FEBS Letters 582 (2008) 1859–1864
Transcriptional regulation of the hepatocyte growth factor gene by pyrrolidine dithiocarbamate Phillip M. Harrisona,*, Farzin Farzanehb a
Kings College London, Department of Liver Studies and Transplantation, Denmark Hill Campus, Bessemer Road, London SE5 9RS, UK b Kings College London, Department of Haematological Medicine, The Rayne Institute, 123 Coldharbour Lane, London SE5 9NU, UK Received 15 March 2008; revised 29 April 2008; accepted 30 April 2008 Available online 12 May 2008 Edited by Beat Imhof
Abstract Hepatocyte growth factor (HGF) mediates cancer cell invasion and metastasis. This study characterised the down-regulation of HGF expression by pyrrolidine dithiocarbamate (PDTC), which markedly reduced HGF mRNA expression and protein production in MRC-5 cells. Reporter gene studies revealed that PDTC inhibited HGF gene transcription and that the response element is located in the region 75 to +42 bp flanking the transcription initiation site. Electrophoretic mobility shift assay identified three specific protein complexes binding in this region, which were abrogated by exposure of cells to PDTC. PDTC deserves further investigation as a novel therapeutic agent for HGF-driven cancers. 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Hepatocyte growth factor; Scatter factor; Pyrrolidine dithiocarbamate; Transcription factor; RNA stability
1. Introduction Hepatocyte growth factor (HGF) is produced by mesenchymal cells and stimulates cell proliferation, motility and morphogenesis in epithelial cells, depending on the target cell and culture conditions [1,2]. Due to its ability to co-ordinate cell proliferation, migration and matrix remodelling, HGF is an important physiological regulator in embryological development [3,4] and has a vital role in the generation and maintenance of normal organ complexity and architecture [5]. Cancer cell growth is an aberrant counterpart of physiological morphogenic growth and HGF is one of the factors stimulating cancer cells to invade locally and to metastasize [6]. The HGF receptor, MET, was originally identified as the product of the oncogene, TPR–MET [7]. Although mutant versions of MET show enhanced signalling, they still require ligand binding to cause cell transformation [8] and HGF has been found to stimulate growth, motility and invasiveness in a number of tumour cell lines [9,10]. The blockade of HGF–
* Corresponding author. Fax: +44 (0) 20 3299 3167. E-mail address:
[email protected] (P.M. Harrison).
Abbreviations: HGF, hepatocyte growth factor; PDTC, pyrrolidine dithiocarbamate; FCS, foetal calf serum; BS, bovine serum; PMA, phorbol 12-myristate 13-acetate; CAT, chloramphenicol acetyl transferase; EMSA, electrophoretic mobility shift assay; IL-1b, interleukin 1 beta; NAC, N-acetylcysteine
MET signalling using a hammerhead ribozyme [11], or neutralizing antibodies against MET [12] or HGF [13], substantially reduced the growth of U-87 cells in nude mice. These findings indicate that HGF availability is crucial for MET-dependent cell growth. Accordingly, inhibitors of HGF production could have clinical application but surprisingly few have been described. Previously, we demonstrated that transforming growth factor-beta inhibits HGF gene expression by reducing HGF mRNA stability [14], and although retinoids [15], glucocorticoids [16] and angiotensin II [17] have all been shown to inhibit secretion of HGF, the mechanisms are unknown. We previously reported that the antioxidant N-acetylcysteine (NAC) increases HGF mRNA expression in MRC-5 cells at the transcriptional level by a mechanism independent of its ability to replete glutathione levels [18]. Pyrrolidine dithiocarbamate (PDTC) is a water soluble, small molecule that is commonly regarded as an antioxidant but it can also bind and transport copper and zinc ions, and is known to modulate several intracellular signalling pathways [19]. Whilst, in some situations NAC and PDTC regulate gene transcription via the same mechanism [20,21], the two compounds have been reported to have antagonistic effects [22,23]. In the present study, we investigate the regulation of HGF expression in MRC-5 cells by PDTC. We demonstrate that rather than increase HGF expression, PDTC is a potent inhibitor of HGF gene transcription leading to a marked reduction in HGF mRNA expression and protein production.
2. Materials and methods 2.1. Materials Foetal calf serum (FCS), bovine serum (BS), actinomycin D, NAC, pyrrolidine dithiocarbamate (PDTC) and phorbol 12-myristate 13-acetate (PMA) were from Sigma Chemical Company, Poole, UK; and the 32 P-adATP and 14C-chloramphenicol were from ICN Flow, UK. 2.2. Culture of cell lines Human embryonic lung fibroblasts (MRC-5 cells) and mouse embryo fibroblasts (NIH3T3), obtained from the European collection of animal cell cultures, were cultured in of Dulbeccos modified Eagle medium with antibiotics (50 units/ml penicillin and 50 lg/ml streptomycin) and 10% FCS or 10% BS, respectively. 2.3. Northern blot analysis Total RNA was isolated according to the method described by Chomczynski and Sacchi [24]. Each test condition was performed in triplicate and the RNA pooled. RNA samples (30 lg) were fractionated by gel electrophoresis (1% agarose and 2.2% formaldehyde) and transferred to Genescreen (Du-Pont-NEN, Boston, USA) in 10· SSC (1.5 M sodium chloride + 0.15 M trisodium citrate, pH 7.0).
0014-5793/$34.00 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.04.055
1860 Northern hybridisation was performed as previously described [14] using a 32P-labelled 668 bp partial cDNA of HGF as a probe. The membrane was exposed to X-ray film with intensifying screens at 70 C; it was then stripped and rehybridised to a 32P-labelled 360 bp partial cDNA probe for GAPDH before being re-imaged. The synthesis of the partial cDNA probes for HGF and GAPDH has been described previously [14]. 2.4. ELISA and scatter factor assay for HGF protein MRC-5 cells were grown in 24-well plates to near confluence, under the same conditions as for the Northern blot analysis, and then exposed to fresh media containing increasing concentrations of PDTC for 3 days. Experiments were performed in triplicate and HGF levels in MRC-5 cell conditioned media were measured by both ELISA (R&D Systems, Abingdon, UK) and scatter factor assay, as described previously [25]. 2.5. HGF gene promoter activity Construction of the HGF-chloramphenicol acetyl transferase (CAT) chimeric reporter genes has been described previously [14]. NIH3T3 cells at 50–60% confluence were transfected with 10 lg of HGF-CAT chimeric plasmid and 2 lg of pSV-b-galactosidase control plasmid (Promega, Madison, USA) using the calcium phosphate method [26] and exposed to 500 lM PDTC for 24 h. The cells were harvested and disrupted by three cycles of freeze–thaw. Aliquots of supernatant were taken for both b-galactosidase and CAT activity; the latter were heated to 60 C for 10 min to inactivate any endogenous acetylase and deacetylase activity. All extracts were stored at 70 C. The b-galactosidase activity was quantified according to the manufacturers instructions (Promega, Madison, USA) in order to assess transfection efficiency, and the volume of supernatant assayed for CAT activity adjusted accordingly. CAT activity was measured according to the manufacturers instructions, as described previously [14]. 2.6. Electrophoretic mobility shift assay (EMSA) MRC-5 cells, grown to near confluence, were scraped with a rubber policeman and pelleted by centrifugation. The cells were washed in 1 ml Tris-buffered saline and pelleted again by centrifugation at 4 C. All further steps were carried out at 4 C, on ice. The cells were resuspended in 0.4 ml of 10 mM Hepes, pH 7.8, 1 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM DTT, 0.1 mM PMSF and incubated for 15 min. The cells were lysed in 25 ll of Nonidet-P40 (10%, v/v) and the nuclei pelleted before being resuspended in 50 ll 50 mM Hepes, pH 7.8, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF and 10% glycerol. After 20 min the extract was centrifuged again and the supernatant stored in aliquots at 70 C. Nuclear protein concentrations were measured by BCA Protein Assay (Sigma, UK), following the manufacturers instructions. The 75 to +42 bp HGF promoter sequence, labelled with c32PdATP using T4 Polynucleotide Kinase for 1 h at 37 C, was used as the DNA probe. The binding buffer, which was kept on ice, consisted of 300 lg/ml bovine serum albumin (BSA), 1 ll poly(dI-dC) Æ poly(dIdC), 2 lg nuclear protein extract and 100-fold molar excess of the unlabelled competitor probe if required, in a final volume of 15 ll. After addition of the labelled probe, the reactions were incubated on ice for 30 min. The unlabelled, double-stranded competitor probes used were: the 75 to +42 HGF sequence; a control designated E3, 5 0 GATCGGTCATGTGGCAAGGCTATTTGGG-3 0 , which has no homology with HGF; and a control designated AP-1, 5 0 CGCTTGATGACTCAGCCGGAA-3 0 , which contains an activator protein 1 consensus binding site. The DNA/protein complexes were resolved on a 6% polyacrylamide gel [1· Tris glycine buffer, 6% Accugel (v/v), 2.5% glycerol (v/v)] electrophoresed at 40 mA for 3 h at 4 C. The gel was dried and exposed to X-ray film with intensifying screens at 70 C.
3. Results 3.1. The effect of PDTC on HGF expression in MRC-5 cells MRC-5 cells were cultured in the presence of PDTC, RNA was extracted and analyzed by Northern hybridization using
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a 668 bp partial cDNA HGF probe, corresponding to exons 6–11 [18]. PDTC profoundly reduced the expression of all HGF mRNA transcripts, in a dose- and time-dependent fashion (Fig. 1a). To determine whether PDTC modulates induced as well as innate HGF mRNA expression, we examined the effect of PDTC on the up-regulation of HGF mRNA in MRC-5 cells by PMA, a protein kinase C activator [27], by human interleukin 1 beta (IL-1b) [28] or by the thiol NAC [18]. HGF mRNA was up-regulated in MRC-5 cells cultured for 24 h in increasing concentrations of PMA, IL-1b (Fig. 1b), or NAC (Fig. 1c) but concomitant exposure to 500 lM PDTC inhibited this increase. To determine whether PDTC increased HGF RNA degradation, we assessed the half-life of the 6.0 kb HGF mRNA by harvesting RNA at time points after the addition of 5 lg/ml actinomycin-D to sub-confluent MRC-5 cells. HGF mRNA levels were analyzed by Northern hybridization (Fig. 1d). The half-life of the 6.0 kb HGF mRNA transcript was around 3 h and was unaffected by exposure to 500 lM PDTC for 6 h, demonstrating that PDTC has no effect on HGF mRNA stability. To look at the effect of PDTC on HGF protein production, HGF levels were measured in MRC-5 cell conditioned media by both immunological assay (ELISA) and the functional scatter factor assay [25]. Exposure to PDTC for 3 days markedly reduced HGF protein production (Fig. 1e). 3.2. PDTC inhibits HGF gene transcription The transcriptional activity of the HGF-CAT constructs demonstrates that the HGF promoter region is highly regulated (Fig. 2a). There are inhibitory regions between 715 to 491 and 375 to 75 bp and an up-regulatory region between 491 and 375 bp. The smallest construct, 75HGFCAT, which contains a potential mammalian C-type LTR TATA box localized at 38 to 20 [29], had the highest transcriptional activity. The activity of the 906HGF-CAT, 715HGF-CAT, 491HGF-CAT, 375HGF-CAT and 75HGFCAT constructs was reduced by exposure to 500 lM PDTC for 24 h to 23%, 21%, 14%, 15% and 22% of baseline values, respectively (Fig. 2a). These results indicate that the main response element mediating the effect of PDTC on HGF gene transcription is located between 75 and +42 bp in the 5 0 flanking region. 3.3. PDTC mediates nuclear protein binding to the HGF gene between 75 and +42 bp EMSA was used to resolve the binding of nuclear proteins to the region of the HGF gene, between 75 and +42 bp. Nuclear protein in control MRC-5 cells produced three specific binding complexes with a 32P-labelled, double-stranded, 75 to +42 bp HGF DNA probe (Fig. 2b). These DNA/protein binding complexes were lost following the addition of an excess of unlabeled HGF DNA probe but were unaffected by an excess of either AP-1 or E3 control probes. The specific DNA/protein binding complexes were also lost when EMSA was performed using nuclear protein extracted from MRC-5 cells that had been exposed to 500 lM PDTC for 24 h, indicating that exposure to PDTC disrupted the assembly of transcription factors binding to this region of the HGF gene (Fig. 2b). We analysed the sequence of the HGF promoter region between 75 and +42 bp to identify possible transcription factor binding sites (MatInspector, Genomatix [29]), which are
P.M. Harrison, F. Farzaneh / FEBS Letters 582 (2008) 1859–1864
1861
Fig. 1. Effect of PDTC on HGF mRNA and protein expression in MRC-5 cells. Total RNA was isolated from MRC-5 cells and 30 lg subjected to sequential Northern blot analysis using: a 32P-labelled 668 bp HGF cDNA probe, 40 h autoradiographic exposure; and a 32P-labelled GAPDH cDNA probe, as a loading control, 6 h autoradiographic exposure. (a) MRC-5 cells were exposed to increasing concentrations of PDTC for 24 h (left panel) or 500 lM PDTC for up to 24 h (right panel). MRC-5 cells were cultured for 24 h with (b) PMA (top panel) or IL-1b (bottom panel) or (c) NAC, in the presence or absence of 500 lM PDTC. (d) MRC-5 cells were exposed to 500 lM PDTC for 6 h and RNA isolated at time points after the addition of 5 lg/ ml actinomycin-D . (e) MRC-5 cells were cultured under the same conditions as for the Northern blot analysis and exposed to increasing concentrations of PDTC for 3 days. HGF levels in the conditioned media were measured by ELISA (left panel) and scatter factor assay [25] (right panel).
shown (Fig. 2c). However, none of the putative transcription factors binding to this region is known to be regulated by
PDTC or to interact with known PDTC-regulated DNA binding proteins, such as AP-1 or NF-jB.
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Fig. 2. Activity of human HGF promoter-CAT chimeric genes and electrophoretic mobility shift assay. (a) NIH3T3 fibroblasts were transfected with HGF-CAT chimeric reporter gene constructs by the calcium phosphate method and exposed to 500 lM PDTC for 24 h. After adjustment for transfection efficiency, standard CAT assay was performed. CMV-CAT and pCAT-basic were used as controls. Each result is the median of four assays. (b) Electrophoretic mobility shift assay was used to demonstrate complex formation between the HGF promoter and nuclear proteins in control and PDTC treated MRC-5 cells. The DNA probe used was the 75 to +42 bp HGF promoter sequence labelled with c32P-dATP. Lanes 1–4, nuclear protein in control MRC-5 cells: lane 1, no competitor; lane 2, unlabelled HGF (75/+42 bp) competitor probe; lane 3, unlabelled non-specific competitor probe, E3; lane 4, unlabelled competitor probe, AP-1; lane 5, nuclear protein in PDTC treated MRC-5 cells, no competitor probe. The specific (displaceable) and non-specific (non-displaceable) nuclear protein–DNA complexes are indicated by arrows. (c) Analysis of the HGF promoter region between 75 and +42 bp identifying putative transcription factor binding sites (MatInspector, Genomatix [29]).
4. Discussion The present study demonstrates that PDTC is a potent inhibitor of basal and induced HGF gene expression in MRC-5 cells resulting in a marked down-regulation of HGF mRNA, in a dose and time-dependent manner. Consequently, HGF protein production by MRC-5 cells was significantly inhibited by all doses of PDTC tested. This is the first study to our knowledge to describe a small molecule inhibitor of HGF expression. PDTC is known to regulate the expression of several genes by modulating the activity of transcription factors. It inhibits
the DNA binding activity of NF-jB, by preventing the degradation of IjB alpha via the ubiquitylation-proteasome proteolytic pathway [30], and of Nuclear Factor of Activated T cells (NFAT), by a mechanism dependent on the stimulation of tyrosine kinase pathways [31]. PDTC also activates AP-1 activity by modulating expression of c-jun and c-fos [32] and Nrf2 DNA binding complexes, containing either JunD or small Maf protein [33]. We analyzed the sequence of the promoter region between 75 and +42 bp to identify potential transcription factor binding sites (MatInspector, Genomatix [29]) but none of the candidate transcription factors binding in this region of the HGF gene, HOXA9, RORA2, OCT1 or HOXC13, is
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known to be regulated by PDTC or to interact with a PDTCregulated DNA binding protein. AP-1 does not appear to regulate this region of the HGF gene, as a probe containing a consensus AP-1 recognition sequence failed to block binding of the nuclear proteins to the HGF promoter. Nevertheless, we identified three specific protein/DNA complexes that bound between 75 and +42 in the 5 0 -flanking region of the HGF gene and these were lost when the MRC-5 cell were exposed to PDTC, suggesting that PDTC exposure disrupts the assembly of the basal transcription apparatus of the HGF gene. However, to date we have not been able to characterize the sequence of the PDTC response element in the human HGF promoter or to confirm the transcription factors abrogated by PDTC. In summary, we show that PDTC inhibits not only innate HGF gene expression in MRC-5 cells but also the up-regulation of HGF mRNA induced by several well-characterized factors, acting via different mechanisms. Furthermore, we demonstrate that PDTC acts by disrupting the assembly of transcription factors to the region of the HGF gene flanking the transcription initiation site. Previous reports have demonstrated that dithiocarbamates protect the liver from N-nitrosodiethylamine-induced tumour development [34] and inhibit the growth of colon cancer cells in mice [35]. PDTC is an orally active, small molecule and, based on the findings of the present study, further work is needed to investigate whether it can inhibit the growth of HGF-dependent tumour cells. Acknowledgements: We would like to thank Prof. Ermano Gherardi for assistance with the scatter factor assay and Dr. Lara Bradley for her assistance in the construction of the HGF Promoter-CAT chimeric reporter genes and reporter gene assays. This study was supported by the Medical Research Council, UK.
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