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Carcinogenesis Advance Access originally published online on May 19, 2006
Carcinogenesis 2006 27(12):2392-2401; doi:10.1093/carcin/bgl078
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Smad–Sp1 complexes mediate TGFß-induced early transcription of oncogenic Smad7 in pancreatic cancer cells

Kerstin Jungert{dagger}, Anita Buck{dagger}, Malte Buchholz, Martin Wagner, Guido Adler, Thomas M. Gress and Volker Ellenrieder*

Department of Gastroenterology, University of Ulm 89081 Ulm, Germany

*To whom correspondence should be addressed at: Department of Internal Medicine, University of Ulm, Robert-Koch Strasse 8, 89081 Ulm, Germany. Tel: +49 731 500 24312; Email: volker.ellenrieder{at}medizin.uni-ulm.de


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
The transcription factor Sp1 has been implicated in cell-type-specific activation of transforming growth factor-ß (TGFß) target genes in normal epithelial cells as well as in aberrant gene activation by TGFß in epithelial tumor cells. Here, we have examined the interaction of Sp1 with components of the Smad signaling cascade and its role in TGFß-induced early gene expression in pancreatic cancer cells. Gene expression profiling was carried out in mithramycin-A-treated cells to identify Sp1-regulated TGFß early response genes. We found that in pancreatic cancer cells Smad proteins and Sp1 cooperatively regulate expression of a distinct set of TGFß target genes potentially involved in tumor progression, including MMP-11, cyclin D1 and Smad7. Mechanistically, TGFß rapidly induces nuclear translocation of Smad proteins and subsequently stimulates Smad–Sp1 complex formation. Using the Smad7 promoter as a model for Smad-/Sp1-induced early gene activation, we demonstrated that this interaction increases Sp1 binding to GC-rich promoter boxes and results in superinduction of Sp1-mediated transcription. Moreover, inhibition of Sp1–DNA binding or transfection of Sp1-specific siRNA prevents TGFß-induced Smad7 expression and consequently enhances Smad signaling in pancreatic cancer cells, as indicated by increased receptor-mediated phosphorylation of Smad3. We thus conclude that Sp1 strongly contributes to the aberrant transcriptional response of transformed epithelial cells to TGFß stimulation.

Abbreviations: qRT–PCR, quantitative real-time PCR; siRNA, small interfering RNA; SBEs, Smad binding elements; SDS, sodium dodecyl sulfate; TGFß, transforming growth factor-ß


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Supplementary material
 References
 
Transforming growth factor-ß (TGFß) controls a wide variety of cellular functions in different cell types through its ability to regulate the expression of specific sets of genes (1,2). In carcinogenesis, TGFß plays a dual role, acting as a tumor suppressor in premalignant stages while promoting tumor outgrowth in advanced stages of the disease (3,4). In normal epithelial cells, TGFß inhibits cell growth chiefly through transcriptional regulation of cell-cycle controlling genes such as the cyclin-dependent kinase inhibitors p15INK4b and p21Waf1/Cip1 (5). During tumor progression, however, many tumor cells change their transcriptional responsiveness to TGFß, escaping from growth inhibition and instead responding to TGFß stimulation with increased expression of genes involved in tumor cell invasion and metastasis (1,6).

TGFß regulates gene transcription primarily through the intracellular Smad signaling cascade, which is initiated by binding of the TGFß ligand to heteromeric complexes of specific type II (TßR-II) and type I (TßR-I) kinase receptors (7). Receptor complexes activate the receptor Smads (R-Smads), Smad2 and Smad3, by phosphorylation, thus enabling the R-Smads to dissociate from the receptor and to induce complex formation with another member of the Smad protein family, Smad4 (7–9). The resulting heteromultimer translocates into the nucleus, where the Smads interact with co-proteins (co-activators or co-repressors) and bind to consensus elements (SBEs, Smad-binding elements) of TGFß target gene promoters (10–15). Interestingly, however, Smad proteins bind DNA with only low affinities and therefore require the association with other sequence-specific transcription factors in order to sufficiently regulate promoter activation (1,6,8). Ultimately, these partnering transcription factors determine the DNA sequences that the Smad complexes will bind, the co-proteins they will recruit and the speed and duration of the active transcription factor complex formation. To date, >20 cell-type specific transcription factors have been identified that recruit Smad complexes to distinct regulatory promoter elements and thus determine the transcriptional outcome and the resulting biological activities induced by TGFß (8,16,17). The availability of these partner proteins, in turn, is regulated by a complex signaling network including mitogenic and anti-mitogenic signaling cascades and varies significantly among different cell types. This explains both the cell-type-dependent and the cell activation state-dependent diversity of TGFß-induced gene selection as seen during carcinogenesis.

In the last 5 years, strong efforts have been made to identify those oncogenic transcription factors that cooperate with the Smads to define the transcriptional TGFß response in late stages of carcinogenesis. An increasing body of evidence suggests a profound role for members of the Stat family, the LEF1/TCF-transcription regulators of Wnt signaling, members of the AP-1 family of proteins, and in particular Sp1, the founding member of the Sp1/KLF-like zinc-finger proteins (18–21). Overexpression of Sp1 or increased Sp1 binding activity, for instance, has been reported in multiple cancer types, including human pancreatic and gastric cancers, in which increased TGFß signaling activity has been associated with a more aggressive phenotype and poor prognosis of the diseases (22–24). Moreover, functional and biochemical analyses revealed that Sp1 directly participates in TGFß-regulated expression of genes associated with tumor cell proliferation, degradation of extracellular matrix and angiogenesis. However, although these studies strongly indicate a key role of Sp1 in gene regulation during TGFß-mediated tumor progression, it has remained elusive as to how and to which extent Sp1 directly participates in TGFß-regulated transcription in pancreatic cancer.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Supplementary material
 References
 
Cell culture and transient transfection
The human pancreatic cell line Panc-1 was obtained from the American Type Culture Collection (ATCC, RMD, USA). IMIM-PC2 cells were provided by F.X. Real (Insitute Municipale de Investigacion Medica, Barcelona, Spain). TD-2 cells were described previously (25) and were provided by R.M. Schmid (Technical University of Munich, Germany). Panc-1 and IMIM-PC2 cells were maintained in Dulbecco's modified minimal essential medium (GIBCO, Invitrogen, NY, USA) and TD-2 cells in RPMI 1640 (Roswell Park Memorial Institute, Buffalo, New York, USA) medium (GIBCO), both supplemented with 10% FCS (GIBCO) and 100 U/ml Streptomycin penicillin/100µg/ml streptomycin (BiochromAG, Berlin, Germany). For transient transfection of Panc-1 and TD-2 cells, the TransfastTM reagent (Promega, Madison, WI, USA) was used, while IMIM-PC2 cells were transfected with jetPEITM (Qbiogene, Irvine, CA, USA), both according to manufacturer's suggestions. Small interfering RNA (siRNA) was transfected into Panc-1 cells with TransmessengerTM (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The targeted sequence to silence the translation of Sp1 was 5'-GGUAGCUCUAAGUUUUGAUtt-3' (Ambion, Austin, TX, USA) The scrambled sequence SilencerTM Negative Control #2 siRNA (Ambion) was used as a negative control.

Plasmid constructs
Standard molecular biology techniques were used to clone the Sp1 open reading frame (ORF), which was obtained from R. Urrutia (GI-Research Unit, Mayo Clinic, MI, USA), into the pCMV-Tag3 vector (Stratagene, La Jolla, CA, USA) for expression as a MYC-tagged protein. The pGL80-wt and pGL80-mut Smad7 constructs were generated by cloning of the following double-stranded oligonucleotides into pGL3 basic plasmid (Promega): pGL80-wt: 5'-GGGGGGGCGGGAAGGAGGCGGCGGCGGCTGGGGGCGGGGGAGGGAGGGGTAGAGGGGGGAGGGAAGGGGGCGGAGGCGGG-3' and its complementary strand; pGL80-mut: 5'-GGGGGAACGGGAAGGAGGCGGCGGCGGCTGGGAACGGGGGAGGGAGGGGTAGAGGGGGGAGGGAAGGGAACGGAAACGGG-3' and its complementary strand. All constructs were verified by sequencing. The p3TP-Lux reporter construct and the FLAG-Smad4 construct were obtained from J. Massague (Sloan Kettering Institute, New York, NY, USA). The FLAG-Smad3 construct was provided by R. Derynck (Departments of Growth and Development, and Anatomy, UCSF, San Fransisco, CA, USA) and the Smad7 reporter plasmids pGL725, pGL725-delSBE and pGL725-delGC were obtained from R. Heuchel (Ludwig Institute for Cancer Research, Uppsala, Sweden) (26). Deletion of the SBE was realized through digestion of the wild-type pGL725 construct with DraI and PauI (BsePI). The GC-rich domain of the Smad7 promoter (pGL725-delGC) was deleted by digestion with PauI (BsePI) and StuI. In the same order the pGL725-delSBE construct was digested with StuI and PauI (BsePI) in order to obtain the pGL725-delSBE/GC reporter gene construct. The 3XSBE reporter construct was purchased from Stratagene.

Luciferase reporter gene assays
For reporter gene assays, cells were seeded in 24-well tissue culture dishes and 24 h later transfected with the indicated constructs. Treatment with 5 ng/ml TGFß (PromoCell GmbH, Heidelberg, Germany) or 100 nM mithramycin A (Sigma-Aldrich, Saint Louis, MI, USA), was carried out 24 h post-transfection. Luciferase assays were performed with a Lumat LB 9501 (Berthold Technologies, Bad Wildbad, Germany) luminometer and the Dual-Luciferase®-Reporter Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase activity and were either expressed as relative luciferase activity (RLA) or as mean ‘fold induction’ with respect to empty vector control. Mean values are displayed ±standard deviations.

cDNA arrays
For expression profiling analyses of pancreatic cancer cells, we used specific candidate gene arrays containing 350 ESTs and 2085 genes selected for a known or suspected role in pancreatic tumor. cDNA clones to be included in the arrays were obtained either from our own cDNA libraries (27) or as IMAGE clones from the RZPD (Resource Center/Primary Database of the German Genome project, Berlin, Germany). Clones were sequence-verified and PCR-amplified in microtitre plates using vector primers. PCR products were spotted in duplicates on 9 x 12 cm Hybond N+ nylon membranes (Amersham Biosciences, Freiburg, Germany) using a MicroGrid II arrayer (BioRobotics LTD, Cambridge, UK).

Hybridizations
Hybridization probes were generated from 10 µg total RNA. First-strand cDNA was synthesized using Oligo(dT)12–18 primers and MMLV reverse transcriptase (Ambion). The first-strand cDNA was labeled by 33P-{alpha}-dATP incorporation. Subsequently, the RNA strand was digested by RNase H and the probes purified by Nucleotide Removal Kit (Qiagen). Probes were competed with 0.25 µg/µl COT-1 DNA (Invitrogen) for 1 h. Hybridizations were done in ULTRArray Hyb Buffer (Ambion) at 50°C for at least 14 h. Filters were washed as follows: 2 x SSC, 0.5% sodium dodecyl sulfate (SDS) at room temperature for 5 min; 0.5 x SSC, 0.5% SDS at 67°C for 15 min; 0.5 x SSC, 0.5% SDS at 67°C for 15 min. All hybridizations were performed in triplicates.

Image analysis, bioinformatics and statistical analysis
Hybridization signals were quantified with a Storm 8600 PhosphorImagerTM (Molecular Dynamics GmbH, Krefeld, Germany). Image analysis was performed with the ArrayVisionTM software (Imaging Research, Ontario, Canada). Raw intensity values were corrected for local background and normalized to the mean signal intensity of all features on an individual array. Mean values and standard deviations of normalized intensity values from triplicate experiments were calculated using Microsoft Excel® (Microsoft, Redmond, WA, USA). Genes were defined as differentially expressed between untreated and TGFß-treated samples if the following criteria were fulfilled: (i) mean normalized signal intensity > 0.5 in at least one of the experimental groups, (ii) ratio of signal intensities > 2 between the sample groups, and (iii) no overlap between standard deviations of the mean intensity values from both sample groups. Cluster analysis was performed using the Genesis software (http://genome.tugraz.at) as described previously (28).

Immunoprecipitation and western blot analysis
For immunoprecipitation, Panc-1 cells were transfected with either FLAG-Smad3 or FLAG-Smad4 expression plasmids. Twenty-four hours post-transfection, cells were incubated in the presence or absence of TGFß for 1 h and cell extracts were obtained as described previously (29). Upon measurement of protein concentration, immunoglobulin G pre-cleared cell extracts were incubated at 4°C for 3 h with agarose-conjugated anti-FLAG M2 antibodies to precipitate FLAG-bound Smad protein complexes (Sigma-Aldrich). After four washes with complete lysis buffer, the immunoprecipitates were eluted by boiling for 5 min in 50 µl of 2x Laemmli sample buffer. For western blotting, the resulting immunoprecipitates or protein extracts from different pancreatic cancer cell lines were electrophoresed through a 6 or 10% SDS-polyacrylamide gel and transferred onto PVDF ImmobilonTM-P membranes (Millipore, Billerica, MA, USA) as described previously (30). PVDF membranes were probed with anti-Smad2/3, anti-phospho Smad3 (both from Upstate Biotechnology, Lake Placid, NY, and Transduction Laboratories, San Diego, CA, USA), anti-Smad4 (Upstate Biotechnology), anti-Sp1 (Santa Cruz Biotechnology, Santa Cruz, California, USA) or anti-ß-actin (Sigma-Aldrich) antibodies washed in TBS washing buffer, and then incubated with peroxidase-conjugated secondary antibodies. Lumi-light western blotting substrate (Roche Applied Science, Mannheim, Germany) was used for visualization.

qRT–PCR analysis
RNA was extracted using the RNeasy Midi Kit (Qiagen GmbH, Hilden, Germany) and first-strand cDNA was synthesized from 10 µg total RNA using random primers and Superscript II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA). The quantitative PCR analysis was done using an ABI PRISM 7700 Sequence Detector System and the SYBR Green PCR Master Mix kit (Applied Biosystems, Wellesley, MA, USA) according to the manufacturer's suggestions. The quantitative real-time PCR (qRT–PCR) was performed with sequence-specific primer pairs determined with the PrimerExpress® program (Applied Biosystems). Cyclophilin A was used as housekeeping gene. The following primer pairs were used: Smad7 forward primer: 5'-TGCTCCCATCCTGTGTGTTAAG-3'; Smad7 reverse primer: 5'-TCAGCCTAGGATGGTACCTTGG-3'. Cyclin D1 forward primer: 5'-TCTTATTGCGCTGCTACCGTT-3'; Cyclin D1 reverse primer: 5'-ACTGATCCTCCAATAGCAGCAAA-3'.

DNAP assay
Panc-1 cells were transfected with the indicated expression plasmids, treated with TGFß for 1 h and lysed as described previously (31,32). Cell extracts were incubated with 2 µg of biotinylated double-strand oligonucleotides corresponding to the Smad7 promoter (box A/B) for 3 h. The sequence used was as follows: 5'-AGGGAAGGGGGCGGAGGCGGGAGGCCTTGC-3'. DNA–protein complexes were collected by precipitation with streptavidin-agarose beads (Sigma-Aldrich) for 1 h, washed three times with RIPA buffer and subjected to western immunoblotting analysis as described above.

Fluorescence microscopy
Panc-1 cells grown on chambered coverslips were treated with TGFß for 1 h, washed, fixed, blocked and probed with anti-Sp1, anti-Smad2/3 or anti-Smad4 antibodies. Smad2/3 or Smad4 were detected with a secondary antibody that was either coupled to Alexa 488 (green) or Alexa 568 (red) (Invitrogen Life Technologies). Sp1 was visualized by a secondary antibody coupled to Alexa 568 (red) (Invitrogen Life Technologies). Cellular DNA was stained for 10 min at 37°C using 0.5 µg of Hoechst 33342 (Molecular Probes/Invitrogen, Karlsruhe, Germany) per milliliter in phosphate-buffered saline. Coverslips were mounted on glass slides and cells were observed with a fluorescence microscope. Hoechst staining was observed with an emission wavelength of 385–470 nm.

Statistical analysis
Statistical analysis of promoter transactivation, as measured by reporter gene assays, as well as of gene expression data was performed by Student's t-test. All reporter experiments and expression analyses were performed in at least three independent experiments.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Supplementary material
 References
 
Characterization of TGFß responsiveness in pancreatic cancer cell lines
In an initial set of experiments, we verified the integrity of the TGFß/Smad signaling pathway in two pancreatic cancer cell lines (human Panc-1 cells and murine TD-2 cells) reported to be Smad signaling-proficient (33). As a control, we additionally analyzed the Smad4-deficient cell line IMIM-PC2 (34). All three cell lines were transiently transfected with the TGFß/Smad-responsive reporter gene constructs p3TP-Lux (containing the –730 CAGA box of the PAI-1 promoter) (35) and 3xSBE-Luc (carrying three SBEs). Transfection of reporter gene plasmids alone caused low basal transcription levels in all tested cell lines. However, as predicted from the Smad4 expression status, stimulation with 5 ng/ml TGFß greatly induced transcription from both reporter plasmids in Panc-1 and TD-2 cells but not in Smad4-deficient IMIM-PC2 cells (Figure 1A). Immunoblotting analyses confirmed the absence of Smad4 expression in IMIM-PC2 cells, while demonstrating similar levels of Smad3 expression in all three cell types. Interestingly, Sp1 protein was detected at comparable levels in all three cell lines as well (Figure 2B). TGFß-induced nuclear translocation of activated Smad complexes in Panc-1 cells was examined by fluorescence microscopy. Consistent with previous observation in other cell types, Smad3 and Smad4 were predominantly found in the cytoplasm of untreated cells, but rapidly translocated into the nucleus following TGFß treatment for 1 h (Figure 2C). Similar results were found in TD-2 cells, in which TGFß treatment induced rapid nuclear translocation of Smad3–Smad4 complexes (data not shown). Together, these experiments confirmed that the normal TGFß/Smad signaling cascade is intact and active in the pancreatic cancer cells lines Panc-1 and TD-2.


Figure 1
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  		Fig. 1 (A) Panc-1, TD-2 and IMIM-PC2 cells were transiently transfected with either p3TP-Lux or 3x SBE-Luc and treated with TGFß (5 ng/ml) for 24 h. Luciferase activity was measured and induction of reporter promoter constructs was expressed as mean fold induction. Mean values were calculated from three independent experiments and are shown as mean ± standard deviation (**P < 0.01 and *P < 0.05). (B) Western blot analysis of Sp1, Smad3 and Smad4 protein expression in the pancreatic cancer cell lines Panc-1, TD-2 and IMIM-PC2. Protein loading was controlled using anti-ß-actin antibodies. (C) Fluorescence microscopy demonstrating TGFß-induced nuclear translocation and distribution of Smad3 and Smad4 in Panc-1 cells. Cells were left either untreated or treated with TGFß for 1 h and stained with anti-Smad2/3 or anti-Smad4 antibodies before visualization. Overlay demonstrates successful cytoplasmic-nuclear shuttling of Smad3 and Smad4 following TGFß treatment. Nuclei were visualized by Hoechst staining.

 

Figure 2
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  		Fig. 2 Inhibition of Sp1 binding activity prevents TGFß-induced regulation of a distinct set of early response genes. Sp1–DNA binding was inhibited by pre-treatment of Panc-1 cells with mithramycin A. Cells were then incubated in the absence or presence of TGFß for 1 h before total RNA was extracted. (A) All genes differentially regulated by TGFß were subjected to hierarchical cluster analysis using uncentered Pearson correlation as the similarity measure. Genes were normalized to equalize signal intensities. Green cells denote low expression, red cells denote high expression and black cells denote intermediate expression. The effects of mithramycin A on TGFß-induced expression of either Smad7 mRNA (B) or cyclin D1 mRNA (C) were detected by real-time PCR. Effects of mithramycin and TGFß treatment on Smad7 mRNA expression levels were calculated relative to basal mRNA expression levels, which were arbitrarily set to 1 for each experiment. Displayed are mean values from three independent experiments ± standard deviations. Statistical analyses were done using Student's t-test (**P < 0.01 and *P < 0.05).

 
Sp1 participates in TGFß-induced early gene transcription
As a first step towards characterizing the role of Sp1 in TGFß-mediated gene regulation, we analyzed early transcriptional changes induced by TGFß treatment of Panc-1 cells in the presence or absence of the Sp1–DNA binding inhibitor mithramycin A. To this end, we performed expression profiling analyses using specifically designed arrays containing 2435 genes selected for their potential involvement in carcinogenesis, including cell-cycle-associated genes, transcription factors, members of various cell signaling pathways and genes encoding for extracellular matrix components. A total of 43 genes were detected as differentially expressed (11 upregulated, 32 downregulated) following TGFß treatment for 1 hour (complete raw data are available as supplementary information). For the majority of these genes, the TGFß effect was considerably attenuated by treatment of the cells with mithramycin A (Figure 2A), suggesting that Sp1 participates in the transcriptional regulation of these genes by TGFß. Most notably, the group of Sp1-regulated TGFß early response genes included several known TGFß-target genes implicated in carcinogenesis, such as the ECM-degrading and metastasis-associated proteinase MMP-11 (also termed stromelysin-3), the cell-cycle regulating cyclin D1 oncogene and the inhibitory Smad protein, Smad7.

To validate the array hybridization results, we assessed the expression levels of Smad7 and cyclin D1 in the treated and untreated cells by quantitative real-time PCR (qRT–PCR). Consistently, treatment of Panc-1 cells with TGFß rapidly induced Smad7 mRNA expression ~4-fold, whereas inhibition of Sp1 by simultaneous mithramycin A treatment completely prevented TGFß inducibility of the Smad7 gene (Figure 2B). Similarly, the 2-fold induction of cyclin D1 by TGFß observed in untreated Panc-1 cells was lost following pre-treatment of the cells with mithramycin A (Figure 2C). Our gene expression analyses thus indicate a significant role of Sp1 in both TGFß-induced and repressed early gene expression. For the purposes of the current study, we have focused on elucidating the precise function of Sp1 in TGFß-induced upregulation of early target genes, using the Smad7 gene promoter as a model.

TGFß-induced Smad7 promoter activation requires integrity of an SBE and a GC-rich Sp1 binding region
The 5'-flanking region of the mouse Smad7 gene comprises a previously described Smad binding element (SBE) and a GC-rich region containing four elements that exactly match the recognition sequence of Sp1 (box A–D) (Figure 3A). TGFß inducibility of the Smad7 promoter was studied by transient reporter gene assays using a 725 bp spanning region of the proximal mouse Smad7 promoter that contains the SBE and the putative GC-rich Sp1 region (pGL725-Luc). TGFß treatment resulted in a 3–4-fold activation of the pGL725-Luc reporter construct in both pancreatic cancer cell lines with functional Smad signaling pathways, but failed to induce transcription from the Smad7 promoter in the Smad4-deficient IMIM-PC2 cell line (Figure 3B). We then investigated the role of Smad3 and Smad4 in Smad7 promoter activation in more detail and performed reporter gene assays following co-transfection of pGL725-Luc with either Smad3 or Smad4 or a combination of both TGFß effector proteins. Individual overexpression of Smad3 or Smad4 alone significantly increased transcription from the pGL725-Luc promoter reporter construct by ~3–4-fold, whereas combined transfection of both Smad proteins led to a 7.5-fold induction of the Smad7 promoter (Figure 3C). Smad3–Smad4 complexes thus very effectively mediated transcription from the Smad7 promoter. Moreover, the activating effect of the Smad3–Smad4 complex was at least in part dependent on the presence of functional Sp1 protein, as evidenced by a 50% reduction of Smad3/4-mediated transcription from the Smad7 promoter following pharmacological inhibition of Sp1 with mithramycin A (Figure 3D). Activated Smad complexes thus appeared to induce transcription from the Smad7 promoter through both Sp1-dependent and independent promoter elements. To test this hypothesis, we used deletion constructs of the Smad7 promoter that lack either the SBE or the GC-rich Sp1 binding region and performed reporter assays to determine the remaining Smad inducibility. As illustrated in Figure 4A, removal of the SBE (pGL725-delSBE) reduced Smad3/4-mediated transcription from the Smad7 promoter, whereas deletion of the GC-rich Sp1-binding region (pGL725-delGC) diminished both basal (by ~50%) and Smad-induced transcription. Moreover, combined mutation of the SBE and the Sp1 binding region (pGL725-delSBE/GC) reduced basal promoter activity as effectively as individual deletion of the GC-rich site and completely shut off Smad inducibility of the Smad7 promoter. Together, these results showed that the integrity of the GC-rich binding region is required not only for basal promoter activation but also for maximal promoter induction by the Smad proteins.


Figure 3
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  		Fig. 3 TGFß-induced Smad7 promoter activation is mediated by Smad3–Smad4 complexes and requires Sp1 binding activity. (A) Schematic representation of the pGL725-Luc (–613/+112) Smad7 reporter gene construct including the predicted GC-rich Sp1 binding region (boxes A–D) and a previously described SBE. (B–D) Pancreatic cancer cells were transfected with the pGL725-Luc reporter gene construct and the relevance of Smads and Sp1 in TGFß-induced promoter induction was studied. (B) Panc-1, TD-2 and IMIM-PC2 cells were treated with TGFß to determine the TGFß inducibility of the pGL725-Luc Smad7 promoter construct depending on the functionality of the Smad signaling pathway. (C) Panc-1 cells were transfected with Smad3 and/or Smad4 expression constructs together with the pGL725-Luc reporter construct. (D) Panc-1 cells were transiently transfected with Smad3 and Smad4 expression plasmids and treated with mithramycin A to block endogenous Sp1 activity. Luciferase activity was measured and induction of pGL725-Luc reporter gene activity was expressed as mean fold induction compared with untreated control that were arbitrarily set to 1. Mean values were calculated from three independent experiments and are shown as mean ± standard deviation. Statistical analyses were done using Student's t-test (**P < 0.01; n.s. = not significant).

 

Figure 4
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  		Fig. 4 TGFß induces Smad7 promoter activation through a SBE and a GC-rich Sp1 binding region. (A) Panc-1 cells were transiently transfected with Smad3 and Smad4 expression plasmids along with pGL725-Luc, pGL725-delSBE, pGL725-delGC or pGL725-delSBE/GC reporter gene constructs. (B) The proximal Smad7 promoter (region –129 to –209 bp) that lacks consensus Smad binding sequences but contains four putative Sp1 binding boxes (A–D) was cloned into the pGL3 basic luciferase vector (pGL80-wt). The functional relevance of the Sp1 binding region in Smad-mediated promoter activation was verified in transient reporter gene assays following combined mutation of the GC-rich boxes (pGL80-mut). Panc-1 cells were transfected with either pGL80-wt or pGL80-mut and the effects of Smads on the proximal Smad7 promoter were measured before and after artificial overexpression of Sp1. Luciferase activity was measured and induction of reporter promoter constructs was expressed as either RLA or mean fold induction. Mean values were calculated from three independent experiments and are shown as mean ± standard deviation. Statistical analyses were done using Student's t-test (**P < 0.01 and *P < 0.05; n.s. = not significant).

 
As Smad proteins themselves do not directly bind to GC-rich promoter sequences (data not shown), we hypothesized that activated Smad complexes might interact with and work through Sp1 to induce transcription from the GC-rich region of the Smad7 promoter. To further explore the functional relevance of the GC-rich region, we performed additional reporter gene assays using an 80 bp Smad7 promoter constructs containing the GC-rich region (–129 to –209) in which mutations (pGL80-mut) of the four GC boxes had been introduced. Figure 4B shows that Smad3/4 complexes increased transcription from the wild-type Sp1 region (pGL80-wt) and illustrates that this induction was further enhanced through additional introduction of Sp1. In contrast, as mutational inactivation of the Gc-rich binding sites strongly lowered both Smad and Sp1 inducibility of the proximal Smad7 promoter, these results clearly suggested that Smad proteins cooperate with Sp1 in TGFß-induced transcription from GC-rich Sp1 binding elements of the proximal Smad7 promoter.

TGFß induces Sp1–Smad complex formation on GC-rich boxes of the proximal Smad7 promoter
We next performed fluorescence microscopy and co-immunoprecipitation studies to examine whether Smad proteins co-localize with and bind to Sp1 in response to TGFß stimulation. For this purpose, we treated Panc-1 cells with TGFß and studied the nuclear localization of endogenous Smad3 and Sp1. Figure 5A demonstrates that Sp1 is constitutively located in the nucleus, whereas successful translocation of Smad3 into the nucleus was observed upon TGFß stimulation. Moreover, in the nucleus Smad3 co-localized well with Sp1 and within discrete regions. We then investigated the ability of activated Smad complexes to associate with nuclear Sp1 by performing co-immunoprecipitation studies using FLAG-tagged Smad expression constructs. Endogenous Sp1 protein was found to co-precipitate with both Smad3 and Smad4 even in the absence of TGFß (Figure 5B, top panel). Treatment with TGFß, however, rapidly increased the physical interaction between Sp1 and the Smad proteins, and these effects were not due to a change in Sp1 protein levels as shown by immunoblot analyses of whole cell lysates (Figure 5B, bottom panel). These findings are in line with previous observations in different cell systems, such as renal epithelial cells, demonstrating Sp1–Smad complex formation upon TGFß (36,37).


Figure 5
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  		Fig. 5 Activated Smad complexes cooperate with and increase DNA binding of Sp1 in pancreatic cancer cells. (A) Fluorescence microscopy demonstrating TGFß-induced nuclear co-localization of endogenous Smad3 and Sp1 in Panc-1 cells. (B) Increased physical association of Smad3 and Smad4 with Sp1 upon TGFß treatment. Panc-1 cells were transfected with FLAG-tagged Smads and incubated in the presence or absence of TGFß for 1 h. Cell lysates were immunoprecipitated with agarose-conjugated anti-FLAG M2 antibodies followed by immunoblotting with an anti-Sp1 antibody to detect Smad-bound endogenous Sp1. Cell lysate were also directly immunoblotted with anti-Sp1 or anti-ß-actin to demonstrate expression of endogenous Sp1 and to control for protein loading. (C) TGFß-induced binding of endogenous Sp1 and Smad complexes to the Sp1 binding boxes A/B was determined by DNAP experiments. Cell extracts from Panc-1 cells untreated or treated with TGFß for 1 h were incubated with biotinylated Smad7 promoter wild-type boxes A/B and streptavidin-agarose beads. Protein–DNA complexes were subjected to western blotting and probed with anti-Smad3, anti-Smad4 and anti-Sp1 antibodies. The expression levels of endogenous Smads and Sp1 were also studied in cell lysates from treated and untreated Panc-1 cells.

 
Moreover, DNA pull-down (DNAP) experiments using the GC-rich box A/B sequence of the Smad7 promoter demonstrated TGFß-dependent binding of Smad3 and Smad4 to the Sp1 consensus site (Figure 5C). While Sp1 readily precipitated with the GC boxes in untreated cells, no DNA binding of endogenous Smad3–Smad4 complexes was observed in the absence of TGFß stimulation. TGFß treatment of the cells, however, not only led to increased Sp1 binding but also facilitated binding of Smad3/4 to the GC-rich promoter element. In contrast, TGFß failed to induce Smad3 and Sp1 binding to the GC-rich element in the Smad4-deficient IMIM-PC2 cells (data not shown).

Together, these results suggested that physical interaction between Smad complexes and Sp1 is required for TGFß-induced transcription from GC-rich promoter sequences and that TGFß induces transcription from the proximal Smad7 promoter through Smad-mediated regulation of Sp1–DNA binding.

Endogenous Sp1 expression is required for TGFß-induced and Smad3/4-mediated expression of Smad7
To further test this hypothesis, we knocked down endogenous Sp1 expression in Panc-1 cells by RNAi technology. qRT–PCR and western blotting analyses demonstrated >90% reduction of Sp1 mRNA and protein levels following transient transfection of Panc-1 cells with appropriate double-stranded RNA oligonucleotides (Figure 6A and B). We then assessed the effects of Sp1 silencing on Smad3 binding to the GC-rich element and performed DNAP experiments using proteins from Sp1-depleted Panc-1 cells (Figure 6C). While significant amounts of endogenous Smad3 were found to precipitate with the proximal GC-rich promoter following TGFß treatment of control siRNA-transfected cells (Figure 6C, lanes 1 and 2), Smad3–DNA complexes were undetectable in Sp1-depleted Panc-1 cells (Figure 6C, lanes 3 and 4). These findings well confirmed our model and demonstrated that Smad3 binding to the GC-rich region of the proximal Smad7 promoter requires the presence of Sp1. In support of this conclusion, Smad complexes successfully increased transcription from the pGL725-Luc promoter construct in control siRNA-transfected cells, whereas depletion of Sp1 lowered Smad responsiveness of the Smad7 promoter (Figure 6D). The remaining Smad inducibility of the full-length Smad7 promoter (pGL725-Luc), on the other hand, might reflect the transactivation of the previously identified SBE located upstream of the GC-rich binding region (see Figure 4A). This finding is in agreement with our observation that neither pharmacological inhibition of Sp1 binding nor siRNA-induced knockdown of endogenous Sp1 resulted in a complete loss of TGFß-induced endogenous Smad7 mRNA expression (see Figure 3D and Figure 6E). Nevertheless, as a consequence of impaired transcription from the Smad7 promoter upon Sp1 depletion, pancreatic cancer cells—at least partially—escape from both basal and TGFß-induced expression of the negative regulator Smad7 (Figure 6E), and thus respond to TGFß with increased activation of the Smad signaling pathway, as reflected by enhanced levels of Smad3 phosphorylation (Figure 6F).


Figure 6
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  		Fig. 6 Sp1 depletion prevents maximal induction of Smad7 promoter activation and expression and increases TGFß–Smad signaling activity. Panc-1 cells were either not transfected, transfected with Sp1-specific siRNA or with non-specific control siRNA. Forty-eight hours later, cells were harvested and total RNA or proteins were extracted to determine the expression of endogenous Sp1 on both the mRNA (A) and the protein level (B). qRT–PCR was conducted with the generated cDNA, which was used as template to perform the PCR using specific primers to Sp1. Immunoblotting was performed using anti-Sp1 antibodies. (C) TGFß-induced binding of endogenous Smad3 to the Sp1 binding box A was determined by DNAP experiments. Protein extracts from TGFß-treated Panc-1 cells transfected with either non-specific control siRNA or Sp1 siRNA were incubated with biotinylated Smad7 promoter wild-type box A/B and streptavidin-agarose beads. Protein–DNA complexes were subjected to western blotting and probed with anti-Smad3 antibodies. Successful knockdown of Sp1 was confirmed in the same cell lysates and protein loading was controlled using anti-ß-actin antibodies. (D) Panc-1 cells were transfected with either non-specific control siRNA or Sp1 siRNA along with the pGL725-Luc reporter gene construct and Smad3 and Smad4 expression vectors. Smad-induced pGL725-Luc promoter activity was expressed as RLA. Mean values were calculated from three independent experiments and are shown as mean ± standard deviation. Statistical analyses were done using Student's t-test (**P < 0.01 and *P < 0.05). (E) Panc-1 cells were transfected with either non-specific control siRNA or Sp1 siRNA before treatment with TGFß for 3 h. cDNA was prepared and subjected to qRT–PCR. Values were calculated relative to basal mRNA expression levels in control siRNA transfected cells, which were arbitrarily set to 1 for each experiment. Displayed are mean values from three independent experiments ± standard deviations (**P < 0.01). (F) Protein extracts from TGFß-treated Panc-1 cells transfected with either non-specific control siRNA or Sp1 siRNA were extracted to determine TGFß-induced phosphorylation of endogenous Smad3. Protein loading was controlled using anti-ß-actin antibodies.

 
Taken together, these results clearly demonstrated an essential role of Sp1 in basal and TGFß-induced expression of the inhibitory Smad7 gene and suggested that receptor-activated Smad complexes induce transcription from GC-rich promoter sequences through functional and physical interaction with DNA-bound Sp1. Knockdown of endogenous Sp1, on the other hand, prevents TGFß-induced Smad binding to, and transcription from, the proximal Smad7 promoter, and thus contributes to higher levels of Smad signaling activity in pancreatic cancer cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Supplementary material
 References
 
In recent years, much progress has been made in understanding the functional implications of Sp1/KLF-like transcription factors during tumorigenesis. While some members of this protein family, for example, KLF10 and KLF11, appear to act as strong tumor suppressors, others promote tumor progression through their ability to regulate gene expression (38,39). Sp1 and the closely related Sp3 and Sp4 proteins, for instance, have been shown to exert oncogenic functions through transcriptional regulation of genes involved in either cell cycle transition, cell migration or angiogenesis (38,40,41). Interestingly, although the high degree of conservation within their DNA binding domains could be taken to indicate that Sp1, Sp3 and Sp4 potentially interact with the same DNA promoter sequences, recent investigations have revealed that all three proteins have preferences for different sequences and thus regulate different sets of specific target genes (38,41). In the context of TGFß-induced tumor progression, Sp1 attracts most attention, as increasing evidence strongly suggests a specific and crucial role of this transcription factor in TGFß-regulated gene expression. Here, we investigated whether Sp1 is involved in TGFß-regulated early gene expression in pancreatic cancer and analyzed the underlying molecular mechanisms.

First evidence for a potential role of Sp1 in TGFß-induced and Smad-mediated early gene expression was gathered from expression profiling analysis in pancreatic cancer cells upon pharmacological disruption of endogenous Sp1-promoter binding. The results of these studies revealed that prevention of Sp1-promoter binding significantly alters the transcriptional response of pancreatic cancer cells to a TGFß stimulus and also demonstrated that Sp1 is actively involved in both gene induction and repression by TGFß. The group of TGFß–Sp1 inducible genes comprised oncogenic transcription factors, cell-cycle controlling genes and members of the matrix metalloproteinase family, known to be involved in matrix degradation and tumor cell invasion (42). One of the most interesting findings, however, was the identification of inhibitory Smad7. In normal epithelial cells, Smad7 forms an important negative TGFß-feedback mechanism that inhibits intracellular signaling through interaction with the activated type I receptor (43,44). This interaction is believed to prevent further R-Smad phosphorylation and thus terminates activation of the Smad signal transduction pathway (45,46). Alterations of Smad7 expression, on the other hand, have been described in a variety of tumor diseases and in association with increased tumorigenesis (47,48). In advanced pancreatic cancer, for example, overexpression of Smad7 leads to increased anchorage-dependent and independent tumor cell growth and renders the malignant cells insensitive to TGFß-mediated growth inhibition. Here, we describe an important mechanism that operates in pancreatic cancer cells to cause maximal TGFß inducibility of the Smad7 promoter. This novel oncogenic function of TGFß requires an intact Smad signal transduction pathway and is mediated through functional cooperation of activated Smad complexes with Sp1. TGFß rapidly induces Smad3–Smad4 complex formation, stimulates their nuclear translocation and subsequently activates transcription from the Smad7 promoter in TGFß-responsive pancreatic cancer cells. The resulting rapid elevation of Smad7 protein levels serves to quickly terminate TGFß signals and thus might contribute to limiting or abolishing the growth inhibitory effects of TGFß (49).

We have further characterized the TGFß-induced Smad7 promoter regulation and identified two responsive elements within a 725 bp spanning promoter region—a previously described SBE and a GC-rich Sp1 binding region—which are both essential for maximal promoter induction by TGFß (26). In fact, TGFß inducibility of the Smad7 promoter was significantly lowered upon mutational inactivation of either the SBE or the proximal GC-rich Sp1 binding region and was completely abolished following combined inactivation of both binding regions. Consistent with these findings, pharmacological inhibition of Sp1–DNA binding or RNAi-mediated knockdown of endogenous Sp1 expression diminished TGFß-induced Smad7 mRNA expression and strongly reduced Smad-mediated induction of the wild-type Smad7 promoter. These observations together with the finding that Smad-mediated transactivation of the proximal GC-rich promoter region was abolished following combined mutation of the Sp1 binding boxes suggested that activated Smads and Sp1 functionally and physically cooperate in TGFß-induced transcription from the proximal Sp1 binding sites. Similar cooperative mechanisms involving Sp1 and Smad proteins have been suggested in the regulation of other TGFß target promoters, although previous studies have largely relied on overexpression and/or pharmacological inhibition of Sp1 (18,26,30,37,50–53).

Combined biochemical studies and fluorescence microscopy revealed that TGFß rapidly induces nuclear co-localization of Smads and Sp1 and subsequently stimulates their physical interaction. Moreover, TGFß-induced Smad–Sp1 interaction increases Sp1 binding to the GC-rich region and results in superinduction of Sp1-mediated transcription from the proximal Smad7 promoter. Surprisingly, however, Smad-mediated superinduction of Sp1 does not require direct DNA binding of the Smads. Usage of RNAi technology in DNAP experiments suggests a mode of Sp1-mediated Smad binding to the Smad7 GC-rich promoter regions in vivo and thus provides evidence for a novel mechanism in TGFß-regulated gene expression, in which Smad proteins primarily function as signal transducers rather than transcription factors. Taken together, our study confirms previous findings indicating that Smad-partnering transcription factors determine TGFß substrate specificity and thus exert profound impact on the cellular response to TGFß. Here, we show that Sp1 significantly participates in TGFß early gene regulation of pancreatic cancer cells. On the basis of our findings, we propose a model in which TGFß induces interaction between Smads and DNA-bound Sp1, leading to increased Sp1 binding activity and transcription. These findings not only extend our knowledge of TGFß-mediated gene expression during carcinogenesis but also implicate that TGFß—via activation of the Smad proteins—might also regulate the expression of genes whose promoters actually lack bonafide Smad binding sites. Future work will be required to analyze whether this novel mechanism is also operative on other TGFß target gene promoters and whether other oncogenic members of the Sp1/KLF-like family, namely Sp3 and Sp4, play a role in TGFß-induced and Smad-mediated gene regulation during pancreatic tumorigenesis.


   Supplementary material
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
References
 
Supplementary material is available at http://www.uni-ulm.de/klinik/medklinik/innere1/forschung/ag-gress/smad_Sp1_complexes.html


   Footnotes
 
{dagger}Both authors have contributed equally to this work. Back


   Acknowledgments
 
This work was supported by the Deutsche Krebshilfe (grant 70-3022-EI 1) to V.E. and by the Deutsche Forschungsgemeinschaft (SFB 518/Project B1) to T.M.G.

Conflict of Interest Statement: None declared.


   References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary material
 References
 

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Received October 19, 2005; revised January 26, 2006; accepted May 10, 2006.


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