Antagonistic functions of tetradecanoyl phorbol acetate-inducible-sequence 11b and HuR in the hormonal regulation of vascular endothelial growth factor messenger ribonucleic acid stability by adrenocorticotropin

Abstract

Expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a potent angiogenic factor, is up-regulated by a variety of factors including hypoxia, growth factors, and hormones. In the adrenal cortex, regulation of VEGF expression by the pituitary hormone ACTH ensures the maintenance of the organ vasculature. We have previously shown that ACTH evokes a rapid and transient increase in VEGF mRNA levels in primary adrenocortical cells through transcription-independent mechanisms. We further demonstrated that the zinc finger RNA-binding protein Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) destabilizes VEGF mRNA through its 3'-untranslated region (3'-UTR) and that Tis11b is involved in the decay phase of ACTH-induced VEGF mRNA expression. In the present study, we attempted to determine the mechanisms underlying ACTH-elicited increase in VEGF mRNA levels in adrenocortical cells. We show that ACTH triggers an increase in the levels of the mRNA-stabilizing protein HuR in the cytoplasm and a concomitant decrease in the levels of HuR in the nucleus. This process is accompanied by an increased association of HuR with the nucleocytoplasmic shuttling protein pp32, indicating that ACTH induces HuR translocation from the nuclear to the cytoplasmic compartment. Leptomycin B, a specific inhibitor of CRM1-dependent nuclear export of pp32, significantly reduced ACTH-induced VEGF mRNA levels. Furthermore, RNA interference-mediated depletion of HuR in adrenocortical cells abrogated ACTH-induced VEGF mRNA expression. Finally, we show that Tis11b and HuR exert antagonistic effects on VEGF 3'-UTR in vitro. Although both proteins could bind simultaneously on VEGF 3'-UTR, Tis11b markedly decreases HuR-binding to this RNA sequence. Altogether, these results suggest that the RNA-stabilizing protein HuR is instrumental to ACTH-induced expression of VEGF mRNA and that the nuclear export of HuR is a rate-limiting step in this process. HuR appears to transiently stabilize VEGF transcripts after ACTH stimulation of adrenocortical cells, and Tis11b appears to subsequently trigger their degradation.

Figure 1. Effect of ACTH on VEGF, Tis11b and HuR mRNAs expression

A, Representative ethidium bromide staining of VEGF, Tis11b and HuR mRNAs amplified by RT-PCR. Primary cultures of BAC cells were treated with 10 nM ACTH for the indicated periods of time. VEGF, Tis11b and HuR mRNA levels were then analyzed by RT-PCR as described in Materials and Methods. B, Quantitation of VEGF, Tis11b and HuR mRNAs levels of independent experiments (n=3 to 5). mRNA level values were normalized to HPRT mRNA levels and are expressed as fold induction over control values at time 0 (unstimulated cells).

Figure 2. Effect of ACTH on Tis11b and HuR protein levels in total cell extracts and subcellular fractions

A, BAC cells were treated with 10 nM ACTH for the indicated periods of time. Tis11b and HuR protein levels of whole cell extracts (10 μg) were analysed by Western blot as outlined in Materials and Methods. The Western blot was subsequently probed with an anti-α-tubulin monoclonal antibody to assess equal loading of samples. B, Quantitation of HuR and Tis11b protein levels of total cell extracts in three independent experiments. Protein level values were normalized to α-tubulin protein levels. C, BAC cells were treated with 10 nM ACTH as indicated in (A). Nuclear (5 μg) and cytoplasmic (20 μg) fractions were prepared as described in Materials and Methods and subjected to Western blot analysis to monitor Tis11b and HuR expression. The same membranes were sequentially probed with antibodies recognizing cytoplasm- and nucleus-specific proteins (α-tubulin and lamin A/C, respectively) to assess the quality of the fractionation process and to check for equal protein loading. D, Cytoplasmic and nuclear HuR protein levels were normalized to α-tubulin and lamin protein levels respectively and are expressed as a fraction of total cytoplasmic or total nuclear protein content (n=2).

Figure 3. Co-immunoprecipitation of HuR from BAC cytoplasmic and nuclear extracts using anti-pp32 antibodies

A, BAC cells were treated with 10 nM ACTH for the indicated periods of time. Cytoplasmic (250 μg protein) and nuclear fractions (70 μg protein) were immunoprecipitated with anti-human pp32 antibodies as mentioned in Materials and Methods. Precipitates were electrophoresed on a 12% denaturing gel, transferred to PVDF membrane, and probed with HuR or pp32 polyclonal antibodies. B, Quantitation of HuR levels in cytoplasmic and nuclear extracts. HuR protein levels were normalized to the IgG light chain bands.

Figure 4. Effect of Leptomycin B (LMB) on ACTH-induced increase in VEGF mRNA levels

A, BAC cells were treated with 10 nM ACTH for the indicated periods of time, in the presence or in the absence of Leptomycin B (5 ng/ml). When used, LMB was added 15 min before ACTH treatment. Cytoplasmic (30 μg) and nuclear fractions (5 μg) were subjected to Western blot analysis of HuR. B, Representative ethidium bromide staining of VEGF mRNA levels amplified by RT-PCR in BAC cells stimulated with ACTH in the presence or in the absence of LMB (5 ng/ml). C, Quantitation of VEGF mRNA levels in BAC cells stimulated with ACTH in the presence or in the absence of LMB, expressed as fold induction of mRNA levels at time 0 (unstimulated cells). Each point is the mean value from two separate experiments.

Figure 5. Effect of HuR repression by RNAi on ACTH-induced increase in VEGF mRNA levels

A, BAC cells were transfected either with HuR specific siRNA or a negative control siRNA as described in Materials and Methods. Forty-eight hours later, culture medium was changed and cells were treated for the indicated periods of time with or without 10 nM ACTH. At each time point of stimulation, total RNA was isolated and RT-PCR analysis was performed to determine HuR, VEGF or HPRT mRNA expression levels. B, Western blot analysis of HuR protein levels in whole-cell extracts (10 μg), showing that HuR siRNA was effective in knocking down HuR protein levels. In this particular experiment, blots for HuR were exposed for 2 min. Despite prolonged exposure of the membrane (15 min), HuR was barely detectable in protein extracts derived from HuR siRNA-treated cells. C, Quantitation of the RT-PCR experiment represented in (A), in which HuR repression was evaluated to 85 %. Results obtained from three independent experiments revealed that ACTH-induced increase in VEGF mRNA levels was altered to a lesser extent when HuR repression was about 70% (data not shown).

Figure 6. Antagonistic effects of Tis11b and HuR in the regulation of VEGF mRNA stability

COS7 cells were transfected as outlined in Materials and Methods. The pLuc-V3′ construct contains the full-length 3′-UTR of the rat VEGF mRNA (2201 bp) (30). A, Dose-dependent effect of Tis11b on pLuc-V3′ reporter gene activity. Results are expressed as relative light units of firefly luciferase activity over relative light units of renilla luciferase activity. B, Dose-dependent effect of HuR on pLuc-V3′ reporter gene activity. C, Effect of Tis11b and HuR co-expression on pLuc-V3′ reporter gene activity. The dose giving the maximal effect of HuR on luciferase activity (0.1 ng) was used with 0.2 or 1 ng of Tis11 b to perform competition studies. Transfections were performed in triplicate and values are means ± S.E from three independent experiments. +, +++, significantly different from control (0 ng of pCMV-Tis11b or pCMV-HuR) with p<0.05 and p<0.001, respectively. There was a statistically significant decrease in luciferase activity for (HuR 0.1 ng + Tis11b 1 ng) compared to HuR 0.1 ng (***, p<0.001), as well as a statistically significant increase in luciferase activity for (HuR 0.1 ng + Tis11b 1 ng) compared to Tis11b 1 ng (**, p<0.01). There was a statistically significant decrease in luciferase activity for (HuR 0.1 ng + Tis11b 0.2 ng) compared to HuR 0.1 ng (**, p<0.01), as well as a statistically significant increase in luciferase activity for (HuR 0.1 ng + Tis11b 0.2 ng) compared to Tis11b 0.2 ng (**, p<0.05). In the lower panel, COS7 cell extracts (10 μg) were immunoblotted using anti-HuR or anti-Tis11 b antibodies to check for HuR and Tis11 b protein expression in transfected cells. HuR and Tis11b are indicated with arrows. In this cell line, Tis11b is indetectable at basal levels. D, Effect of Tis11b and HuR co-expression on pLuc-V3′ reporter gene mRNA levels. COS7 cell total RNA (20 μg) were analyzed by Northern blot as indicated in Materials and Methods.

Figure 7. Binding of HuR and Tis11 b to VEGF 3′-UTR RNA

A, Restriction map of the 2201 bp-long 3′-UTR of VEGF mRNA. Tis11b binding element (TBE) is located between nucleotides 1161 and 1235 (30). The 40-bp functional HuR binding site is located between nucleotides 1285 and 1325 (32). Flags with white circles represent the nonameric ARE motifs UUAUUUA(A/U)(A/U) and those with black circles represent the pentameric motif AUUUA. B, VEGF full length 3′UTR RNA probe was mixed either with bacterial cell extracts containing Tis11b (2 μg) and increasing doses of purified GST-HuR (0, 1, 2, or 5 μg) or purified GST-HuR (1μg) and increasing doses of Tis11b (0, 2, 4, or 6 μg). The reaction mixtures were treated with UV radiation and were analyzed by electrophoresis as outlined in Materials and Methods. The positions of migration of the HuR and Tis11 b RNA-protein complexes are indicated with arrows, (ns) represents a non-specific band observed with the control bacterial extract. C, Quantitation of Tis11 b binding to VEGF 3′-UTR in the presence of increasing doses of HuR (squares) and of HuR binding to VEGF 3′-UTR in the presence of increasing doses of Tis11b (circles) in 2 to 4 independent experiments. 100 % represents either the binding of Tis11b in the absence of HuR or the binding of HuR in the absence of Tis11 b.