Table 1.
Custom TaqMan Assay gene-specific primers and reporter probes.
Table 2.
Primers used for FGFR2 promoter constructs.
Figure 1.
Effect of cytokines treatment on MCF-7 cell proliferation.
Immunofluorescence analysis with a polyclonal antibody directed against Ki67 in MCF-7 cells that were left untreated, treated with 10 ng/ml KGF as a positive control or treated with 10 ng/ml IL1β, IL2, IL6, TNFα and IFNγ for 24, 48 and 72 h. The percentage of Ki67-positive cells was determined by counting the number of Ki67-positive nuclei versus total number of nuclei in ten different areas randomly taken from three different experiments. Error bars represent standard deviations. #P<0.05, *P<0.01.
Figure 2.
Effect of cytokines on the regulation of KGFR mRNA and protein expression in MCF-7 cells.
(A–D) Quantitative real-time PCR analysis of KGFR mRNA expression in MCF-7 cells following treatment with 10 ng/ml IL1β, IL2, IL6, TNFα or IFNγ for 8, 24, 48 and 72 h. Relative KGFR mRNA levels are shown as fold value of the level of KGFR mRNA in untreated cells. Each experiment was performed in triplicate, and mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. #P<0.05. (E–H) Western blot analysis of KGFR protein levels in MCF-7 cells untreated or treated with 10 ng/ml IL1β, IL2, IL6, TNFα or IFNγ for 8, 24, 48 and 72 h. KGFR protein expression was evaluated by blotting with an anti-Bek antibody. Western blot with anti-Tubulin antibody was used as loading control. The images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells. Densitometric analysis was also performed for each experiment and reported as a graph. Error bars represent standard deviations. #P<0.05.
Figure 3.
Effect of higher doses of cytokines on KGFR mRNA and protein expression in MCF-7 cells.
(A–B) Quantitative real-time PCR analysis of KGFR mRNA expression in MCF-7 cells following treatment with 100 ng/ml IL1β, IL2 or TNFα for 24 and 48 h. Relative KGFR mRNA levels are shown as fold value of the level of KGFR mRNA in untreated cells. Each experiment was performed in triplicate, and mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. *P<0.01. (C–D) Western blot analysis of KGFR protein levels in MCF-7 cells untreated or treated with 100 ng/ml IL1β, IL2 and TNFα for 24 and 48 h. KGFR protein expression was evaluated by blotting with an anti-Bek antibody. Western blot with anti-Tubulin antibody was used as loading control. The images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells. Densitometric analysis was also performed for each experiment and reported as a graph. Error bars represent standard deviations. #P<0.05.
Figure 4.
Effect of cytokines on KGF and FGFR2-IIIc mRNA expression in human primary fibroblasts.
(A) Quantitative real-time PCR analysis of KGF mRNA expression following treatment with 100 ng/ml IL1β, IL2 or TNFα for 24 h. Relative KGF mRNA levels are shown as fold value of the level of KGF mRNA in untreated cells. (B) Quantitative real-time PCR analysis of FGFR2-IIIc mRNA expression following treatment with 100 ng/ml IL1β, IL2 or TNFα for 24 h. Relative FGFR2-IIIc mRNA levels are shown as fold value of the level of FGFR2-IIIc mRNA in untreated cells. Each experiment was performed in triplicate, and mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. #P<0.05, *P<0.01. (C) Quantitative real-time PCR analysis to compare the amount of FGFR2-IIIc mRNA expression in HF and in MCF-7 cells, both untreated or treated with 100 ng/ml TNFα for 24 h. Relative FGFR2-IIIc mRNA levels are shown as fold value of the level of FGFR2-IIIc mRNA in untreated HF cells. Each experiment was performed in triplicate, and mRNA levels were normalized to GAPDH mRNA expression. Error bars represent standard deviations. *P<0.01. (D) Western blot analysis of FGFR2-IIIc protein levels in HF cells untreated or treated with 100 ng/ml IL1β, IL2 and TNFα for 48 h. FGFR2-IIIc protein expression was evaluated by blotting with an anti-Bek antibody. Western blot with anti-Tubulin antibody was used as loading control. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells.
Figure 5.
Role of TNFα in the stimulation of FGFR2 promoter activity.
(A) Schematic representation of the FGFR2 promoter construct, in which a 1.5 kb cassette of FGFR2 gene around the transcription initiation site is linked to the luciferase reporter gene. Putative binding sites for STAT and E2F transcription factors families in the promoter sequence are shown as white or grey ovals, respectively. (B) Luciferase reporter assays were performed in HEK293 cells. The recombinant pGL3-basic-1.5 kb FGFR2 promoter (−1139/+459) construct was transfected into HEK293 cells. 6 h after transfection, cells were left untreated or treated with 100 ng/ml IL1β, 100 ng/ml IL2 or 50 ng/ml TNFα, and luciferase activities were determined 24 h after treatment. Luciferase reporter assay data are expressed as percentage of control (untreated cells) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. *P<0.01.
Figure 6.
Role of E2F1 in TNFα-induced stimulation of FGFR2 promoter.
(A) Luciferase reporter assays were performed in HEK293 cells. The recombinant pGL3-basic-1.5 kb FGFR2 promoter (−1139/+459) construct was co-transfected into HEK293 cells with pRcCMV (empty vector) or pRc-CMV-E2F1, and luciferase activities were determined 24 h after trasfection. Data are expressed as percentage of control (cells transfected with pRcCMV alone) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. *P<0.01. (B) Western blot analysis of E2F1 protein levels and pRb phosphorylation status in MCF-7 cells untreated or treated with 100 ng/ml TNFα for 3 h. E2F1 protein expression was assessed by blotting with an anti-E2F1 polyclonal antibody. pRb phosphorylation was evaluated by blotting with an anti-phospho-pRb antibody. Tubulin was used as loading control. The images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells. Densitometric analysis was also performed for each experiment and reported as a graph. Error bars represent standard deviations. #P<0.05. (C) Western blot analysis of p38 and C-Raf phosphorylation status in MCF-7 cells untreated or treated with 100 ng/ml TNFα for 1 and 3 h. p38 and C-Raf phosphorylation was evaluated by blotting with anti-phospho-p38 and anti-phospho-C-Raf antibodies, respectively. Western blot with anti-p38 or anti-C-Raf antibodies, respectively, was used as loading control. The images are representative of at least three independent experiments. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells. Densitometric analysis was performed for each experiment and reported as a graph. Error bars represent standard deviations. #P<0.05, *P<0.01. (D) Co-immunoprecipitation assay was performed to study in vivo interaction between pRb and E2F1 proteins. MCF-7 cells, untreated or treated with 100 ng/ml TNFα, were immunoprecipitated with anti-pRb antibody and blotted with anti-E2F1 antibody. Western blot with anti-pRb antibody was used as loading control. (E) Western blot analysis of pRb phosphorylation status in MCF-7 cells untreated or treated with 100 ng/ml TNFα for 3 h, alone or in the presence of the p38 inhibitor SB202190 (10 µM), the C-Raf inhibitor GW5074 (1 µM ) or both of them. pRb phosphorylation was evaluated by blotting with an anti-phospho-pRb antibody. Tubulin was used as loading control. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells. (F) Western blot analysis of KGFR protein levels in MCF-7 cells untreated or treated with 100 ng/ml TNFα for 48 h, alone or in the presence of the p38 inhibitor SB202190 (10 µM), the C-Raf inhibitor GW5074 (1 µM) or both of them. KGFR protein expression was evaluated by blotting with an anti-Bek antibody. Western blot with anti-Tubulin antibody was used as loading control. The intensity of the bands was evaluated by densitometric analysis, normalized and reported as relative expression with respect to the untreated cells.
Figure 7.
Role of proinflammatory cytokines in the stimulation of FGFR2 promoter activity.
(A) Schematic representation of the second FGFR2 promoter construct, in which a 1.3 kb cassette of FGFR2 gene upstream of the previously used 1.5 kb cassette is linked to the luciferase reporter gene. Putative binding sites for STAT and E2F transcription factors families in the promoter sequence are shown as white or grey ovals, respectively. (B) Luciferase reporter assays were performed in HEK293 cells. The recombinant pGL3-basic-1.3 kb FGFR2 promoter (−2235/−909) construct was transfected into HEK293 cells. 6 h after transfection, cells were left untreated or treated with 100 ng/ml IL1β, 100 ng/ml IL2 or 50 ng/ml TNFα, and luciferase activities were determined 24 h after treatment. Luciferase reporter assay data are expressed as percentage of control (untreated cells) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. #P<0.05, *P<0.01.
Figure 8.
Identification of E2F1-responsive region in the FGFR2 promoter.
(A) Truncation analysis of FGFR2 promoter activity in HEK293 cells. Cells were co-transfected with different pGL3-basic-FGFR2 promoter truncated constructs and pRcCMV (empty vector) or pRc-CMV-E2F1, and luciferase activities were determined 24 h after transfection. Constructs with different FGFR2 promoter lengths are depicted along the left. Putative binding sites for E2F transcription factors family in the promoter sequence are shown as grey ovals. Luciferase reporter assay data are expressed as percentage of control (cells transfected with pRcCMV alone) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. #P<0.05, *P<0.01. (B) Lysates of HEK293 cells were subjected to ChIP with indicated antibodies (E2F1 or IgG). Immunoprecipitated DNA and input DNA were subjected to PCR amplification of the FGFR2 promoter region between −48 and +245. The image is representative of three independent experiments.
Figure 9.
Mutation analysis of FGFR2 promoter activity in HEK293 cells.
(A) Constructs in which pGL3-basic was linked to different FGFR2 promoter regions (−81/+58 or −81/+5) or to synthetic sequences with sequentially mutated 7 nucleotides stretches in the E2F1 responsive region (+5/+58) were co-transfected with pRcCMV empty vector or with pRcCMV-E2F1 into HEK293 cells, and luciferase activities were determined 24 h after transfection. Normal and mutated constructs of FGFR2 promoter are depicted along the left. Luciferase reporter assay data are expressed as percentage of control (cells transfected with pRcCMV alone) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. *P<0.01. (B) Constructs in which pGL3-basic was linked to FGFR2 promoter regions containing the wild-type +5/+11 sequence (WT), the mutated +5/+11 stretch (MutX) or seven mutated sequences, in each of which one nucleotide was replaced with a T in different positions of the +5/+11 sequence, were co-transfected with pRcCMV empty vector or with pRcCMV-E2F1 into HEK293 cells, and luciferase activities were determined 24 h after transfection. Normal and mutated constructs of FGFR2 promoter are depicted along the left. Luciferase reporter assay data are expressed as percentage of control (cells transfected with pRcCMV alone) and represent the means of three separate experiments after correcting for differences in transfection efficiency by pRL-TK activities. Error bars represent standard deviations. *P<0.01.
Figure 10.
Model for TNFα effects in vivo.
(A) TNFα acts on fibroblasts by stimulating an increase in FGFR2-IIIc expression and in KGF production. KGF can then bind to and activate KGFR, whose expression on keratinocytes has been at the same time stimulated by TNFα. (B) TNFα treatment in keratinocytes induces the hyper-phosphorylation of pRb, and the subsequent release of transcriptionally active E2F1, that can translocate from cytosol to the nucleus, where it binds FGFR2 promoter and allows for KGFR biosynthesis to occur. See text for details.