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Published ahead of print on July 15, 2004, doi:10.1165/rcmb.2004-0117OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 538-543, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2004-0117OC

Mechanistic Similarities between Cultured Cell Models of Cystic Fibrosis and Niemann-Pick Type C

Nicole M. White, Deborah A. Corey and Thomas J. Kelley

Departments of Pediatrics and Pharmacology, Case Western Reserve University and Rainbow Babies and Children's Hospital, Cleveland, Ohio

Address correspondence to: Thomas J. Kelley, Ph.D., Department of Pediatrics, Case Western Reserve University, 8th floor BRB, 10900 Euclid Ave., Cleveland, OH 44106-4948. E-mail: tjk12{at}cwru.edu


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Discussion
 References
 
Recent data demonstrate that inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase restores normal signal transducer and activator of transcription-1 and inducible nitric oxide synthase expression regulation in cystic fibrosis (CF) cells through the modulation of RhoA function. These findings lead to the hypothesis that alterations in the cholesterol synthesis pathway may be an initiating factor in CF-related cell signaling regulation. A disease with a known lesion in the cholesterol synthesis pathway is Niemann-Pick type C (NPC). The hypothesis of this study is that CF cells and NPC fibroblasts share a common mechanistic lesion and should exhibit similar cell signaling alterations. NPC fibroblasts exhibit similar alterations in signal transducer and activator of transcription-1, RhoA, SMAD3, and nitric oxide synthase protein expression that characterize CF. Further comparison reveals NPC-like accumulation of free cholesterol in two cultured models of CF epithelial cells. These data identify novel signaling changes in NPC, demonstrate the cholesterol-synthesis pathway is a likely source of CF-related cell signaling changes, and that cultured CF cells exhibit impaired cholesterol processing.

Abbreviations: acid sphingomyelinase, ASM • cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • nitric oxide synthase, NOS2 • Niemann-Pick type C, NPC • phosphate-buffered saline, PBS • reverse transcriptase/polymerase chain reaction, RT-PCR • signal transducer and activator of transcription-1, STAT1


   Introduction
Top
 Abstract
 Introduction
Material and Methods
 Results
 Discussion
 References
 
Several studies indicate that inherent inflammatory cell signaling pathways exhibit altered regulation in cystic fibrosis (CF) epithelial cells. Our laboratory has identified altered expression of individual proteins mostly related to inflammatory signaling in CF epithelium, including reduced expression of the inducible form of nitric oxide synthase (NOS2) (1–3), reduced expression of SMAD3 (a transforming growth factor-ß1 signaling protein) (4), altered regulation of signal transducer and activator of transcription-1 (STAT1) activity (5, 6), and elevated expression of the small GTPase RhoA (7). Our overall hypothesis has been that each of these CF-related alterations is due to changes in a single process regulated by CF transmembrane conductance regulator (CFTR) function that is capable of influencing a broad range of signaling interactions. We have recently demonstrated that STAT1 activation and NOS2 expression can be normalized in CF epithelial cells by interfering with isoprenoid/cholesterol synthesis by influencing RhoA signaling through 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibiton with mevastatin (7). These data strongly implicate isoprenoid/cholesterol-dependent pathways as the triggering event in mediating altered CF cellular responses.

Niemann-Pick is primarily a neurologic disease characterized by endosomal and lysomal accumulation of unesterified cholesterol and sphingomyelin (reviewed in Ref. 8). There are multiple variations of Niemann-Pick disease that exhibit similar phenotypes. Niemann-Pick Types A (NPA) and B (NPB) are the result of mutations in the gene for acid sphingomyelinase (ASM). NPA is characterized by an almost complete loss of ASM activity, whereas NPB exhibits approximately a 90% reduction in ASM function. Reduced ASM activity results in an accumulation of sphingomyelin and other lipids, including unesterified cholesterol in endosomes and lysosomes. Niemann-Pick type C (NPC) is the direct result of impaired cholesterol transport due to gene mutations in NPC1 (9). The hypothesis of this study is that CF cells and NPC fibroblasts share a common mechanistic lesion and should exhibit similar cell signaling alterations.

Circumstantial evidence based on disease phenotypes suggests the presence of some shared mechanisms between CF and NPC. In addition to neurologic symptoms, one of the more pronounced phenotypes in NPC is excessive lipid accumulation in the liver and spleen leading to hepatosplenomegaly and progressive liver failure. Interestingly, patients with CF are also prone to developing hepatosplenomegaly, although this is typically attributed to ductal occlusion and only affects ~ 20% of patients (10). However, at least one report describes CF liver disease as being characterized by excessive lysosomal accumulation of lipids (11). In addition to hepatosplenomegaly, nonpulmonary radiographic features of Niemann-Pick disease include metacarpal widening and osteoporosis (12), both secondary features of CF. CFTR is not expressed in the brain to any great extent, accounting for a lack of CF-related neurologic symptoms in CF. However, Niemann-Pick disease is characterized in part by severe pulmonary manifestations. A recent study examining the more rare form of NPC, NPC2, reported that six of seven patients followed in the study died of respiratory failure due to severe airway disease (13). Pulmonary involvement has been reported in each type of Niemann-Pick disease, but airway disease appears to be more common in NPB and NPC2.

Data in this manuscript provide evidence of shared cell signaling regulatory mechanisms in cell models of NPC and CF. These data show that NPC and CF cell models share a number of cell-regulatory alterations, including reduced NOS2 and SMAD3 expression, and elevated STAT1 and RhoA expression. Data also indicate that secondary effects of lost CFTR function influences normal cholesterol processing pathways.


   Material and Methods
Top
 Abstract
 Introduction
 Material and Methods
Results
 Discussion
 References
 
Cell Culture
NPC fibroblasts (GM03123A) containing two missense mutations in the NPC1 gene were obtained from Coriell Cell Repository (Camden, NJ). Control human fibroblasts (CRL-2076) were obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were grown at 37°C in 95% O2–5% CO2 on Falcon 10 cm diameter tissue culture dishes (Biosource International, Camarillo, CA) in modified Eagle's medium (MEM) with 2 mM L-gluatmine containing 15% fetal bovine serum and 1 µ/ml penicillin/streptomycin. IB3–1 cells ({Delta}F508/W1282X) and S9 cells (IB3–1 cell stably transfected with the full-length wild-type CFTR as controls) were developed by Pamela L. Zeitlin (Johns Hopkins University, Baltimore, MD). These cells were grown at 37°C in 95% O2–5% CO2 on Falcon 10 cm diameter tissue culture dishes in LHC-8 Basal Medium (Biofluids, Biosource International) with 5% fetal bovine serum. Human epithelial 9/HTEo- cells overexpressing the CFTR regulatory (R) domain (pCEPR; CF phenotype) and mock-transfected 9/HTEo- cells (pCEP, wild type phenotype) were a generous gift from the lab of Dr. Pamela B. Davis (Case Western Reserve University, Cleveland, OH). Cells were cared for as previously described (14).

Mice
Mice lacking CFTR expression (Cftrtm1Unc) were obtained from Jackson Laboratories (Bar Harbor, ME). CFTR wild-type mice were siblings of Cftr–/– mice. All mice were used between 6 and 8 wk of age. CF mice were fed a liquid diet as described by Eckman and colleagues (15). Mice were cared for in accordance with the Case Western Reserve University IACUC guidelines by the CF Animal Core Facility. Excised mouse nasal epithelium was obtained from both wild-type and CF animals and was processed as previously described (5).

Western Immunoblotting
Antibodies against RhoA (mouse), Smad3 (rabbit), NOS2 (mouse), and STAT1 (mouse) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibody against actin (rabbit) was obtained from Sigma-Aldrich (St Louis, MO). Protein samples were prepared by homogenizing excised nasal epithelium or 60-mm dishes of cultured cells in ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 200uM Na2VO4, and 10 µg/ml pepstatin and leupeptin) for 30 min at 4°C while shaking. Cell lysates were microcentrifuged at 4°C at 14,000 rpm for 10 min. Proteins were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis containing 20–40 µg of protein of 6–12% acrylamide gel. The samples were transferred to an Immobilon-P membrane (Millipore, Bedford, MA) at 15 V for 30 min. The blots were blocked overnight in phosphate-buffered saline (PBS: 138 mM NaCl, 15 mM Na2HPO4, 1.5 mM KCl, and 2.5 mM KH2PO4) containing 5% nonfat dehydrated milk and 0.1% Tween-20 at 4°C. Blots were incubated overnight for Smad3 or NOS2 or 1–2 h for other primary antibodies (all at 1:1,000 dilution) in PBS containing 5% nonfat dehydrated milk and 0.1% Tween-20. Blots were washed three times in PBS and incubated in secondary antibody conjugated to horseradish peroxidase for 1 h at room temperature (dilution 1: 4,000; Sigma). Blots were washed again in PBS before visualizing using Super Signal chemiluminescent substrate (Pierce, Rockford, IL) and exposing the membrane to Kodak scientific imaging film (Kodak, Rochester, NY). Quantification of protein expression was accomplished by densitometry software on the VersaDoc (Quality One; BioRad, Hercules, CA).

Filipin Staining
Cells were treated as previously described by Kruth and coworkers (16). Briefly, cells were grown to 75–90% confluence on Fisherbrand coverslips. Cells were rinsed three times with PBS and then fixed with 2% paraformaldehyde for ~ 30 min. Cells were rinsed once more with PBS and then incubated with 0.05 mg/ml filipin (Sigma-Aldrich) in PBS for 1 h on a shaker in the dark. Filipin was dissolved fresh in dimethylformamide before each experiment. Cells were rinsed in PBS before mounting using SlowFade Light antifade (Molecular Probes, Eugene, OR) on slides. A Leica DMIRE2 confocal microscope (Leica Imaging Systems, Mannheim, Germany) using the HCX PL AP x63 1.4 oil objective was used to view fixed samples of cells at room temperature using Leica light software.

Acid Sphingomyelinase Assay
ASM assay was obtained from Molecular Probes and the protocol was followed accordingly on 9HTEo- cells using the two-step ASM assay with 45 µg protein. Fluorescence was measured using a Molecular Devices Fmax fluorescent plate reader.

Real-Time Reverse Transcriptase/Polymerase Chain Reaction Analysis of NPC1 mRNA Expression
NPC1 mRNA levels were determined on a LightCycler quantitative polymerase chain reaction (PCR) machine (F. Hoffmann-La Roche Ltd, Basel, Switzerland). Primers used to amplify a portion of the human NPC1 gene were 5'-ACACCTTCTCTCTCTTTGCGGG-3' and 5'-GCTTGTTCCATCTTCAGCACCTC-3'. Total RNA was obtained from cells by the TRIzol method and cDNA was produced using Moloney murine leukemia virus with random primers according to the manufacturer's instructions (GIBCO-BRL, Gaithersburg, MD).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Discussion
 References
 
CF-Like Cell Signaling Alterations in NPC Fibroblasts
NOS2 induction. The observation of reduced NOS2 expression in the presence of apparently aggressive inflammatory signaling provides an intriguing insight into cell signaling alterations in CF cells (1–3). We have previously demonstrated that altered NOS2 regulation was dependent on CFTR function (3), but how CFTR influences cell signaling is still unknown. To determine if impaired cholesterol regulation contributes to altered NOS2 expression, we examined NPC as a model system. As seen in CF cells, human NPC fibroblasts fail to induce NOS2 protein expression in response to cytomix (1 ng/ml tumor necrosis factor-{alpha}, 0.5 mg/ml interleukin-1ß, 100 U/ml interferon-{gamma}). However, robust NOS2 expression is seen in control wt fibroblasts in response to identical stimulation (3.4- ± 0.5-fold) (Figure 1). These results demonstrate that NPC cells exhibit the same impaired ability to induce NOS2 expression characteristic of CF epithelial cells.



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  		Figure 1. Reduced induction of (NOS2) in NPC fibroblasts compared with wild-type (wt) control fibroblasts. (A) NOS2 protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium. (B) Western blot of NOS2 and actin expression in NPC and wt fibroblasts in the presence (+) or absence (-) of cytomix (CM). (C) Densitometry analysis of NOS2 expression relative to actin protein content (NOS2/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number (n) of samples is shown in parentheses above each bar. Significance determined by t test; *P = 0.002. Error bars represent SEM.

 
STAT1 expression. Previous studies indicated that alterations in STAT1 regulation contribute to impaired NOS2 induction in CF cells. Despite a reduction in STAT1-mediated signaling, STAT1 protein levels were elevated in multiple models of CF epithelium (5). To further determine if CF-related cell signaling changes are conserved in NPC fibroblasts, STAT1 protein levels were evaluated. When evaluating STAT1 levels in NPC fibroblasts, an increase in protein expression (1.9- ± 0.3-fold) above control cells was found (Figure 2). STAT1 activity in NPC cells is yet to be determined. These results further indicate that similar pathways are altered in NPC cells as seen in CF model cells.



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  		Figure 2. Increased STAT1 protein expression in NPC fibroblasts compared with wt control fibroblasts. (A) STAT1 protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium. (B) Western blot of STAT1 and actin expression in NPC and wt fibroblasts. (C) Densitometry analysis of STAT1 expression relative to actin protein content (STAT1/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number (n) of samples is shown in parentheses above each bar. Significance determined by t test; *P = 0.03. Error bars represent SEM.

 
RhoA expression. We have recently reported that CF cells exhibit increased protein expression of RhoA and that RhoA inhibition with the HMG-CoA reductase inhibitor mevastatin normalized STAT1 and NOS2 regulation (7). Similar to findings in CF model systems, investigation of RhoA protein levels in NPC cells showed increased expression (4.3- ± 1.2-fold) compared with controls (Figure 3). RhoA is modified by prenylation and an increase in protein expression may possibly be the result of increased isoprenoid/cholesterol synthesis reported in NPC (17, 18). The effectiveness of mevastatin in normalizing CF-related signaling alterations and the known lesion in this system in NPC models suggest that increased isoprenoid/cholesterol synthesis maybe an upstream mediator of the signaling alterations reported in CF model systems.



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  		Figure 3. Western blot of increased RhoA expression in NPC and CF model systems. (A) RhoA and actin expression in NPC and non-NPC cells. RhoA protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium. (B) Western blot of RhoA and actin expression in NPC and wt fibroblasts. (C) Densitometry analysis of RhoA expression relative to actin protein content (RhoA/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number of replicates is shown in parentheses above each bar. Significance determined by t test; *P = 0.02. Error bars represent SEM.

 
SMAD3 expression. Although not directly related to NOS2 regulation, another CF-related cell signaling alteration we have observed is decreased expression of the transforming growth factor-ß1 signaling protein SMAD3 (4). An examination of NPC fibroblasts revealed that SMAD3 protein expression is reduced 64% relative to wild-type controls (Figure 4). These results indicate that multiple inflammatory pathways affected in NPC cells parallel the signaling changes seen in CF model systems.



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  		Figure 4. Decreased Smad3 expression in NPC and CF model systems. (A) SMAD3 protein expression in Cftr+/+ (wt) and Cftr–/– (CF) mouse nasal epithelium. (B) Western blot of SMAD3 and actin expression in NPC and wt fibroblasts. (C) Densitometry analysis of SMAD3 expression relative to actin protein content (SMAD3/actin) in NPC and wt fibroblasts. Data represent averages of three separate experiments. Number of samples is shown in parentheses above each bar. Significance determined by t test; *P < 0.001. Error bars represent SEM.

 
An Examination of NPC-Like Phenotypes in CF Model Cells
The above data demonstrate that the protein expression of four proteins, NOS2, STAT1, RhoA, and SMAD3, are identically altered in NPC fibroblasts as they are in CF model systems. These results show that NPC fibroblasts are similar to CF cells from the standpoint of cell signaling regulation and support a role for cholesterol-mediated regulation of signaling in CF. The following experiments focus on determining if CF model cells exhibit NPC-like phenotypes.

Filipin staining. Because NPC fibroblasts exhibit CF-like cell signaling changes, we examined whether CF model cells shared any phenotypes characteristic of NPC. The defining characteristic of NPC cells is elevated content of free cholesterol as determined by filipin staining (19, 20). Filipin from Streptomcyces filipinensis is a bacterial protein that binds to unesterified cholesterol and fluoresces in the ultraviolet range. A representative staining pattern of control and NPC fibroblasts is shown in the top panel of Figure 5. The 9/HTEo-pCEPR (CF phenotype) cells exhibit a nearly identical filipin staining pattern as observed in NPC controls. Similar results were obtained with a second CF model cell line, IB3 cells, compared with S9 controls. There is some degree of staining in S9 cells that may indicate incomplete rescue of this phenotype by transgene expression of CFTR. However, there is a clear difference in staining intensity and pattern between S9 and IB3 cells. The elevated levels of unesterified cholesterol and the punctuated staining pattern point to an inherent flaw in intracellular cholesterol processing in two models of CF epithelial cells.



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  		Figure 5. Increased filipin staining in CF epithelial cells compared with controls. Increased filipin staining in NPC fibroblasts compared with wt fibroblasts are used for staining control and for comparison. 9/HTEo- pCEP (wt) and pCEPR (CF phenotype) images are representative of results observed with 16 coverslips/cell type over four separate experiments. S9 (CFTR corrected) and IB3 (CF) images are representative of results obtained with 14 coverslips/cell type over three separate experiments.

 
The possible incomplete rescue of cholesterol processing in S9 cells as indicated by filipin staining calls into question whether signaling differences are preserved in this model system. We have shown above signaling differences in CF mouse nasal epithelium as a comparison to NPC model cells. We have also previously reported the NOS2, RhoA, STAT1, and SMAD3 signaling changes in mice and in the pCEP/pCEPR cells (1–7). However, no direct examination of these pathways have been performed in the S9/IB3 cell model. We examined RhoA and STAT1 porotein expression in these cell lines since these changes appear to be upstream in the signaling consequences related to cholesterol processing. As shown in Figure 6, STAT1 exhibits elevated expression in IB3 cells compared with CFTR-corrected S9 controls, which is consistent with our other CF models. However, RhoA expression is robust in both IB3 and S9 cells, suggesting that the partial correction of cholesterol esterification in S9 cells is insufficient to completely modulate RhoA regulation. These data do not address whether RhoA is constitutively active in S9 and IB3 cells as we have previously reported in mouse models of CF (7). Further characterization of the ability of ectopic CFTR expression to influence cholesterol processing and related signaling cascades in CF cells is needed.



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  		Figure 6. RhoA and STAT1 protein expression in IB3 (CF) and SP (CFTR-corrected) cells. (A) Western blot of RhoA, STAT1, and actin protein expression in IB3 (hatched bars) and S9 cells (closed bars). (B) Densitometry analysis of RhoA and STAT1 expression relative to actin protein content in IB3 and S9 cells. Data represent averages of three separate experiments. Significance determined by t test and error bars represent SEM.

 
Potential Mechanisms of Increased Filipin Staining in CF Model Cells
ASM activity. There are various forms of Niemann Pick disease, two of which are characterized as ASM deficiencies that cause a secondary accumulation of lipids. Sphingomyelinase converts sphingomyelin to ceramide at the plasma membrane. To narrow the focus of a true model system and illuminate a possible mechanism of why and how de novo synthesis of cholesterol is upregulated in CF cell models, ASM activity was investigated using 9/HTEo-pCEP (control) and pCEPR (CF-phenotype) cells because filipin staining differences were more distinct in this model system. Two different experiments with a total of eight samples for each cell type were averaged and reported in Figure 7. There appears to be no significant difference in ASM activity between 9/HTEo- pCEPR (CF phenotype) and pCEP2 (control) cells. Two positive controls were used; one to measure sphingomyelinase from Staphyloccus sp. and hydrogen peroxide to ensure the assay was functional. A negative control of reaction buffer was also measured and used to subtract background from each sample. This in vitro study implies that the ASM is functional in CF cells and not the cause of the cholesterol accumulation seen with filipin staining.



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  		Figure 7. ASM activity in pCEPR (CF phenotype) and pCEP (wt phenotype) 9/HTEo cells. Negative (neg cont) and positve controls (ASM and H2O2) were performed as described in MATERIALS AND METHODS. The bars are averages of two separate experiments. Number of replicates is shown in parentheses above each bar. Significance determined by t test and no significant difference was found. Error bars represent SEM.

 
NPC1 mRNA expression. NPC is caused by a direct lipid transport problem resulting from a mutation in the NPC1 gene. A potential cause of elevated filipin staining in CF model cells is an indirect decrease of NPC1 expression leading to the development of NPC-like phenotypes. Therefore, NPC1 mRNA content was examined using quantitative real-time reverse transcriptase (RT)-PCR. Baseline expression levels of NPC1 show a 2.7- ± 0.4-fold increase in pCEPR 9/HTEo (CF phenotype) cells compared with controls (n = 5, Figure 8). Similarly, IB3 cells exhibit a 1.7- ± 0.2-fold increase in NPC1 mRNA content compared with control S9 cells. These data suggest that reduced NPC1 expression is not a direct cause of elevated filipin staining and possibly indicate that CF-phenotype cells are responding to an increase in free cholesterol by upregulating the expression of a cholesterol transport protein.



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  		Figure 8. Increased NPC1 mRNA levels in CF model cells compared with respective controls. (A) Representative melting curve real-time RT-PCR traces showing 106 copies/µl standard, samples from pCEP and pCEPR cells, and a water control. (B) Quantified real-time RT-PCR determination of NPC1 mRNA expression in pCEP/pCEPR and S9/IB3 cell models were examined. Results are given as a ratio of NPC1 mRNA and GAPDH mRNA (NOS2/GAPDH) normalized to wt phenotype cells (pCEP and S9, respectively). Number of replicates is given in parentheses. Significance determined by t test with *P = 0.005 and **P = 0.04.

 

   Discussion
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
References
 
Understanding the relationship between CFTR function and the regulation of cell signaling cascades is an ongoing goal. Alterations in the regulation of NOS2, STAT1, SMAD3, and RhoA protein expression in CF models have previously been identified (1–7). The observation that inhibition of HMG-CoA reductase activity with mevastatin normalized STAT1 and NOS2 regulation in CF cells through RhoA-dependent mechanisms lead to the hypothesis that alterations within the isoprenoid/cholesterol synthesis pathway represent a potential source of cell signaling changes in CF cells. To address this hypothesis, we turned to a model system that has a well-defined increase in endogenous isoprenoid/cholesterol synthesis, NPC. Our data demonstrate that NPC fibroblasts exhibit identical alterations in NOS2, STAT1, RhoA, and SMAD3 that we have reported in CF epithelial cells. The reproducibility of these protein expression profiles between CF and NPC model systems support our hypothesis that an alteration in cholesterol-related pathways is a potential source of improper cellular responses in CF.

The above studies reveal that NPC fibroblasts resemble CF cells from the standpoint of altered expression of various signaling proteins. We next examined two cultured models of CF epithelial cells for similarities to typical NPC phenotypes. Increased staining of free, unesterified cholesterol by filipin is a defining characteristic of NPC cells. Filipin staining of free cholesterol revealed increased staining in CF-phenotype pCEPR 9/HTEo- cells and in immortalized IB3 CF cells compared with respective controls. The staining patterns obtained in CF model cells were very similar from those obtained from NPC fibroblasts. These data indicate that inherent cholesterol processing mechanisms are altered in CF cells in a manner similar to what is observed in NPC.

In an effort to identify a cause of impaired cholesterol processing in CF model cells, two Niemann-Pick–related mechanisms were examined. An examination of ASM activity revealed no significant change between 9/HTEo- pCEP (control) and pCEPR (CF-phenotype) model cells. These findings are consistent with those reported, but not shown, by Grassme and colleagues (21). However, our results are obtained in an in vitro assay and do not speak to in vivo substrate availability or ceramide transport to the plasma membrane. Another mechanistic possibility is that an indirect influence of CFTR function on NPC1 expression leads to NPC characteristics in CF cells. Expression of NPC1 message was examined in two models of CF epithelia and a significant increase in NPC1 expression compared with wt controls was observed. These data do not address function, but do suggest that CF cells are responding to a lesion in normal cholesterol processing by increasing expression of a cholesterol transport protein. A caveat to these studies is that both the CF cell line models used are immortalized, clonal cell lines and global conclusions about CF pathogenesis have to be made with caution. However, signaling changes in these cell lines have been consistent with data from CF mouse models and human samples making them useful models to begin examining these novel processes.

An inherent flaw in cholesterol processing in CF has multiple potential consequences. In addition to cell signaling regulation, the impairment of cholesterol processing may also influence CF susceptibility to bacterial infection. Recent results by Kowalski and Pier demonstrate that CFTR clusters in lipid rafts in response to bacterial challenge, but the expression of {Delta}F508 CFTR impedes this process (22). Although inherent problems in {Delta}F508 CFTR localization may be responsible for this result, a Niemann-Pick–like impairment in intracellular lipid transport may also contribute to this observation. The findings by Kowlaski and Pier are similar to those by Grassme and coworkers, who recently reported that ceramide-rich lipid rafts were necessary for innate defense against Pseudomonas aeruginosa (21). Evidence continues to mount in support of the hypothesis that alterations in lipid-related processes contribute to poorly explained aspects of CF pathogenesis.

How a loss of CFTR function would lead to the development of NPC-like phenotypes is not obvious, and alterations is ASM activity or NPC1 expression do not seem to be involved. One possibility is the direct regulation of cholesterol or other lipid transport by CFTR. Circumstantial support for this possibility is the considerable structural similarity shared between CFTR and the ATP binding cassette protein A1 (ABCA1) (23). ABCA1 is primarily a cholesterol transport protein important in regulating cholesterol efflux across the plasma membrane, and the protein whose dysfunction is associated with Tangier's disease (24). In an interesting parallel with our CF-related data, one report demonstrates that RhoA protein expression is elevated in cells from patients with Tangier's disease (24), suggesting a further link between cholesterol transport and Rho GTPase regulation. More direct evidence of lipid transport mediated by CFTR has been published showing CFTR-mediated transport of sphingosine-1-phosphate, although no cholesterol transport through CFTR was observed (25). Another possibility is an alteration in the intracellular environment caused by CFTR dysfunction. Picciano and colleagues demonstrate that CFTR undergoes endosomal recycling that is dependent on the C-terminal tail of CFTR (26). Schweibert and colleagues have demonstrated that CF-related cell signaling alterations can also be influenced by interactions at the CFTR C-terminus (27). Related to CFTR presence in endosomes, Poschet and coworkers have shown that endocytic vesicles are hyperacidified in CF lung epithelial cells due to the loss of CFTR function (28). Altered endosomal environment due to lost CFTR function may cause an indirect impairment of cholesterol processing mechanisms. Finally, impaired fatty acid processing in CF has been demonstrated in multiple reports (29–31). Increased filipin staining in our CF model cells suggests that cholesterol is not being re-esterified. Possible explanations for this observation are that cholesterol is not properly reaching the endoplasmic reticulum (ER), as is the case in NPC, or that there is a lack of fatty acid substrate to be used for cholesterol esterification.

Understanding how CFTR function influences cholesterol processing will require considerable investigation. Data presented in this manuscript demonstrate that NPC and CF are very similar at the cellular level. A comparison of CF and NPC may be useful in increasing our understanding of pathogenic mechanisms in both diseases.


   Acknowledgments
 
This work is supported by NIH/NHLBI grant HL64899. The authors thank Dr. P. Davis for providing materials necessary for the completion of this study, and P. Bead for technical assistance.


   Footnotes
 
Conflict of Interest Statement: N.M.W. has no declared conflicts of interest; D.A.C. has no declared conflicts of interest; and T.J.K. has no declared conflicts of interest.

Received in original form April 8, 2004

Received in final form June 21, 2004


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Discussion
 References
 

  1. Kelley, T. J., and M. L. Drumm. 1998. Inducible nitric oxide synthase expression is reduced in CF murine and human airway epithelial cells: possible involvement in CF-related sodium hyperabsorption and susceptibility to bacterial infection. J. Clin. Invest. 102:1200–1207.[Medline]
  2. Meng, Q., D. R. Springall, A. E. Bishop, K. Morgan, T. J. Evans, S. Habib, D. C. Gruenert, K. M. Gyi, M. E. Hodson, M. H. Yacoub, and J. M. Polak. 1998. Lack of inducible nitric oxide synthase in bronchial epithelium: a possible mechanism of susceptibilty to infection in cystic fibrosis. J. Pathol. 184:323–331.[CrossRef][Medline]
  3. Steagall, W. K., H. L. Elmer, K. G. Brady, and T. J. Kelley. 2000. Cystic fibrosis transmembrane conductance regulator-dependent regulation of epithelial inducible nitric oxide synthase expression. Am. J. Respir. Cell Mol. Biol. 22:45–50.[Abstract/Free Full Text]
  4. Kelley, T. J., H. L. Elmer, and D. A. Corey. 2001. Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells. Am. J. Respir. Cell Mol. Biol. 25:732–738.[Abstract/Free Full Text]
  5. Kelley, T. J., and H. L. Elmer. 2000. In vivo alterations of interferon regulatory factor-1 and protein inhibitor of activated Stat1 protein levels in cystic fibrosis epithelium. J. Clin. Invest. 106:403–410.[Medline]
  6. Zheng, S., B. P. De, S. Choudhary, S. A. Comhair, T. Goggans, R. Slee, B. R. Williams, J. Pilewski, S. J. Haque, and S. C. Erzurum. 2003. Impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis. Immunity 18:619–630.[CrossRef][Medline]
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