Is it for the money? She says that they hide there, they do not need a lot of money and they are the only ones that can do this.
She shows a scar on her leg and says that her inlaws did that to her because she did not die after her husband. he died only three days after their marriage. If she did not run away then she might also be dead already even though seh didn't even remember her husbands face. But her story is not that bad.
She is happy that she met her boss and they live nicely now. But everyone outside this place does not live nicely. So they started helping all the widows. Nokdu asks if they killed the military minister becuase he did bad things to widows?
She says no, it was for a better world for people without power, like them. She believes that.
She continues walking ahead.
Ssook and Nokdu sit with Cheon to fill her in on everything. They say that the military minister died by falling. They had a lot of help from widow Kim.
Cheon tells Nokdu that she promised it to her and they cannot delay her joining anymore, so welcome to the team widow Kim. She says that another person is waiting outside for her but Ssook will wait and talk with Cheon.
Nokdu leaves so Ssook asks cheon if there was any problem?
Cut to Dongju who is thinking about the governor. The people that work there said that he was not just a pervert, he was a governor of an island. Dongju hurries off somewhere.
Meanwhile, Aengdoo talks to Yool-moo. It appears that she is his little informant now and tells him all that happens in the house between Nokdu and Dongju. She says that Nokduu did not come home and Dongju eunnie was in and out of the house all night. She was sighing and looking outside. She went to the front yard and walked around. Then she said YOU STAY OUTSIDE OVERNIGHT AND NOT TELL ME! That is what she did.
Yool-moo looks a bit serious ad looks over at Donju who is at the gate fretting.
Elsewhere, Nokdu gets the official tour of all the assassin things. That night Ssook and Jung tell Nokdu that she did a good job today. Jung then asks Ssook why they don't guard together? But Ssook says no, then she tells Nokdu that she does not have to work too hard from the beginning.
Dongju wakes up in a very dark room. This might be her home. The door opens so she asks if it is you? But it is not, it is Yool-moo. She tells him that he cannot come in like this. But he says that it is okay, he can. She is all like what?
In the town, the assassins start to leave, Nokdu runs off as well.
In the temple meeting ground, Heo Yoon meets with his supporters to talk about what is happening with the little prince.
Cut to yoolmoo leaving Dongju's place with a sigh.
Nokdu walks around the corner and sees him leaving.
In the temple, Heo Yoon tells his people that they are prepared for the coup! One of them thinks that the King is still sharp now. What if everything we prepared becomes nothing because we are too careful? This might be the perfect time!
Someone yells, Dont' do it!
Then Yool Moo comes swaggering in with a smile. Everyone turns their heads to look at him. Heo Yoon is shocked.
Yool-moo tells them that they should not hurry. I took care of the urgent matter. he throws bloody clothes on the table. It looks like he already killed the little prince?!
Yool-moo tells him that peolple are outside, but they all serve him.
They ask him what he did for the prince? Yool-moo says that he killed him himself.
Flashback to Yool-moo showing up to Cheon when Cheon killed that governor. She tells him, can you really keep the promise? He says yes and tells her that her role starts now. She starts to sign a parchment and he smiles.
On the island, the little prince cries for his mother when an assassin comes in and kills the guards. The lady assassins show up so this assassin gives them the letter that Cheon wrote. They look at each other and then turn and leave.
The assassin then goes inside and kills the little prince.
In the temple, one of the politicians yells at Yool-moo and is about to hit him. Yool-moo immediately runs his blade thorugh him and splatters the wall with his blood.
In her room, Dongju is terrified as she holds her legs and rocks back and forth.
Nokdu comes in so she hops up right away and says that she was waiting for him. He tells her that they should leave. the head out. She asks why he is not making eye contact with her? He tells her to go somewhere bright. He will explain it all later.
She asks why he said that customer was a pervert? Why did you come to this widow town? he tells her that he will tell her later and heads out.
In the temple, the remaining men look at their fallen colleague. Yool-moo smiles and tells them that he does not like noise.
Nokdu goes to the temple and looks at it from outside.
Heo Yoon is about to pull his sword. But then one of his men pulls a dagger to his neck. Then Yool-moo's people come in with their swords.
Yool-moo casually walks over to Heo Yoon and tells him that he does not want the young Prince to be King, he wants him to make him the King. Ah, so Yool-moo wants to be King! It is the war of the princes!
Heo Yoon asks if he thinks this cheap threat will work? Yool-moo says, of course not, his threat will not end with this. Today is a little greeting.
Heo Yoon asks, if you want to be the King, then do you think you deserve it?
They stare at each other, the camera goes back and forth between them.
Nokdu opens a door to wherever he is at. A man is standing there outside. It is the King! The King lifts his head up to look at NOkdu and Nokdu looks down at the King.
Oh wow! This is a crazy amazing episode! Ah, it gives me chills! How can this show be this good and unexpected? I really did not think that Yool-moo was anything close to being bad. But now it makes sense as to why he could pull rank on all those people and have all those guards at the ready and everything, because he is a prince!
I love this turn of events. It is the battle of the princes y'all. So it seems like Nokdu is a prince (at least I think he is even though the preview makes it seem like his brother could be). He looks just like the King which I did not even realize until the end of the episode when they were both looking at each other.
So, one prince is killed (poor little guy) and another prince is using his power to get Heo Yoon to make him the new King. While yet another prince is parading around like a woman as he tries to find out why some assassins want to kill him and his family. Though I think that storyline was a Macguffin and we have moved into a much meatier and juicier storyline with the crazy King and palace princes.
I love how this writer has led us to this point with lots of funny moments and thrills and twists and turns thrown in. It is so good!
VO – The gisaeng house that feeds the widow town. The owner and all the widows close to them.
HT – if I am really the Kings son, then hiding like this my entire life wont be too regretful
YM – I heard you joined the assassin group
ND – Are you preparing a coup?
Caption – The War is…
Cheon – Tomorrow morning we all have to leave the widow town
YM – if I kill him, would you still be okay?
Caption – Starting
ND – i am not asking you to like me, just don't disappear, please.
The Tale of Nokdu receives 7.1% and 6.7% viewership ratings for episodes 3 and 4
The Tale of Nokdu Reaches 7.1% and 8.3% Viewership in its Premiere Week
Couple poster for The Tale of Nokdu
Flower Posters for the Flower Boy (and Girl) of The Tale of Nokdu
Happy Chuseok Greetings from The Tale of Nokdu Cast
Script Reading Alert for KBS2 drama series The Tale of Nokdu
The Tale of Nokdu: Episode 5 (9 & 10) Live Recap
The Tale of Nokdu: Episode 4 (7-8) Live Recap
The Tale of Nokdu: Episode 2 (3-4) Recap
The Tale of Nokdu
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Table122000
October 21, 2019 / 10:37 am
Oh My! What an episode. Turns out Yool Mu is a cold blooded killer and royalty who wants to take the throne. Did not see that coming, so well done writers! So Yool Mu and Nokdu are actually cousins. Still confused about why Dong Ju's family were killed if she was betrothed to royalty YM in the past? Can't wait for the next episode. I really hope it doesn't get cancelled.
I really want to know what happened in the past too! I think it might be related to the King and how he never feels secure in the palace. Perhaps there was another coup attempt or maybe Dongju's father was on the opposition side against the Prince before he became king? I think it is something to do with that!
I am soooo excited to watch this later when sub is available! By reading the recap it looks like there were a lot of crazy things that happened and will take me on a roller-coaster ride 😊
Thanks again V for the recap! 😊
Yes, this episode was a big roller coaster!
missjb
October 21, 2019 / 3:04 pm
It's abit jarring imo. The first half is romantic comedy, meanwhile the second half turn into melodrama. I'm not against twist and turns, but this kind of trick of writers to hooked viewers is abit cheap. But no going back, no way after this, the tone will go back into romcom for couples period episode given we know now Yeol Mo's facade and the politics start to kick out. Hopefully the writers play the politics more fun and have more depth.
It is definitely as sudden turn from rom-com to almost thriller?
October 22, 2019 / 8:28 am
This kings and prince part is way to complicated, i just don't get it whose son is young prince, who is Yul mu 's father how he is a price, what about nokdu is he the real son of present king? If yes why he wanted to kill his own son 😣😣😣😣😣
I am a bit confused as well. But Ii think it goes like this:
Nokdu – The Kings son of the present King
Yool-moo – The Kings nephew. His father is the Kings brother so they are all princes.
I have no idea why the King wanted to kill his own son, but it seems like it might be for superstitious reasons?

redpajama_set_name: RedPajamaCommonCrawl


Marigold and other beautiful flowers.
Berchtesgaden, where we are now. As you can see, it is a right dump! View from the balcony.
Marvellous day travelling through into the Bavarian Alps into, but only just, Austria. Lots of cows with big bells on and outside the hotel are a gaggle of goats or is it herd. Anyway got lost due to bossy boots sat nav sending us up a track into a farm, where the farmer looked nasty with a pitchfork in his hand. We waved and scarpered quick, laughing when we felt safe. Back to maps, G says, just as long as I don't have to read them.
Drove through forests and meadows, heading for the mountains, all morning. Absolutely beautiful and what a perfect place to live. Germany is a land of such variety, but everywhere we passed looked prosperous and cared-for. Saw two wild boar, fields full of pheasants and a bison. G said it was not a bison, just a massive bullock, but I know what I saw. He's just talking bullocks.
All day on back roads, over the thousand mile stage today, but much better than taking the direct route. Where we ended up today is a town called Berchtesgaden, close to where Adolph Hitler had his summer palace, known as the Eagles' Nest. This has been the only place we have booked to stay for the whole trip as I wanted to go here for G's landmark birthday. Yes, he really is 100 tomorrow.
Anyway, stopped at a gorgeous town which the book says we would never forget, forgotten the name already. Had a coffee and an Appel something or other, needed a wee. Went inside, a vicious woman said something in German pointing to the bar. There were loads of people with little hoops going upstairs, I thought for an exercise class. Found out ages later the hoops you had to collect with a key on to get into the bogs and out. How ridiculous. Felt very traumatised. Lots of people in lederhosen, suppose because it was weekend. Like to see them getting out of that lot for a wee in a rush.
Hotel very old fashioned with a glorious view of the Alps. Houses all have flowers on them.
A Russian woman staying in the room next door came for a chat. Perfect English. She is a guide in St Petersberg. Really nice. Name is Monika.
Should be going to Eagles Nest tomorrow but will have to check on weather. We have to pre book a bus up. My best friend Moniker is on the case as she is so professional in these matters.
After consulting G, who knows everything, the lovely town we stopped at for drink was called Rosenheim. A wonderful brewer's dray, festooned with flowers and pulled by four huge Shire horses was touring the town. I posed for a picture next to he horses, a bit nervous actually in case one kicked me. Turned out to be much, much worse as one of them did an enormous dump right next to me. G wanted a photo of my expression, but was laughing too much to get camera out.
How was I to know this was the toilet key?


A recent addition to Polar's quality control process is the installation of 10 No. steel holding rigs. Each rig provides a solid and square platform in which a range of healthcare windows can be held securely in place using toggle clamps.
The rigs are used in the final stages of window fabrication which includes lock hardware operation, hardware set up and the application of an anti pick seal. The set up allows up to 10 No. windows to be worked on at any one time and ensures that a multi point quality control check is carried out prior to site delivery and final installation.
The image used shows Polar's Humber Secure windows, which incorporate a multi-positional locking system and removable mesh.


text:
Q: Add multiple time from multiple objects in array and display using pipe in angular 5 in HTML In object array i have multiple objects in nested array, each object has its own value of time e.g ""call_duration": "0:21"" ,I want to add all the times and display it in proper time format from HTML side.
Array is
[
  {
    "phone_number": "1905",
    "interactions": [
      {
        "agentId": "hassan1",
        "call_duration": "0:21"
      },
      {
        "agentId": "shabber",
        "call_duration": "1:22"
      }
    ]
 }
]

I want to add those call duration and display the final time, all the things should be handle in HTML

A: It would be difficult to get total time, only through HTML.
I would suggest you use a function in javascript to get total time. Like below :
HTML
...
<div *ngFor="let item of data ">
      Phone: {{item.phone_number}} - Total Duration {{totalDuration(item.interactions)}}
</div>
...

TS
...
totalDuration(interactions: { 'agentId': string, 'call_duration': 'string' }[]) {
    let totalDuration = 0;
    interactions.forEach(item=>{
      // fetch seconds
      const seconds = +item.call_duration.split(':')[1];
      // fetch minutes and convert them into seconds
      const minutes = +item.call_duration.split(':')[0];
      const secondsOfMinutes = minutes * 60;
      // add all seconds
      totalDuration += seconds + secondsOfMinutes;
    })
    // convert totalDuration to readable format
    return Math.floor(totalDuration / 60) + ":" + (totalDuration % 60 ? totalDuration % 60 : '00')
  }
...

I have also created example on stackblitz.

A: suppose you have an array like:
myObject = [
  {
    "phone_number": "1905",
    "interactions": [
      {
        "agentId": "hassan1",
        "call_duration": "0:21"
      },
      {
        "agentId": "shabber",
        "call_duration": "1:22"
      }
    ]
 }
]

to Display this data you can use Nested Loop e.g:
<div *ngFor="let data of myObject ">
      Phone: {{data.phone_number}} - Intractions <span *ngFor="let intrac of data.interactions">{{intrac.call_duration}}</span>
</div>

Nested loop through your interactions Array.

redpajama_set_name: RedPajamaStackExchange


text:
Cauliflower Heart - A Romantic Wrestler
-London Book Festival Runner Up-Romance
-Hollywood Book Festival Runner-Up Best Genre Fiction
-Silver Readers' Favorite International Book Award for Best Fiction-Sports
-Southern California Book Festival Honorable Mention-Best Fiction
Claudine Bellamy grew up in a strong family of wrestlers and married a champion fighter. She feels protected and cared for and she likes it that way. When circumstances change, however, Claudine finds herself unprotected and alone. First in the Cauliflower Heart trilogy, A Romantic Wrestler, introduces Claudine Bellamy, who thinks she has the perfect life. She has a superstar wrestling husband, and her family enjoys all of the perks of celebrity. But when life grows bitter and Claudine's husband needs help, will she be strong enough to save him?
Cauliflower Heart: A Romantic Wrestler
"With Cauliflower Heart Diana Hart Smith allows us to not only to enjoy the lines of her fine first novel, but to read between those lines to get a better feel for what it was like to grow up with the wrestling business as essential to life as the air she breathed." --Mick Foley, New York Times #1 best-selling author Member WWE Hall of Fame
"There's no doubt the Cauliflower Heart trilogy carries more than a grain of truth." --Patrick Lennon, Fightin Talk Editor, Daily Star, UK
"… an authentic and poignant journey of a wife and mother who comes from a respected wrestling lineage and tells the unique tale of courage and love from a place of deep understanding." --Gail Kim, Former WWE Divas Champion and former TNA Knockouts Champion, Pro Wrestling Illustrated 2012 Top Female Wrestler in the World
In the blink of an eye, the idyllic world that Claudine Bellamy knew, brutally turns upside down. While her family struggles with tragedy and permanent loss, their professional wrestling business is publicly exposed. Acutely vulnerable, is Claudine resilient enough to resist being exploited by corrupt vultures of the celebrity world and protect her precious family?
© 2016 by Diana Hart.


The home-buying journey can be uncertain, full of twists and turns you don't see coming. Even if you're using a Veterans Affairs loan—and taking advantage of the unique benefits that come along with it—you could still run into some big roadblocks along the way.
That's why you need a savvy real estate agent to guide you. And while any agent technically can help with a VA purchase, an agent who's particularly experienced with military buyers can help you better navigate the process. From pre-approval to closing, a military-friendly real estate agent will know what's on the path ahead, and remove any obstacles in your way so you can get the house of your dreams.
During the home-buying process, an agent should help you nail down criteria such as square footage, number of bedrooms, price range, and school district. But for many veterans, housing needs go far beyond that, says Twila Lukavich, a Realtor® with Russell Real Estate Services in Cleveland who has specialized in helping buyers use VA loans since 2009.
An agent who has experience working with military clients can help find the right home, with a special eye toward a veteran's specific situation. For instance, a VA-savvy agent can help disabled veterans find housing grants or a home with adaptive renovations so that they can live independently in a barrier-free environment.
Lukavich also cites many of her clients who suffer from post-traumatic stress disorder and have specialized needs that most agents might not understand.
Active-duty service members may have to relocate often and quickly, and that sometimes means buying and selling on tight timelines. Lukavich is used to streamlining the process for her military clients who are moving to the area, frequently performing a walk-through of homes via Skype and video for clients who may end up purchasing a home sight unseen.
And it's not just about buying—if you have to sell your home quickly, a military-friendly agent can help get it done while not sacrificing on a profit.
Most lenders will say they do VA loans, but those who lack experience often don't understand the special circumstances or documentation required, says Michael Garcia, broker and owner of TQS Realty in Palm Beach, FL.
"I've had numerous veterans who used a conventional mortgage lender almost get denied at closing because they didn't understand some nuance," he says.
In most purchase situations, buyers understand that they may need to make some repairs to the home after purchase to get it move-in ready—from replacing electrical systems to repairing or replacing a worn roof. But to qualify for a VA loan, the house must meet a set of VA-designated Minimum Property Requirements.
While most buyers—and sometimes even agents—are focused on a home's bells and whistles, a VA specialist has an eye for spotting the red flags that might need to be addressed before a VA loan can close, from broken window panes and rotten exterior wood, to torn flooring or missing light fixtures.
"If you are well-versed in MPRs, you can often tell upfront that in your professional opinion the house won't pass the appraisal," says Benny Dinsmore, a Realtor® with Coldwell Banker in Frisco, TX, who has 20 years of experience with military clients.
Property condition problems aren't automatic deal breakers, but repairs will often need to be made to keep the deal moving forward.
While you can find the list of approved properties on the Department of Veterans Affairs website, a real estate agent who doesn't regularly deal with veterans may not know to look.
"You don't want to visit a place and fall in love, then realize you've wasted time and possibly money because your agent didn't do their research," Garcia says.
The good news is lenders may be able to help buyers get an unapproved condo development on the approved list. Talk with your lender for more details.
A local company proud to support our local community…..
We love these fun and creative ideas!


text:
R&B Singer Donell Jones Survives Car Crash with 'No Injuries' After Falling Asleep While Driving
Donell Jones has endure a fender bender subsequent to nodding off while driving, uncovered the R&B vocalist in an Instagram post shared Tuesday.
The performer, known for hit melodies including 1999's "U Know What's Up" and 2000's "Where I Want to Be," shared a progression of pictures from the episode to the web-based entertainment stage and told fans in the post's subtitle that he experienced no wounds.
"I nodded off while driving yesterday and wound up in a trench," composed Jones, 49, close by photographs of his Mercedes-Benz stopped into a green channel.
Donell Jones (@donelljonesforever)'in paylaştığı bir gönderi
"I leave without any wounds yet I took in a significant example, That in the event that you're drained simply leave the vehicle cause this might have been terrible… ."
Proceeding with the inscription, Jones refered to his confidence for guarding him. "I realize that I am genuinely secured however I wanted this experience," he said. "Much thanks to you God for folding your arms over me as you have consistently finished… My adoration and steadfastness is to you.. 🙏🏾👌🏾"
Under the post, a few individual performers left steady remarks for the vocalist. "My brother trust you alright 🙏🏽❤," composed Fat Joe. "Dam yo. Im happy you're alright," remarked Da Imp.
The Instagram represent R&B bunch Next expressed, "Your karma and great energy will constantly safeguard you my brother. Gifts generally 🔑🤟🫡"
"I'm happy you're ok!!!🙏🏾❤" said SWV part Cheryl "Coko" Bet, while Mario Winans stated, "Express gratitude toward GOD you okay!"
Born to Wedded to Medication star Renee Byrd and gospel vocalist Roy Jones, Donell was brought up in Chicago and composed melodies for Usher and 702 preceding delivering his presentation collection My Heart through L.A. Reid's LaFace Records in 1996. Over his long term vocation, Donell has scored Board Hot 100 hits including "In the Hood," "Thumps Me Off My Feet" (initially by Stevie Marvel), "U Know What's Up," "Where I Want to Be" with Lisa Lopes of attention, "It's So Difficult" with Big Quip, "You Realize That I Love You" and "Put Me Down" with Styles P.


When Sachin announced his retirement from International one-dayers, I thought to myself 'Good Riddance'.
You have to admit that he is no longer the Fastest Indian between the pitches. It appeared like his foot was set in concrete when he could barely play a stroke but then Indian cricket fans are quick to forgive.
About 200 days ago, when he scored his 100th ton, he dismissed all rumors of a retirement and we asked for divine mercy for even suggesting that to the god of Indian cricket.
For a cricketer way past his expiry date, we surely made sure that we squeezed every bit off him. Our cricket administrators in the time-tested tradition of politic myopia have failed to groom and induct younger and more talented youngsters and we are back to our loosing ways.
Everybody loves to throw their dirty underwear at poor ol' Dhoni, who is now doing the rounds of every known shrine in India in search for answers.
As a nation, we have very poor skills in estimating and preparing for major sporting events.
2012 has been a bad year of attrition for Indian Cricket and the damage has been done.
So what's ahead for Indian Cricket?
Difficult to say. With Sachin on the rolls, it appeared that our team was always playing for him. They always wanted him to get another ton, another milestone before he hangs up his boots. His retirement could actually be exactly what Indian cricket needs right now.
Dhoni, your moment is waiting.
With the below par results our team has shown at recent matches, I doubt if we'll even get to the quarter-finals at the next ICC World Cup.


The Funniest Town Names in the U.S.
What goes into picking the name of a city? Quite a lot of work, actually. Some scroll backwards for inspiration to find something of historical significance (i.e., Lincoln, Nebraska), some find inspiration in natural landmarks (i.e., Palm Springs, California), others plagiarize (i.e., Lebanon, New Hampshire). Although the aforementioned strategies sure make for respectable names, an article about them is, well … boring.
Today on Travelversed, we shine a light on the wacky, odd, and hilariously named places in the U.S. that very clearly forgot to read The City-Naming Handbook. Let's just say that I don't envy the tourism boards of the following cities, towns, and municipalities.
So, hop in our wood-paneled family sedan and get your camera ready. These are the funniest town names in the United States.
How very meta of you, Unalaska.
Unalska, Alaska is the largest city in the Aleutian Islands, located on Unalaska Island. The name is a derivative of "Ounalashka," which means "near the peninsula." Unalaska is the home of the ever-steaming Makushin Volcano, and it is staggeringly beautiful.
Unfortunately for tourists, though, it's impossible to ask for directions to Unalaska without sounding like a burnt-out philosophy student.
No, Why, Arizona, wasn't named by a snarky teen. In fact, the name is a direct reference to the community's roads. You see, Why is home to two major highways (State Route 85 and State Route 86) that once intersected to form the shape of a Y. And, due to a now-abolished state law requiring city names to have at least three letters, decision-makers chose Why. The "Y" intersection no-longer exists, but the "Why" name lives on!