Tristetraprolin inhibits Ras-dependent tumor vascularization by inducing vascular endothelial growth factor mRNA degradation - PubMed (original) (raw)
Tristetraprolin inhibits Ras-dependent tumor vascularization by inducing vascular endothelial growth factor mRNA degradation
Khadija Essafi-Benkhadir et al. Mol Biol Cell. 2007 Nov.
Abstract
Vascular endothelial growth factor (VEGF) is one of the most important regulators of physiological and pathological angiogenesis. Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway and overexpression of VEGF are common denominators of tumors from different origins. We have established a new link between these two fundamental observations converging on VEGF mRNA stability. In this complex phenomenon, tristetraprolin (TTP), an adenylate and uridylate-rich element-associated protein that binds to VEGF mRNA 3'-untranslated region, plays a key role by inducing VEGF mRNA degradation, thus maintaining basal VEGF mRNA amounts in normal cells. ERKs activation results in the accumulation of TTP mRNA. However, ERKs reduce the VEGF mRNA-destabilizing effect of TTP, leading to an increase in VEGF expression that favors the angiogenic switch. Moreover, TTP decreases RasVal12-dependent VEGF expression and development of vascularized tumors in nude mice. As a consequence, TTP might represent a novel antiangiogenic and antitumor agent acting through its destabilizing activity on VEGF mRNA. Determination of TTP and ERKs status would provide useful information for the evaluation of the angiogenic potential in human tumors.
Figures
Figure 1.
Expression of VEGF mRNA in normal and tumor cell lines. (A) Twenty micrograms of total protein extracts from control or Ras Val 12 (clone 5C)-transformed cells were subjected in duplicate to Western blotting by using a monoclonal pERK antibody. The amounts of actin are shown as a loading control. Average ERK activity normalized to actin amounts obtained by quantification of the two independent samples is also shown. (B) Total RNA isolated from normal Chinese hamster fibroblasts and Ras Val 12 (clone 5C) cells were analyzed by Northern blotting for the expression of VEGF mRNA after DRB treatment (25 μg/ml). 36B4 RNA is shown as a loading control. (C) The amount of VEGF mRNA was quantified with a PhosphorImaging system (Storm 840; Amersham Biosciences, Roosendaal, The Netherlands). VEGF mRNA half-lives, deduced from two independent experiments, are indicated (p < 0.05).
Figure 2.
VEGF mRNA expression is regulated by the ERK pathway. (A) Raf1-ER cells were serum starved and treated with 1 μM tamoxifen for 3 h. Then, cells were treated or not with 10 μM U0126. Protein extracts (20 μg) were analyzed by Western blotting by using a monoclonal anti-pERK antibody. Data are representative of two independent experiments. (B) Raf1-ER cells were serum starved and treated with 1 μM tamoxifen for 3H. Cells were treated with 25 μg/ml DRB in the presence or absence of 10 μM U0126. VEGF mRNA was detected by Northern blotting and normalized against 36B4 RNA. (C) The amount of VEGF mRNA was quantified with a PhosphorImaging system. VEGF mRNA half-lives, deduced from two independent experiments, are indicated (p < 0.05).
Figure 3.
ERK activation induces the expression of TTP and VEGF mRNA. (A) Raf1-ER cells were serum deprived, treated with 1 μM tamoxifen for the indicated time, and total RNAs were isolated. Quantitative PCR was performed using specific primers for VEGF and TTP. Fold induction of VEGF and TTP RNA is relative to time 0 after normalization to 36B4 in each sample. (B) Protein extracts (30 μg) prepared in parallel, were analyzed by Western blotting by using a polyclonal anti-TTP, monoclonal anti-pERK, and ERK antibodies. Data are representative of three independent experiments.
Figure 4.
TTP interacts with the VEGF mRNA 3′-UTR in vitro. (A) Schematic map of the VEGF 3′-UTR illustrating the templates used for generation of riboprobes for REMSA. (B) REMSAs were performed by incubating the radiolabeled VEGF 3′-UTR transcripts (full length, NsiI, and ΔNsiI) with purified GST-TTP or GST. The arrows point to RNA–protein complexes. (C) Competition with specific competitor (100 M excess) corresponding to unlabeled NsiI and ΔNsiI transcripts, respectively, was also performed in the presence of GST-TTP. The arrows point to RNA–protein complexes.
Figure 5.
TTP interacts with VEGF mRNA in vivo. (A) Raf1-ER cells were stably transfected with an expression vector encoding a tetracycline inducible myc-tagged TTP. Cells were stimulated for various times with 1 μg/ml tetracycline. Inducible overexpression of myc-TTP was then verified either by Western blotting or (B) by immunostaining, by using a monoclonal anti-myc antibody. (C) Tamoxifen-treated Raf1-ER/TTP cell extracts were immunoprecipitated with no antibody, with the anti-myc antibody, or with an irrelevant antibody (anti-Ras antibody) in the presence or absence of MEK inhibitor 10 μM U0126. Equal aliquots of purified total RNA isolated from the immunoprecipitates (P), and from the supernatants (S), were assayed by reverse transcriptase-PCR to detect the VEGF (top) and 36B4 (bottom) transcripts.
Figure 6.
TTP overexpression decreases VEGF mRNA half-life in vivo. (A) Raf1-ER/TTP cells were serum-starved and stimulated (+) or not (−) with 1 μg/ml tetracycline for 24 h. After 3 h of stimulation with tamoxifen, cells were incubated in the absence or presence of 10 μM U0126 for one supplemental hour, and then in the presence of 25 μg/ml DRB for the indicated times. During the DRB chase, cells were maintained or not in the presence of U0126. The amounts of VEGF mRNA remaining were quantified by real-time PCR. The values are normalized to 36B4, and the values at time 0 were taken as 100%. VEGF mRNA half-lives were deduced from the curves (n = 3; p < 0.05). (B) Raf1-ER/TTP cells were serum-starved and stimulated with 1 μg/ml tetracycline for 24 h before stimulation or not with tamoxifen for indicated time. Left, time course of tamoxifen stimulation. Arrow and bracket indicate the unshifted and the retarded bands, respectively. Right, cell extracts were treated or not with CIP. Protein extracts (30 μg) were then analyzed by Western blotting by using anti-myc, pERK and ERK antibodies. This experiment is representative of two independent experiments. (C) Raf1-ER or Raf1-ER/TTP cells were serum starved, stimulated with 1 μM tamoxifen and incubated in the absence or presence of 10 μM U0126 for one supplemental hour. Protein extracts (30 μg) were analyzed by Western blotting using anti-TTP, myc, and ERK antibodies. This experiment is representative of two independent experiments. (D) Exponentially growing Raf1-ER or Raf1-ER/TTP cells were stimulated with or without 1 μg/ml tetracycline for 24 h. Secreted VEGF was measured by ELISA. VEGF levels were normalized to the cell number. -Fold inhibition of secreted VEGF are presented as a mean of three independent experiments performed in triplicate.
Figure 7.
TTP knockdown increases VEGF mRNA level. (A) Cells were transfected with siRNAs against TTP or with a control siRNA (Dharmacon RNA Technologies). Forty-eight hours after the second transfection, cells were serum starved overnight and stimulated with tamoxifen for 3 h in the absence or presence of 10 μM U0126. Total RNA was isolated and real-time PCR analysis was performed to determine TTP and (B) VEGF mRNA expression levels. The data shown represent the mean ± SE of three independent experiments.
Figure 8.
TTP induced an inhibitory effect on luciferase activity. (A) 293 Raf1-ER cells were cotransfected with a VEGF mRNA-3′-UTR-luciferase reporter construct and different amounts of TTP expression plasmids in the presence or absence of 1 μM tamoxifen. Luciferase assays were conducted 24 h after transfection. Relative luciferase activity was normalized to total protein amount. The luciferase counts obtained with the construct Luciferase/VEGF 3′-UTR in the absence of TTP was taken as the value of reference (100%). The percentages of inhibition exerted by TTP are also indicated on the figure. Results are reported as the mean ± SE of three independent experiments performed in triplicate. (B) Mapping of different sequences in VEGF mRNA 3′-UTR used for TTP inhibition of reporter gene activity. (C) Transfections were performed as described in A in the presence or absence of 100 ng of TTP. The luciferase values obtained for each construct in the absence of TTP were taken as the value of reference (100%). Plotted are the percentages of remaining luciferase activity in the presence of TTP. Results are reported as the mean ± SE of three independent experiments performed in triplicate.
Figure 9.
Inducible expression of TTP inhibits tumor angiogenesis in vivo. (A) Left, Raf-ER/TTP cells were transfected with a plasmid coding for Ha-Ras. Cells from stable clones were stimulated for various times with 1 μg/ml tetracycline before lysis. Total cell extracts were used to detect both Ras and myc-TTP proteins by Western blot analysis by using anti-Pan Ras and Myc antibodies, respectively. Protein levels of total ERK are shown as loading control. Right, supernatants from stable clones cultured in the presence or absence of 1 μg/ml tetracycline for 24 h were collected and analyzed by mouse VEGF-specific ELISA. Results are reported as the mean ± SE of three independent experiments each run in duplicate. (B) Left, eight nude mice per condition were inoculated with cells from stable clone or negative and positive control. Animals were randomized into two groups. One group received weekly 750 μg/ml doxycycline. Tumor size was measured at the indicated times and tumor volume was calculated. The medians of the tumor size (cubic millimeters) are indicated. Right, tumors were excised from animals at day 35 after tumor cell inoculation when the largest tumor had reached a diameter of 4 cm. Representative examples for each experimental group are shown. (C) Tumor tissues were lysed and intratumoral hemoglobin and VEGF contents were measured by Drabkin reagent kit 525 (Sigma) and ELISA (R&D systems), respectively. The data were corrected to the protein concentration. (D) Immunofluorescence staining of frozen tumor sections with anti-CD31 antibody. Data are representative of two independent experiments.
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