help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on March 25, 2004, doi:10.1165/rcmb.2003-0447OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0447OCv1
31/2/234    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, J. Y.
Right arrow Articles by Kelley, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, J. Y.
Right arrow Articles by Kelley, T. J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 234-240, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0447OC

Isoprenoid-Mediated Control of SMAD3 Expression in a Cultured Model of Cystic Fibrosis Epithelial Cells

Jenny Y. Lee, Heather L. Elmer, Kristie R. Ross 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}po.cwru.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Several cellular signaling alterations have been identified in cystic fibrosis (CF) epithelium. One of these alterations is reduced SMAD3 protein expression and a corresponding reduction in SMAD3-mediated transforming growth factor-ß1 (TGF-ß1) signaling in CF epithelial cells compared with wild-type (wt) controls. The goal of this study was to identify a mechanism leading to reduced SMAD3 protein expression in CF epithelium. Based on previous work demonstrating isoprenoid-mediated regulation of CF-related alterations in signal transducer and activator of transcription-1 (Stat1) and inducible nitric oxide synthase (NOS2) expression, the hypothesis of this study is that inhibition of isoprenoid-dependent signaling will restore SMAD3 expression and signaling in a model of CF epithelium. Presented data will demonstrate that inhibition of both farnesyl and geranylgeranyl transferase activities partially restores SMAD3-mediated TGF-ß1 signaling and normalizes SMAD3 protein expression in one cultured model of CF cells. Analysis of the human SMAD3 promoter demonstrates that isoprenoid regulation of SMAD3 expression is dependent on Sp1/Sp3 activity, although farnesyl-mediated pathways may be acting through a secondary mechanism as well. Isoprenoid-mediated regulation of SMAD3 expression, coupled with previous data demonstrating isoprenoid control of Stat1 and NOS2 expression, suggest that the isoprenoid/cholesterol synthesis pathway is a critical intermediate in influencing CF-related cell signaling changes.

Abbreviations: cystic fibrosis, CF • CF transmembrane conductance regulator, CFTR • plasminogen activator inhibitor-1, PAI-1 • phosphate-buffered saline, PBS • transforming growth factor, TGF • wild-type, wt


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cystic fibrosis (CF) is a genetic disease that is caused by an absent or deficient cystic fibrosis transmembrane conductance regulator (CFTR) (1). The loss of CFTR function manifests as abnormal airway clearance of thick mucous causing obstruction and susceptibility to infections, especially Pseudomonas aeruginosa, which is generally nonpathogenic in the normal host. Airway inflammation plays an important role in the pathogenesis of CF lung disease (2–7). The cycle of airway obstruction, infection, and inflammation ultimately leads to lung tissue destruction (8, 9). One hypothesis to explain the excessive inflammatory response is that a lack of anti-inflammatory signaling leads to an imbalance in responses to stimuli (10–13).

Transforming growth factor ß (TGF-ß) family members have a broad array of biological activities such as inhibiting cell growth, promoting proliferation of fibroblasts, and deposition of collagen. TGF-ß1 is also potentially involved in regulating the balance between inflammation and fibrosis (14–16). Patients with CF who have a high TGF-ß1–producing genotype have exhibited a more rapid deterioration in lung function compared with those with a low TGF-ß1–producing genotype (17). Furthermore, samples from liver biopsies taken from patients with CF-related liver disease have shown a similar relationship between TGF-ß1 expression and increased hepatic fibrosis and worse liver disease (18).

TGF-ß1 signaling is initiated when type I and type II serine/threonine receptors are activated (19, 20). Various Smad proteins mediate intracellular signaling in response to TGF-ß family members. Smads 1, 2, 3, 5, and 8 are receptor-regulated Smads (R-Smads) that are directly phosphorylated by type I receptors and translocate to the nucleus where regulation of target gene transcription occurs. We have previously reported reduced expression of TGF-ß1 signaling protein SMAD3 in CF epithelial cells (21). Reduced SMAD3 protein expression occurs in both a cultured cell model of CF as well as in the nasal epithelium of cftr–/– mice. Decreased SMAD3 protein content subsequently leads to reduced SMAD3-mediated signaling stimulated by TGF-ß1, although Smad2/Smad4 activation remains intact (21). The diminished magnitude of SMAD3-dependent signaling potentially impacts anti-inflammatory responses in CF epithelium (21).

We have recently demonstrated that inhibition of isoprenoid/cholesterol synthesis normalizes Stat1 and NOS2 regulation in CF epithelial cells through inhibition of the small GTPase RhoA (22). Most Ras and Rho family GTPases require modification with either farnesyl or geranylgeranyl isoprene moieties (23–25). Previous reports have demonstrated an upregulation of TGF-ß expression and signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase when the cholesterol metabolic pathway is inhibited using 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase inhibitors (25). Farnesylpyrophosphate (FPP) serves as a branchpoint in the cholesterol metabolic pathway and is a precursor for cholesterol and geranylgeranylpyrophosphate (GGPP). FPP and GGPP are important lipid attachments for posttranslational modification of Ras and Rho. The hypothesis of this study was that inhibition of the prenylation pathway will correct reduced SMAD3 protein expression and signaling in CF epithelial cells. Given the multiple points of isoprenoid signaling interactions, this manuscript focuses strictly on farnesyl and geranylgeranyl transferase manipulation to discern any differences between farnesyl- and geranylgeranyl-dependent pathways. Presented data will demonstrate restoration of SMAD3 protein expression in a model of CF epithelium in response to isoprenyl transferase inhibition and an increase in SMAD3-dependent TGF-ß1 signaling. Data will also demonstrate that isoprenyl transferase inhibition influences SMAD3 expression at the level of promoter activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
SMAD3 Promoter Constructs
A 1,892-bp fragment corresponding to –1879 to +13 relative to the ATG start site of the human SMAD3 gene (Accession no. AF025293) was amplified from genomic DNA isolated from A549 cells with the 5'-primer 5'-AAT GGT ACC GCT AGC TGC GGA TTG GCT TGT GG-3' and the 3'-primer 5'-AGA AAG CTT AGA TCT AGG ATG GAC ATG GCT G-3'. The 5'-primer has restriction sites for Acc65I and NheI incorporated into the 5' end. The 3' primer has restriction sites for HindIII and BglII incorporated into the 5' end. This amplified region was subcloned into the pGL3basic firefly luciferase plasmid using Acc651 and HindIII restriction sites to create a human SMAD3 promoter–luciferase (SMAD3p-Luc) construct.

Sequential 5' truncations of the promoter were constructed by digesting the 1,892-bp SMAD3p fragment (–1879, +13) with PvuII and EcoRI, with PvuII and BstXI, with PvuII and BlpI, with NheI, or with PvuII and PstI to generate promoter fragments of 1581, 1025, 861, 421, and 177 bp, respectively. Fragments were blunt ended with T4 DNA polymerase (NEB, Beverly, MA) and ligated into pGL3basic.

The SMAD3 promoter Sp1/Sp3 region was amplified using the 5' primer 5'-TCAAGATCTCTGCGAGTCCAACCCTCTCC-3' and the 3'-primer 5'-GTAAAGCTTGGGAAGGAAAGTCCAACCC-3'. The 5'-primer has a BglII restriction site incorporated into the 5' end and the 3' primer has a HindIII restriction site incorporated into the sequence. The Sp1 region was amplified from the SMAD3p-luc vector using Pfx polymerase (Invitrogen, Carlsbad, CA). The PCR product was subcloned into pGL3 basic at the HindIII and BglII sites (Sp1SMAD3p-luc). The SMAD3 promoter with the Sp1/Sp3 region deleted from the construct ({Delta}Sp1SMAD3p-luc) was made by inverse PCR. Primers flanking the Sp1/Sp3 cluster were chosen and oriented to amplify in the opposite direction. The 5' ends of the primers were phosphorylated to allow the inverse PCR product to recircularize. The 5' primer used was 5'-/5Phos/CAGAGGCGGAGGGATCTGCG-3'. The 3' primer used was 5'-/5Phos/AGTCCACACTCGGGCGGCTC-3'. Gel purified inverse PCR product was recircularized using fast ligation kit (Roche, Indianapolis, IN) according to manufacturer's instructions to generate {Delta}Sp1SMAD3p-luc.

Cell Culture
The human alveolar type-II epithelial adenocarcinoma cell line (A549) is grown at 37°C in 95% O2/5% CO2 on Falcon 10-cm-diameter tissue culture dishes in Ham's F-12 Kaign's Modification (Biofluids, Inc., Rockville, MD) with 10% fetal bovine serum, 2 mM L-glutamine, 1 U/ml penicillin, and streptomycin. 9/HTEo- pCEP and pCEPR cells, where pCEPR cells overexpress the CFTR regulatory (R) domain resulting in a CF phenotype and pCEP2 cells, which express a 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 (26).

Transfection Protocol and Evaluation of Luciferase Expression
We used the 3tp-lux reporter construct available from Dr. Joan Massague (La Jolla, CA) to test for SMAD3-mediated TGF-ß1 activation in A549 cells and in 9/HTEo- pCEP and pCEPR cells using protocols previously described (21). A549 cells were co-transfected with the human SMAD3 promoter region in the pGL3B firefly (FF) luciferase vector (SMAD3p-luc) and Thymidine kinase promoter driven Renilla luciferase construct (pRL-TK) (25:1 ratio ff/renilla) or with the pHRG renilla luciferase construct (50:1 ratio). Renilla luciferase expression is used as an internal control for transfection efficiency. A549 cells were seeded at a density of 50,000 cells/well in 96-well tissue culture dishes 24 h before transfection. For each transfection, 0.1µg of DNA was placed into 10 µl of serum-free culture medium with 1.0 µl Lipofectamine Plus Reagent (Gibco BRL, Gaithersburg, MD) and incubated for 15 min at room temperature. Transfected cells were treated with FTI-277 (10 µM) for 24 h, GGTI-298 (10 µM) for 24 h, or mevastatin (50 µM) for 24 h. These concentrations have been previously shown to completely inhibit respective isoprenyl transferase activities in culture (27, 28), as well as bring about functional changes in actin rearrangement in smooth muscle cells (29). Cells were then lysed in 1x Cell Culture Lysis Reagent (Promega, Madison, WI) at room temperature for 20–30 min and assayed for luciferase activity according to manufacturer instructions (Promega) with a Molecular Devices (Sunnyvale, CA) Lmax luminometer. Results are normalized to Renilla luciferase activity and expressed in relative light units (RLU).

Western Immunoblotting
9/HTEo- PCEPR and PCEP2 cells were plated on 10-cm cell culture dishes and grown to 80–85% confluence at 37°C in 95% O2/5% CO2. Cells were treated with FTI-277 (10 µM) and GGTI-298 (10 µM) for 24 h and TGF-ß1 (2 ng/ml) for 12 h. Cells were lysed in 300 µl of ice-cold lysis buffer (50 mM Tris, pH 7.5, 1% Triton X-100, 100 mM NaCl, 50 mM NaF, 200 µM Na3VO4, 10 µg/ml pepstatin, and leupeptin (Sigma Chemical Co, St. Louis, MO) for 30 min at 4°C. Plates were scraped to suspend cells and transferred to 1 ml micro centrifuge tubes and centrifuged for 10 min at 14,000 rpm at 4°C. The supernatant was extracted and frozen at –80°C for at least 24 h. Protein concentration of samples was measured using the Bio-Rad Dc protein assay system (Bio-Rad, Hercules, CA). Proteins were separated using sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) through 7.5% acrylamide gel. Gels were transferred to Immobilon-P membrane (Millipore, Bedford, MA). Blots were blocked in phosphate-buffered saline (PBS) solution mixed with 5% nonfat dehydrated milk and 0.1% Tween-20 (Sigma Chemical Co) overnight at 4°C. Antibodies against SMAD3 (rabbit) and Erk1 (rabbit) were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA). Blots were incubated with 1:1,000 dilution SMAD3 for 3 h or Erk1 (1:1,000) for 1 h at room temperature and washed three times with PBS with 0.1% Tween-20. Secondary antibody conjugated to horseradish peroxidase (1:4,000 dilution) was added to the blots for 1 h at room temperature and washed at least three times with PBS with 0.1% Tween-20. Signal was visualized by incubating with Super Signal chemiluminescent substrate (Pierce, Rockford, IL) for 8 min at room temperature. The membranes were exposed to Fuji scientific imaging film (Fuji, Tokyo, Japan). Protein expression was quantified by densitometry using Kodak Digital Science 1D software and designated by mean pixel density.

Electromobility Shift Assay
Protocols and reagents for the electromobility shift assay were provided by Panomics (Redwood City, CA) and manufacturer instructions were followed. A biotinylated probe corresponding to sequence of a putative Sp1/Sp3 binding site from the SMAD3 promoter was used with the sequence 5'-GGG AGC GGG CGG GAG CGG GAG-3'. Specificity of binding is controlled for by competing with unlabeled probe and with probe containing mutations within the Sp1/Sp3 binding region with the sequence 5'-GGG AGC GGG CAG AAG CGG GAG-3'. Samples are run on a 6% polyacrylamide/glycerol gel in Tris-Borate-EDTA (TBE) buffer at 4°C in the presence of 25 ng/µl recombinant human Sp1 (rhSp1). Separated samples are then electroblotted onto Biodyne B membrane (Panomics) and ultraviolet crosslinked. Biotinylated oligos are detected by probing with strepavidin conjugated to horseradish peroxidase and visualized by enhanced chemiluminescence.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Augmentation of TGF-ß1 Signaling by Isoprenyl Transferase Inhibition
The first goal of these studies was to determine if inhibition of isoprenyl transferase activity could enhance SMAD3-mediated TGF-ß1 signaling in wild-type (wt) and CF-phenotype airway epithelial cells. Using A549 cells, the ability of the farnesyl transferase inhibitor FTI-277 (FTI) and the geranylgeranyl transferase inhibitor GGTI-286 (GGTI) to influence the plasminogen activator inhibitor-1 (PAI-1) promoter (3tp-lux) was examined in the presence and absence of TGF-ß1 (2 ng/ml). In the absence of TGF-ß1, both FTI and GGTI stimulated a 4.3-fold increase in luciferase expression (n = 24 and P < 0.001 for each) (Figure 1). In the presence of TGF-ß1, FTI and GGTI each augmented luciferase expression compared with TGF-ß1 alone with FTI being far more effective (Figure 1).



View larger version (24K):
[in this window]
[in a new window]
  		Figure 1. Isoprenoid transferase inhibition enhances PAI-1 promoter activation in the presence and absence of TGF-ß1 in A549 cells. Addition of FTI-277 (FTI, 10 µM) or GGTI-286 (GGTI, 10 µM) stimulate an ~ 5-fold increase in luciferase expression driven by the PAI-1 promoter (3tp-lux) normalized to renilla luciferase expression in the absence of TGF-ß1; n = 24, *P < 0.0001 (solid bars). Both FTI and GGTI significantly augment 3tp-lux activation in the presence of TGF-ß1 (TGF), with FTI having a greater effect (n = 24; *P < 0.0001 and **P < 0.05) (slashed bars). Data are presented as the relative light units (RLU) of the ratio firefly/renilla. Error bars represent SEM; n = 24 for each condition. Significance determined by Duncan's multiple range test. Slashed bars, TGF; solid bars, no TGF.

 
Because inhibition of isoprenoid transfer augments TGF-ß1 activity, we examined the effects of FTI and GGTI on SMAD3-mediated TGF-ß1 signaling in a model of CF epithelial cells in which we previously reported reduced SMAD3 expression. 9/HTEo-cells overexpressing the CFTR regulatory (R) domain (pCEPR cells, CF phenotype) were treated with FTI or GGTI. Parallel treatments were performed on control transfected 9/HTEo-pCEP cells (wt phenotype). Correspondingly, we have also shown that pCEPR cells have diminished overall SMAD3-mediated TGF-ß1 signaling compared with pCEP 9/HTEo- cells, although fold increases in 3tp-lux activity are similar in response to TGF-ß1 in both cell types. Treatment of CF-phenotype pCEPR cells with either FTI or GGTI resulted in increased TGF-ß1 responsiveness, whereas only FTI augmented TGF-ß1 signaling in control pCEP cells (Figure 2). Data in Figure 2A are normalized to control pCEP cells treated with TGF-ß1 equaling 100% activity. These data show that the combination of TGF-ß1 and GGTI treatment in CF-phenotype pCEPR cells brings activity up to basal values observed in control cells, but does not restore full activation. Data in Figure 2B are presented as fold increase normalized to untreated cells in each respective cell type. These data demonstrate that treatment with FTI and GGTI, particularly GGTI, augment TGF-ß1 activation of the 3tp-Lux construct to a greater degree in CF phenotype pCEPR cells than observed in control pCEP cells and that SMAD3-mediated TGF-ß1 signaling is partially restored in CF model cells by the inhibition of isoprenyl transferase activity.



View larger version (22K):
[in this window]
[in a new window]
  		Figure 2. Isoprenoid transferase inhibition differentially stimulates PAI-1 promoter activation in 9/HTEo- pCEP (wt) and pCEPR (CF phenotype) cells. (A) Addition of FTI-277 (FTI, 10 µM) significantly increases 3tp-lux activation in control pCEP cells both in the presence and absence of TGF-ß1, whereas GGTI-286 (GGTI, 10 µM) has no effect (*P < 0.001 compared with untreated control and **P < 0.0001 compared with TGF-ß1 alone–treated samples [solid bars]). Both FTI and GGTI significantly augment 3tp-lux activation in CF-phenotype pCEPR cells (#P < 0.001 compared with untreated control and ##P < 0.0001 compared with TGF-ß1 alone–treated samples [striped bars]). Data are presented as the relative light units (RLU) of the ratio firefly/renilla. Error bars represent SEM; n = 14 for each condition. Significance determined by t test. (B) The same data in A are shown as fold stimulation. Error bars represent SEM; n = 14 for each condition. Significance determined by t test comparing identical treatments in pCEP cells (solid bars) and pCEPR cells (slashed bars) (P values shown in parentheses).

 
Regulation of SMAD3 Protein Expression by Isoprenyl Transferase Inhibitors
We have previously reported that reduced SMAD3-mediated TGF-ß1 signaling in CF phenotype pCEPR cells compared with control pCEP cells is due to diminished SMAD3 protein expression (21). Based on previous data demonstrating isoprenoid/cholesterol regulation of Stat1 and NOS2 regulation in CF cells (22), we hypothesized that elevated TGF-ß1 signaling in pCEPR cells in response to FTI and GGTI may be in part due to restoration of SMAD3 protein expression. CF phenotype pCEPR cells were treated with either FTI-277 (10 µM) or GGTI-286 (10 µM) for 24 h and analyzed for SMAD3 protein expression. SMAD3 protein expression in pCEPR cells was restored to control pCEP levels in response to both FTI and GGTI exposure (Figure 3). These data show that isoprenoid-dependent mechanisms are responsible for reduced SMAD3 expression in CF epithelial cells, and that restoration of SMAD3 expression is one contributing mechanism by which isoprenyl transferase inhibitors increase TGF-ß1–stimulated signaling in CF-phenotype cells. These data are consistent with our previous findings that isoprenoid/cholesterol dependent signaling regulates CF-related alterations in Stat1 and NOS2 regulation.



View larger version (31K):
[in this window]
[in a new window]
  		Figure 3. Restoration of SMAD3 protein expression in CF-phenotype pCEPR cells in response to farnesyl and geranylgeranyl transferase inhibition. (A) SMAD3 protein expression in 9/HTEo- pCEP2 and pCEPRF cells. Cells were treated with FTI (10 µM) or GGTI (10 µM) for 24 h as indicated. Erk1 content is determined for protein loading control. (B) Densitometry analysis of SMAD3 protein content. Data are presented as a SMAD3/Erk1 ratio. Values are an average of six replicates, and error bars represent SEM (*P < 0.001). Significance determined by t test compared with untreated pCEP cells. Solid bars, pCEP; slashed bars, pCEPR.

 
SMAD3 Promoter
To more effectively identify mechanisms necessary for the regulation of SMAD3 expression, we amplified a 1,892-bp region of genomic DNA upstream of the human SMAD3 gene. This amplified region was subcloned into the pGL3basic (pGL3B) firefly luciferase plasmid to create a human SMAD3 promoter-luciferase (SMAD3p-Luc) construct. A diagram of the promoter region with potential regulatory elements is shown in Figure 4. To confirm that this region possessed promoter capabilities, basal luciferase expression of pGL3basic and SMAD3p-Luc were compared. A549 cells were transfected using a lipofectamine protocol with either pGL3B or SMAD3p-luc along with pRL (renilla luciferase expressing vector) as an internal control for transfection efficiency. Luciferase expression driven by the putative SMAD3 promoter region was 116-fold greater than was observed with the parent pGL3basic construct (Figure 5A) in A549 cells, indicating promoter capabilities.



View larger version (63K):
[in this window]
[in a new window]
  		Figure 4. Human SMAD3 promoter. (A) Sequence of the full-length –1892 to +13 human SMAD3 promoter region used in the SMAD3p-Luc promoter construct. (B) Diagram of putative regulatory sites within the human SMAD3 promoter region. Data indicate that the cluster of Sp1 sites between –408 and –849 play an important regulatory role in SMAD3 promoter activation. Sites of restriction digests used to make reporter constructs of various lengths are numbered by bp length and indicated by the gray circles.

 


View larger version (11K):
[in this window]
[in a new window]
  		Figure 5. SMAD3 promoter driven luciferase expression in A549 cells. (A) Luciferase expression driven by insertion of the SMAD3 promoter region into pGL3basic compared with pGL3B alone. (B) SMAD3 promoter function is increased by treatment of A549 cells with either farnesyl transferase inhibitor (FTI) or geranylgeranyl transferase inhibitor (GGTI) compared with nontreated cells (NT). Data are normalized to pRL renilla luciferase expression used as an internal control for transfection efficiency. Data are presented as the relative light units (RLU) of the ratio firefly/renilla. Error bars represent SEM; n = 16 for each condition. Significance determined by Duncan's multiple range test (*P < 0.001).

 
Prenylation-Dependent Regulation of the SMAD3 Promoter
We tested the effects of FTI-277 (10 µM) and of GGTI-286 (10 µM) on SMAD3 promoter function. Both FTI and GGTI significantly increased SMAD3 promoter function by ~ 2-fold, respectively, as determined by luciferase expression in A549 cells (Figure 5B). These studies show that farnesyl and geranylgeranyl transferases act to decrease SMAD3 promoter activity.

To identify a mechanism leading to isoprenyl transferase-mediated regulation of SMAD3 expression, various promoter constructs were made to identify sites important to FTI and GGTI function. Sequential truncations of the full-length SMAD3 promoter were made (Figure 6A) and analyzed for FTI and GGTI regulation. Basal activity and GGTI-mediated induction of promoter activity clearly requires the region between –408 and –849 bp (Figure 6B). FTI-mediated effects on SMAD3 promoter are not apparently limited to that region, possibly suggesting a secondary or nonspecific interaction. The region of the SMAD3 promoter between –408 and –849 bp contains a cluster of six GC-rich Sp1/Sp3-binding sites. To specifically examine the importance of this region, two more constructs were made. The first construct consists of the full-length promoter with the Sp1/Sp3-rich region deleted ({Delta}Sp1SMAD3p-luc) (Figure 7A). The second construct is the Sp1/Sp3 region isolated from the SMAD3 promoter driving luciferase expression (Sp1SMAD3p-luc) (Figure 7A). GGTI stimulates the Sp1/Sp3-containing construct, whereas GGTI has no effect on the Sp1/Sp3-deletion construct (Figure 7B). FTI stimulates both constructs, indicating some influence on Sp1/Sp3-mediated signaling, but also indicating that another interaction is taking place.



View larger version (39K):
[in this window]
[in a new window]
  		Figure 6. Identification of isoprenoid responsive regions within the human SMAD3 promoter. (A) Schematic diagrams of full-length SMAD3 promoter construct (SMAD3p-Luc) and sequential truncations of the promoter constructed to identify functional regions of the promoter. (B) Isoprenoid transferase inhibitor-mediated regulation of the SMAD3 promoter was tested on truncations of the promoter region compared with nontreated (NT) samples. GGTI-mediated activation of the SMAD3 promoter is dependent on sequence elements between 849 and 408 bp, a region with a cluster of 8 Sp1 sites. Data are presented as the relative light units (RLU) of the ratio firefly/renilla. Error bars represent SEM; n = 16 for each condition. Significance determined by t test (*P < 0.001 and #P < 0.05 compared with respective NT control). Solid bars, NT; wide striped bars, FTI; thin striped bars, GGTI.

 


View larger version (22K):
[in this window]
[in a new window]
  		Figure 7. Isoprenoid regulation of Sp1 binding region of the SMAD3 promoter. (A) Schematic diagrams of full-length SMAD3 promoter construct (SMAD3p-Luc), a construct lacking a region rich in Sp1 sites ({Delta}Sp1SMAD3 -Luc), and a construct comprised solely of the Sp1 rich region (Sp1SMAD3-Luc). (B) FTI- and GGTI-mediated regulation of the SMAD3 promoter was tested on SMAD3p-Luc, {Delta}Sp1SMAD3-Luc, and Sp1SMAD3-Luc constructs and compared with nontreated (NT) samples. Data are presented as the relative light units (RLU) of the ratio firefly/renilla. Error bars represent SEM; n = 16 for each condition. Significance determined by t test (*P < 0.001 compared with respective NT control). Solid bars, NT; wide striped bars, FTI; thin striped bars, GGTI.

 
Sp1/Sp3 Regulation of SMAD3 Expression
To verify that Sp1/Sp3-mediated interactions indeed influence SMAD3 expression regulation, the effect of mithramycin on both SMAD3 promoter function and SMAD3 protein expression in A549 cells were examined. Mithramycin has been demonstrated to be an effective, although relatively nonspecific, inhibitor of Sp1/Sp3 transcription activity by interfering with their binding to DNA (30–32). SMAD3 promoter function was inhibited by ~ 50% in the presence of mithramycin (Figure 8A). To specifically verify that Sp1 is capable of interacting with the SMAD3 promoter, an EMSA was performed using a probe comprised of an Sp1/Sp3 binding site within the SMAD3 promoter. Figure 8B demonstrates that purified recombinant human Sp1 (rhSp1) binds specifically to this region of the SMAD3 promoter. To confirm that promoter regulation correlated with SMAD3 protein expression, A549 cells were exposed to mithramycin for 24 h and exhibited a reduction in SMAD3 protein expression (Figure 8C). These data are consistent with the proposal that Sp1/Sp3 function is involved in the regulation of SMAD3 expression.



View larger version (17K):
[in this window]
[in a new window]
  		Figure 8. Sp1-mediated regulation of SMAD3 expression. (A) Sp1-dependent TGF-ß1–mediated stimulation of the SMAD3 promoter. (A) The effect of the Sp1 inhibitor mithramycin (Mith) was examined to confirm the role of Sp1 in SMAD3 promoter function. Asterisk indicates values significantly lower than NT samples. Significance determined by t test (*P < 0.01). (B) Purified recombinant human (rh)Sp1 was used as a positive control for binding. Lane 1: no protein; lane 2: rh Sp1; lane 3: rhSp1 with unlabeled probe; lane 4: rhSp1 with unlabeled probe containing binding site mutations. (C) Inhibition of Sp1 activity diminishes SMAD3 protein content in A549 cells. Cells were treated for 18 h with mithramycin (Mith, 8 µM) and control cells were left not treated (NT). Shown samples are representative of triplicate experiments. Erk1 content is determined for protein loading control.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously reported diminished expression of the TGF-ß1 signaling protein SMAD3 in CF epithelial cells (21). The goal of this study was to identify a mechanism responsible for CF-related reductions in SMAD3 protein. We have recently demonstrated that isoprenoid/cholesterol-dependent mechanisms are responsible for altered Stat1 and NOS2 regulation in CF epithelial cells (22). The hypothesis of this study was that isoprenoid-dependent signaling would also be responsible for the regulation of SMAD3 expression.

Precedent for a relationship between isoprenoid-dependent pathways and TGF-ß1 signaling has been reported. Kucich and colleagues reported that inhibition of geranylgeranyl transferase I activity significantly augments fibronectin transcription in response to TGF-ß1 (33). Similarly, Bulus and coworkers demonstrated that inhibition of farnesyl transferase activity reverses Ras-dependent negative regulation of TGF-ß type II (TGFßRII) function (34). Neither of these studies addressed SMAD3 regulation directly. We have found that inhibition of the prenylation pathway results in increased TGF-ß1 responsiveness in CF epithelial cells. Furthermore, inhibition of isoprenyl transferase activity (either farnesyl or geranylgeranyl transferases) leads to restoration of SMAD3 protein expression in a cultured cell model of CF epithelium. One unresolved finding, however, is that apparently complete restoration of SMAD3 protein expression by isoprenyl transferase inhibition results in only partial TGF-ß1–mediated SMAD3 signaling improvement. These data suggest that reduced SMAD3 expression may not be the only lesion in the TGF-ß1 signaling pathway in this model of CF epithelium. One possible explanation for these findings is that TGFßRII function is also affected in this model of CF epithelial cells and our conditions are insufficient to modify this phenotype. A role for TGFßRII, a Ras regulated protein, may also explain the differential influence of FTI compared with GGTI. The present study and our previous work have focused solely on SMAD3 regulation in CF epithelium. The incomplete correction of SMAD3 signaling, despite corrected SMAD3 protein expression, indicates that a more comprehensive examination of TGF-ß1-mediated signaling in CF cells is warranted.

Using a SMAD3 promoter reporter construct, it was determined that both farnesyl and geranylgeranyl transferase inhibition stimulated an ~ 2-fold increase in promoter activity. Sequential truncations of the promoter revealed that a cluster of Sp1/Sp3 sites within the promoter is critical for promoter function and isoprenoid regulation. More specific analysis of these sites further demonstrates the importance of Sp1/Sp3 signaling and also indicates differing mechanisms for farnesyl- and geranylgeranyl-dependent pathways. The {Delta}Sp1SMAD3p-luc construct is unaffected by GGTI, whereas the full-length SMAD3p-luc and the Sp1SMAD3p-luc constructs are stimulated by the presence of GGTI. These data are consistent with a report by Adnane and colleagues who demonstrate activation of Sp1 with GGTI-286 (24). However, all SMAD3 promoter constructs are stimulated to an extent by FTI. These data do not rule out a role of farnesyl-related regulation of Sp1 signaling, but do suggest a secondary mode of regulation. The regulation of TGFßRII by Ras and farnesylated proteins reported by Bulus and coworkers represents a candidate mechanism for the secondary pathway we observe with FTI.

The restoration of SMAD3 protein expression in CF epithelial cells by inhibition of isoporenyl transferases is consistent with our previous findings demonstrating normalization of Stat1 and NOS2 regulation by inhibiting the isoprenoid/cholesterol pathway (22). These combined data strongly indicate that inherent changes within the isoprenoid/cholesterol pathway significantly contribute to secondary cell signaling alteration in CF epithelial cells. The source of these isoprenoid/cholesterol pathway alterations is currently unclear. Reports of lipid imbalances in CF cells suggest that impairment of proper lipid transport pathways may be a source of altered isoprenoid-dependent signaling (35, 36). The above data also imply that Sp1/Sp3 signaling may be impaired in CF epithelial cells. There is no direct evidence of CF-related impairment of Sp1/Sp3 signaling in the literature, and we are currently pursuing these studies. Circumstantial evidence does indicate a potential decrease in Sp1/Sp3 function in CF. Sp1/Sp3 activity is essential for interleukin-10 expression, and multiple articles demonstrate reduced expression of the anti-inflammatory cytokine interleukin-10 in CF epithelium, although no specific mechanism for reduced expression has been identified (12, 37–38).

In conclusion, this study demonstrates improvement of SMAD3-mediated TGF-ß1 signaling and restoration of SMAD3 protein expression in a specific model of CF epithelial cells in response to isoprenyl transferase inhibition. One caveat to this study is the use of a single-cell model of CF epithelium. The primary goal of this study was to examine SMAD3 regulatory process; therefore, a model system with clear SMAD3 expression differences was chosen. The pCEP and pCEPR 9/HTEo- cells have been a consistent and useful model system for the study of CF-related cell signaling changes. However, the use of a single, clonal model system that may not be completely representative of CF physiology due to potential selection pressures of being in culture do limit universal conclusions about the applicability to all CF cells. The results of this study illuminate new SMAD3 regulatory pathways that are clearly discernable in this specific model system. The regulation of SMAD3 expression by isoprenoids is mediated at least in part by Sp1/Sp3 activity as determined by SMAD3 promoter studies. These data are consistent with our previous findings demonstrating isoprenoid-mediated control of Stat1 and NOS2 regulation in CF epithelial cells and suggest that the isoprenoid/cholesterol pathway is a potential site of novel therapeutic intervention to restore secondary cell signaling abnormalities identified in CF.


   Acknowledgments
 
This work is supported by a grant from the Cystic Fibrosis Foundation and a Clinical Fellowship Award from the Cystic Fibrosis Foundation to J.Y.L. The authors thank Drs. P. Davis and J. Massague for providing materials necessary for the completion of this study, and P. Bead for technical assistance.

Received in original form December 17, 2003

Received in final form February 6, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. MacLusky, I., and H. Levison. 1998. Cystic fibrosis. In Kendig's Disorders of the Respiratory Tract in Children, 6th ed. V. Chernick and T. Boat, editors. W. B. Saunders Company, Philadelphia. 838–876.
  2. Davis, P., M. Drumm, and M. Konstan. 1996. Cystic fibrosis: state of the art. Am. J. Respir. Crit. Care Med. 154:1229–1256.[Medline]
  3. Konstan, M., and M. Berger. 1993. Infection and inflammation of the lung in cystic fibrosis. In Cystic Fibrosis. Lung Biology in Health and Disease, Vol. 64. P. Davis, editor. Marcel Dekker, New York. 219–276.
  4. Balough, K., M. McCubbin, M. Weinerger, W. Smits, R. Ahrens, and R. Fick. 1995. The relationship between infection and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr. Pulmonol. 20:63–70.[Medline]
  5. Konstan, M., and M. Berger. 1997. Current understanding of the inflammatory process in cystic fibrosis: onset and etiology. Pediatr. Pulmonol. 24:137–142.[CrossRef][Medline]
  6. Armstrong, D., K. Grimwood, R. Carzino, J. Gutierrez, J. Hull, A. Olinsky, E. Phelan, C. Roberston, and P. Phelan. 1995. Lower respiratory infection and inflammation in infants with newly diagnosed cystic fibrosis. BMJ 310:1571–1572.[Free Full Text]
  7. Muhlebach, M., P. Stewart, M. Leigh, and T. Noah. 1999. Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients. Am. J. Respir. Crit. Care Med. 160:186–191.[Abstract/Free Full Text]
  8. Berger, M., and M. Konstan. 1999. Immunopathogenesis in cystic fibrosis lung disease. In Cystic Fibrosis in Adults. T. Yankaskas and M. Knowles, editors. Lippincott-Raven, Philadelphia. 115–141.
  9. Khan, T., J. Wagener, T. Bost, J. Martinez, F. Accurso, and D. Riches. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151:1075–1082.[Abstract]
  10. Chmiel, J., M. Konstan, J. Knesebeck, J. Hilliard, T. Bonfield, D. Dawson, and M. Berger. 1999. IL-10 attenuates excessive inflammation in chronic Pseudomonas infection in mice. Am. J. Respir. Crit. Care Med. 160:2040–2047.[Abstract/Free Full Text]
  11. Bonfield, T., J. Panuska, M. Konstan, K. Hilliard, J. Hilliard, H. Ghnaim, and M. Berger. 1995. Inflammatory cytokines in cystic fibrosis lungs. Am. J. Respir. Crit. Care Med. 152:2111–2118.[Abstract]
  12. Bonfield, T., M. Konstan, P. Burfeind, J. Panuska, J. Hilliard, and M. Berger. 1995. Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine inerleukin-10, which is downregulated in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 13:257–261.[Abstract]
  13. Osika, E., J. Cavaillon, K. Chadelat, M. Boule, C. Fitting, G. Tournier, and A. Clement. 1999. Distinct sputum cytokine profiles in cystic fibrosis and other chronic inflammatory airway disease. Eur. Respir. J. 14:339–346.[Abstract]
  14. Robers, A., and M. Sporn. 1993. Physiological action and clinical applications of transforming growth factor beta. Growth Factors 8:1–9.[Medline]
  15. Barnard, J., R. Lyons, and H. Mosies. 1990. The cell biology of transforming growth factor ß. Biochim. Biophys. Acta 1032:79–87.[Medline]
  16. Whitman, M. 1998. Smads and early developmental signaling by the TGF-ß superfamily. Genes Dev. 12:2445–2462.[Free Full Text]
  17. Arkwright, P., S. Laurie, M. Super, V. Pravica, M. Schwarz, A. Webb, and I. Hutchinson. 2000. TGF-ß1 genotype and accelerated decline in lung function of patients with cystic fibrosis. Thorax 55:459–462.[Abstract/Free Full Text]
  18. Lewindon, P., T. Pereira, A. Hoskins, K. Bridle, R. Williamson, R. Shepherd, and G. Ramm. 2002. The role of hepatic stellate cells and transforming growth factor-ß1 in cystic fibrosis liver disease. Am. J. Pathol. 160:1705–1715.[Abstract/Free Full Text]
  19. Massague, J., and Y. G. Chen. 2000. Controlling TGF-ß signaling. Genes Dev. 14:627–644.[Free Full Text]
  20. Heldin, C., K. Miyazono, and P. Dijke. 1997. TGF-ß signaling from cell membrane to nucleus through Smad proteins. Nature 390:465–471.[CrossRef][Medline]
  21. Kelley, T., H. Elmer, and D. Corey. 2001. Reduced SMAD3 protein expression and altered transforming growth factor-ß1-mediated signaling in cystic fibrosis epithelial cells. Am. J. Respir. Cell Mol. Biol. 25:732–738.[Abstract/Free Full Text]
  22. Kreiselmeier, N. E., N. C. Kraynack, D. A. Corey, and T. J. Kelley. 2003. Statin-mediated correction of STAT1 signaling and inducible nitric oxide synthase expression in cystic fibrosis epithelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol. 285:L1286–L1295.[Abstract/Free Full Text]
  23. Molnar, G., M. Dagher, M. Geiszt, J. Settleman, and E. Ligeti. 2001. Role of prenylation in the interaction of rho-family small GTPases with GTPase activating proteins. Biochemistry 40:542–549.
  24. Adnane, J., F. Bizouarn, Y. Qian, A. Hamilton, and S. M. Sebti. 1998. P21 (WAF1/CIP1) is up regulated by the geranylgeranyltransferase I inhibitor GGTI-298 through a transforming growth factor beta- and Sp1-responsive element: involvement of the small GTPase rhoA. Mol. Cell. Biol. 18:6962–6970.[Abstract/Free Full Text]
  25. Park, H., and J. Galper. 1999. 3-Hydroxy-3-methylglutaryl CoA reductase inhibitors up-regulate transforming growth factor-ß signaling in cultured heart cells via inhibition of geranylgeranylation of RhoA GTPase. Proc. Natl. Acad. Sci. USA 96:11525–11530.[Abstract/Free Full Text]
  26. Perez, A., K. Risma, E. Eckman, and P. Davis. 1996. Over expression of R domain eliminates camp-stimulated Cl-secretion in 9/HTEo-cells in culture. Am. J. Physiol. 217:L35–L92.
  27. Lerner, E. C., Y. Qian, M. A. Blaskovich, R. D. Fossum, A. Vogt, J. Sun, A. D. Cox, C. J. Der, A. D. Hamilton, and S. M. Sebti. 1995. Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J. Biol. Chem. 270:26802–26806.[Abstract/Free Full Text]
  28. Lerner, E. C., Y. Qian, A. D. Hamilton, and S. M. Sebti. 1995. Disruption of oncogenic K-Ras4B processing and signaling by a potent geranylgeranyltransferase I inhibitor. J. Biol. Chem. 270:26770–26773.[Abstract/Free Full Text]
  29. Lesh, R. E., C. W. Emala, H. T. Lee, D. Zhu, R. A. Panettieri, and C. A. Hirshman. 2001. Inhibition of gernylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells. Am. J. Physiol. Lung Cell Mol. Physiol. 281:L824–L831.[Abstract/Free Full Text]
  30. Blume, S. W., R. C. Snyder, R. Ray, S. Thomas, C. A. Koller, and D. M. Miller. 1991. Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo. J. Clin. Invest. 88:1613–1621.
  31. Greenwel, P., Y. Inagaki, W. Hu, M. Walsh, and F. Ramirez. 1997. Sp1 is required for the early response of alpha2(I) collagen to transforming growth factor-beta1. J. Biol. Chem. 272:19738–19745.[Abstract/Free Full Text]
  32. Krikun, G., F. Schatz, N. Mackman, S. Guller, R. Demopoulos, and C. J. Lockwood. 2000. Regulation of tissue factor gene expression in human endometrium by transcription factors Sp1 and Sp3. Mol. Endocrinol. 14:393–400.[Abstract/Free Full Text]
  33. Kucich, U., J. C. Rosenbloom, G. Shen, W. R. Abrams, A. D. Hamilton, S. M. Sebti, and J. Rosenbloom. 2000. TGF-beta1 stimulation of fibronectin transcription in cultured human lung fibroblasts requires active geranylgeranyl transferase I, phosphatidylcholine-specific phospholipase C, protein kinase C-delta, and p38, but not erk1/erk2. Arch. Biochem. Biophys. 374:313–324.[CrossRef][Medline]
  34. Bulus, N. M., H. M. Sheng, N. Sizemore, S. M. Oldham, J. V. Barnett, R. J. Coffey, D. R. Beauchamp, and J. A. Barnard. 2000. Ras-mediated suppression of TGFbetaRII expression in intestinal epithelial cells involves Raf-independent signaling. Neoplasia 2:357–364.[CrossRef][Medline]
  35. Boujaoude, L. C., C. Bradshaw-Wilder, C. Mao, J. Cohn, B. Ogretmen, Y. A. Hannun, and L. M. Obeid. 2001. Cystic fibrosis transmembrane regulator regulates uptake of sphingoid base phosphates and lysophosphatidic acid: modulation of cellular activity of sphingosine 1-phosphate. J. Biol. Chem. 276:35258–35264.[Abstract/Free Full Text]
  36. Freedman, S. D., M. H. Katz, E. M. Parker, M. Laposata, M. Y. Urman, and J. G. Alvarez. 1999. A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in cftr(–/–) mice. Proc. Natl. Acad. Sci. USA 96:13995–14000.[Abstract/Free Full Text]
  37. Soltys, J., T. Bonfield, J. Chmiel, and M. Berger. 2002. Functional IL-10 deficiency in the lung of cystic fibrosis (cftr(–/–)) and IL-10 knockout mice causes increased expression and function of B7 co-stimulatory molecules on alveolar macrophages. J. Immunol. 168:1903–1910.[Abstract/Free Full Text]
  38. Tone, M., M. J. Powell, Y. Tone, S. A. Thompson, and H. Waldmann. 2000. IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3. J. Immunol. 165:286–291.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
N. M. White, D. Jiang, J. D. Burgess, I. R. Bederman, S. F. Previs, and T. J. Kelley
Altered cholesterol homeostasis in cultured and in vivo models of cystic fibrosis
Am J Physiol Lung Cell Mol Physiol, February 1, 2007; 292(2): L476 - L486.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. R. Hallows, A. C. Fitch, C. A. Richardson, P. R. Reynolds, J. P. Clancy, P. C. Dagher, L. A. Witters, J. K. Kolls, and J. M. Pilewski
Up-regulation of AMP-activated Kinase by Dysfunctional Cystic Fibrosis Transmembrane Conductance Regulator in Cystic Fibrosis Airway Epithelial Cells Mitigates Excessive Inflammation
J. Biol. Chem., February 17, 2006; 281(7): 4231 - 4241.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2003-0447OCv1
31/2/234    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, J. Y.
Right arrow Articles by Kelley, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, J. Y.
Right arrow Articles by Kelley, T. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2004 American Thoracic Society.