Von Hippel-Lindau gene product modulates TIS11B expression in renal cell carcinoma: impact on vascular endothelial growth factor expression in hypoxia - PubMed (original) (raw)

Von Hippel-Lindau gene product modulates TIS11B expression in renal cell carcinoma: impact on vascular endothelial growth factor expression in hypoxia

Sutapa Sinha et al. J Biol Chem. 2009.

Abstract

TIS11B belongs to a group of RNA-binding proteins (including TIS11/tristetraprolin and TIS11D) that share characteristic tandem CCCH-type zinc-finger domains and can be rapidly induced by multiple stimuli. TIS11B has been shown to regulate vascular endothelial growth factor (VEGF) mRNA stability in adrenocorticotropic hormone-stimulated primary adrenocortical cells. TIS11B has also been documented as a negative regulator of VEGF during development, but nothing has yet been reported in the context of human cancers. The Von Hippel-Lindau (VHL) tumor suppressor protein regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia. However, whether it can do so in hypoxia is still unclear. Here, we report a unique regulatory function of VHL in VEGF expression in hypoxia that is mediated through modulation of TIS11B protein levels in renal cancer cells. In normoxia, we detected increased expression of the microRNA hsa-miR-29b in the VHL-overexpressing renal cancer cell line 786-O. We also show that this increased expression of hsa-miR-29b decreased TIS11B protein expression by post-transcriptional regulation in normoxia. In contrast, in hypoxia, increased TIS11B expression paralleled an increased TIS11B mRNA stability in VHL-overexpressing 786-O cells. This VHL-mediated TIS11B up-regulation in hypoxia may be important for TIS11B-regulated gene expression: we observed a down-regulation of VEGF mRNA in hypoxia in VHL-overexpressing cells compared with parental 786-O cells, and this effect was reversible by silencing TIS11B expression.

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Figures

FIGURE 1.

FIGURE 1.

VEGF-A mRNA levels in 786-O-neo and 786-O-VHL cells. RNA was collected from 786-O-neo and 786-O-VHL cells incubated under normoxic and hypoxic conditions (16–50 h). Real-time PCR for VEGF-A was done with the cDNAs from the respective samples, and β-actin was used for cDNA normalization. Subjecting both cell lines to hypoxic growth conditions resulted in significant VEGF mRNA accumulation. However, the level of VEGF-A mRNA was still considerably higher (p = 0.02) in 786-O-neo cells than in 786-O-VHL cells even after 50 h of hypoxia. Data represent the average of three independent determinations. N, normoxia; H 16, hypoxia, 16 h; H 24, hypoxia, 24 h; H 42, hypoxia, 42 h; H 50, hypoxia, 50 h.

FIGURE 2.

FIGURE 2.

TIS11B protein levels under normoxic and hypoxic growth conditions. A, Western blot analysis was performed with lysates from 786-O-neo and 786-O-VHL cells under normoxia or hypoxia. A significantly high level of TIS11B protein expression was detected in 786-O-neo cells (p = 0.003) compared with 786-O-VHL cells in normoxia. However, increased TIS11B expression in 786-O-VHL cells compared with 786-O-neo cells was observed after 16 and 24 h (p = 0.004) of hypoxia. α-Tubulin was used as the loading control. NIH Image quantitation data normalized with respect to α-tubulin are shown. The amount of VHL expression in 786-O-VHL cells in normoxia is also indicated. B, an up-regulation of TIS11B protein levels was also found in kidney proximal tubular epithelial cells (HK-2) after 24 h of hypoxia. Data are representative of three separate experiments, which gave similar results. N, normoxia; H 16, hypoxia, 16 h; H 24, hypoxia, 24 h.

FIGURE 3.

FIGURE 3.

Effect of VHL overexpression in 786-O cells under normoxic and hypoxic growth conditions. 786-O cells were transfected with pBABe-puro-VHL and incubated under normoxia or hypoxia. A, Western blotting was performed to detect the level of VHL after transfection in 786-O cells in normoxia. α-Tubulin was used as the loading control. B and C, after 24 h of hypoxia, RNA was collected, and real-time PCR was performed to detect TIS11B and VEGF-A mRNA levels. Data represent the average of three independent determinations. N, normoxia; H 24, hypoxia, 24 h.

FIGURE 4.

FIGURE 4.

A, differential expression of miR-29b. The expression levels of mature miR-29b in 786-O-neo, 786-O-VHL, and HK-2 cells were measured by real-time PCR using specific primers. Single-tube TaqMan miRNA assays were performed to quantify mature miR-29b, and they were normalized to RNU6B in triplicate. Relative expression (-fold) was calculated using the comparative Ct method. Real-time PCR analysis showed an increased expression of miR-29b in 786-O-VHL and HK-2 cells compared with 786-O-neo cells under normoxia. Under hypoxia, the expression level in all three cell lines did not change significantly. Data represent the average of three independent determinations. B, introduction of miR-29b reduces TIS11B levels in normoxia. 786-O-neo cells were transfected with 1 n

m

scrambled control (Conmi) or miR-29b (29bmi) mimic and analyzed for the expression of TIS11B by Western blotting. α-Tubulin was used as the loading control. Data are representative of three separate experiments, which gave similar results. The relative -fold expression of TIS11B and p values (control versus treated sample) have been included. C and D, the inhibitor of miR-29b up-regulates TIS11B expression in normoxia. 786-O-VHL and HK-2 cells transfected with the 200 n

m

non-targeting control (ConIn) or antisense inhibitor (29bIn) of miR-29b were analyzed for TIS11B by Western blotting. α-Tubulin was used as the loading control. Data are representative of three separate experiments, which gave similar results. The relative -fold expression of TIS11B and p values (control versus treated sample) have been indicated. N, normoxia; H 24, hypoxia, 24 h.

FIGURE 5.

FIGURE 5.

TIS11B is a target of miR-29b in normoxia. The 3′-UTR of TIS11B enables miR-29b regulation. A, the complementarity between TIS11B cDNA and miR-29b in humans is shown. B, 293T and 786-O-neo cells were cotransfected with the miR-29b mimic or the non-targeting control and the full-length 3′-UTR of TIS11B gene downstream of the luciferase gene. A significant down-regulation of luciferase activity was recorded 36 h after transfection. Experiments were performed twice in triplicate (n = 6).

FIGURE 6.

FIGURE 6.

VHL regulates TIS11B mRNA stability. The half-life of TIS11B mRNA was monitored in 786-O and 786-O+VHL cells under normoxia (A) and hypoxia (B). 786-O and 786-O+VHL cells were cultured under either normoxia or hypoxia for 24 h, and then actinomycin D (5 μg/ml) was added to the growth medium. Immediately after the addition of actinomycin D, the cells were returned to the same culture conditions (normoxia or hypoxia). During the following 5 h, total RNA was prepared at different time points, and real-time PCR was performed. Data represent the average of three independent determinations.

FIGURE 7.

FIGURE 7.

TIS11B down-regulates VEGF-A expression. 786-O-VHL cells were transfected with the scrambled control (Consi) or TIS11B (TIS11Bsi) siRNA using DharmaFECT 1 and incubated under normoxia and hypoxia. After 72 h, total RNA was collected, and real-time PCR was performed to detect TIS11B and VEGF-A mRNA expression. After TIS11B knockdown (B and D), increased expression of VEGF-A was observed in both normoxia (A) and hypoxia (C). Data represent the average of three independent determinations. N, normoxia; H 24, hypoxia, 24 h.

FIGURE 8.

FIGURE 8.

Model for VHL-modulated TIS11B expression in regulation of VEGF-A in RCC. A, in normoxia, RCC 786-O cells express an increased level of TIS11B protein, overriding the active positive regulators (e.g. HIF-2α and HuR) of VEGF-A, and a moderately high level of VEGF-A is detected. B, in hypoxia in 786-O cells, TIS11B expression decreases, which allows for increased VEGF-A synthesis. C, in normoxia, VHL overexpression down-regulates the positive regulators of VEGF synthesis and increases the level of the miRNA miR-29b in RCC. An elevated level of miR-29b in VHL-expressing cells targets the 3′-UTR of the RNA-binding protein TIS11B and down-regulates its translation. VEGF-A expression then remains low in VHL-overexpressing 786-O cells in normoxia. D, in hypoxia, VHL-induced miR-29b expression remains unaltered. However, mRNA stabilization contributes significantly to maintaining high levels of TIS11B in 786-O-VHL cells. This stabilized TIS11B keeps VEGF-A expression low compared with the VEGF-A levels found in 786-O null cells under hypoxic stress.

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