help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on January 27, 2005, doi:10.1165/rcmb.2004-0345OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0345OCv1
32/4/350    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 Han, S.
Right arrow Articles by Roman, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Han, S.
Right arrow Articles by Roman, J.
American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 350-359, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0345OC

Peroxisome Proliferator–Activated Receptor-{gamma} Ligands Inhibit {alpha}5 Integrin Gene Transcription in Non–Small Cell Lung Carcinoma Cells

ShouWei Han, Hilda N. Rivera and Jesse Roman

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, Emory University School of Medicine; and Atlanta Veterans Affairs Medical Center, Atlanta, Georgia

Correspondence and requests for reprints should be addressed to ShouWei Han, M.D., Ph.D., Division of Pulmonary, Allergy and Critical Care Medicine, Emory University School of Medicine, Whitehead Bioresearch Building, 615 Michael Street, Suite 205-M, Atlanta, GA 30322. E-mail: shan2{at}emory.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
We previously showed that fibronectin stimulates the growth of non–small cell lung carcinoma (NSCLC) cells through integrin {alpha}5ß1–dependent signals. We also demonstrated that peroxisome proliferator–activated receptor (PPAR){gamma} ligands inhibit lung carcinoma cell growth. Because {alpha}5ß1 activation elicits cellular signals linked to cell survival and regulation of cell cycle progression, we studied the effects of PPAR{gamma} ligands on its expression. We found that PPAR{gamma} ligands decreased mRNA and protein expression of the {alpha}5 subunit of the {alpha}5ß1 heterodimer in NSCLC; this was associated with reduced NSCLC adhesion to fibronectin. The suppressive effect of the PPAR{gamma} ligands BRL 49653 and GW1929, but not PGJ2, on {alpha}5 gene expression were reversed by GW9662, an antagonist of PPAR{gamma}. GW1929 activated the extracellular regulated kinase (Erk), and an inhibitor of the Erk pathway (PD98095) prevented its effect on {alpha}5. PPAR{gamma} ligands also reduced {alpha}5 gene promoter activity, and this was blocked by Erk antisense oligonucleotides. PPAR{gamma} ligands GW1929 and BRL49653inhibited AP-1 DNA binding, whereas 15d-PGJ2 inhibited Sp1 DNA binding; both effects were blocked by Erk antisense oligonucleotides. GW1929 partially blocked fibronectin-induced NSCLC cell growth, but did not affect cell growth induced by epidermal growth factor. These results suggest that PPAR{gamma} ligands inhibit {alpha}5 expression in NSCLC through Erk-related signals.

Key Words: {alpha}5 integrin • Erk • human lung carcinoma • PPAR{gamma} • transcription factors


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In general, cell growth and survival requires the interaction of cell surface integrin receptors with the surrounding extracellular matrix (1, 2). Integrins comprise a family of heterodimeric transmembrane glycoproteins consisting of noncovalently linked {alpha} and ß subunits that regulate cell–matrix interactions, cell–cell adhesion, and cellular differentiation, among other processes, and that have been implicated in wound healing. Integrins are also thought to be partly responsible for the altered invasive properties of tumor cells (3, 4). In many tumors, the overexpression of the fibronectin integrin receptor {alpha}5ß1 is associated with a more malignant phenotype (5). {alpha}5ß1 is generally not found in normal lung tissue, but it is expressed in a considerable fraction of lung carcinomas (6). The expression of {alpha}5ß1 in non–small cell lung carcinomas (NSCLC) correlates with tumor progression, and its overexpression is associated with decreased survival (6). We recently demonstrated that fibronectin stimulates the proliferation and the survival of NSCLC cells through {alpha}5ß1-mediated signals that trigger the induction of COX-2 expression and the production of the autocrine factor PGE2 (7).

In view of the above, we are in search of agents that affect {alpha}5ß1 expression, and this search led us to the peroxisome proliferator–activated receptor (PPAR){gamma} ligands. Here, we explore the effects of PPAR{gamma} ligands on {alpha}5 gene expression, and the contribution of this mechanism to the ability of PPAR{gamma} ligands to suppress NSCLC cell growth. The PPARs are members of the steroid-thyroid hormone superfamily of ligand-activated transcription factors (8). PPARs, like other hormone nuclear receptors, heterodimerize with the retinoid X receptor (RXR) and bind to specific DNA response elements termed DR-1, which consist of a direct repeat of two AGGTCA half-sites separated by a single intervening nucleotide (8, 9). Of the three PPAR isoforms identified, PPAR{alpha}, ß (previously referred to as {delta}), and {gamma}, PPAR{gamma} has been the most intensively investigated. This receptor participates in fundamental biological processes related to cellular differentiation, insulin sensitivity, and cell cycle control (8, 9). Several studies have implicated PPAR{gamma} in lung cancer as well. PPAR{gamma} is expressed in lung carcinomas, and PPAR{gamma} agonists induce growth arrest and promote changes associated with differentiation as well as apoptosis in a variety of lung carcinoma cell lines. For example, PPAR{gamma} agonists have been found to inhibit the growth of A549 adenocarcinoma cells due to G0/G1 cell cycle arrest through the downregulation of G1 cyclins D and E (10). Most notably, the treatment of NSCLC tumor–bearing SCID mice with a PPAR{gamma} ligand inhibited tumor growth and metastasis (10).

A connection between PPAR{gamma} activation and {alpha}5 integrin expression has not been reported, although PPAR{gamma} ligands have been shown to regulate other integrin genes (11). Here, we investigate the effects of PPAR{gamma} ligands on {alpha}5 integrin gene expression in human NSCLC cells. Furthermore, we identify potential novel interactions of ligand-activated PPAR{gamma} with the transcription factors AP-1 and Sp1 in the {alpha}5 gene promoter. Our results demonstrate that the inhibitory effect of PPAR{gamma} ligand on lung carcinoma cell growth is mediated, in part, through decreased {alpha}5 integrin expression, and suggest that targeting the integrin {alpha}5 receptor gene might provide a novel mechanism by which PPAR{gamma} ligands inhibit human lung carcinoma cell growth.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Culture and Chemicals
The NSCLC cell lines H1838 and H2106 were obtained from The American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, HEPES buffer, 50 IU/ml penicillin/streptomycin, and 1 µg amphotericin (complete medium) as previously described (12). Rosiglitazone (BRL49653 15d-PGJ2 and GW9662 were obtained from Caymen Chemical Co (Ann Arbor, MI). Epidermal growth factor (EGF) and GW1929 were purchased from Sigma Chemical Inc. (St. Louis, MO). Poly (dI-dC) and [methyl-3H] thymidine were purchased from Amersham Biosciences (Piscataway, NJ). {gamma}-32P-dATP was purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA). The Mek-1/Erk inhibitor PD98095 was purchased from Calbiochem (San Diego, CA). Antibodies against Erk1, Erk2, phosphor-Erk, Sp1, c-Jun and c-Fos were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). C/EBPß was purchased from Cell Signaling Technology, Inc. (Beverly, MA). The integrin {alpha}5 monoclonal antibody (CD49e) was purchased from BD Biosciences Pharmingen (San Diego, CA). The Gel Shift Assay System and the Dual-Luciferase Report Assay kit were obtained from Promega (Madison, WI). LightCycler-FastStart DNA Master SYBR Green I kit and the 5' DNA Terminus Labeling System were purchased from Roche Molecular Biochemicals (Indianapolis, IN). All RT-PCR kit components were obtained from Perkin Elmer Co. (Foster City, CA). All other chemicals were purchased from Sigma unless otherwise indicated.

Antisense Erk Oligonucleotide Treatment of Cells
The sequences of phosphorothioate oligodeoxynucleotides (ODN) that target the translation initiation codons of Erk1 and Erk2 mRNAs were as follows: the antisense-ODN sequence was 5'-GCC-GCC-GCC-GCC-GCC-AT-3' (referred to as AS ERK2) and the corresponding sense-ODN sequence was 5'-ATG-GCG-GCG-GCG-GCG-GC-3' (referred to as S ERK2) according to published data (13). For the transfection of Erk ODN, cells were grown to 70% confluence, and a 1 µM concentration of Erk phosphorothioate ODN mixed with 3 µl of FuGENE 6 transfection reagent per well of serum-free medium was added to the cells for 24 h at 37°C. The medium was changed into regular culture medium in the morning, and the cells were treated with PPAR{gamma} ligands for an additional 24 h for Luciferase activity assays, gel mobility shift assays, and Western blot analyses.

[Methyl-3H] Thymidine Incorporation Assay
Human NSCLC cells were incubated with 1 Ci/mL [methyl-3H] thymidine (specific activity 250 Ci/mmol; Amersham, Piscataway, NJ), fibronectin (20 µg/ml), or EGF (20 ng/ml) in the presence or absence of GW1929 (10 µM) for up to 24 h. The medium was removed and the attached cells were washed with 1x PBS. Afterwards, the attached cells were treated with ice-cold 6% trichloroacetic acid (TCA) at 4°C for 20 min and washed once with 6% TCA. The cells were then solubilized with 0.1 N NaOH and counted in a liquid scintillation counter in 4 ml of scintillation fluid.

Western Blot Analysis
The procedure was performed as previously described (14). Protein concentrations were determined by the Bio-Rad protein assay (Hercules, CA). Equal amounts of protein from whole cell lysates (50 µg) were solubilized in 2x SDS-sample buffer and separated on SDS–6% polyacrylamide gels. Blots were incubated with monoclonal antibodies raised against mouse {alpha}5 (1:5,000 dilution), Erk1/2, phosphor-Erk, phosphor-c-Jun, Sp1, c-Jun and c-Fos (1:2,000), or C/EBPß antibodies (1:1,000). The blots were washed and followed by incubation with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (1:2,000 dilution; Cell Signaling Technology). The blots were washed, transferred to freshly made ECL solution (Amersham, Arlington, IL) for 1 min, and exposed to X-ray film. In controls, the {alpha}5 antibody was omitted or replaced with a control rabbit IgG.

Reverse Transcriptase PCR
Total RNA was prepared from human NSCLC cells with TRIzol Reagent (GIBCO, BRL, Rockville, MD) according to the manufacturer's instructions. To amplify the 171-bp {alpha}5 and 200-bp GAPDH cDNA fragments, the DNA sequence of the primers used for the amplification of the human {alpha}5 transcript were: forward primer: 5'-GGC AGC TAT GGC GTC CCA CTG TGG-3', reverse primer: 5'-GGC ATC AGA GGT GGC TGG AGG CTT-3' (171-bp PCR product); and for GAPDH sense (5'-CCATGGAGAAGGCTGGGG-3'), antisense (5'-CAAAGTTGTCATGGATGACC-3') according to published data (15, 16). RT-PCR was performed as previously described (16). Analysis of amplicons was accomplished on 1% agarose gel containing 0.2 µg/µl ethidium bromide and visualized under ultraviolet transiluminator.

Real-Time RT-PCR
This procedure was described previously (16). Final results were expressed as n-fold differences in {alpha}5 gene expression relative to the GAPDH gene. All PCR reactions using LightCycler-FastStart DNA Master SYBR Green I kit were performed in the Cepheid Smart-Cycler real-time PCR cycler (Sunnyvale, CA) (16). Experiments were performed in triplicate for each data point.

Cell Adhesion Assay
Human NSCLC cells were seeded on plates precoated with fibronectin (20 µg/ml) in the presence or absence of GW1929 or rosiglitazone (10 µM each). After 24 h, nonadherent cells were washed, and adherent cells were quantified with a colorimetric assay that detects the intracellular enzyme hexosaminidase as described before (17).

Plasmids
All promoter sequences were taken from the p{alpha}5-CAT reporter vectors as described previously (17). Briefly, the promoters from the p{alpha}5-CAT constructs (–923, –178, –92, –41, and –27 bp) were digested with the appropriate restriction endonucleases, purified by agarose gel electrophoresis, and ligated into the pGL2 luciferase (Luc) reporter vector (Promega) with T4 DNA ligase. All p{alpha}5-Luc promoter constructs were sequenced to ensure that no mutations occurred during construction. Synthetic Renilla Luciferase Report Vector (phRL-TK) was obtained from Promega.

Transient Transfection Assays
Human NSCLC cells were seeded at a density of 1 x 105 cells/well in 6-well dishes and grown to 60% confluence. For each well, the plasmid DNA containing wild-type and a series of deleted {alpha}5 promoter constructs (1.2 µg/well) and 0.2 µg of the internal control plasmid phRL-TK Synthetic Renilla Luciferase Reporter Vectors were cotransfected into the cells using FUGENE 6 lipofection reagent as described in our earlier work (18). After 24 h of incubation, cells were treated with PPAR{gamma} ligands for an additional 24 h. The preparation of cell extracts and the measurement of luciferase activities were performed using the Dual-Luciferase Reporter Kit according to recommendations by the manufacturer. Changes in firefly luciferase activity were calculated and plotted after normalization with changes in Renilla luciferase activity in the same sample.

Electrophoretic Mobility Shift Assay
Nuclear protein extracts were prepared for electrophoretic mobility shift assay (EMSA) as described earlier (19). The protein content of the nuclear extract was determined using the Bradford protein assay kit (Sigma). EMSA experiments were performed as described before (20). The probes of double-stranded oligonucleotides for AP-1, Sp1, and C/EBP that were synthesized by Sigma-Genosys based on the human {alpha}5 integrin promoter sequence (21) were: wild-type Sp1 (5'-GCAAACTCCTCCCCGCGTTGAGT-3'), mutant Sp1 (5'-GCAAACTCCTAACGTTGAGT-3'); wild-type C/EBP (5'-GGGAGTTTGGCAAACTCCTC-3'), C/EBP mutant (5'-GGGAGCCAGGCTAACTCCTC-3'); wild-type AP-1 (5'-CGGGTTTGATCATTCGCCT-3'); mutated Ap-1 (5'-CGCGTTGAGTCATTCGCCT-3'). The underlined bases indicate mutations. The complimentary oligonucleotides were annealed and purified following the manufacturer's instructions. The AP-1, Sp1, and C/EBP oligonucleotides were end-labeled with [{gamma}-32P] ATP using T4 polynucleotide kinase as recommended by the manufacturer. Nuclear proteins (5 µg) from control and treated cells were incubated with 32P-labeled oligonucleotide probe under binding conditions (Promega) in a final volume of 20 µl. When applicable, 2 µg of anti–c-Jun or Sp1 antibodies were added to each binding reaction. For cold competition, a 100-fold excess of the respective unlabeled consensus oligonucleotides was added in reaction buffer contained nuclear protein before adding probe. The same amount of mutated oligonucleotides probe was used as another control. After binding, protein–DNA complexes were electrophoresed on a native 4.5% polyacrylamide gel using 1x Tris-Glycine buffer. Each gel was then dried and subjected to autoradiography at –80°C.

Statistical Analysis
All experiments were repeated a minimum of three times. All data collected from electrophoresis gel mobility shift assays (EMSA), luciferase activity assays, RT-PCR or real-time RT-PCR, Western blot, and [Methyl-3H] thymidine incorporation assay were expressed as means ± SD. The data presented in some figures are from a representative experiment, which was qualitatively similar in the replicate experiments. Statistical significance was determined with Student's t test (two-tailed) comparison between two groups of data sets. Asterisks shown in the figures indicate significant differences of experimental groups in comparison with the corresponding control condition (P < 0.05; see figure legends).


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Effects of PPAR{gamma} Ligands on {alpha}5 Gene Expression
Previously, we showed that {alpha}5 mRNA was expressed in several human lung carcinoma cell lines (7). Here, we show that {alpha}5 protein is also detected in the two NSCLC cell lines tested (Figure 1A). We have also demonstrated the expression of PPAR{gamma} mRNA and protein in these two NSCLC cell lines in our previous study (22). In this study, we report that the PPAR{gamma} ligand GW1929 reduced {alpha}5 protein expression in a dose- and time-dependent manner with optimal concentrations of 10 µM at 24 h (Figures 1B and 1C). Similar dose- and time-dependent responses were observed in cells exposed to the PPAR{gamma} ligands rosiglitazone and 15d-PGJ2 (not shown). The reduction in {alpha}5 protein was associated with a decrease in {alpha}5 messenger RNA. Both rosiglitazone (not shown) and GW1929 (10 µM each) reduced the {alpha}5 mRNA levels in a time-dependent manner with maximal reduction at 8 h as determined by RT-PCR analysis in H1838 cells (Figure 2A). This finding was confirmed with real-time RT-PCR analysis (Figure 2B). As expected, the decrease in {alpha}5 protein expression was associated with a reduction in cell adhesion to fibronectin (Figure 2C).



View larger version (30K):
[in this window]
[in a new window]
  		Figure 1. Dose- and time-dependent inhibition of {alpha}5 protein by PPAR{gamma} ligands in human NSCLC cells. (A) NSCLC cells express {alpha}5. Cellular protein was isolated from two NSCLC cell lines (H1838 and H2106), processed, and submitted to Western blotting with an antibody against the {alpha}5 subunit of the integrin {alpha}5ß1. (B) GW1929 suppresses {alpha}5 protein expression in a dose-dependent manner. Cellular protein was isolated from H1838 cells treated with increased concentrations of GW1929 for up to 24 h and processed for Western blotting for {alpha}5. (C) GW1929 suppresses {alpha}5 expression in a time-dependent manner. Cellular protein was isolated from H1838 cells treated with GW1929 (10 µM) for the indicated time periods. Western blot analysis was performed using a monoclonal antibody against {alpha}5. For all Western blots, actin was used as control for normalization purposes. The right panels in B and C depict densitometric results of several Western blot gels.

 


View larger version (24K):
[in this window]
[in a new window]
  		Figure 2. Effect of PPAR{gamma} ligands on {alpha}5 expression. (A) Inhibition of {alpha}5 mRNA expression by GW1929. Total RNA was isolated from H1838 cells that were cultured for the indicated time periods in the presence or absence GW1929 (10 µM). Afterward, {alpha}5 mRNA expression was tested using RT-PCR. (B) Inhibition of {alpha}5 mRNA expression by PPAR{gamma} ligands. Real-time PCR was used to examine the effects of GW1929 and rosiglitazone (10 µM each) on {alpha}5 mRNA expression after culturing the cells for 8 h. The bar graphs represent the mean ± SD of {alpha}5/GAPDH of three independent experiments. (C) Inhibition of cell adhesion to FN by PPAR{gamma} ligands. H1838 cells were layered on fibronectin-coated 96-well plates (20 µg/ml) in the presence or absence of GW1929 or rosiglitazone (10 µM each) for 24 h. Afterwards, the cells were washed and the adherent cells quantified (C). All data are depicted as means ± SD. *Significant differences as compared with the vehicle control (Con). "Con" indicates untreated cells.

 
PPAR{gamma}-Dependent and -Independent Signaling Pathways Are Involved in Regulation of {alpha}5 Gene Expression
PPAR{gamma} ligands have been shown to affect a number of genes via both PPAR{gamma}-dependent and -independent signals depending upon the cell type studied (23, 24). We examined if this was also the case in our system. We found that the inhibitory effect of rosiglitazone and GW1929 on {alpha}5 protein expression was prevented by GW9662, a specific antagonist of PPAR{gamma} (Figure 3A). In contrast, another PPAR{gamma} ligand, 15d-PGJ2, also decreased {alpha}5, but this effect was not blocked by GW9662 (Figure 3B). These observations suggest that the effect of PPAR{gamma} ligands on {alpha}5 expression is mediated through both PPAR{gamma}-dependent (for rosiglitazone and GW1929) and -independent signaling pathways (for 15d-PGJ2).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 3. PPAR{gamma} ligands reduce {alpha}5 expression via PPAR{gamma}-dependent and -independent signals. Cellular protein was isolated from H1838 cells cultured for 1 h in the presence or absence of GW9662 (20 µM) before exposing the cells to GW1929 and rosiglitazone (A) or 15d-PGJ2 (B) (10 µM each) for an additional 24 h. Afterward, the samples were subjected to Western blot analysis to detect {alpha}5 protein. Actin served as internal control for normalization purposes. Panels on the right show densitometry analysis of {alpha}5 protein blots. *Significant differences as compared with the no treatment. **Significance of combination treatment as compared with rosiglitazone or GW1929 alone.

 
Erk Phosphorylation Is Involved in Inhibition of {alpha}5 Expression by PPAR{gamma} Ligands
Next, we examined the intracellular mechanisms involved in the reduction of {alpha}5 by PPAR{gamma} ligands. Specifically, we tested whether regulation of {alpha}5 by PPAR{gamma} ligands was mediated by activation of the Erk pathway. As shown in Figure 4A, the PPAR{gamma} ligand GW1929 stimulated Erk1/2 phosphorylation in a time-dependent manner. Total protein levels of Erk1/Erk2 were not affected and longer treatment periods had no further effects (data not shown). Similar results were obtained when 15d-PGJ2 was tested (not shown). Consistent with a role for Erks, treatment with PD98095, an inhibitor of the Erk pathway, blocked the inhibitory effect of GW1929 and rosiglitazone on {alpha}5 protein expression (Figure 4B).



View larger version (45K):
[in this window]
[in a new window]
  		Figure 4. Role of Erk in reduction of {alpha}5 expression by PPAR{gamma} ligands. (A) Effects of PPAR{gamma} ligands on Erk phosphorylation. Cellular protein was isolated from H1838 cells cultured for up to 60 min in the presence or absence of GW1929 (10 µM), then subjected to Western blot analysis for Erk1, Erk2, and phosphorylated Erk1/2. Panel on the right shows densitometry analysis of p-Erk1/2 blots. (B) Effects of MEK-1 inhibitor on the response to PPAR{gamma} ligands. Cellular protein was isolated from H1838 cells cultured for 1 h in the presence or absence of PD98095 (25 µM) before exposing the cells to GW1929 or rosiglitazone (10 µM each) for an additional 24 h, then subjected to Western blot analysis. Actin served as internal control for normalization purposes. Panel on the right shows densitometry analysis of {alpha}5 protein blots. *Significant differences as compared with the control (Con). **Significance of combination treatment as compared with rosiglitazone or GW1929 alone. "Con" indicates untreated cells.

 
The Effect of PPAR{gamma} Ligands on {alpha}5 Gene Promoter Activity
The human {alpha}5 gene promoter contains multiple transcription factor binding sites including C/EBP, Sp1, and AP-1 located between –92 and +23 (Figure 5A), and these sites have been shown to respond to different stimuli (21, 25, 26). To elucidate the cis-acting elements in the {alpha}5 gene promoter that mediate the inhibitory effects of PPAR{gamma} ligands, transient transfection assays were performed with wild-type {alpha}5 and several {alpha}5 deletion constructs connected to a luciferase reporter gene (17). As shown in Figure 5B, the PPAR{gamma} ligands 15d-PGJ2, rosiglitazone, and GW1929 (each 10 µM) suppressed {alpha}5 promoter (–923/+23) activity in H1838 cells. We found that the suppressive effects of the PPAR{gamma} ligands were detected only in three {alpha}5 promoter constructs (–932/+23, –179/+23, and –92/+23). In contrast, there was little response to PPAR{gamma} ligands in the smallest deletion construct (–41/+23), and, as expected, no response with complete deletion of the promoter (–26/+23). These observations suggested that the region between –92 and –41 bp mediated the inhibitory effect of PPAR{gamma} ligands on {alpha}5 gene expression (Figure 5C).




View larger version (65K):
[in this window]
[in a new window]
  		Figure 5. Effect of PPAR{gamma} ligands on {alpha}5 promoter activity. (A) Promoter map. This image depicts the 5'-flanking region of the human {alpha}5 gene including several transcription factor sites such as NF-{kappa}B, NF-IL6, Sp1, AP-1, AP-2, and others. Among these, only three sites located within the –92 to +23 region of {alpha}5 gene promoter [C/EBP site (–73), Sp1 site (–60), and AP-1 site (–50)] are shown. (B) Inhibition of {alpha}5 promoter activity by PPAR{gamma} ligands. H1838 cells were cotransfected with {alpha}5 promoter constructs (1.6 µg/well) ligated to luciferase and 0.1 µg/well of control plasmid phRL-TK Synthetic Renilla Luciferase Reporter Vectors. After transfection for 24 h, the cells were cultured in the presence or absence of 15d-PGJ2, GW1929, or rosiglitazone (each 10 µM) for an additional 24 h, followed by measurement of luciferase activity, which was determined using a dual-reporter luciferase kit. (C) Effects of PPAR{gamma} ligands on deletion promoter activities. H1838 cells were transfected with the full-length {alpha}5 promoter (–932/+23) or several deletion constructs (–178/+23, –92/+23, –41/+23, –26/+23) ligated to luciferase as indicated and 0.01 µg of control Renialla hpRL-SV-40 plasmid, and luciferase activity measured after treatment as indicated above. (D) Inhibition of Erks by Erk antisense oligonucleotides. Cellular protein was isolated from H1838 cells transfected with control oligonucleotide or Erk antisense oligonucleotide (1 µM each) for 24 h, then subjected to Western blot analysis. Panel on the right shows densitometry analysis of Erk1 and Erk2 blots. (E) Effects of Erk antisense oligonucleotides (ODN) on {alpha}5 promoter activity. H1838 cells were cotransfected with the {alpha}5 promoter deleted construct (–92/+23) and 0.1 µg of control plasmid phRL-TK Synthetic Renilla Luciferase Reporter Vectors in the presence or absence of Erk antisense and sense oligonucleotides (1µM each) for 24 h before exposing the cells to 15d-PGJ2, rosiglitazone and GW1929 (each 10 µM) for an additional 24 h, followed by measurement of luciferase activity as indicated above. A representative experiment out of at least three performed is shown. *Significant difference from control (Con). "Con" indicates untreated cells.

 
We also tested if the activation of Erk signals by PPAR{gamma} ligands was involved in inhibition of the {alpha}5 promoter activity. First, we tested if Erk1/2 antisense knocked down the expression of Erk. As shown in Figure 5D, in H1838 cells, Erk1/2 antisense oligonucleotide (1 µM) completely blocked Erk1 and slightly reduced Erk2 production. The levels of Erk1/2 and actin were unaffected in untransfected cells and in cells treated with Erk sense oligonucleotides (Figure 5D). Next, Erk antisense oligonucleotides (1 µM) transfected into the cells were found to block the effects of 15d-PGJ2, rosiglitazone, and GW1929 on {alpha}5 gene promoter activity, whereas Erk sense oligonucleotides had no effect (Figure 5E).

AP-1 and Sp1 Sites, but Not C/EBP Sites, in the {alpha}5 Gene Promoter Mediate the PPAR{gamma} Ligand Effects on {alpha}5 Gene Expression
EMSAs were performed to identify the transcription factors that mediate the inhibition of {alpha}5 gene expression by PPAR{gamma} ligands. As shown in Figure 6, the PPAR{gamma} ligands rosiglitazone and GW1929 caused a reduction in the binding of AP-1 (Figure 6A). However, they had little effect on Sp1 (Figure 6B) and C/EBP (Figure 6C). In contrast, 15d-PGJ2 reduced DNA binding by Sp1 (Figure 6B), while slightly increasing DNA binding by AP-1 (Figure 6A). PD98095 (25 µM), the inhibitor of Erk, and the Erk antisense oligonucleotide (1 µM) transfected into H1838 cells blocked the effects of GW1929 and rosiglitazone on AP-1 DNA binding (Figures 6D and 6E). PD98095 and the Erk antisense oligonucleotide also prevented the reduction of Sp1 DNA binding in cells treated with 15d-PGJ2 (Figures 6F and 6G). The addition of c-Jun (Figure 6H) or Sp1 (Figure 6I) antibodies produced one supershifted band. There were no binding activities when either AP-1, Sp1, or C/EBP oligonucleotides were mutagenized. As a result of competition assays, specific bands for AP-1, Sp1, or C/EBP were attenuated by 100-fold molar excess of unlabeled respective oligonucleotides.







View larger version (252K):
[in this window]
[in a new window]
  		Figure 6. EMSA to examine AP-1, Sp1, and C/EBP protein binding to {alpha}5 promoter and the effects of PPAR{gamma} ligands. (A–C) Effects of PPAR{gamma} on transcription factors. Oligonucleotides containing the AP-1 (A), Sp1(B), and the C/EBP (C) sites were end-labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells treated with rosiglitazone, GW1929, or 15d-PGJ2 (10 µM each) for 24 h. (D) Effect of the Mek-1/Erk inhibitor on AP-1. Oligonucleotides containing the AP-1 sites were end-labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells treated with PD98095 (25 µM) for 1 h before exposing the cells to rosiglitazone, GW1929 (10 µM each) for an additional 24 h. (E) Effect of Erk antisense oligonucleotide on AP-1. Oligonucleotide containing the AP-1 site was end-labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells transfected with Erk antisense or sense oligonucleotide (1µM each) for 24 h before exposing the cells to GW1929 (10 µM) for an additional 24 h. (F) Effect of the Mek-1/Erk inhibitor on Sp1. Oligonucleotides containing the Sp1 site were end-labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells treated with PD98095 (25 µM) for 1 h before exposing the cells to 15d-PGJ2 (10 µM) for an additional 24 h. (G) Effect of Erk antisense oligonucleotide on Sp1. Oligonucleotide containing the Sp1 site was end-labeled with {gamma}32P-ATP and incubated with nuclear extracts (5 µg) from H1838 cells transfected with Erk antisense or sense oligonucleotide (1 µM each) for 24 h before exposing the cells to 15d-PGJ2 (10 µM) for an additional 24 h. (H and I) Supershift of AP-1 and Sp1. Supershift of the complexes was achieved by adding antibodies raised against c-Jun (H) or Sp1 (I) to the binding reaction. Note that for competition assays presented in most of the figures presented above, a molar excess (x100) of consensus AP-1, Sp1, or C/EBP oligonucleotides were added to the reaction. Oligonucleotides containing a mutated AP-1 (Mut AP-1), Sp1 (Mut Sp1), or C/EBP site (Mut C/EBP) that were end-labeled with {gamma}32P-ATP were used to confirm the binding specificity. All experiments were repeated at least three times. Control ("Con") indicates untreated cells.

 
We further determined whether PPAR{gamma} ligands affected the AP-1 subunits c-Jun and c-Fos, or Sp1 and C/EBPß proteins. As shown in Figure 7, rosiglitazone (10 µM) reduced c-Jun (Figure 7A) and 15d-PGJ2 (10 µM) reduced Sp1 (Figure 7B) in a time-dependent manner, whereas c-Fos and C/EBPß proteins remained essentially unchanged (Figure 7C).



View larger version (41K):
[in this window]
[in a new window]
  		Figure 7. The effect of PPAR{gamma} ligands on nuclear proteins c-Jun, c-Fos, Sp1, and C/EBP. (A) Effect of rosiglitazone on c-Jun protein. Protein from cell lysates was isolated from H1838 cells that were treated with rosiglitazone for the indicated time periods. Afterward, Western blot analysis was performed using polyclonal antibodies against c-Jun. (B) Effect of 15d-PGJ2 on Sp1 protein. Protein from cell lysates was isolated from H1838 cells that were treated with 15d-PGJ2 for the indicated time periods. Afterward, Western blot analysis was performed using polyclonal antibodies against sp1. (C) Effect of PPAR{gamma} ligands on c-Fos and C/EBPß. Protein from cell lysates was isolated from H1838 cells that were treated with rosiglitazone, 15d-PGJ2, or GW1929 (10 µM each) for 24 h followed by Western blot analysis as indicated above using polyclonal antibodies against c-Fos and C/EBPß, respectively. Panels on the right of each image show densitometry analysis of c-Jun, Sp1, c-Fos, or C/EBPß protein blots. Actin was used as an internal control for normalization purposes. *Significant differences as compared with the zero time treatment. "Con" indicates untreated cells.

 
The Effect of PPAR{gamma} Ligands on Cell Growth Induced by Fibronectin or EGF
Data from our laboratory and that of others have shown that fibronectin stimulates cell growth through {alpha}5 integrin signaling (7, 27). Because PPAR{gamma} ligands were shown to inhibit {alpha}5 expression and bind to fibronectin (FN), then we would expect that pretreatment with PPAR{gamma} ligands would result in a decrease in FN-induced cell proliferation. To test if PPAR{gamma} ligands prevent fibronectin-induced cell growth, H1838 cells were treated with GW1929 (10 µM) for 1 h before exposing the cells to fibronectin (20 µg/ml) or EGF (20 ng/ml) for an additional 24 h. As expected, both fibronectin and EGF increased cell growth (Figure 8). Of note, GW1929 prevented fibronectin-induced cell growth, but it had no effect on EGF-stimulated cell proliferation. Furthermore, we examined whether blockade of the PPAR{gamma} signal prevents the effect of GW1929 on cell growth induced by fibronectin. As shown in Figure 8, the PPAR{gamma} antagonist GW9662 blocked the antagonistic effect of GW1929 on fibronectin-induced cell growth, whereas GW9662 alone had no effect on cell growth, nor did it affect EGF-induced cell proliferation.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 8. Effect of PPAR{gamma} ligands on human lung carcinoma cell growth induced by fibronectin or epidermal growth factor (EGF). H1838 cells were incubated with or without GW9662 (20 µM) for 1 h, then with or without GW1929 (10 µM) for 2 h before exposing the cells to fibronectin (20 µg/ml) or EGF (20 ng/ml) and 1 µci [Methyl-3H] thymidine for an additional 24 h. Afterwards, radioactivity was measured as an indication of cell proliferation. Note that FN induced growth that was blocked by the PPAR{gamma} ligand GW1929, but not by the PAPR{gamma} antagonist GW9662. EGF was not affected by the PPAR{gamma} ligand. *Significant differences as compared with the vehicle control. **Significance of combination treatment as compared with fibronectin alone. ***Significance of combination treatment as compared with fibronectin plus GW1929. "Con" indicates untreated cells.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Integrin {alpha}5ß1 expression in tumor cells has been linked to important biological process such as cell migration and invasion (3–6). In human lung carcinoma cells, the overexpression of {alpha}5ß1 has been associated with increased invasiveness and malignant progression (6). How exactly {alpha}5ß1 modulates these tumor-related processes is unknown, but they are likely related to its ability to mediate interactions with its ligand, the matrix protein fibronectin. We have found that by binding to fibronectin, {alpha}5ß1 elicits signals that stimulate COX-2 gene expression with subsequent production of the tumor cell autocrine factor PGE2, resulting in increased cell growth (7). Others have reported that the adhesion of lung carcinoma cells to fibronectin (presumably through {alpha}5ß1) enhances tumorigenicity and confers resistance to apoptosis induced by standard chemotherapeutic therapy (28). In view of the importance of {alpha}5ß1 in lung carcinoma cells, we explored the effects of PPAR{gamma} ligands on its expression. PPAR{gamma} ligands have been shown to promote cell growth arrest (9, 10, 12), but the mechanisms mediating this effect are poorly understood.

First, we demonstrated that the PPAR{gamma} ligands rosiglitazone and GW1929 inhibited {alpha}5 mRNA and protein expression, suggesting a role for PPAR{gamma} ligands in control of {alpha}5 expression in tumor cells. Then, we attempted to delineate the signaling pathways involved in this process in NSCLC cells. We focused on intracellular kinases because PPAR{gamma} ligands have been shown to regulate cell growth through effects on several kinase-signaling pathways (29–31). We found that the treatment of cells with the PPAR{gamma} ligand GW1929 was associated with rapid phosphorylation of Erk. In addition, an inhibitor of the MEK-1/Erk signaling pathway prevented the effects of GW1929 and rosiglitazone on {alpha}5 protein expression. Similar results were obtained with 15d-PGJ2, suggesting a common signal pathway that mediates the ability of PPAR{gamma} ligands to regulate {alpha}5. This finding was similar to that of Kim and coworkers, who showed that PD98059 (the same Mek-1/Erk inhibitor used here) prevented the inhibitory effects of PGJ2 on neuroblastoma cell growth (30). Also, Takeda and colleagues found that the PPAR{gamma} ligand PGJ2 and thiazolidinediones such as pioglitazone and troglitazone elicited activation of Erk followed by induction of c-fos mRNA expression in cultured vascular smooth muscle cells (31). Although Erk is thought to play a key role in regulation of cell proliferation, it has been suggested that activation of Erk might also mediate cell cycle arrest and apoptosis (32). Consistent with this, in our system, it appears that Erk signaling mediates the suppressive effects of PPAR{gamma} ligands on {alpha}5 gene expression.

Several studies have demonstrated that PPAR{gamma} ligands can act through PPAR{gamma}-dependent and -independent signals (23, 24). This appears to be the case in our system as well. We showed that a specific inhibitor of PPAR{gamma}, GW9662, reversed the effects of the PPAR{gamma} ligands GW1929 and rosiglitazone on {alpha}5 expression. In contrast, GW9662 did not prevent the effects of 15d-PGJ2. 15d-PGJ2 has been shown to act through PPAR{gamma}-independent mechanisms in several cell systems, and this is likely due to the activation of unrelated transcription factors (33–35). Cultured rat mesangial cells transfected with a peroxisome proliferator responsive element (PPRE) luciferase reporter did not respond to 15d-PGJ2 exposure (34).

The regulation of {alpha}5 gene transcription appears to be a key mechanism for the control of {alpha}5ß1 surface expression (25, 26). To investigate whether the downregulation of {alpha}5 by PPAR{gamma} ligands reflects alterations in the transactivation of the {alpha}5 gene promoter, we performed transient transfection experiments using {alpha}5 full-length and deletion promoter–reporter constructs. We found that 15d-PGJ2, GW1929, and rosiglitazone reduced {alpha}5 promoter activity. Consistent with a role for Erk, we found that the Erk antisense oligonucleotide blocked the suppression of {alpha}5 gene expression induced by PPAR{gamma} ligands. Furthermore, the region between –92 and –41 in the {alpha}5 promoter was demonstrated to play a major role in mediating the effects of PPAR{gamma} ligands on the {alpha}5 gene in our system. Several transcription factor–binding sites within the {alpha}5 promoter have been characterized, including regulatory elements for the NF-IL6 family member C/EBP, Sp1, and AP-1 (17, 21, 26). Corbi and coworkers showed that the transcription factor AP-1 contributes to the induction of {alpha}5 gene transcription (21). Gingras and colleagues found that Sp1, via both the -42/-92 and -92/-132 {alpha}5 promoter segments, played an important role in the regulation of the {alpha}5 gene in rabbit corneal epithelial cells (26). We showed that the treatment of H1838 cells with rosiglitazone or GW1929 inhibited DNA binding by AP-1, but had little effect on Sp1 and C/EBP DNA binding. In contrast, 15d-PGJ2 reduced Sp1 DNA binding. Erk antisense oligonucleotides blocked the effects of PPAR{gamma} ligands on AP-1 and Sp1 DNA binding, yet again confirming the role of Erk activation in the regulation of the {alpha}5 gene by PPAR{gamma} ligands.

Rosiglitazone suppressed nuclear c-Jun and 15d-PGJ2 suppressed Sp1 protein expression, but neither affected c-Fos nor C/EBPß protein. The role of cellular phosphor Erk1/2 and AP-1 in the regulation of integrin gene expression has been shown in several studies (17, 36, 37). c-Jun is a target of ERK1/2, and an increase in c-Jun phosphorylation by Erk has been found to accompany an increase in AP-1 activity (36–38). Also, inhibition of Mek-1 was found to diminish H2O2-induced phosphorylation of c-Jun and DNA binding activity of AP-1 in H1299 cells, a p53-deficient human lung carcinoma cell line (37).

We also showed that 15d-PGJ2 reduced Sp1 binding and inhibited Sp1 expression, and this appeared to mediate the effects of this ligand on {alpha}5 gene expression. Similarly, Zhang and coworkers showed that 15d-PGJ2 inhibited PPAR{gamma}-dependent expression of Sp1 in human umbilical vein endothelial cells (39). Sugawara and colleagues found that the suppression of transcription of the thromboxane receptor gene by 15d-PGJ2 was linked with inhibition of Sp1 DNA binding in vascular smooth muscle cells (40). It should be noted that PPAR{gamma} ligands can interact with Sp1 bound to GC-rich DNA suggesting that its effects can occur independently of PPAR/RXR heterodimers (41). Also, and consistent with our results, 15d-PGJ2 has been shown not to affect Jun-NH2-terminal kinase activity in human mesangial cells (42).

Because PPAR{gamma} ligands were shown to inhibit {alpha}5 expression and bind to fibronectin, we would expect that pretreatment with PPAR{gamma} ligands would result in a decrease in fibronectin-induced cell proliferation. Consistent with this idea, we found that GW1929 prevented fibronectin-induced cell growth, but it had no effect on cell proliferation induced by EGF. The inhibitory effect of GW1929 on fibronectin-induced cell growth was prevented by the PPAR{gamma} antagonist GW9662. In contrast, GW9662 had no effect on EGF-induced cell growth; this suggests a different signal pathway in mediating the EGF effect. In NSCLC cells, like other cells, EGF-stimulated cell growth is mediated through EGF receptors that have intrinsic tyrosine kinase activity and induce cellular signaling (43). However, their might be some degree of overlap between these pathways, because functional EGF receptor activation has been linked to {alpha}5ß1-mediated epithelial cell proliferation in the presence of fibronectin (27). Camp and Tafuri found that EGF, by stimulation of Mek/Erk phosphorylation, decreased ligand-activated PPAR{gamma} transcriptional activity in NIH 3T3 cells (44). PPAR{gamma} ligands have also been shown to decrease the expression of heparin-binding EGF-like growth factor (HB-EGF) in rat intestinal epithelial cells (45). The data presented in this report support the idea that both soluble (e.g., growth factors) and insoluble (e.g., extracellular matrices) factors control tumor cell proliferation. Furthermore, they are consistent with observations linking cell–matrix interactions with regulation of the cell cycle in malignant and nonmalignant cells. The disruption of these interactions not only affects cellular growth, but might trigger apoptotic cell death (46).

Overall, our study demonstrates that PPAR{gamma} ligands inhibit integrin {alpha}5 gene expression in human NSCLC cells through PPAR{gamma}-dependent and -independent signals. The suppression of {alpha}5 is mediated through activation of Erk and a decrease in c-Jun protein (with subsequent effects on AP-1) and Sp1 DNA binding to elements located in the promoter region of the {alpha}5 gene. These mechanisms explain, at least in part, the inhibitory effects of PPAR ligands on fibronectin-induced NSCLC cell growth. These novel findings suggest a new mechanism by which PPAR{gamma} ligands can control lung carcinoma cell growth.


   Footnotes
 
This work was supported by American Cancer Society Institutional Research Grant 6-47083, by the Aerodigestive and Lung Cancer Program of the Winship Cancer Institute at Emory University (S.W.H), by a Merit Review Grant from the Department of Veterans Affairs (J.R.), and by a grant from the National Institutes of Health (J.R).

Conflict of Interest Statement: S.H. has no declared conflicts of interest; H.N.R. has no declared conflicts of interest; and J.R. has no declared conflicts of interest.

Received in original form November 6, 2004

Received in final form January 15, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Parise LV, Lee J, Juliano JL. New aspects of integrin signaling in cancer. Semin Cancer Biol 2000;10:407–414.[CrossRef][Medline]
  2. Kanwar YS, Wada J, Lin S, Danesh FR, Chugh SS, Yang Q, Banerjee T, Lomasney JW. Update of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis. Am J Physiol Renal Physiol 2004;286:F202–F215.[Abstract/Free Full Text]
  3. Hood JD, Cheresh DA. Role of integrins in cell invasion and migration. Nat Rev Cancer 2002;2:91–100.[CrossRef][Medline]
  4. Pignatelli M. Integrins, cadherins, and catenins: molecular cross-talk in cancer cells. J Pathol 1998;186:1–2.[CrossRef][Medline]
  5. Pellat-Deceunynck C, Barille S, Puthier D, Rapp MJ, Harousseau JL, Bataille R, Amiot M. Adhesion molecules on human myeloma cells: significant changes in expression related to malignancy, tumor spreading, and immortalization. Cancer Res 1995;55:3647–3653.[Abstract/Free Full Text]
  6. Adachi M, Taki T, Higashiyama M, Kohno N, Inufusa H, Miyake M. Significance of integrin alpha5 gene expression as a prognostic factor in node-negative non-small cell lung cancer. Clin Cancer Res 2000;6:96–101.[Abstract/Free Full Text]
  7. Han S, Sidell N, Roser-Pager S, Roman J. Fibronectin stimulates human lung cancer cell growth by inducing cyclooxygenase-2 (COX-2) gene expression. Int J Cancer 2004;111:322–331.[CrossRef][Medline]
  8. Cabrero A, Laguna JC, Vazquez M. Peroxisome proliferator-activated receptors and the control of inflammation. Curr Drug Targets Inflamm Allergy 2002;1:243–248.[CrossRef][Medline]
  9. Rocchi S, Auwerx J. Peroxisome proliferator-activated receptor-gamma: a versatile metabolic regulator. Ann Med 1999;31:342–351.[Medline]
  10. Keshamouni VG, Reddy RC, Arenberg DA, Joel B, Thannickal TJ, Kalemkerian GP, Standiford TJ. Peroxisome proliferator-activated receptor-gamma activation inhibits tumor progression in non-small-cell lung cancer. Oncogene 2004;23:100–108.[CrossRef][Medline]
  11. Vaidya S, Somers EP, Wright SD, Detmers PA, Bansal VS. 15-Deoxy-Delta12, 14-prostaglandin J2 inhibits the beta2 integrin-dependent oxidative burst: involvement of a mechanism distinct from peroxisome proliferator-activated receptor gamma ligation. J Immunol 1999;163:6187–6192.[Abstract/Free Full Text]
  12. Han S, Roman J. Suppression of prostaglandin E (2) receptor subtype EP2 by PPARgamma ligands inhibits human lung carcinoma cell growth. Biochem Biophys Res Commun 2004;314:1093–1099.[CrossRef][Medline]
  13. Lefevre G, Calipel A, Mouriaux F, Hecquet C, Malecaze F, Mascarelli F. Opposite long-term regulation of c-Myc and p27Kip1 through overactivation of Raf-1 and the MEK/ERK module in proliferating human choroidal melanoma cells. Oncogene 2003;22:8813–8822.[CrossRef][Medline]
  14. Han SW, Lei ZM, Rao CV. Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin during the differentiation of human endometrial stromal cells into decidua. Endocrinology 1996;137:1791–1797.[Abstract]
  15. Proulx S, Guerin SL, Salesse C. Effect of quiescence on integrin alpha5beta1 expression in human retinal pigment epithelium. Mol Vis 2003;9:473–481.[Medline]
  16. Han S, Inoue H, Flowers LC, Sidell N. Control of COX-2 gene expression through peroxisome proliferator-activated receptor gamma in human cervical cancer cells. Clin Cancer Res 2003;9:4627–4635.[Abstract/Free Full Text]
  17. Boles BK, Ritzenthaler J, Birkenmeier T, Roman J. Phorbol ester-induced U-937 differentiation: effects on integrin alpha (5) gene transcription. Am J Physiol Lung Cell Mol Physiol 2000;278:L703–L712.[Abstract/Free Full Text]
  18. Han S, Wada RK, Sidell N. Differentiation of human neuroblastoma by phenylacetate is mediated by peroxisome proliferator-activated receptor gamma. Cancer Res 2001;61:3998–4002.[Abstract/Free Full Text]
  19. Dignam JD, Lebovitz RM, Roeder RG. Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res 1983;11:1475–1489.[Abstract/Free Full Text]
  20. Han S, Sidell N. RU486-induced growth inhibition of human endometrial cells involves the nuclear factor-kappab signaling pathway. J Clin Endocrinol Metab 2003;88:713–719.[Abstract/Free Full Text]
  21. Corbi AL, Jensen UB, Watt FM. The alpha2 and alpha5 integrin genes: identification of transcription factors that regulate promoter activity in epidermal keratinocytes. FEBS Lett 2001;474:201–217.
  22. Han S, Sidell N, Fisher FB, Roman J. Up-regulation of p21 gene expression by peroxisome proliferator-activated receptor gamma in human lung carcinoma cells. Clin Cancer Res 2004;10:1911–1919.[Abstract/Free Full Text]
  23. Motojima K. Peroxisome proliferator-activated receptor (PPAR)-dependent and -independent transcriptional modulation of several non-peroxisomal genes by peroxisome proliferators. Biochimie 1997;79:101–106.[Medline]
  24. Chen CW, Chang YH, Tsi CJ, Lin WW. Inhibition of fibronectin-gamma-mediated inducible nitric oxide synthase induction by the peroxisome proliferator-activated receptor gamma ligand, 15-deoxy-delta 12, 14-prostaglandin J2, involves inhibition of the upstream Janus kinase/STAT1 signaling pathway. J Immunol 2003;171:979–988.[Abstract/Free Full Text]
  25. Larouche K, Leclercm S, Salesse C, Guerin SL. Expression of the alpha 5 integrin subunit gene promoter is positively regulated by the extracellular matrix component fibronectin through the transcription factor Sp1 in corneal epithelial cells in vitro. J Biol Chem 2000;275:39182–39192.[Abstract/Free Full Text]
  26. Gingras ME, Larouche K, Larouche N, Leclerc S, Salesse C, Guerin SL. Regulation of the integrin subunit alpha5 gene promoter by the transcription factors Sp1/Sp3 is influenced by the cell density in rabbit corneal epithelial cells. Invest Ophthalmol Vis Sci 2003;44:3742–3755.[Abstract/Free Full Text]
  27. Kuwada S, Li X. Integrin alpha5/beta1 mediates fibronectin-dependent epithelial cell proliferation through epidermal growth factor receptor activation. Mol Biol Cell 2000;11:2485–2496.[Abstract/Free Full Text]
  28. Rintoul RC, Sethi T. Extracellular matrix regulation of drug resistance in small-cell lung carcinoma. Clin Sci (Lond) 2002;102:417–424.[Medline]
  29. Goetze S, Bungenstock A, Czupalla C, Eilers F, Stawowy P, Kintscher U, Spencer-Hansch C, Graf K, Nurnberg B, Law RE, et al. Leptin induces endothelial cell migration through Akt, which is inhibited by PPARgamma-ligands. Hypertension 2002;40:748–754.[Abstract/Free Full Text]
  30. Kim EJ, Park KS, Chung SY, Sheen YY, Moon DC, Song YS, Kim KS, Song S, Yun YP, Lee MK, et al. Peroxisome proliferator-activated receptor-gamma activator 15-deoxy-Delta12, 14-prostaglandin J2 inhibits neuroblastoma cell growth through induction of apoptosis: association with extracellular signal-regulated kinase signal pathway. J Pharmacol Exp Ther 2003;307:505–517.[Abstract/Free Full Text]
  31. Takeda K, Ichiki T, Tokunou T, Iino N, Takeshita A. 15-Deoxy-delta 12,14-prostaglandin J2 and thiazolidinediones activate the MEK/ERK pathway through phosphatidylinositol 3-kinase in vascular smooth muscle cells. J Biol Chem 2001;276:48950–48955.[Abstract/Free Full Text]
  32. Baek SJ, Wilson LC, His LC, Eling TE. Troglitazone, a peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, selectively induces the early growth response-1 gene independently of PPAR gamma: a novel mechanism for its anti-tumorigenic activity. J Biol Chem 2003;278:5845–5853.[Abstract/Free Full Text]
  33. Okano H, Shiraki S, Inoue H, Yamanaka Y, Kawakita T, Saitou Y, Yamaguchi Y, Enokimura N, Yamamoto N, Sugimoto K, et al. 15-deoxy-delta-12–14–PGJ2 regulates apoptosis induction and nuclear factor-kappaB activation via a peroxisome proliferator-activated receptor-gamma-independent mechanism in hepatocellular carcinoma. Lab Invest 2003;83:1529–1539.[CrossRef][Medline]
  34. Sawano H, Haneda M, Sugimoto T, Inoki K, Koya D, Kikkawa R. 15-Deoxy-Delta12, 14-prostaglandin J2 inhibits IL-1beta-induced cyclooxygenase-2 expression in mesangial cells. Kidney Int 2003;61:1957–1967.
  35. Perez-Sala D, Cernuda-Morollon E, Canada FJ. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-Delta 12,14-prostaglandin J2. J Biol Chem 2003;278:51251–51260.[Abstract/Free Full Text]
  36. Ren Z, Kar S, Wang Z, Wang M, Saavedra JE, Carr BIJS-K. A novel non-ionic diazeniumdiolate derivative inhibits Hep 3B hepatoma cell growth and induces c-Jun phosphorylation via multiple MAP kinase pathways. J Cell Physiol 2003;197:426–434.[CrossRef][Medline]
  37. Chung YW, Jeong DW, Won JY, Choi EJ, Choi YH, Kim IY. H2O2-induced AP-1 activation and its effect on p21 (WAF1/CIP1)-mediated G2/M arrest in a p53-deficient human lung cancer cell. Biochem Biophys Res Commun 2003;293:1248–1253.
  38. Drobna Z, Jaspers I, Thomas DJ, Styblo M. Differential activation of AP-1 in human bladder epithelial cells by inorganic and methylated arsenicals. FASEB J 2003;17:67–69.[Abstract/Free Full Text]
  39. Zhang J, Fu M, Zhao L, Chen YE. 15-Deoxy-prostaglandin J (2) inhibits PDGF-A and -B chain expression in human vascular endothelial cells independent of PPAR gamma. Biochem Biophys Res Commun 2002;298:128–132.[CrossRef][Medline]
  40. Sugawara A, Uruno A, Kudo M, Ikeda Y, Sato K, Taniyama Y, Ito S, Takeuchi K. Transcription suppression of thromboxane receptor gene by peroxisome proliferator-activated receptor-gamma via an interaction with Sp1 in vascular smooth muscle cells. J Biol Chem 2002;277:9676–9683.[Abstract/Free Full Text]
  41. Krey G, Mahfoudim A, Wahlim W. Functional interactions of peroxisome proliferator-activated receptor, retinoid-X receptor, and Sp1 in the transcriptional regulation of the acyl-coenzyme-A oxidase promoter. Mol Endocrinol 1995;9:219–231.[Abstract]
  42. Wilmer WA, Dixon C, Lu L, Hilbelink T, Rovin BH. A cyclopentenone prostaglandin activates mesangial MAP kinase independently of PPARgamma. Biochem Biophys Res Commun 2001;281:57–62.[CrossRef][Medline]
  43. Carpenter G. Receptors for epidermal growth factor and other polypeptide mitogens. Annu Rev Biochem 1987;56:881–914.[CrossRef][Medline]
  44. Camp HS, Tafuri SR. Regulation of peroxisome proliferator-activated receptor gamma activity by mitogen-activated protein kinase. J Biol Chem 1997;272:10811–10816.[Abstract/Free Full Text]
  45. Kitamura S, Miyazaki Y, Hiraoka S, Nagasawa Y, Toyota M, Takakura R, Kiyohara T, Shinomura Y, Matsuzawa Y. PPARgamma ligands inhibit cell growth and suppress the expression of cyclin D1 and EGF-like growth factors in ras-transformed rat intestinal epithelial cells. Int J Cancer 2001;94:335–342.[CrossRef][Medline]
  46. Danen EH, Yamada KM. Fibronectin, integrins, and growth control. J Cell Physiol 2001;189:1–13.[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
S. Han, J. D. Ritzenthaler, B. Wingerd, H. N. Rivera, and J. Roman
Extracellular Matrix Fibronectin Increases Prostaglandin E2 Receptor Subtype EP4 in Lung Carcinoma Cells through Multiple Signaling Pathways: THE ROLE OF AP-2
J. Biol. Chem., March 16, 2007; 282(11): 7961 - 7972.
[Abstract] [Full Text] [PDF]

Home page
 JCOHome page
F. R. Hirsch and S. M. Lippman
Advances in the Biology of Lung Cancer Chemoprevention
J. Clin. Oncol., May 10, 2005; 23(14): 3186 - 3197.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0345OCv1
32/4/350    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