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To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Gugg 1321C, Mayo Clinic College of Medicine, 200 First St. SW, Rochester, MN 55905. Tel.: 507-538-3581; Fax: 507-233-1058
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.
Angiogenesis, the formation of new blood vessels from pre-existing vessels, is an essential process for establishing a closed circulatory system and for supplying oxygen and nutrients to tissues. Under normal circumstances, angiogenesis is a highly ordered and tightly regulated process of positive and negative regulatory pathways (
The immediate-early protein tristetraprolin family consists of three known members in mammals (ZFP36 or tristetraprolin, ZFP36L1 or TIS11B (tetradecanoylphorbol acetate-inducible sequence 11B), and ZFP36L2 or TIS11D) and a fourth member found only in the mouse and rat (ZFP36L3). They share characteristic tandem CCCH-type zinc-finger domains, and although they are rapidly induced by multiple stimuli, their basal mRNA level varies (
). However, its role in tumor angiogenesis is largely unknown.
In this study, we investigated the role of VHL in the regulation of VEGF through TIS11B in RCC. We observed that VHL overexpression regulated TIS11B in the renal cancer cells (786-O). We also found that the microRNA (miRNA) hsa-miR-29b was overexpressed in 786-O cells expressing exogenous VHL, which could then target the TIS11B transcript to repress its expression under normoxia. However, under hypoxic stress, TIS11B mRNA became stabilized in the VHL-expressing 786-O cells and targeted the VEGF transcript for degradation.
RCC and other tumors that arise in patients with the VHL syndrome are characteristically well vascularized, a property that has been attributed to their consistent overexpression of the potent angiogenic factor VEGF (
). A number of factors have been reported to regulate VEGF in various tumors and in non-tumorigenic cells. One of the well studied regulators is hypoxic stress, and VHL has emerged as a key factor in cellular responses to hypoxia. It has been well established that VHL down-regulates VEGF gene expression at both the transcriptional and post-transcriptional levels in normoxia (
). However, the effect of VHL on VEGF regulation under hypoxic stress remains to be determined. In this study, we observed that the VEGF level in VHL-expressing 786-O cells remained significantly lower than that in the parental 786-O cells lacking endogenous VHL expression, even after prolonged hypoxia. Here, we have provided evidence as to how VHL regulates VEGF expression in hypoxia.
In normoxia, 786-O cells expressing exogenous VHL express a low level of TIS11B. However, in these same cells under hypoxia, we detected an increased level of TIS11B expression in contrast to the level in the VHL-deficient renal cancer cell line 786-O. Our results suggest that VHL regulates VEGF synthesis through TIS11B primarily by two mechanisms depending on whether the environment is normoxic or hypoxic. VHL overexpression increases the level of the miRNA miR-29b in RCC. In normoxia, miR-29b in VHL-expressing cells targets the 3′-UTR of the RNA-binding protein TIS11B to down-regulate its translation without affecting mRNA stability. Although VHL-induced miR-29b expression remains unaltered by hypoxic stress, it seems to be functionally silent, as evidenced when the introduction of an anti-miR-29b oligonucleotide did not significantly affect the level of TIS11B in hypoxia. Therefore, it appears that VHL can regulate TIS11B through an alternative mechanism in a hypoxic environment. We detected a stabilization of the TIS11B transcript for an extended period in VHL-expressing cells compared with that in the parental VHL-null 786-O cells under hypoxic conditions. Thus, enhanced mRNA stabilization contributes significantly to maintain high levels of TIS11B in 786-O-VHL cells under hypoxia. Future studies are under way to delineate the mechanism of VHL-mediated regulation of the RNA-binding protein involved in TIS11B mRNA stabilization in hypoxia. Of importance, knockdown of TIS11B in hypoxia led to a significant increase in the VEGF mRNA level. These observations present a novel inhibitory role of VHL in VEGF regulation in hypoxia through increased TIS11B expression.
Hypoxia is the major inducer of VEGF synthesis in both physiological and tumor angiogenesis. Although there are checks and balances during the physiological process of hypoxia-mediated VEGF-A synthesis, such tight control is absent in pathological angiogenesis. Hypoxia-inducible factor (HIF) and HuR are two molecules contributing mainly to the control of VEGF expression (
). One possible reason for this high level of TIS11B expression in normoxia is to override the active positive regulators (e.g. HIF-2α and HuR) of VEGF synthesis. However, in VHL-expressing cells, even a low level of TIS11B is important for maintaining VEGF expression under a threshold value in normoxia because knockdown of TIS11B in these cells also shows an up-regulation of VEGF.
) showed that TIS11B mutant embryos exhibited extraembryonic and intraembryonic vascular defects, cardiac abnormalities, and an elevated level of VEGF. Additionally, TIS11B has been shown to be an important regulator of myogenic differentiation (
). Therefore, in VHL-expressing cells, we believe that a balanced TIS11B-regulated VEGF level in both normoxia and hypoxia is important for its physiological function. As shown here with the loss of VHL, TIS11B-regulated VEGF synthesis is also impaired and this, in turn, favors unrestricted expression of VEGF and its subsequent pathological effects.
Our findings on regulation of VEGF by the VHL/TIS11B axis in RCC are summarized in Fig. 8. In normoxia, RCC 786-O cells express an increased level of TIS11B protein to override the active positive regulators (e.g. HIF-2α and HuR) of VEGF-A and synthesize a moderately high level of VEGF-A. In hypoxia, however, a decrease in TIS11B expression relieves its inhibitory effects on VEGF-A, allowing a further increase in VEGF-A synthesis under the control of HIF and HuR in 786-O cells. Interestingly, VHL overexpression in 786-O cells keeps the TIS11B level low by inhibiting TIS11B translation through miR-29b in normoxia. VEGF-A expression also remains low in VHL-overexpressing 786-O cells in normoxia because of VHL-mediated inhibition of HuR and HIF activity. Although miR-29b expression remains unaltered in VHL-overexpressing 786-O cells under hypoxia, an increased stability of TIS11B mRNA has been observed, and stabilized TIS11B maintains VEGF-A expression at ∼30% lower levels compared with the VEGF-A levels found in 786-O null cells under hypoxic stress. Here, our data uncovered a novel inhibitory pathway regulating VEGF synthesis through interplay between the VHL/miR-29b/TIS11B axis and HIF/HuR positive regulatory axis under hypoxia.