Volume 126, Issue 7, Pages 1828-1843 (June 2004) Activin A stimulates vascular endothelial growth factor gene transcription in human hepatocellular carcinoma cells  Karola Wagner, Michael Peters, Arne Scholz, Christoph Benckert, Hugo Sanchez Ruderisch, Bertram Wiedenmann, Stefan Rosewicz  Gastroenterology  Volume 126, Issue 7, Pages 1828-1843 (June 2004) DOI: 10.1053/j.gastro.2004.03.011

Figure 1 Expression of VEGF and activin A and its receptors in human HCC. Immunostaining with polyclonal antibodies against VEGF (A), activin A (B), ActR-I (C), ActR-IB (D), ActR-II (E), and ActR-IIB (F) was performed in paraffin-embedded HCC tissue sections by using the avidin-biotin-peroxidase method. The antigen-antibody complexes stain red. To confirm the specificity of the observed immunohistochemical signals, we used preimmune goat immunoglobulin G as a first antibody, which failed to show relevant staining. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 2 Expression of activin A and its receptors in HCC cell lines: RT-PCR analysis. Sequence-specific primers are shown in Table 1. RT-PCR for VEGF was performed by using primers that recognize all known splice variants. Alternating lanes represent reactions with (+) or without (−) the addition of reverse transcriptase (RT). RT-PCR for β-actin was used for comparison of input mRNA integrity. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 3 Effects of activin A on cell growth and VEGF expression. (A) HuH7, HepG2, and Hep3B cells were incubated in serum-free medium in the absence or presence of activin A (25 ng/mL), and cells were counted after 96 hours of treatment. Data are expressed as the percentage of untreated controls and are the means ± SEM from at least 3 separate experiments, each performed in triplicate. ∗P = 0.0065 (HuH7); ∗P < 0.001 (HepG2). (B) Cells were treated with vehicle or activin A 25 ng/mL for 96 hours, and the VEGF protein concentration in supernatants was determined by enzyme-linked immunosorbent assay and normalized to cell number. Shown are the means ± SEM of at least 4 independent experiments, each conducted in triplicate. ∗P = 0.009 (HuH7). (C) HuH7 cells were incubated in serum-free medium with or without activin A (25 ng/mL) for the indicated time periods, and the VEGF protein concentration in supernatants was analyzed by enzyme-linked immunosorbent assay. Data represent the percentage of untreated controls and are the means ± SEM derived from 4 experiments, each performed in triplicate. ∗P = 0.008. (D) Cells were incubated in serum-free medium with or without activin A (25 ng/mL) for 96 hours, and VEGF protein was analyzed in supernatants by enzyme-linked immunosorbent assay and normalized to cell number. Data shown represent the mean ± SEM of at least 4 experiments, each performed in triplicate. ∗P = 0.025 (EGR-1); ∗P = 0.022 (TFK-1); ∗P = 0.022 (DU145). (E) EGR-1, TFK-1, and DU145 cells were treated with activin A (25 ng/mL) or vehicle for 96 hours, and cell numbers were determined. Data represent the percentage of vehicle-treated controls and are the means ± SEM from at least 5 separate experiments, each conducted in triplicate. ∗P < 0.001 (EGR-1); ∗P = 0.011 (TFK-1);∗P = 0.0062 (DU145). Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 4 Activin A transactivates the VEGF gene promoter. (A) HuH7, HepG2, and Hep3B cells were transiently transfected with a -2018-VEGF-Luc construct, which contains the VEGF promoter region (−2018 to +50) upstream of the luciferase gene, and treated with activin A 25 (ng/mL) for 24 hours. Luciferase activity was determined and normalized for transfection efficiency. Data show the mean relative light units of at least 3 experiments, conducted in hexaplicate. ∗P = 0.004 (HuH7); ∗P = 0.02 (HepG2); ∗P = 0.01 (Hep3B). (B) HuH7 cells were transiently transfected with the -2018-VEGF-Luc construct and incubated for 24 hours with the indicated concentrations of activin A. Luciferase activity was measured and expressed as a percentage of vehicle-treated cells. Data represent the mean of 6 experiments, each performed in triplicate. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 5 Mapping of the activin A-responsive region of the human VEGF promoter. (A) HuH7 cells were transiently transfected with the indicated 5′-deletion reporter gene constructs of the human VEGF promoter and subsequently incubated with activin A (25 ng/mL) or vehicle. After 24 hours, luciferase activity was measured in relative light units, and data are expressed as the mean ± SEM of at least 3 experiments. (B) HuH7 cells were transiently transfected with a heterologous enhancerless herpes simplex thymidine kinase promoter reporter construct fused to region −85/−50 of the human VEGF promoter and incubated with activin A (25 ng/mL) or vehicle for 24 hours. Luciferase activity was determined in relative light units, and data represent the mean of 3 experiments. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 6 Identification of the transcription factor-binding sites required for activin A-induced transactivation of the human VEGF promoter. (A) Schematic representation of the -85/-50 region of the human VEGF promoter with its putative binding sites for transcription factors Egr-1, AP-2, Sp1, and Sp3, which bind to GC boxes, and Smads, which bind to repeats of perfect (GTCT) and degenerate (GnCn) Smad boxes. The perfect Smad box is absent from the proximal VEGF promoter region, but 3 direct repeats of degenerate Smad boxes, each arranged in right-left orientation, as indicated by arrows, can be identified. (B) HuH7 cells were transiently transfected with the indicated VEGF luciferase constructs encoding mutationally inactivated Egr-1- and AP-2-binding sites or GC box mutations, respectively (substituted nucleotides are shown in small letters). Cells were treated with vehicle or activin A (25 ng/mL), luciferase activity was measured in relative light units, and data are expressed as the mean ± SEM of 5 independent experiments, each conducted in hexaplicate. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 7 Identification of the transcription factors binding to the −88/−50 region of the VEGF promoter. (A) Nuclear extracts derived from HuH7 cells, treated with activin A (25 ng/mL) for the indicated time points, were incubated with a 32P-labeled oligonucleotide spanning the −88/−50 VEGF promoter sequence, and DNA/protein complexes were separated in gel shift assays. For competition experiments, nuclear extracts were preincubated with a 100× molar excess of wild-type or mutant Sp oligonucleotides (B), AP-2 or Egr-1 oligonucleotides (C), or CAGA wild-type or mutant oligonucleotides (D). For supershift experiments, nuclear extracts were incubated with antibodies specific for Sp1 and Sp3 (B), AP-2 and Egr-1 (C), and Smad2/3 (Sd2/3) and Smad4 (Sd4) (D) before being incubated with the 32P-labeled VEGF −88/−50 probe. Incubation of HuH7 nuclear extracts together with a 32P-labeled CAGA box containing oligonucleotides (lanes 31–37) served as a positive control for DNA binding of Smad proteins. Lanes 5 and 31 represent control lanes in which the oligonucleotide probes were not incubated with any nuclear extracts. Each condition was repeated at least 3 times, and representative gels are shown. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 8 Effects of activin A on Sp1 transactivation. (A) HuH7 cells were stimulated with activin A for the indicated time periods. Nuclear extracts were prepared, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subsequently incubated with antibodies against Sp1 and Sp3. Data represent a typical result obtained from a series of 3 independent experiments. (B) HuH7 cells were transiently co-transfected with Gal4-Luc, containing 5 Gal4 binding sites in front of the luciferase gene along with the indicated Gal4 fusion proteins. After 24 hours of treatment with activin A (25 ng/mL) or vehicle, luciferase activity was measured and corrected for transfection efficiency. Results are shown as the mean ± SEM from 3 independent experiments, each conducted in hexaplicate. ∗P = 0.036. (C) HuH7 cells were incubated with activin A (25 ng/mL) for the indicated time points, nuclear extracts were prepared, and Sp1 was immunoprecipitated from aliquots of 250 μg. Immunoblots with the indicated antibodies were performed to determine activin A-induced changes in Sp1 phosphorylation and glycosylation and Sp1/p53 interaction. To ensure that equal amounts of protein had been examined, Western blots were subsequently stripped and reprobed with Sp1 antibody. Shown are representative blots of 3 independent experiments. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 9 Effects of activin A on Smad/Sp1 interaction. (A) HuH7 cells were incubated with activin A (25 ng/mL) for the indicated time periods, and nuclear extracts were prepared. Aliquots of 2 mg of nuclear proteins were immunoprecipitated with a polyclonal Sp1 antibody. The resulting immunocomplexes were subsequently separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by immunoblotting with the indicated antibodies. No complexes were detected in nuclear extracts immunoprecipitated with normal rabbit immunoglobulin G (data not shown). To ensure that equivalent amounts of protein had been analyzed, immunoblots were reprobed with anti-Sp1 antibody. Nuclear (B) and cytosolic (C) extracts and whole-cell lysates (D) were analyzed in parallel. Shown are representative blots of 3 independent experiments. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 10 Functional relevance of Smads for activin A-induced transactivation of the VEGF promoter. (A) Wild-type HuH7 cells and HuH7 cells transiently transfected with Smad2 or a carboxy-terminally truncated mutant of Smad2 (Smad2ΔC) were incubated with activin A (25 ng/mL) for 24 hours. Whole-cell lysates (Lys) and nuclear extracts (NE) were prepared and separated by 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and analyzed by immunoblotting with a monoclonal antibody against Smad2. Data shown represent a typical result obtained from a series of 3 independent experiments. (B) HuH7 cells were transiently transfected with 30 ng of the -85-VEGF-Luc reporter construct and the indicated amount of Smad2, Smad3, or Smad4. Luciferase activity was measured in relative light units, and data represent the mean ± SEM from at least 6 experiments. (C) HuH7 cells were co-transfected with 30 ng of the -85-VEGF-Luc reporter construct and increasing amounts of Smad2ΔC, Smad3ΔC, or Smad4ΔC, as indicated, and subsequently incubated with activin A (25 ng/mL) or vehicle for 24 hours. Luciferase activity was determined, and results are expressed as fold induction over untreated cells. Bars represent the mean ± SEM of 6 experiments. In all assays, the total amount of DNA was maintained constant by co-transfection of empty expression vector, and all assays were conducted in hexaplicate. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)

Figure 11 Functional relevance of the Smad2 signaling pathway for activin A-mediated accumulation of endogenous VEGF protein. (A) HuH7 cells were transiently transfected with Smad2 or a control empty vector (pRK5). After 96 hours, the VEGF protein concentration in supernatants was analyzed with an enzyme-linked immunosorbent assay and normalized to cell number. Data represent the mean ± SEM of 5 separate experiments, each performed in triplicate. ∗P = 0.049. (B) HuH7 cells transiently transfected with a carboxy-terminally truncated dominant negative mutant of Smad2 (Smad2ΔC) or an empty control vector (pRKs) were incubated with or without activin A (25 ng/mL) for 96 hours. VEGF protein in supernatants was determined by enzyme-linked immunosorbent assay and normalized to cell number. Data are expressed as the mean ± SEM of 7 experiments, each conducted in triplicate. ∗P = 0.0059. Gastroenterology 2004 126, 1828-1843DOI: (10.1053/j.gastro.2004.03.011)