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Institut National de la Santé et de la Recherche Médicale (INSERM), Equipe Mixte 01-05; and Commissariat à l’Energie Atomique (CEA), Department of Cellular Responses and Dynamics, F-38054 Grenoble, France
Address all correspondence and requests for reprints to: Dr J. J. Feige, Institut National de la Santé et de la Recherche Médicale (INSERM), Equipe Mixte 01-05, Département Réponses et Dynamique Cellulaires, laboratoire ANGIO, CEA-Grenoble, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9, France. E-mail: jjfeige{at}cea.fr.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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and AP-1 for the hypoxic response (7, 8) and Sp1 and AP-2 for the growth factor response (9, 10). Second, VEGF mRNA stability is regulated in response to some of these effectors through the binding of stabilizing and destabilizing proteins to AU-rich elements (AREs) located in the 3'-untranslated region (3'-UTR) of VEGF mRNA (11, 12, 13, 14). Third, translation of VEGF mRNA into protein is also a controlled mechanism implying several alternative initiation codons (15). The choice of the preferential initiation codon is modified under hypoxic conditions (16) or under oncogenic transformation (17). These different levels of regulation may cooperate to amplify the effects of such or such regulator. For example, hypoxia-induced stimulation of VEGF expression results from both HIF-1-mediated stimulation of transcription (7) and HuR-mediated stabilization of VEGF mRNA (11).
HuR is a ubiquitously expressed member of the ELAV (embryonic-lethal abnormal visual in Drosophila melanogaster) family of RNA binding proteins, which also comprises the neuron-specific proteins HuD, HuC, and Hel-N1 (18, 19). The protein products of all four genes bind with high affinity and specificity to AU-rich elements (AREs) in a variety of mRNAs, such as those encoding VEGF, c-fos, c-jun, IL-3, TNF, and granulocyte macrophage colony-stimulating factor, and are believed to increase mRNA stability, mRNA translation, or both (20, 21). Although the precise mechanisms regulating HuR function in mRNA stabilization are poorly understood, increasing evidence indicates that HuR function is intimately linked to its subcellular localization (22, 23, 24). Indeed, HuR contains three classical RNA-binding domains [RNA recognition motifs (RRM)]: the first two (RRM1 and RRM2) have been implicated in ARE recognition whereas the third (RRM3) has been suggested to bind the poly A tail of target mRNAs (25). The region located between RRM2 and RRM3, known as the hinge region, is essential for HuR subcellular localization. This region contains a shuttling domain, HNS (for HuR nucleocytoplasmic sequence) that is involved in the interaction of HuR with the acidic phosphoproteins ligands, pp32, SET/ß, and APRIL (26). Like HuR, these proteins are primarily nucleoplasmic but shuttle between the nucleus and the cytoplasm. pp32 and APRIL contain domains homologous to nuclear export signals (NES) known to interact with CRM1 (chromosomal region maintenance 1), the nuclear export receptor for the HIV-1 Rev protein. It has been shown recently that pp32 and APRIL mediate CRM1-dependent export of HuR in response to heat shock (27).
We have previously reported that ACTH rapidly and transiently stimulates VEGF expression by primary cultures of bovine adrenocortical (BAC) cells (28). Interestingly, we observed that this increase in VEGF mRNA was transcription independent because it was still observed in the presence of 5,6-dichloro-1-b-ribofuranosylbenzimidazole (DRB), a potent transcription inhibitor (28). We then characterized Tis11b (tetradecanoyl phorbol acetate-inducible-sequence 11b) as a zinc finger RNA-destabilizing protein the synthesis of which is also induced by ACTH, although slightly later than that of VEGF (29). We hypothesized that Tis11b might play a role in the control of VEGF mRNA stability. Indeed, we recently characterized the interaction between Tis11b and VEGF mRNA 3'-UTR and identified a 75-bp sequence in this 3'-UTR, containing two AU-rich elements, as the Tis11b binding-element (TBE) (30). We could show that Tis11b binding to TBE induces destabilization of VEGF mRNA, resulting in a reduction of VEGF mRNA half-life from 130 min down to 60 min. In the present work, we attempted to identify the mechanism by which ACTH induces VEGF mRNA. We identified HuR as a potential regulator and could establish that ACTH induces a rapid increase of HuR levels in the cytoplasm and a concomitant decrease of HuR levels in the nucleus. Moreover, coimmunoprecipitations experiments revealed that ACTH induces a molecular complex between the nucleocytoplasmic shuttling protein pp32 and HuR in the cytoplasm, suggesting that pp32 is involved in HuR accumulation in this compartment. Finally, as the Tis11b- and HuR-binding sites are quite close to each other on the VEGF mRNA 3'-UTR, we wondered whether these two proteins might antagonize each other in the control of VEGF mRNA stability.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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illustrates an RT-PCR analysis of VEGF, Tis11b, and HuR mRNA levels in adrenocortical cells stimulated with 10 nM ACTH for various periods of time ranging from 0–5 h. As previously reported (29), VEGF mRNA levels peaked after 2–3 h, whereas the hormone induced the expression of Tis11b between 3 and 5 h of treatment (Fig. 1, A and B). In contrast, HuR mRNA levels were unchanged over the period of ACTH stimulation (Fig. 1, A and B). Northern blot analysis of HuR mRNA levels in ACTH-treated cells confirmed that they were very stable for up to 24 h of hormone treatment (maximal value reaching 137 ± 40% of mRNA levels measured in nontreated cells, n = 3, data not shown).
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, A and B, after exposure of BAC cells to 10 nM ACTH, no significant changes were observed in HuR protein content of whole-cell extracts. In contrast, ACTH induced a progressive increase in Tis11b protein content (Fig. 2, A and B). This increase was apparent as early as 1 h after stimulation and peaked after 5–6 h. Tis11b protein expression remained slightly elevated 24 h after ACTH treatment (data not shown).
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, HuR was weakly present in the cytoplasm (20 µg of cytoplasmic extracts) but was abundant in the nucleus (5 µg of nuclear extracts) of unstimulated cells. Quantitation of cytoplasmic and nuclear HuR revealed that ACTH treatment led to a 2- to 3-fold increase of cytoplasmic HuR as early as 30 min after stimulation and a concomitant decrease in nuclear HuR (Fig. 2D). Cytoplasmic HuR levels remained elevated for up to 3 h and then decreased by 4 h of stimulation. In unstimulated cells, HuR was estimated to be about 5- to 6-fold more abundant in the nucleus. Hybridization of the same membranes with antibodies recognizing cytoplasm- and nucleus-specific proteins (-tubulin and lamin A/C, respectively) allowed us to verify that nuclear proteins did not leak into the cytoplasmic fractions during cell fractionation (Fig. 2C). To determine the subcellular localization of Tis11b in BAC cells, the same blots were probed with affinity-purified polyclonal anti-Tis11b antibodies (30). As shown on Fig. 2C, Tis11b was exclusively detected in the cytoplasmic fractions. Upon prolonged exposure of the membrane (15 min), a very weak Tis11b signal could be detected in the nuclear fractions (data not shown). Exposure of BAC cells to ACTH induced a robust increase in Tis11b protein levels in the cytoplasm (Fig. 2C), which was correlated to the induction of Tis11b that we observed in whole-cell extracts (Fig. 2A). Altogether, these results indicate that ACTH treatment of BAC cells leads to a rapid increase of HuR protein content in the cytoplasm with a concomitant decrease of HuR protein content in the nucleus and to a delayed increase of Tis11b protein in the cytoplasm.
HuR Interacts with the Nucleocytoplasmic Shuttling Protein pp32 in BAC Cells
In light of the observation that increases in cytoplasmic HuR are largely due to the CRM1-dependent export of nuclear HuR, which involves complexes between HuR and the nucleocytoplasmic shuttling proteins pp32 and/or APRIL (26, 27), we further tested whether these proteins might contribute to ACTH-induced accumulation of HuR in the cytoplasm using coimmunoprecipitation experiments. Because we could not detect APRIL in whole BAC cell extracts (data not shown), we focused on pp32. Cytoplasmic and nuclear BAC extracts were exposed to anti-pp32 antibody. Probing the immunoprecipitates with anti-HuR antibody revealed that pp32 is associated with HuR in the cytoplasm and that ACTH treatment increases this association (Fig. 3, A and B). The same blot was probed with anti-pp32 antibody to check for pp32 immunoprecipitation. In the nucleus, HuR-pp32 complexes are less abundant as compared with the cytoplasm and are not significantly altered by hormone treatment (Fig. 3, A and B). These results suggest that ACTH-induced increase in cytoplasmic HuR levels involves pp32-mediated HuR delocalization from the nucleus to the cytoplasm of BAC cells.
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, the hormone-induced increase of HuR in the cytoplasm was abolished at all time points of stimulation in the presence of LMB (5 ng/ml).
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). Quantitation of two independent experiments showed that after 2 h of ACTH treatment, VEGF mRNA levels were induced up to 290 ± 60% of control levels and that, in the presence of LMB, maximal induction reached only 147 ± 60% of control levels. LMB also had a discrete effect by itself on the basal expression of VEGF mRNA.
HuR Knockdown Inhibits ACTH-Induced Increase in VEGF mRNA Levels
To provide further evidence of the involvement of HuR in the regulation of VEGF mRNA expression by ACTH, we used RNA interference (RNAi) to more selectively target HuR. Using a specific short interfering RNA (siRNA) targeting exon 2 in HuR gene, we showed by both RT-PCR and Western blotting that this siRNA was effective in decreasing HuR mRNA and protein levels in BAC cells after 48 h of treatment (Fig. 5, A and B, time point 0). Quantitation of HuR mRNA levels in three independent experiments revealed that HuR gene expression was knocked down by 70–85% (data not shown). In these experiments, HuR protein levels were barely detectable upon prolonged exposure of the Western blot (data not shown). BAC cells pretreated either in the presence of a negative control siRNA or in the presence of HuR siRNA were further stimulated with 10 nM ACTH, and VEGF mRNA levels were analyzed by RT-PCR. In this particular experiment, cells pretreated with negative control siRNA displayed a 2.5-fold increase in VEGF mRNA expression after 4 h of exposure to ACTH (Fig. 5, A and C). In contrast, HuR siRNA impaired ACTH-elicited up-regulation of VEGF mRNA levels (Fig. 5, A and C). Moreover, silencing HuR expression led to a substantial reduction in basal VEGF mRNA expression down to 39 ± 19% of controls (n = 3, data not shown).
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shows that luciferase activity was decreased in a dose-dependent manner by Tis11b, a statistically significant decrease being observed with a plasmid amount as low as 0.5 ng (62.5 ± 7.9% of controls; P < 0.01, n = 3). In contrast, HuR expression led to an increase in the reporter gene activity over a narrow window of the cotransfected HuR plasmid (Fig. 6B). A statistically significant increase of luciferase activity was observed with 0.1 ng of pCMV-HuR (156.8 ± 13.8% of controls; P < 0.001, n = 3; Fig. 6A). Experiments were then performed to determine the competitive effects of HuR and Tis11b coexpression on VEGF mRNA 3'-UTR. As shown in Fig. 6C, the luciferase activity recorded after cotransfection of 1 ng of pCMV-Tis11b and 0.1 ng of pCMV-HuR was significantly lower than the luciferase activity obtained with 0.1 ng of pCMV-HuR alone (81.8 ± 2.3% as compared with 152.2 ± 6.3% of controls, respectively; P < 0.001, n = 3). This inhibitory effect of Tis11b on HuR-induced luciferase activity was observed with a concentration of pCMV-Tis11b as low as 0.2 ng. Conversely, the luciferase activity measured after cotransfection of 1 ng of pCMV-Tis11b and 0.1 ng of pCMV-HuR was significantly higher than the one measured with pCMV-Tis11b alone (81.8 ± 2.3% as compared with 48.6 ± 2.1% of controls, respectively; P < 0.01, n = 3). Western blot analysis of cell extracts from these transfection experiments revealed that COS7 cells constitutively express HuR protein but not Tis11b protein (Fig. 6C, lower panel). As expected, transfection of either 0.05 ng or 0.1 ng of pCMV-HuR plasmid led to an increased expression of HuR. By contrast, Tis11b expression was barely detectable with 0.2 ng of pCMV-Tis11b plasmid but clearly detected after transfection of 1 ng of plasmid.
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). Northern blot analysis of COS7 total RNA revealed that transfection of 0.1 ng of pCMV-HuR increased Luc-V3' mRNA levels similarly to the reporter gene activity. Cotransfection of 0.2 and 1 ng of pCMV-Tis11b with 0.1 ng of pCMV-HuR abolished HuR-induced increase in Luc-V3' mRNA. Finally, Luc-V3' mRNA level was decreased in a dose-dependent manner by Tis11b. Altogether, these results suggest that Tis11b and HuR could antagonize each other to regulate VEGF mRNA expression through its 3'-UTR.
Tis11b and HuR Compete for Binding to the 3'-UTR of VEGF mRNA
Because Tis11b and HuR appeared to antagonize each other in the regulation of VEGF mRNA expression, experiments were performed to determine whether these proteins could compete with each other for the binding to VEGF 3'-UTR. Tis11b-expressing bacterial extract was mixed with increasing amounts of purified glutathione-S-transferase (GST)-HuR fusion protein and purified GST-HuR was mixed with increasing amounts of Tis11b-expressing bacterial extract. These mixtures were exposed to UV light in cross-linking assays with VEGF 3'-UTR RNA probe. As shown in Fig. 7B, a covalent ribonucleoprotein complex with an apparent molecular mass of 38 kDa was detected when Tis11b-expressing bacterial extract was incubated with VEGF 3'-UTR (lane 2). This complex was not observed in the presence of control bacterial extracts (lane 1). On the other hand, a covalent ribonucleoprotein complex with an apparent molecular mass of 64 kDa was detected when GST-HuR fusion protein was incubated with VEGF 3'-UTR (lane 6). When 1 µg of GST-HuR was added to 2 µg of Tis11b, both complexes of 38 and 64 kDa were detected, indicating that Tis11b and HuR could bind simultaneously to the VEGF 3'-UTR RNA probe (lane 3). Increasing the amount of GST-HuR in the presence of 2 µg of Tis11b resulted in a slight decrease in the intensity of the Tis11b-RNA complex and a corresponding increase in HuR-RNA complex (lanes 4 and 5). Increasing the amount of Tis11b in the presence of 1 µg of HuR resulted in a marked decrease in the intensity of the HuR-RNA complex (lanes 7–9). Unexpectedly, no increase in Tis11b-RNA complex was observed, suggesting that optimal Tis11b binding to VEGF 3'-UTR requires additional factors, which remain to be identified. Indeed, we could observe an increase of Tis11b binding to VEGF 3'-UTR over a narrow window of Tis11b doses ranging from 1–3 µg, followed by a decrease of Tis11b binding to VEGF 3'-UTR at higher doses (data not shown). Quantitation of two to four independent experiments shows that increasing doses of Tis11b markedly decreased HuR binding to VEGF 3'-UTR (to 23.2 ± 9.5% of HuR binding in the absence of Tis11b; Fig. 7C, n = 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transcriptional regulation of VEGF expression by hormones in endocrine tissues, including endometrium, ovaries, and adrenal cortex, has been extensively studied (34, 35, 36, 37, 38, 39). In contrast, the possible involvement of posttranscriptional regulation in hormonally regulated expression of VEGF has received much less attention to date. We have previously shown that the trophic hormone ACTH triggers a rapid and transient increase in VEGF mRNA levels in adrenocortical cells via transcription-independent mechanisms (28). We could further demonstrate that the decay phase of ACTH-induced VEGF mRNA levels involves the RNA-destabilizing protein Tis11b (30). In the current study, we aimed at investigating the molecular mechanisms of ACTH-elicited increase in VEGF mRNA levels. Four major conclusions can be drawn from the present work: 1) ACTH induces a rapid delocalization of the RNA-stabilizing protein HuR from the nucleus to the cytoplasm in adrenocortical cells without affecting total cellular HuR mRNA and protein levels; 2) Blocking nuclear export of NES-bearing proteins by LMB impairs ACTH-induced expression of VEGF mRNA; 3) Silencing the expression of HuR by siRNA markedly inhibits ACTH-mediated induction of VEGF mRNA levels; and 4) Tis11b and HuR exert an antagonistic action on VEGF mRNA in vitro.
First, our finding that the levels of cytoplasmic and nuclear HuR are potently influenced by the trophic hormone ACTH provides a new insight into the regulation of HuR in adrenocortical cells, suggesting a connection between the ability of HuR to stabilize ACTH-induced labile mRNAs and the subcellular targeting of these mRNAs. Various other stimuli can shift the nucleo-cytoplasmic distribution of HuR. Cytoplasmic localization of HuR is associated with conditions of cellular stress including heat shock (40), UV irradiation (41), and amino acid starvation (42), as well as stimulation with lipopolysaccharide in the case of macrophages (43). HuR can bind to AU-rich regions in c-fos mRNA, a CU-rich region in c-jun mRNA, and a U-rich domain in c-myc and VEGF mRNAs (11, 20, 44, 45), all of these genes being early-response genes to ACTH in the adrenal cortex (28, 46). Our observation that ACTH induces an increase in cytoplasmic levels of HuR suggests that HuR may play a role in stabilizing these labile mRNAs. Our results showing that 1) HuR coimmunoprecipitates with the nucleocytoplasmic shuttling protein pp32 in the cytoplasm of ACTH-stimulated cells and 2) ACTH increases transiently the association of HuR with pp32 in the cytoplasm argue that the hormone triggers nuclear export of HuR to the cytoplasm. Interestingly, there is accumulating evidence that the nuclear-cytoplasmic localization of HuR is modulated by signal transduction pathways (47, 48, 49, 50, 51). ACTH is known to increase intracellular cAMP levels in cultured adrenocortical cells. We have previously shown that forskolin, an activator of adenylate cyclase, was as potent as ACTH in increasing VEGF mRNA levels (28). In the present study, stimulation of BAC cells with ACTH in the presence of H89 (10 µM), a potent and specific inhibitor of protein kinase A, completely abolished the hormone-induced increase of HuR in the cytoplasm (data not shown). Moreover, stimulation of BAC cells with forskolin (10 µM) led to a substantial increase of HuR levels in the cytoplasm [
165% of HuR protein content of nonstimulated cells after 1–2 h of stimulation (data not shown)]. These observations suggest that cAMP and protein kinase A are involved in the hormone-induced delocalization of HuR to the cytoplasm. The potential targets of protein kinase A, however, remain to be determined in future studies. Because pp32 is a phosphoprotein (26), it is tempting to speculate that pp32 shuttling or its interaction with HuR could be regulated by phosphorylation. This hypothesis is worth testing because we failed to identify phosphorylated forms of HuR in response to ACTH (data not shown).
Second, we observed that LMB, a specific inhibitor of the nuclear export receptor CRM1, significantly impaired ACTH-induced increase in VEGF mRNA. Although it is possible that LMB inhibits other components of the nucleocytoplasmic trafficking machinery, we hypothesize that ACTH-induced association of pp32 and HuR most likely confers CRM1-dependent export on HuR. Therefore, disruption of the nuclear export of HuR protein partners by LMB might lead to retention of HuR in the nucleus and thereby might impair ACTH-induced increase in VEGF mRNA levels. Indeed, we observed that LMB prevented the ACTH-mediated increase of HuR in the cytoplasm of BAC cells as well as the hormone-induced increase in VEGF mRNA levels, thus indicating that HuR export to the cytoplasm is instrumental in ACTH-induced VEGF mRNA.
Because BAC cells already have a high level of HuR, defining the functions of HuR in the cellular response to ACTH using the overexpression approach could lead to effects that might not reflect the true function of the endogenous protein. To clearly establish the role of HuR in ACTH-induced VEGF mRNA, we disrupted the expression of endogenous HuR in BAC cells using RNAi. Interestingly, RNAi-mediated depletion of HuR leads to a complete inhibition of hormone-induced expression of VEGF, indicating that HuR is a limiting factor in the induction phase of VEGF mRNAs by ACTH. HuR expression was significantly reduced (by 80–85%) 48 h before ACTH treatment. In our RNAi experiments in which HuR expression was reduced by only 70%, inhibition of ACTH-elicited VEGF mRNA levels was not complete (data not shown), suggesting that low levels of HuR were sufficient to stabilize VEGF mRNA. These results, together with those we reported on the involvement of the mRNA-destabilizing protein Tis11b in the decay phase of ACTH-elicited VEGF mRNA levels, indicate that ACTH controls VEGF expression in adrenocortical cells mainly by posttranscriptional mechanisms. The pivotal role of HuR in the stabilization of VEGF mRNA by hypoxia has been extensively studied (52). To our knowledge, this work is the first to report a hormonal up-regulation of VEGF mRNA expression that is mediated by the RNA-stabilizing protein HuR.
Fourth, concomitant overexpression of Tis11b and HuR revealed that Tis11b completely abrogated HuR-induced luciferase activity on a heterologous transcript consisting of luciferase cDNA cloned upstream of VEGF mRNA 3'-UTR (Luc-V3'). Our results showing that HuR increases luciferase activity derived from Luc-V3' in vitro are similar to those reported by Goldberg-Cohen et al. (32). Using a similar reporter construct, these authors have shown further that the increase in reporter activity was related to HuR binding to a 40-bp RNA element (nucleotides 1285–1325) within VEGF mRNA 3'-UTR, which confers increased stability to the heterologous transcript. Tis11b and HuR bind to distinct but very close RNA elements on VEGF mRNA 3'-UTR [nucleotides 1161–1235 (30) and 1285–1325 (32) for Tis11b and HuR, respectively]. Our UV cross-linking experiments indicate that Tis11b and HuR can simultaneously, as well as individually, bind to VEGF 3'-UTR RNA probe. However, Tis11b potently prevents HuR binding to VEGF 3'-UTR, a finding that is consistent with the inhibitory effect of Tis11b on HuR-mediated increase in reporter gene activity and mRNA level (cotransfection experiments; Fig. 6, C and D). For as yet unidentified reasons, the decrease in HuR binding to VEGF 3'-UTR in the presence of increasing doses of Tis11b was not paralleled by an increase in Tis11b binding. Similar results showing that HuR and tristetraprolin (TTP, also named Tis11), the most studied member of the Tis11 protein family, can bind simultaneously and competitively to granulocyte macrophage colony-stimulating factor 3'-UTR have been reported by Raghavan et al. (53). More recently, Lal et al. (54) provided evidence that HuR and the mRNA-destabilizing factor AUF1 can bind target transcripts on both distinct, nonoverlapping sites, and on common sites in a competitive fashion. They propose that the fate of the mRNA target depends on HuR and AUF1 abundance, the target RNA sequence, and the subcellular compartment investigated. Experiments aiming at dissecting the molecular mechanisms governing the binding of Tis11b and HuR to specific or common sites on VEGF 3'-UTR are under way.
At the functional level, after stimulation of adrenocortical cells by ACTH, HuR, which predominates in the nucleus, may shuttle to the cytoplasm where it may stabilize VEGF mRNA, allowing it to be translated. Subsequently, Tis11b expression is induced and the cytoplasmic level of Tis11b increases. The relative levels and binding affinities of HuR and Tis11b for their specific sequences in VEGF mRNA 3'-UTR may determine the fate of VEGF transcripts, with HuR predominance promoting VEGF mRNA stabilization and Tis11b predominance promoting VEGF mRNA degradation. At some point (between 4 and 6 h of stimulation by ACTH, according to our data), Tis11b may predominate and facilitate VEGF mRNA degradation. This model provides a mechanism by which VEGF gene could be transiently expressed in BAC cells.
In conclusion, this work reports for the first time that ACTH triggers a rapid nuclear export of HuR into the cytoplasm of adrenocortical cells followed by an induction of Tis11b protein synthesis and cytoplasmic accumulation, two processes that appear to be responsible for the transient stimulation of VEGF mRNA and protein accumulation. In an in vivo model of adrenal cortex tissue regression triggered by the suppression of pituitary ACTH secretion, we recently observed that adrenocortical VEGF expression is dramatically reduced during this process, resulting in massive disorganization and regression of the capillary network (55). It will therefore be of great interest to establish the respective contributions of HuR and Tis11b to the in vivo regulation of adrenal vasculature by ACTH in future studies.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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BAC Cell Culture and Treatments
Bovine adrenal glands were obtained from a local slaughterhouse. Zona fasciculata-reticularis cells were prepared by enzymatic dispersion with trypsin, and primary cultures were established as described in detail elsewhere (56). BAC cells were kept at 37 C in Ham’s F12 medium supplemented with 10% horse serum, 2.5% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, 20 µg/ml gentamycin, under 5% CO2-95% air atmosphere. On d 4, cells cultured in 10-cm petri dishes (3 x 106 cells per dish) were stimulated with 10 nM ACTH for the indicated periods of time before subcellular fractionation or processing for total RNA isolation as described hereafter. In experiments designed to silence HuR expression (RNAi), BAC cells were transfected on d 2 of culture.
Preparation of Subcellular Fractions
Total cell extracts, as well as nuclear and cytoplasmic fractions, were prepared using the Protein and RNA Isolation System (Ambion, Inc., Austin, TX) according to the manufacturer’s instructions. A protease inhibitor cocktail [10 µg/ml of leupeptin, 1 µg/ml of aprotinin, 1 µg/ml of pepstatin, and 25 µg/ml of 4-(2-aminoethyl-benzenesulfonyl fluoride] was added to the provided buffers. Briefly, 10-cm petri dishes (3 x 106 BAC cells per dish) were lysed on ice in 400 µl of Cell Disruption buffer and collected with a rubber spatula. A portion of the total cell lysate was used for RNA isolation (300 µl), and the remainder was kept for protein analysis (100 µl).
For nuclear and cytoplasmic lysate preparation, BAC cells (3 x 106 cells per 10-cm petri dish) were trypsinized, washed in PBS, and then suspended in 400 µl of Cell Fractionation Buffer. After a 15-min incubation on ice, nuclear and cytoplasmic fractions were separated by centrifugation (5 min at 500 x g). The nuclear pellet was lysed in 400 µl of Cell Disruption buffer and kept 10 min on ice to ensure complete disruption before processing the sample for protein analysis.
Protein concentration was determined using a Micro BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL).
RNA Isolation and RT-PCR
BAC cell total RNA was extracted using the RNAgents kit (Promega Corp., Charbonnières, France) according to the manufacturer’s instructions. This system consistently yields 50–80 µg total RNA/3 x 106 cells. For RT-PCR analysis of Tis11b, VEGF, HuR, or HPRT gene expression, 1 µg of total RNA was reverse transcribed with Superscript II reverse transcriptase (Invitrogen) and PCR amplified using Taq polymerase (QBiogen, Illkirch, France). Amplification of VEGF mRNA isoforms was performed using the primers and amplification conditions described by Gaillard et al. (28). The size of the expected amplified fragments was 462 bp and 332 bp for the two major VEGF transcripts, VEGF165 and VEGF121, respectively. The primers for PCR of Tis11b were as follows: 5'-CGAAGAAAACGGTGCCTGTAAG-3' and 5'-AGTAGGTGAGCCCAAGAGGTCATC-3'. This primer pair sequence amplifies a 354-bp fragment. The amplification conditions were as follows: 94 C for 5 min followed by 25 amplification cycles, each consisting of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and 72 C for 5 min for final extension. The primers for hypoxanthine phosphoribosyl transferase (HPRT) amplification were as follows: 5'-GCCATCACATTGTAGCCCTCT-3' and 5'-TGCGACCTTGACCATCTTTGG-3'. This primer pair sequence amplifies a 305-bp fragment. The amplification conditions were as follows: 94 C for 5 min followed by 25 amplification cycles, each consisting of 94 C for 1 min, 55 C for 1 min, 72 C for 1 min, and 72 C for 5 min for final extension. The primers for HuR amplification were as follows: 5'-ATGACCCAGGATGAGTTACGAAGC-3' and 5'-GTTCACAAAGCCATAGCCCAAG-3'. This primer pair sequence amplifies a 111-bp fragment. The amplification conditions were as follows: 94 C for 5 min followed by 25 amplification cycles, each consisting of 94 C for 1 min, 52 C for 1 min, 72 C for 1 min, and 72 C for 5 min for final extension. PCR products were analyzed on 2% agarose ethidium bromide-containing gels, visualized using a Vistra FluorImager (Molecular Dynamics, Sunnyvale, CA) and quantitated using ImageQuant software (Molecular Dynamics). Preliminary experiments were done to select the starting amounts of RNA so that there was a linear relationship between the amount of input RNA and the OD of the HPRT band on ethidium bromide-stained gel.
SDS-PAGE
SDS-PAGE was performed according to Laemmli (57). Total proteins or subcellular fraction extracts (5–20 µg/lane) were solubilized in sample buffer [60 mM Tris-HCl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol, 10% glycerol, 0.01% bromophenol blue], boiled for 5 min and loaded onto a 12% SDS-PAGE minigel (Mini Protean II System; Bio-Rad Laboratories, Hercules, CA). Electrophoresis was performed at 150 V for 1 h.
Western Blot Analysis
SDS-PAGE-resolved proteins were electrophoretically transferred onto a polyvinylidine difluoride (PVDF) membrane according to Towbin et al. (58). After transfer, the membrane was incubated in a blocking buffer (PBS buffer containing 0.1% Tween 20 and 5% nonfat dry milk) for 1 h at room temperature. The blots were probed sequentially with antibodies to a peptide fragment (amino acids 49–63) of Tis11b protein (1:500; CovalAb, Lyon, France), antihuman HuR (1:1000, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), monoclonal anti--tubulin (1:200,000, a generous gift from Dr. D. Job, CS-U366 INSERM, CEA-Grenoble, France), and antilamin A/C (1:15000, a generous gift from Dr. Deloulme, TS-EMI 01–04 INSERM, CEA-Grenoble, France) for 2 h in PBS containing 0.1% Tween. The membrane was thoroughly washed with the same buffer (3 x 10 min), and then incubated for 1 h with either horseradish peroxidase (HRP)-labeled goat antirabbit IgG (immunodetection of Tis11b and HuR) or HRP-labeled goat antimouse IgG (immunodetection of tubulin), or HRP-labeled goat antiguinea pig IgG (immunodetection of lamin A/C). The PVDF sheet was washed as above, and the antigen-antibody complex was revealed by Enhanced Chemiluminescence, using the Western blotting detection kit from Amersham Biosciences (Buckinghamshire, UK) and BioMax Kodak films (Sigma).
Immunoprecipitation Assay
BAC cells cultured in 10-cm petri dishes (3 x 106 cells per dish) were stimulated with 10 nM ACTH for the indicated periods of time. Subcellular fractions (cytoplasm and nuclei) were prepared as indicated previously. Lysates was cleared by centrifugation for 5 min at 12,000 x g at 4 C. The antihuman pp32 (I1PP2A) polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was added at a concentration of 2 µg/ml to whole supernatants (
250 µg protein for cytoplasm and 70 µg protein for nuclei), which were then gently rocked for 2 h at 4 C, before being incubated for 30 min with Protein A/G Sepharose beads. Immunoprecipitates were pelleted, washed four times with radioimmunoprecipitation buffer, analyzed by SDS-PAGE, and transferred to a PVDF membrane. Blots were probed with anti-HuR or anti-pp32 antibodies, which were used at 1:1000.
RNAi
Expression of HuR (ELAVL1, GenBank accession no. NM_001419) was inhibited by transfection of a predesigned siRNA duplex targeted to exon 2 of human HuR gene (Ambion). siRNA sense and antisense sequences were 5'-GGAUGAGUUACGAAGCCUGtt-3' and 5'-CAGGCUUCGUAACUCAUCCtt-3', respectively. Adrenocortical cells were transfected 1 d after plating with 10 nM of either HuR siRNA duplex or negative control siRNA (Ambion), using siPORT lipid reagent (Ambion). Typically, cells were analyzed for the loss of HuR mRNA and protein expression 48 h after transfection using RT-PCR and immunoblotting. At this time point, culture medium was changed, and cells were treated for the indicated periods of time with or without 10 nM ACTH.
Transient Transfections and Dual Luciferase Activity Assay
COS7 cells were grown in DMEM supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 25 µg/ml gentamycin and transfected in 12-well plates using lipofectamine (Invitrogen) according to the manufacturer’s recommendations. Plasmid pLuc-V3' contains the firefly luciferase cDNA cloned upstream of the rat VEGF 3'-UTR and downstream of the thymidine kinase (TK) promoter (30). Plasmid pCMV-murine HuR was provided by Dr. Jonathan LaMarre (University of Guelph, Ontario, Canada). pCMV-Tis11b plasmid was described previously (30). pRL-TK encoding Renilla luciferase was obtained from Promega Corp.
Various amounts of either pCMV-Tis11b, pCMV-HuR, or both (0.05–1 ng) were cotransfected with 500 ng pLuc-V3', 25 ng of pRL-TK, and pUC19 up to a total of 700 ng plasmid DNA into 1.5 x 105 cells. Renilla and firefly luciferase activities were measured sequentially 48 h after transfection with the Dual-Luciferase reporter assay system (Promega Corp.) on a LUMAT LB 9507 luminometer (EGG-Berthold, Bad Wildbad, Germany). Results are expressed as relative light units of firefly luciferase activity over relative light units of Renilla luciferase activity to compensate for variations in transfection efficiency. Each transfection condition was performed in triplicate.
Northern Hybridization
Total RNA from COS7 cells was extracted using the RNAgents kit (Promega) according to the instructions of the manufacturer. RNA (15–20 µg) was size fractionated on a 1% formaldehyde agarose gel, vacuum transferred onto Hybond-n + membranes (Amersham Biosciences) and fixed by UV cross-linking. Northern blots were pre-hybridized in Rapid Hybridization Buffer (Amersham Biosciences) at 65 C for 30 min. [32P]dCTP-labeled luciferase cDNA probe (2 x 106 cpm/ng DNA; Rediprime random primer labeling kit, Amersham Biosciences) was then added, and the incubation was continued for 2 h at 65 C. Blots were washed for 5 min and 15 min successively at room temperature in 2x saline sodium citrate (SSC), 0.1% SDS, and then for 15 min in 1x SSC, 0.1% SDS. The final wash was performed at 65 C for 15 min in 0.5x SSC, 0.1% SDS. RNA-cDNA hybrids were visualized on phosphor screen (Molecular Dynamics, Inc.) after a 12- to 24-h exposure period. Blots were stripped and reprobed with 18S cDNA probe to assess RNA loading.
Recombinant Protein Expression and Purification
Tis11b recombinant protein was produced as reported elsewhere (30). GST-HuR fusion protein was produced using Escherichia coli BL21 strain transformed with pGEX-5X2-HuR plasmid (provided by Dr. J. A. Steitz, Yale University school of Medicine, New Haven, CT). Bacteria were grown at 37 C to an A600 nm of 0.6 in 2YT medium (16 g/liter tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) containing 100 µg/ml of ampicillin. The GST-HuR fusion protein was induced with 0.1 mM isopropyl-ß-D-thiogalactopyranoside for 2 h and purified using MicroSpin GST Purification Module (Amersham Biosciences) according to the manufacturer’s instructions. Briefly, cells were harvested by centrifugation (2500 x g at 4 C for 5 min). The pellet was resuspended with ice-cold PBS containing 0.1 mg/ml lysozyme and lysed by repeated freeze/thawing (10 times). Clarified lysate was mixed for 10 min with Glutathione Sepharose 4B Microspin column. After two washes with PBS, GST-HuR fusion protein was eluted with 10 mM reduced glutathione. Purity of GST-HuR protein was examined by Coomassie blue staining after SDS-PAGE analysis.
RNA-Protein UV Cross-Linking Assay
[32P]UTP-labeled and unlabeled riboprobes were synthesized in vitro using pSp64 plasmid containing the entire VEGF 3'-UTR and the Riboprobe SP6 in vitro Transcription System (Promega). Integrity of RNA transcripts was visualized on 1% agarose ethidium bromide-containing gel. RNA transcripts (1 x 106 cpm) were incubated for 20 min at room temperature with 1–6 µg of either Tis11b-containing bacterial extract or GST-HuR fusion protein, in 10 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, 5% glycerol, 0.5% Nonidet P-40, and 2 mM dithiothreitol. Yeast tRNA (50 ng/µl) and heparin (2 µg/µl) were then added for 10 min. Mixtures were exposed to UV light for 30 min on ice. of RNase T1 (100 U) (Invitrogen) were then added for 20 min, and RNA-protein complexes were analyzed by 12% SDS-PAGE and autoradiography.
Statistical Analysis
Results are expressed as means ± SE. The mean values were compared by ANOVA using Fisher’s test. A value of P < 0.05 was considered as statistically significant. Quantitation of immunoblots and autoradiograms was performed using a Molecular Imager FX and Quantity One software (Bio-Rad).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 23, 2005
Abbreviations: ARE, AU-rich element; BAC cell, bovine adrenocortical cell; CRM1, chromosomal region maintenance 1; ELAV, embryonic-lethal abnormal visual; GST, glutathione-S-transferase; HPRT, hypoxanthine phosphoribosyl transferase; HRP, horseradish peroxidase; LMB, leptomycin B; NES, nuclear export signal; PVDF, polyvinylidine difluoride; RNAi, RNA interference; RRM, RNA recognition motif; SDS, sodium dodecyl sulfate; siRNA, short interfering RNA; SSC, saline sodium citrate; TBE, Tis11b-binding element; Tis11b, tetradecanoyl phorbol acetate-inducible-sequence 11b; TK, thymidine kinase; 3'-UTR, 3'-untranslated region; VEGF, vascular endothelial growth factor.
Received for publication March 11, 2005. Accepted for publication November 14, 2005.
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and ß. Proc Natl Acad Sci USA 97:10972–10977 and ß. Cancer Res 62:4977–4984 mRNA is a target of the mRNA-stabilizing factor HuR. Mol Cell Biol 21:721–730This article has been cited by other articles:
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