Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover - PubMed (original) (raw)

Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover

S Lin et al. Mol Cell Biol. 2000 Nov.

Abstract

Prostaglandin A(2) (PGA(2)), an experimental chemotherapeutic agent, causes growth arrest associated with decreased cyclin D1 expression in several cancer cell lines. Here, using human non-small-cell lung carcinoma H1299 cells, we investigated the mechanisms whereby PGA(2) down-regulates cyclin D1 expression. Transcription rates of the cyclin D1 gene, studied using a cyclin D1 promoter-luciferase construct and nuclear run-on assays, were not affected by PGA(2) treatment. Instead, the cyclin D1 mRNA was rendered unstable after exposure to PGA(2). Since the stability of labile mRNA is modulated through binding of proteins to specific mRNA sequences, we sought to identify protein(s) recognizing the cyclin D1 mRNA. In electrophoretic mobility-shift assays using radiolabeled RNA probes derived from different regions of cyclin D1 mRNA, we observed that (i) lysates prepared from PGA(2)-treated cells exhibited enhanced protein-cyclin D1 RNA complex formation; (ii) the kinetics of complex formation correlated closely with that of cyclin D1 mRNA loss; and (iii) binding occurred within a 390-base cyclin D1 3' untranslated region (UTR) (K12). This binding activity could be cross-linked, revealing proteins ranging from 30 to 47 kDa. The RNA-binding protein AUF1, previously associated with the degradation of target mRNAs, bound cyclin D1 mRNA, because anti-AUF1 antibodies were capable of supershifting or immunoprecipitating cyclin D1 mRNA-protein complexes. Finally, insertion of K12 in the 3'UTR of reporter genes markedly reduced the expression and half-life of the resulting chimeric mRNAs in transfected, PGA(2)-treated cells. Our data demonstrate that PGA(2) down-regulates cyclin D1 expression by decreasing cyclin D1 mRNA stability and implicates a 390-base element in the 3'UTR in this regulation.

PubMed Disclaimer

Figures

FIG. 1

PGA2 treatment results in cell growth arrest. H1299 cells were either left untreated or were treated with 30 μM PGA2. Cell numbers were determined every 24 h using a hemacytometer. Open squares, untreated cells; filled squares, PGA2-treated cells. Values shown are the means ± SEM from three independently performed experiments.

FIG. 2

Cyclin D1 expression after exposure to PGA2. (A) Dose-response analysis of PGA2 treatment on cyclin D1 expression. H1299 cells were treated for the times shown with the indicated doses of PGA2. RNA was then isolated, and the expression of cyclin D1 and p21 was assessed by Northern blot analysis. (B) Kinetics of cyclin D1 mRNA down-regulation. Cyclin D1 mRNA levels in H1299 cells that were either treated with the indicated doses of PGA2 for 16 h (right) or treated with 30 μM PGA2 for the times shown (left) were examined by Northern blot analysis. Cyclin D1 mRNA signal was normalized to 18S rRNA signal. Data represent the means ± SEM from three independently performed experiments. (C) Western blot analysis of cyclin D1 expression was carried out using 40 μg of whole-cell lysates prepared from either untreated or PGA2-treated H1299 cells, as described in Materials and Methods.

FIG. 3

Influence of PGA2 on transcriptional activation of the cyclin D1 gene. (A) Effect of PGA2 on cyclin D1 promoter activity. Cells stably transfected with a plasmid harboring a luciferase reporter gene driven by the human cyclin D1 promoter (mg37) were either left untreated or were treated with 30 μM PGA2 for the times indicated. Luciferase activity was then determined on cell lysates (30 to 50 μg of protein) and expressed as percentages relative to luciferase activity in untreated cells. (B) Effect of PGA2 on the transcription rate of cyclin D1. Cells that were either left untreated or treated with 30 μM PGA2 for 8 h were subjected to nuclear run-on assay. Membranes were blotted with cDNAs encoding β-actin, cyclin D1, and a control plasmid without an insert (pBlueScript). The signals were visualized with a PhosphorImager.

FIG. 4

Binding to cyclin D1 transcripts. (A) Schematic representation of the full-length cyclin D1 cDNA and various transcripts derived from the 5′UTR, coding region, and 3′UTR used in this study. Transcripts were synthesized after preparation of DNA templates by RT-PCR, as described in the Materials and Methods section. The 5′ end of each fragment contains the T7 RNA polymerase promoter sequence. (B) Detection of RNA-protein binding activity by EMSA analysis. PCR-amplified cDNA fragments were used as templates to synthesize 32P-radiolabeled RNA probes (A1, B2, B3, and C4). Following treatment of H1299 cells with 30 μM PGA2 for the times indicated, cytoplasmic and nuclear fractions (10 μg each) were assayed for binding to the indicated RNA probes. Reaction mixtures were resolved on 7% native polyacrylamide gels. Signals were visualized with a PhosphorImager. Brackets indicate complexes forming with B2 and B3 fragments in a PGA2-dependent fashion. f, radiolabeled RNA digested with RNase T1 without incubation with cell lysate. (C) Correlation between PGA2-mediated degradation of cyclin D1 mRNA and PGA2-induced RNA-protein-binding activity. Cyclin D1 mRNA levels in H1299 cells that were either left untreated or treated with 30 μM PGA2 for the times indicated were determined by Northern blot analysis (top); lysates (cytoplasmic fraction) prepared from H1299 cells treated in parallel were incubated with 32P-radiolabeled B2 transcript and then subjected to EMSA (bottom). Radioactive signals were visualized with a PhosphorImager.

FIG. 5

Binding region. (A) Schematic representation of regions within the B2 fragment of the cyclin D1 mRNA assayed for protein binding. (B) Binding of cytoplasmic proteins (10 μg) derived from either PGA2-treated or untreated cells, to various RNA transcripts encompassing the B2 region of cyclin D1 mRNA. Complex formation was assayed by EMSA as described in the legend of Figure 4. Note that fragment K12 is composed of fragments K9 and I12. f, radiolabeled transcript not incubated with protein lysate, then digested with RNase T1. (C) Competition assays. Cytoplasmic lysates from PGA2-treated cells (24-h exposure to the drug) were incubated with a 5-, 10-, or 50-fold molar excess of either cold K12 or cold A1 RNA on ice for 20 min, then with radiolabeled B2 RNA on ice for an additional 20 min. After RNase T1 digestion, reactions were analyzed by EMSA. (D) Schematic representation of regions within the K12 fragment (left) and transcripts assayed by EMSA after incubation either without (−) or with protein lysates (lysate) from PGA2-treated (30 μM, 8 h) H1299 cells (right). Transcripts not incubated with protein lysate, then subjected to RNase T1 digestion.

FIG. 6

Detection of AUF1 in the cyclin D1 RNA-protein complexes. (A) Detection of AUF1-cyclin D1 associations by immunoprecipitation. Cytoplasmic lysates from cells that were either left untreated (0 h) or treated with 30 μM PGA2 for 8 or 24 h were first allowed to bind radiolabeled RNA (either K12 or A1), and then the RNA-protein complexes were UV cross-linked. Complexes were either resolved directly by electrophoresis through SDS–12% polyacrylamide gels or first immunoprecipitated using protein A beads (Beads) or protein A beads coated with anti-AUF1 antibody (Beads+AUF1) or after no immunoprecipitation (No IP), and then resolved by electrophoresis through SDS–12% polyacrylamide gels. f, free radiolabeled RNA, not incubated with protein lysate. (B) Supershift analysis. Cytoplasmic proteins isolated from cells treated with PGA2 for 24 h were preincubated with anti-AUF1, anti-p27Kip1, anti-JNK1, or anti-p53 antibodies for 30 min on ice, prior to addition of radiolabeled K12 RNA. Complexes were subjected to EMSA analysis through 5% native gels. Native and SDS-containing polyacrylamide gels were dried and signals visualized with a PhosphorImager. Arrow indicates the band supershifted by the AUF1 antibody. f, radiolabeled transcript not incubated with protein lysate but digested with RNase T1.

FIG. 7

PGA2-induced AUF1 expression. (A) Forty-microgram aliquots of whole-cell lysate prepared from H1299 cells that were either left untreated (0 h) or treated with PGA2 for 8 or 24 h were subjected to Western blot analysis using anti-AUF1 antibody (64). Molecular weights of AUF1 isoforms are shown. (B) Forty-microgram aliquots of cytoplasmic lysates from cells treated as described in panel A were examined for the expression of p45AUF1 using an antibody that specifically recognizes a unique 19-amino-acid region present in the p45 isoform. This antibody also recognizes p40AUF1 (not shown). control, lysate from K562 cells. (C) Gel mobility shift of recombinant p45AUF1 (1 to 50 nM) binding either K16 or O14 radiolabeled transcripts. NP, no protein.

FIG. 8

Influence of the K12 region of the cyclin D1 mRNA on the expression of a chimeric luciferase reporter construct after PGA2 treatment. Three micrograms of each pGL3-promoter (pGL3), pGL3-K12, and pGL3-CR plasmid was transiently transfected into H1299 cells along with 1 μg of β-galactosidase reporter as a control. Transfected cells were either treated with PGA2 for 24 h or left untreated. Luciferase activities were determined and normalized against β-galactosidase measurements. Values, shown as the means ± SEM from five independently performed experiments, represent the luciferase values in PGA2-treated populations relative to those measured in untreated cells (to which a value of 100% was assigned).

FIG. 9

Influence of the K12 region of the cyclin D1 mRNA on the expression of a chimeric EGFP reporter construct after PGA2 treatment. Five micrograms of each pTRE-d2EGFP, pTRE-d2EGFP-CR, or pTRE-d2EGFP-K12 plasmid was transiently transfected into H2 cells (previously transfected with pTet and selected based on its strong induced and high doxycycline-dependent repression of reporter gene expression), along with 1 μg of β-galactosidase reporter as a control. Twenty hours after transfection, cells were treated either with doxycycline alone (control) or with a combination of doxycycline and PGA2 (30 μM, PGA2). At the times indicated following addition of the drug(s), RNA was prepared for Northern blot analysis; assessment of the expression levels of the EGFP, EGFP-CR, or EGFP-K12 transcripts was followed by that of cyclin D1 expression (not shown) and 18S rRNA, through sequential rounds of stripping and hybridization. Representative Northerns are shown. Graphs depict the relative abundance of each EGFP-derived transcript at various times after addition of doxycycline (time 0, 100%), represented on a logarithmic scale. Dashed line, 50% mRNA, which served to obtain the half-life values for each transcript.

FIG. 10

Expression of cyclin D1 mRNA in MDA-MB-453 and MCF-7 cells following PGA2 treatment. (A) MDA-MB-453 and MCF-7 cells were treated with PGA2 for the times indicated, and Northern blot analysis was used to assess cyclin D1 expression. (B) Quantitation of cyclin D1 mRNA signals after normalization to 18S rRNA signals on the same blot. Values shown are the means ± SEM from three independently performed experiments.

References

1. Albanese C, Johnson J, Watanabe G, Eklund N, Vu D, Arnold A, Pestell R G. Transforming p21ras mutants and c-Ets-2 activate the cyclin D1 promoter through distinguishable regions. J Biol Chem. 1995;270:23589–23597. - PubMed
1. Amici C, Palamara A T, Santoro M G. Induction of thermotolerance by prostaglandin A in human cells. Exp Cell Res. 1993;207:230–234. - PubMed
1. Atwater J A, Wisdom R, Verma I M. Regulated mRNA stability. Annu Rev Genet. 1990;24:519–541. - PubMed
1. Baldin V, Lukas J, Marcote M J, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993;7:812–821. - PubMed
1. Beier F, Lee R J, Taylor A C, Pestell R G, LuValle P. Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chrondrocytes. Proc Natl Acad Sci USA. 1999;96:1433–1438. - PMC - PubMed

Down-regulation of cyclin D1 expression by prostaglandin A(2) is mediated by enhanced cyclin D1 mRNA turnover - PubMed (original) (raw)