Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability - PubMed (original) (raw)

Identification of a human VPF/VEGF 3' untranslated region mediating hypoxia-induced mRNA stability

K P Claffey et al. Mol Biol Cell. 1998 Feb.

Free PMC article

Abstract

Hypoxia is a prominent feature of malignant tumors that are characterized by angiogenesis and vascular hyperpermeability. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) has been shown to be up-regulated in the vicinity of necrotic tumor areas, and hypoxia potently induces VPF/VEGF expression in several tumor cell lines in vitro. Here we report that hypoxia-induced VPF/VEGF expression is mediated by increased transcription and mRNA stability in human M21 melanoma cells. RNA-binding/electrophoretic mobility shift assays identified a single 125-bp AU-rich element in the 3' untranslated region that formed hypoxia-inducible RNA-protein complexes. Hypoxia-induced expression of chimeric luciferase reporter constructs containing this 125-bp AU-rich hypoxia stability region were significantly higher than constructs containing an adjacent 3' untranslated region element without RNA-binding activity. Using UV-cross-linking studies, we have identified a series of hypoxia-induced proteins of 90/88 kDa, 72 kDa, 60 kDa, 56 kDa, and 46 kDa that bound to the hypoxia stability region element. The 90/88-kDa and 60-kDa species were specifically competed by excess hypoxia stability region RNA. Thus, increased VPF/VEGF mRNA stability induced by hypoxia is mediated, at least in part, by specific interactions between a defined mRNA stability sequence in the 3' untranslated region and distinct mRNA-binding proteins in human tumor cells.

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Figures

Figure 1

Figure 1

Hypoxia-induced VPF/VEGF expression in M21 melanoma cells. (A) M21 melanoma cells were exposed to normoxic (N) or hypoxic (H) conditions for the indicated times. Total RNA was extracted and analyzed using Northern blot for VPF/VEGF (VEGF) and 36B4 (36B4), ribosome-associated protein, as a control. Hybridization signals for each are indicated and ethidium bromide (Eth. Br.) stained gel is shown. (B) Quantification of the fold induction of VPF/VEGF mRNA signal at the indicated times. VPF/VEGF signals were normalized to 36B4 control for each lane and hypoxic conditions were compared with normoxic conditions. (C) Rate of VPF/VEGF secretion from M21 cells under hypoxic conditions. Conditioned media analyzed for VPF/VEGF using enzyme-linked immunosorbent assay was evaluated for increased VPF/VEGF secretion for the indicated time periods.

Figure 2

Figure 2

Hypoxia-induced VPF/VEGF transcription and mRNA stability in M21 melanoma cells. (A) Transcription run-off assay of M21 cells exposed to 24 h normoxic (N) or hypoxic (H) conditions. Total radiolabeled RNAs were hybridized to denatured cDNA templates encoding GAPDH, VPF/VEGF, Glut-1, and 36B4. Fold induction by quantification is indicated. (B) Representative Northern blot analysis of M21 cells preincubated for 20 h under normoxic or hypoxic conditions. Actinomycin D was added at time 0 and total RNA was isolated after up to 3 h. Mature VPF/VEGF mRNA signal and 36B4 control are indicated by arrows. VPF/VEGF mRNA decay curves and mRNA half-life (_t_1/2) are presented below each Northern blot.

Figure 3

Figure 3

Complete VPF/VEGF165 3′ UTR cDNA sequence and potential regulatory elements. (A) Complete 3′ UTR cDNA sequence beginning with base 1 as the first base after the translation stop codon in the coding region. The polyadenylation site at the 3′ end consists of 13 adenosine residues but is not preceded by a consensus AAUAAA sequence. Two consensus internal polyadenylation sequences are encircled. Two destabilizing elements with the consensus sequence 5′-UUAUUUA(U/A)(U/A)-3′ are indicated (solid boxes). Five underlined sequences show partial consensus sequences that include the AUUUA sequence. Broken boxes indicate AU-rich elements consisting of at least five AU pairs in a row (AU 1–4). The human VPF/VEGF165 3′ UTR sequence is accessible in GenBank accession no. AF022375. (B) Schematic map of VPF/VEGF165 FL cDNA and the RNA transcripts used in this study. The numbers indicate base location in the 3′ UTR, and the numerical bar on the bottom represents the 3′ UTR sequence given in A.

Figure 4

Figure 4

Identification of a 500-bp sequence in the 3′ UTR of VPF/VEGF mRNA that forms hypoxia-inducible RNA–protein complexes. (A) M21 cell extracts were subjected to RNA binding/EMSA with the FL VPF/VEGF, 5′ UTR and coding region (5′+ CR), the 5′ UTR (5′ UTR), and the 3′ UTR (3′ UTR) RNA probes. RNase T1 was not added to a FL probe, indicating minimal endogenous RNase activity (−). RNA–protein complexes are indicated by arrows and lines. RNase T1-resistant bands are indicated (T1 Resist). (B) RNA binding/EMSA of VPF/VEGF subfragment 3′ UTR 1.5-kb probe (3′ 1.5 kb) and the 0.5-kb probe (3′ 0.5 kb). Arrow indicates major complex that is present in normoxic (N) and increased in hypoxic (H) M21 cell extracts.

Figure 5

Figure 5

Hypoxia-inducible RNA–protein complexes form with the VPF/VEGF AU-rich 3′ HSR sequence in M21 cells. (A) RNA binding/EMSA was performed with an RNA transcript probe (3′ HSR). RNA–protein complexes were detected in the presence of both RNase T1 and RNase A. Orientation-specific RNA–protein complexes were detected by using sense (S) and antisense (AS) 3′ HSR probes and 24-h normoxic (N) and hypoxic (H) extracts. RNase-resistant complexes are indicated by arrows. Note the hypoxic induction of the faster mobility complex. (B) RNA–protein complexes competed with sense but not antisense RNA containing the 3′ HSR sequence. A 3′ HSR probe was used to perform RNA binding/EMSA with M21 normoxic cell extract without (−) and with a 50-fold excess of sense (S) or antisense (AS) 3′ HSR or 3′ 0.5-kb transcripts.

Figure 6

Figure 6

Stem loop secondary structure of the VPF/VEGF AU-rich 3′ HSR. Secondary structure of the 125-bp 3′ HSR sequence within the VPF/VEGF 3′ UTR as determined by MUFOLD (Jaeger et al., 1989a,b; Zuker, 1989). Potential RNA loop structure energies for the AU-rich regions 1 and 2 alone and the whole 3′ HSR are indicated.

Figure 7

Figure 7

VPF/VEGF 3′ HSR confers increased hypoxia expression to a constitutively expressed luciferase-VPF/VEGF chimeric construct. (A) Schematic model of cytomegalovirus-enhancer/promoter-driven luciferase-VPF/VEGF reporter constructs. VPF/VEGF 3′ UTR elements in the sense orientation were cloned 3′ to luciferase sequence. (B) Luciferase activity of transient transfected M21 cells under normoxic or hypoxic conditions for 12 h (n = 3). Luciferase activity was normalized to normoxic light units/mg of protein to account for differences in transfection efficiency. *p value for t test of hypoxic compared with normoxic conditions.

Figure 8

Figure 8

Identification of hypoxia-induced RNA-binding proteins using EMSA/UV-cross-linking and RNA affinity chromatography. (A) Normoxic (N) and hypoxic (H) M21 cell extracts bound to the 3′ HSR probe were subjected to UV-cross-linking/RNase treatment and analyzed by reducing SDS-PAGE. Hypoxia-induced proteins covalently cross-linked to the 3′ HSR probe are indicated by arrows. (B) Competitive inhibition of RNA–protein complex formation by excess cold VPF/VEGF 3′ HSR. M21 24-h hypoxic cell extracts EMSA/UV cross-linked to VPF/VEGF 3′ HSR mRNA (−) was competed with unlabeled VEGF 3′ HSR (0.05, 0.5, and 1 μg 3′ HSR, lanes 2–4, respectively). The arrows indicate 3′ HSR mRNA protein complexes.

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