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. 2009 Oct 28;4(10):e7639.
doi: 10.1371/journal.pone.0007639.

Retinoic acid mediates long-paced oscillations in retinoid receptor activity: evidence for a potential role for RIP140

Kelly C Heim  1 , Joshua J Gamsby, Mary P Hever, Sarah J Freemantle, Jennifer J Loros, Jay C Dunlap, Michael J Spinella
Affiliations

Retinoic acid mediates long-paced oscillations in retinoid receptor activity: evidence for a potential role for RIP140

Kelly C Heim et al. PLoS One. .

Abstract

Background: Mechanisms that underlie oscillatory transcriptional activity of nuclear receptors (NRs) are incompletely understood. Evidence exists for rapid, cyclic recruitment of coregulatory complexes upon activation of nuclear receptors. RIP140 is a NR coregulator that represses the transactivation of agonist-bound nuclear receptors. Previously, we showed that RIP140 is inducible by all-trans retinoic acid (RA) and mediates limiting, negative-feedback regulation of retinoid signaling.

Methodology and findings: Here we report that in the continued presence of RA, long-paced oscillations of retinoic acid receptor (RAR) activity occur with a period ranging from 24 to 35 hours. Endogenous expression of RIP140 and other RA-_target genes also oscillate in the presence of RA. Cyclic retinoid receptor transactivation is ablated by constitutive overexpression of RIP140. Further, depletion of RIP140 disrupts cyclic expression of the RA _target gene HOXA5. Evidence is provided that RIP140 may limit RAR signaling in a selective, non-redundant manner in contrast to the classic NR coregulators NCoR1 and SRC1 that are not RA-inducible, do not cycle, and may be partially redundant in limiting RAR activity. Finally, evidence is provided that RIP140 can repress and be induced by other nuclear receptors in a manner that suggests potential participation in other NR oscillations.

Conclusions and significance: We provide evidence for novel, long-paced oscillatory retinoid receptor activity and hypothesize that this may be paced in part, by RIP140. Oscillatory NR activity may be involved in mediating hormone actions of physiological and pathological importance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. RAR-dependent transactivation is cyclic in the continued presence of RA and is sensitive to RIP140 depletion and overexpression.
A, Actively proliferating NT2/D1 cells were transiently tranfected with an RARE-tk-luciferase reporter and either a control insertless expression plasmid (RAR-LUC + RA, RARE-LUC no RA), a RIP140 expression plasmid driven by the SV40 promoter-enhancer (RARE-LUC + RA + HA-RIP140) or RIP140 siRNA (RARE-LUC + RA + siRNA-RIP140). Cells were then treated with RA (1 µM) or vehicle and luciferase activity was monitored in live cells every 10 minutes for 74 hours using a LumiCyle luminometer. Each date line represents the average of three biological replicate plates. The experiment was performed twice with similar results. B, Two of the data lines in (A), RARE-LUC + RA and RARE-LUC + RA + siRNA-RIP140 are depicted with zoomed scaling of y-axis. Data points are represented by the error bars (S.E.M.) of the replicate plates. C, Hypothetical model representing one interpretation of results in (A), see text for details.
Figure 2
Figure 2. Endogenous RIP140 transcripts oscillate in the continued presence of RA.
NT2/D1 cells were pretreated with alpha-amanitin (2.5 µM) for 2 hours and then treated with RA (1 µM) for the indicated time points. RIP140 expression was monitored by quantitative real-time PCR using primers in A, the processed transcript or B, in an intron of the unspliced heteronuclear transcript. Data was normalized to GAPDH. Trend line is the 5th order polynomial. One experiment of three with similar results is presented.
Figure 3
Figure 3. Endogenous expression of RA _target genes oscillate in the continued presence of RA.
Actively proliferating NT2/D1 cells were pretreated with alpha-amanitin (2.5 µM) for 2 hours and then treated with RA (1 µM) for the indicated time points. RARB (A, C) and HOXA5 (B, D) expression was monitored by quantitative real-time PCR using primers in the processed transcript (A, B) or in an intron on the unspliced heteronuclear transcript (C, D). Data was normalized to GAPDH. Trend line is the 5th order polynomial except for B, 6th polynomial. The experiment was performed three times with similar results.
Figure 4
Figure 4. RIP140 depletion alters the cyclic pattern of HOXA5 expression.
A, NT2/D1 cells were treated with RIP140 siRNA or control siRNA and exposed to RA (1 µM) for the indicated time points. HOXA5 expression was measure by quantitative real-time PCR specific for unspliced heteronuclear transcripts. B, Data in (A) for control siRNA treated cells with zoomed scaling of y-axis. Data was normalized to GAPDH. Trend line is the 6th order polynomial.
Figure 5
Figure 5. RAR _target genes differentially respond to RIP140 depletion.
A, Northern analysis depicting the effect of RIP140 shRNA on expression of RIP140 and the known RA _target gene RARB. NT2/D1 cells were treated with RIP140 shRNA lentivirus or control shRNA lentivirus and selected in puromycin. Three individual stable pools of RIP140 shRNA treated NT2/D1 cells and one control shRNA treated pool were exposed to RA (1 µM) for 24 hours. The 28S ribosomal RNA served as a loading control. B–D, Quantitative real-time PCR analysis of NT2/D1 shRNA control and NT2/D1 shRNA RIP140 cells exposed to RA (1 µM) for the indicated time points. Data was normalized to GAPDH. Error bars represent the range of the average of duplicate experiments for the expression of B, RIP140; C, RARB; and D, RARA.
Figure 6
Figure 6. Comparison of the effects of RIP140, NCoR1 and SRC1 depletion on RA-induced RARB expression.
A, Northern analysis depicting the effect of control, RIP140, NCoR1 and SRC1 siRNA on expression of the known RA _target gene RARB. NT2/D1 cells treated with the indicated siRNA were exposed to RA (1 µM) for 24 hours. The 28S ribosomal RNA served as a loading control. Blot was hybridized to probes specific for RARB, RIP140, NCoR1 and SRC1. Note, only RIP140 siRNA resulted in enhanced RA induction of RARB and only RIP140 is induced with RA. B–D, Quantitative real-time PCR analysis of NT2/D1 cells treated as in (A) and assayed for expression of RARB (B), NCoR1 (C) and SRC1 (D). B and D are the average of triplicate experiments and error bars are S.E.M. *, p<.02 with the paired, two-tailed T test. **, p<.05, with the unpaired, two-tailed T test. (C) is the average of duplicate experiments and error bars depict the range of the two values.
Figure 7
Figure 7. RIP140 expression is induced with RA, estrogen, dexamethasone, and rosiglitazone in MCF7 cells.
A, Schematic of the RIP140 gene locus. The protein-coding region of RIP140 is contained within exon 5. A 3.5 kb highly evolutionarily conserved region (ECR) is indicated by the arrowheads. B and C, MCF7 cells were treated with vehicle controls, DMSO or ethanol (EtOH), or RA (1 µM), estrogen (0.1 µM, E2), dexamethasone (1 µM, Dex) or rosiglitazone (1 µM, Rosi) for 24 hours and expression of RIP140 was measured by quantitative real-time PCR with primers within exon 5 (B) or exon 1b (C). Data was normalized to GAPDH. Depicted is that average of triplicate PCR reactions of the same samples and error bars are S.D. D, COS-1 cells were transfected with an ER-alpha expression plasmid and a control thymidine kinase (tk)-liciferase reporter (TK-Luc) or a reporter containing a 550 bp segment of the RIP140 ECR region containing a consensus estrogen receptor response (ERE) (RIP-ECR-TK-Luc). Cells were treated with vehicle or estrogen (0.1 µM) for 24 hours and firefly luciferase activity was determined and normalized to renilla luciferase activity. Bars are the average of triplicate transfections and error bars are S.D. *, p<.0001 with the unpaired two-tailed T test as compared to vehicle treated RIP-ECR-TK-Luc cells. One of two experiments with similar results.
Figure 8
Figure 8. RIP140 represses ligand dependent GR, PPARγ, and VDR activity.
A, COS-1 cells were transfected with a GRE-tk-luciferase reporter, a GR expression plasmid and an insertless expression plasmid (empty vector) or RIP140 expressing plasmid (HA-RIP140). Cells were treated with vehicle or dexamethasone (1 µM, Dex) for 24 hours and firefly luciferase activity was measured and normalized to renilla luciferase. Data are the average of triplicate transfections and error bars are S.D. *, p<.005 with the unpaired two-tailed T test. Experiment was repeated with similar results. B, COS-1 cells were transfected with a PPRE-tk-luciferase reporter, PPARγ and RXR expression plasmids and an insertless expression plasmid (empty vector) or RIP140 expressing plasmid (HA-RIP140). Cells were treated with vehicle or rosiglitazone (1 µM, Rosi) for 24 hours and firefly luciferase activity was measured and normalized to renilla luciferase. Data are the average of triplicate transfections and error bars are S.D. *, p<.005 with the unpaired two-tailed T test. One of two experiments with similar results. C, Semi-quantitative PCR analysis of T47D cells transfected with control siRNA or siRNA to RIP140. Cells were treated with Vitamin D3 (1 µM) for 24 hours and expression of the VDR _target gene, CYP24A1 was determined. Actin expression was used as a control.
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References

    1. Niles RM. Signaling pathways in retinoid chemoprevention and treatment of cancer. Mutat Res. 2004;555:81–96. - PubMed
    1. Freemantle SJ, Spinella MJ, Dmitrovsky E. Retinoids in cancer therapy and prevention: Promise meets resistance. Oncogene. 2003;22:7305–7315. - PubMed
    1. Buletic Z, Soprano KJ, Soprano DR. Retinoid _targets for the treatment of cancer. Crit Rev Eukaryot Gene Exp. 2006;16:193–210. - PubMed
    1. Mark M, Ghyselinck NB, Chambon P. Function of retinoic acid receptors during embryonic development. Nucl Recept Signal. 2009;7:e002. - PMC - PubMed
    1. Perissi V, Rosenfeld MG. Controlling nuclear receptors: the circular logic of cofactor cycles. Nat Rev Mol Cell Biol. 2005;7:542–54. - PubMed

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