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Molecular Biology and Genetics |
Induces Angiogenic Factor Up-Regulation in Malignant Glioma Cells
Birmingham Veterans Affairs Medical Center, Birmingham, Alabama 35233 [L. B. N., E. S., Y. H., X. Y., P. H. K.], and Departments of Neurology [L. B. N., P. H. K.], Clinical Pharmacology [M. J. J.], and Physiology and Biophysics [P. H. K.], University of Alabama at Birmingham, Birmingham, Alabama 35294-0007
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ABSTRACT |
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(TNF-), interleukin 8, and, to a lesser extent, vascular endothelial growth factor in U251 glioma cells after stimulation with TNF-. RNA kinetic studies indicated that TNF- induced the stabilization of all three transcripts. Using a luciferase reporter assay, we demonstrate that the AU-rich elements (AREs) in the 3'-untranslated region of these genes significantly contribute to this posttranscriptional regulation. UV cross-linking and immunoprecipitation with glioma extracts indicate that HuR binds to all three AREs. When HuR is overexpressed in glioma cells, there is enhanced RNA stabilization of all three angiogenic factor transcripts with a concomitant increase in mRNA and protein expression (up to 7-fold). These findings indicate that TNF- up-regulates angiogenic factor expression in MG cells and that RNA stabilization, via the AREs in the 3'-untranslated region, contributes to this up-regulation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(2, 3, 4, 5, 6)
. RNA stabilization is an important control point for expression of these and other labile genes, often in response to extracellular signals such as hypoxia, cytokines, growth factors, ions, or hormones (7, 8, 9, 10, 11, 12, 13, 14)
. A major determinant of RNA half-life for these genes is the presence of AREs in the 3'-UTR of the transcript (7
, 9)
. The longevity of the transcript is governed by the interaction of cellular factors that bind to the ARE. These factors include TTP, AUF1, and butyrate response factor 1, which are linked to RNA destabilization, and HuR, which promotes stabilization (9
, 15)
. We recently reported that HuR is overexpressed in primary MGs and thus postulated that this RNA-binding protein plays a role in the up-regulation of angiogenic factors in gliomas by binding to the ARE and stabilizing the transcript (16)
. We investigate this hypothesis by analyzing the posttranscriptional regulation of VEGF, IL-8, and TNF- mRNAs in a MG cell line after TNF- stimulation. This inflammatory cytokine is relevant because it is expressed within the glioma and surrounding parenchyma and may exert paracrine or autocrine effects on the glioma (3
, 17, 18, 19)
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, and VEGF [as described previously (16)
] were ligated into the pflM1 site downstream from the luciferase coding sequence in the PGL2 control plasmid (Promega, Madison, WI). The TNF- 3'-UTR in the antisense orientation was used as the negative control. All plasmids were sequenced to verify correct orientation of the inserts. The FLAG-HuR cDNA was generated by PCR using the following oligonucleotides: 5'-TCAAGCTTGCGGCCGCATGGACTACAAGGACGACGATGACAAGTCTAATGGTTATGAAGAC-3' (upstream) and 5'-TAGACCTTGATATC TTATTTGTGGGACTTGTT-3' (downstream). The PCR product was digested with NotI and EcoRV and ligated into the same restriction sites in the pTRE2-hyg plasmid (Clontech, Palo Alto, CA). Probes for UV cross-linking were synthesized with [32P]UTP using a kit (Ambion, Austin, TX). Templates for the probes were generated by PCR, and each upstream primer included the T7 transcriptional recognition sequence (5'-TAATACGACTCACTATAGGG-3'). The primers were as follows: IL-8, 5'-TAAGTTTTTTCATCATAACAT-3'; and TNF-, 5'-ctgcaggacttgagaagac-3'. For VEGF, the probe template was synthesized from a plasmid that contained the segment of the VEGF 3'-UTR regulatory region (VRS) 3' to the EcoRI site (20)
.
Cell Culture and Transfections.
U251 MG cells were kindly provided by Dr. Yancey Gillespie. The U251 Tet-On cells were a gift from Dr. Erwin Van Meir. For stable transfections, pTRE2 plasmids were transfected into U251 Tet-On cells, and the clones were selected with hygromycin. The luciferase-3'-UTR plasmids were cotransfected with pRSV-Neo into U251 MG cells, and the clones were selected with neomycin. For transient transfections, U251 MG cells were seeded in a 96-well plate at a density of 20,000 cells/well. On the following day, plasmids were transfected into the cells using the transIT kit (Mirus, Madison, WI) using a total volume of 100 µl. A ß-galactosidase plasmid was cotransfected with the test plasmids as described previously (21)
. After 6 h, media containing 20% fetal bovine serum were added to the wells for an additional 4–6 h. The cells were then placed in serum-free media overnight. On the following morning, cells were treated with TNF- at a dose of 10 ng/ml or vehicle (PBS). The cells were harvested directly in the wells at different time points with lysis buffer (Promega). Luciferase activity was measured as described previously (21)
, except that luminescence was measured in a Spectrafluor plus machine (Tecan U.S., Durham, NC). ß-Galactosidase activity was measured using the FluoReporter LacZ/Galactosidase kit (Molecular Probes, Eugene, OR). All transfections were done in triplicate, and luciferase values were normalized to ß-galactosidase activity. For RNA kinetics, U251 MG cells were plated to 75% confluence. The cells were serum-starved for 12 h and then stimulated with TNF-. After 6 or 24 h, the cells were pulsed with actinomycin D (10 µg/ml) for different time periods. For U251 Tet-On clone induction, cells were treated with DOX 24–36 h before TNF- stimulation at a dose of 3 µg/ml. Mock-induced cells were treated with an equal volume of ethanol (vehicle). Induction of HuR was assessed by Western blot of total cell extract with the M5 anti-FLAG antibody (Sigma, St. Louis, MO) according to the manufacturer’s specifications.
RNA Isolation and Quantitative Real-Time PCR.
Total RNA was extracted with Trizol (InVitrogen, Carlsbad, CA), purified on RNeasy columns (Qiagen, Valencia, CA), and quantitated with the RiboGreen kit (Molecular Probes). The Gene Amp 7700 Sequence Detection system (Applied Biosystems) was used for the detection of real-time PCR products amplified from reverse-transcribed total RNA (25 ng). PCR reactions were done in triplicate for each sample on a 96-well plate that included separate wells to quantitate an internal RNA control (S9) and a standard curve. The cycling conditions were 48°C for 30 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Taqman primers (Applied Biosystems, Foster City, CA) were synthesized as follows: IL-8 upstream, 5'-CTGGCCGTGGCTCTCTTG-3'; IL-8 downstream, 5'-CCTTGGCAAAACTGCACCTT-3'; VEGF upstream, 5'-CTTGCCTTGCTGCTCTACC-3'; VEGF downstream, 5'-CACACAGGATGGCTTGAAG; luciferase upstream, 5'-TGGCCCTTCCGCATAGAAC-3'; luciferase downstream, 5'-GATTTGATTGCCAAAAATAGGATCTC-3'; c-myc upstream, 5'-CGTCTCCACACATCAGCACAA-3'; c-myc downstream, 5'-TCTTGGCAGCAGGATAGTCCTT-3'; S9 upstream, 5'-ATCCGCCAGCGCCAT-3'; and S9 downstream, 5'-TCAATGTGCTTCTGGGAATCC-3'. Taqman probes were as follows: IL-8, 5'-CAGCCTTCCTGATTTCTGCAGCTCTGTG-3'; VEGF, 5'-AGTTCATGGATGTCTATCAGCGCAGCT-3'; luciferase, 5'-CCTGCGTCAGATTCTCGCATGCC-3'; c-myc, 5'-ACGCAGCGCCTCCCTCCACTC-3'; and S9, 5'-AGCAGGTGGTGGTGAACATCCCGTCCTT-3'.
RNA Kinetics.
After TNF- stimulation, the cells were treated with actinomycin D (10 µg/ml), and RNA was collected at various time points. The RNA quantities were expressed as "percentage of RNA remaining" compared with the time point when actinomycin D was added. Degradation curves were estimated based on a model of exponential decay (GraphPad Software, San Diego, CA).
UV Cross-Linking and Immunoprecipitation.
Nuclear extracts were prepared from U251 MG cells using the Nu-Per kit (Pierce Endogen, Rockford, IL), and the protein concentration was determined with the BCA protein assay kit. The UV cross-linking was performed as described previously (14)
. The samples were electrophoresed on a 4–15% Tris gradient gel (Bio-Rad, Hercules, CA), dried, and exposed on a phosphorimager. For immunoprecipitation, anti-Hu IgG was added to the UV cross-linked sample in immunoprecipitation buffer as described previously (14)
.
Analysis of Cytokine Protein Expression.
The glioma cell line U251 MG and Flag-HuR clones were plated at a density of 1 x 106 cells. Cells were changed to serum-free media for 24 h and stimulated with TNF- (10 ng/ml) or PBS control for various time intervals. The media were collected and analyzed by ELISA for VEGF, IL-8, and TNF- using commercially available kits (R&D Systems, Minneapolis, MN). The values were normalized to total protein concentration of the cell pellet as measured with a commercial assay (Pierce Endogen).
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RESULTS |
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Stimulation Up-Regulates Angiogenic Growth Factor Expression in Glioma Cells. at different time intervals and then analyzed for mRNA and protein expression of IL-8, TNF-, and VEGF. Control cells were mock-stimulated with PBS. As shown in Fig. 1
, TNF- mRNA expression increased by approximately 7-fold at 6 h (compared with control) and rose to >78-fold at 24 h. IL-8 expression peaked in 6 h at a 13-fold induction and tapered to 8-fold over control at 24 h. VEGF mRNA expression, on the other hand, showed only a small induction (1.7-fold) at 24 h. An earlier report by Ryuto et al. (22)
indicated a marked up-regulation of VEGF mRNA during the initial 3 h after TNF- stimulation, but this waned rapidly to baseline by 6 h, which was the initial time point we examined. This transient increase was attributed to a transcriptional effect rather than RNA stabilization, although later time points (beyond 1 h) for RNA kinetics were not analyzed. Secreted TNF- and IL-8, as measured by ELISA, also significantly increased over all three time intervals, peaking at 24 h (18-fold over control for TNF- and 12-fold for IL-8). Similar to the RNA pattern, VEGF secretion increased only modestly at 24 h (1.6-fold) and may partly relate to the relatively high basal expression of the protein in unstimulated cells.
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. stimulation coupled with the presence of stability elements within the 3'-UTRs of IL-8, TNF-, and VEGF led us to investigate the contribution of posttranscriptional mechanisms to this up-regulation. U251 MG cells were stimulated with TNF- for 6 or 24 h followed by analysis of RNA kinetics (Fig. 2)
. The RNA half-life was extrapolated from the decay curve at the time point representing 50% of the initial RNA level before actinomycin D treatment. We observed a marked increase in half-lives for all three angiogenic factor mRNAs after 24 h of TNF- stimulation compared with 6-h interval. The decay curves had two phases: a rapid decay phase (in the initial 1–2 h); followed by a plateau phase, where there was little change in RNA levels. Prolonged stimulation (24 h) with TNF- blunted the rapid decay phase, resulting in an extended mRNA half-life. The effect was greatest for IL-8, where the half-life increased from 0.5 h to >6.0 h. We observed a 2.5-fold increase in half-life for TNF-(0.2 to 0.5 h) and a 2-fold increase for VEGF (1.0 to 1.9 h). These results represent the mean of three separate experiments, each done in triplicate. Comparisons of the rapid decay phase with mock-stimulated cells could not be done because of the low basal RNA levels for these genes and the lack of any significant induction with PBS stimulation.
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stimulation. This mRNA has a class I ARE, with AUUUA motifs dispersed in the 3'-UTR and no degradation nonamer (UUAUUUA(A/U)(A/U) (7)
. IL-8, TNF-, and VEGF AREs, on the other hand, have more AUUUA or AUUUUA motifs and one or more conserved nonamers (20
, 23)
. Differential RNA stabilization of TNF- and other lymphokine mRNAs versus c-myc has been observed previously in human T lymphocytes after stimulation with phorbol myristate acetate or anti-CD28 antibody (24
, 25)
. Moreover, because the experiments in this report analyzed the kinetics of the whole mRNA, it is also possible that stabilizing or destabilizing elements located in the open reading frame or 5'-UTR may contribute to these differences.
The ARE-rich 3'-UTR Enhances Gene Expression in TNF--stimulated Glioma Cells by Prolonging mRNA Half-Life.
To address the potential contribution of the 3'-UTR to TNF--induced up-regulation of angiogenic factor expression, we transiently transfected U251 MG cells with plasmids containing AREs for IL-8, TNF-, and VEGF downstream from the luciferase open reading frame (Fig. 3A)
. The TNF- ARE in reverse orientation was used as a control. We then compared luciferase expression in TNF--stimulated cells with mock-stimulated cells (Fig. 3B)
. For all three constructs, we observed a marked induction of luciferase expression with TNF- stimulation that increased at each time interval and peaked at 24 h (2.5-fold for IL-8, 3.3-fold for TNF-, and 2.1-fold for VEGF). The results are an average of six independent transfections. The increase in luciferase activity for the three constructs is not likely to be related to a transcriptional effect of TNF- (i.e., on the SV40 promoter) because the control construct, containing the TNF- 3'-UTR in the reverse orientation, showed no induction at any time interval. We postulated that the induction resulted from a posttranscriptional effect due to the presence of AREs in the 3'-UTR. To address this possibility, we stably transfected U251 MG cells with the IL-8 and VEGF ARE constructs (Fig. 3A)
and examined the kinetics of luciferase mRNA degradation with quantitative real-time PCR (Fig. 4)
. Unlike the wild-type IL-8 and VEGF genes, the constitutively active SV40 promoter present in the ARE clones produced similar levels of RNA transcript with both TNF- and mock stimulation, allowing for a direct comparison of RNA degradation. We observed a marked stabilization of luciferase mRNA (half-life > 6.0 h) after 24 h of TNF- stimulation for both the VEGF and IL-8 ARE clones (Fig. 4)
; the half-lives for mock-stimulated cells, on the other hand, were 0.6 and 2.5 h, respectively. In the TNF--stimulated cells, we observed that the RNA levels after actinomycin D treatment were often greater than those at time 0 for the four different clones. Thus, for the VEGF clones, the decay curve extrapolated back to an initial value greater than 100% (dashed line). Thus, the addition of the IL-8 or VEGF AREs led to stabilization of the luciferase mRNA in glioma cells and indicates that posttranscriptional mechanisms contribute to the up-regulation of angiogenic growth factor expression induced by TNF- stimulation.
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, and VEGF in Gliomas.
. Each of the probes contained the AREs as shown in Fig. 3A
. For the VEGF and IL-8 ARE probes, we observed a similar 4-band pattern of binding. The bands were approximately 35, 50–55, 80, and 95–100 kDa in size. The pattern was similar in cytoplasmic extracts, although the bands were much less intense (data not shown). For TNF-, the banding pattern was similar, except for the intermediate-sized bands that were fainter and slightly larger (60–70-kDa range). Interestingly, the upper two bands for all three AREs were similar in size to those cross-linked to a cyclooxygenase 2 ARE probe in colon cancer (14)
. All of the bands in the size range described degraded with proteinase K, indicating that they were a protein complex. Several lower bands did not degrade in the VEGF cross-linking and most likely represent RNA complexes. We also analyzed nuclear and cytoplasmic extracts by UV cross-linking from U251 MG cells stimulated with TNF- for 6 and 24 h but found no qualitative or obvious quantitative differences in binding patterns (not shown). To determine whether the lowest band (35 kDa) represented HuR, we immunoprecipitated the UV cross-linked U251 MG extracts with an anti-HuR antibody. We found that HuR bound to each ARE (Fig. 5
, Lane 3 for each gel) and that the size of the band corresponded to the 35-kDa band observed in the UV cross-linked extracts. No bands were identified with the control antibody (Fig. 5
, Lane 4 for each gel). Work is currently under way to identify the molecular nature of the other dominant bands observed by UV cross-linking.
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-stimulated Glioma Cells Overexpressing HuR Have Marked RNA Stabilization and Up-Regulation of VEGF, IL-8, and TNF-.
. By Western blot, there was marked induction of the transgene with DOX (DOX+) but no induction with vehicle alone (DOX-). We then analyzed these clones for angiogenic factor expression after TNF- stimulation. The results shown in Fig. 6
represent the average of both clones. By 24 h, there was a marked up-regulation of all three angiogenic growth factors, with IL-8 being the strongest (7.2-fold) followed by VEGF (6.4-fold) and TNF- (5.4-fold). These inductions represent a 175–320% increase compared with unstimulated cells (Fig. 6
, 0 h versus 24 h). Interestingly, the baseline values of the angiogenic factors in DOX+ cells were substantially higher than those in DOX- cells (2.7–3.1-fold), indicating that overexpression of HuR alone was sufficient to increase their expression. We also analyzed the expression pattern up to 72 h after TNF- stimulation and found that IL-8 expression increased dramatically to 590% over the 0 h baseline (data not shown).
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for 24 h and measured RNA kinetics (Fig. 7)
. When HuR was overexpressed (DOX+), there was a marked blunting of the rapid decay phase of the curve, as with the wild-type cells. The half-life for all three transcripts was prolonged compared with DOX- cells, with IL-8 showing the most pronounced effect (t1/2 > 6.0 h). There was an approximately 2–3-fold increase in the half-lives of VEGF and TNF- transcripts. Interestingly, the half-life for c-myc in the DOX+ cells was similar to that in wild-type U251 MG cells (Fig. 2)
and slightly reduced compared with that in the DOX- cells, indicating that HuR overexpression had no effect on c-myc RNA kinetics after TNF- stimulation (data not shown). This finding is consistent with the wild-type U251 MG cells, supporting the observation that lymphokine and growth factor mRNAs are differentially stabilized in response to extracellular stimuli (24
, 25)
.
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in the DOX- cells were similar to the wild-type values (Fig. 2)
; however, the IL-8 half-life was shorter (3.8 h), which may be related to the double transfection and selection methods necessary to generate the clones. To exclude an effect of DOX itself on RNA kinetics, we treated U251 Tet-On cells alone under similar experimental conditions and found that the mRNA kinetics of VEGF, IL-8, TNF-, and c-myc were similar to those of wild-type U251 MG cells (data not shown). |
DISCUSSION |
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is a critical step for neovascularization and rapid growth (26
, 27)
. We have identified TNF- as a potent extracellular signal for enhancing the RNA stability and expression of these angiogenic growth factors in glioma cells, and HuR substantially augments this response. Whereas this study focused on one cell line, the findings nonetheless indicate a potential role for TNF- in glioma progression.
The presence of AREs in the 3'-UTRs of IL-8, TNF-, and VEGF and our observation that HuR is overexpressed in gliomas formed the basis of our hypothesis that these angiogenic factors may be posttranscriptionally regulated in these tumors (16
, 28)
. After TNF- stimulation, we observed a time-dependent increase in RNA half-life (6 versus 24 h) for all three transcripts. This time frame is similar to previous findings with hypoxia-induced VEGF mRNA stabilization in a C6 glioma model and indicates that posttranscriptional effects can be delayed (29)
. Interestingly, protein expression also peaked at 24 h, suggesting a link to the prolonged RNA half-lives. This possibility is strengthened by results from the heterologous reporter experiments, where substantial induction of luciferase activity coincided with RNA stabilization of the luciferase transcript. The blunting of the rapid degradation phase of the decay curve by TNF- stimulation, however, was not observed with c-myc, indicating either additional regulatory elements outside of the ARE (e.g., the open reading frame or 5'-UTR) or subtle differences within the ARE (30, 31, 32)
. The mere presence of AUUUA motifs in the 3'-UTR, as demonstrated here and in other cell types, is insufficient to predict a RNA stabilizing effect (24
, 25
, 33)
.
Because the AREs of these angiogenic growth factor transcripts could confer the RNA stabilizing effects of TNF- to the luciferase transcript, we analyzed this element for differences in binding patterns in stimulated and unstimulated glioma extracts. The UV cross-linking results, however, did not reveal any qualitative or quantitative differences. In other cell systems, such as T lymphocytes or preadipocytes, alterations in RNA binding have been observed after stimulation, with the appearance of a 43-kDa AU-binding complex (25
, 34)
. Such a complex was not observed in our analysis with U251 MG cells. Additionally, there was no shift of nuclear factors, including HuR, to the cytoplasm after stimulation as observed in association with other mRNAs such as p21 or cyclins (35, 36, 37, 38)
. These observations suggest that TNF- may induce subtle posttranslational modifications (e.g., phosphorylation) of HuR or other RNA-binding factors or promote novel protein-protein interactions that would not have been identified by our binding assay. A number of cellular factors bind to the ARE in addition to the Elav proteins, including AUF and TTP (8
, 9
, 39
, 40)
. Our UV cross-linking data indicated three to four RNA-protein complexes larger than AUF or TTP that have yet to be characterized. The band in the 50–55-kDa range is similar to one identified in a malignant epithelial cell line that bound to the TNF- 3'-UTR (41)
. The interaction between these factors to confer stability or instability to the transcript is likely to be complex. AUF1, for example, has been clearly linked to RNA destabilization (9)
, but recent studies analyzing its overexpression in a transgenic mouse revealed an up-regulation of several ARE-bearing mRNAs (42)
. In our study, on the other hand, overexpression of HuR in TNF--stimulated glioma cells significantly augmented the RNA stabilization of the three angiogenic growth factors but not c-myc, yet HuR has been shown to bind avidly to the 3'-UTR of c-myc by a filter binding assay and by ELISA (20
, 43)
.4
Taken together, these findings suggest that the determinants of RNA stability or instability cannot be predicted by any one individual RNA-binding factor.
The dysregulation of cytokine expression in malignancies can promote neoplastic progression by influencing critical cellular processes such as angiogenesis, cell cycle regulation, or immunomodulation (44, 45, 46)
. The relevance of our findings to glioma biology is 3-fold. First, TNF- has been detected in glioma tumors, both in neoplastic cells and in infiltrating inflammatory cells (3
, 17, 18, 19)
. Thus, there are both paracrine and autocrine pathways for the induction of TNF-. Second, TNF- has previously been shown to exert its angiogenic effect on human microvascular cells by modulating VEGF and IL-8 expression (4)
. We have demonstrated that TNF- can up-regulate the expression of these angiogenic growth factors by glioma cells. Third, we have shown that HuR is overexpressed in glioma tumors in vivo (16)
. Therefore, the glioma tumor, as with our HuR-tet clones, is primed for RNA stabilization. A microenvironment of inflammatory cells, reactive glial cells, and glioma tumor cells expressing TNF- thus provides both paracrine and autocrine loops for promoting the angiogenic phenotype through stabilization of growth factor mRNAs.
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FOOTNOTES |
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1 L. B. N. was supported by a Department of Veterans Affairs Medical Research Entry Program Award. P. H. K. was supported by a Merit Review from Department of Veterans Affairs. 
2 To whom requests for reprints should be addressed, at Department of Neurology, University of Alabama at Birmingham, 1235 Jefferson Tower, 625 South 19th Street, Birmingham, AL 35294-0007. Phone: (205) 975-8116; Fax: (205) 934-0928; E-mail: pking{at}uab.edu
3 The abbreviations used are: MG, malignant glioma; TNF-, tumor necrosis factor ; IL-8, interleukin 8; VEGF, vascular endothelial growth factor; TTP, tristetraprolin; ARE, AU-rich element; UTR, untranslated region; DOX, doxycycline.
4 L. Burt Nabors and Peter H. King, unpublished observation.
Received 11/18/02. Accepted 5/ 9/03.
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