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MDM2 binds and inhibits vitamin D receptor a

a

a

b

Kristina Heyne , Tessa-Carina Heil , Birgit Bette , Jörg Reichrath & Klaus Roemer

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Internal Medicine I and José Carreras Center, Bldg. 45.3, University of Saarland Medical Center, 66421 Homburg (Saar), Germany b

Dept. of Dermatology, University of Saarland Medical Center, 66421 Homburg (Saar), Germany Accepted author version posted online: 13 May 2015.

Click for updates To cite this article: Kristina Heyne, Tessa-Carina Heil, Birgit Bette, Jörg Reichrath & Klaus Roemer (2015): MDM2 binds and inhibits vitamin D receptor, Cell Cycle, DOI: 10.1080/15384101.2015.1044176 To link to this article: http://dx.doi.org/10.1080/15384101.2015.1044176

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MDM2 binds and inhibits vitamin D receptor

Kristina Heyne1, Tessa-Carina Heil1, Birgit Bette1, Jörg Reichrath2 and Klaus Roemer 1* 1

Internal Medicine I and José Carreras Center, Bldg. 45.3, University of Saarland

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Medical Center, 66421 Homburg (Saar), Germany; 2Dept. of Dermatology, University of Saarland Medical Center, 66421 Homburg (Saar), Germany. Keywords: vitamin D; vitamin D receptor; MDM2; transcription; tumor suppression

*Correspondence to: Klaus Roemer; Email: [email protected] Abstract The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level of VDR whereas knockdown of endogenous MDM2 increased the level of VDR. In addition to ubiquitin-marking proteins for degradation, MDM2, once recruited to promoters by DNA-binding interaction partners, can inhibit the transactivation of genes. Transient transfections with a VDR-responsive luciferase reporter revealed that low

levels of MDM2 potently suppress VDR-mediated transactivation.

Conversely, knockdown of MDM2 resulted in a significant increase of transcript from the CYP24A1 and p21 genes, noted cellular targets of transactivation by liganded VDR. Our findings suggest that MDM2 negatively regulates VDR in some analogy to p53. 1

Introduction

The active form of the hormone vitamin D, 1a,25-dihydroxyvitamin D3 (1,25D), exerts almost all of its known effects through the binding to the vitamin D receptor (VDR), a DNAbinding transcription factor of the nuclear receptor superfamily. Upon association with 1,25D through its C-terminal ligand binding domain VDR typically forms a heterodimer with the retinoid X receptor (RXR) to contact, via the N-terminal DNA binding domain, vitamin D response elements (VDRE) with the consensus sequence (PGGTCA)3n(GPGTTCA) (P, Downloaded by [University of Leeds] at 18:37 17 May 2015

purine base; n, any nucleotide). Nanomolar quantities of 1,25D are sufficient to activate VDR in this manner and to regulate a plethora of genes

1, 2

; however, un-liganded VDR, VDR not

associated with RXR, and VDR tethered to other DNA-binding factors rather than binding to DNA directly, may also function as transcriptional regulator on subsets of genes

3-5

. Global

chromatin immunoprecipitation (ChIP) analyses have suggested that in the absence of hormone less than 1,000 sites in the human genome are occupied by VDR whereas up to 8,000 sites may be occupied when VDR is liganded

5, 6

. Genes may be activated or repressed

upon VDR binding. The underlying mechanisms are not yet fully comprehended. Altogether, while the initiation of transactivation is relatively well understood, much less is known about the termination of transactivation and the suppression of genes by VDR 3, 4, 6.

MDM2 (Murine Double Minute 2 homolog) is a multifunctional E3 ubiquitin ligase that acts as an important negative regulator of several transcription factors 7-10. For instance, MDM2 is a major inhibitor of the p53 and its relative p73, which like VDR fulfil essential functions in metabolism, homeostasis and tumor suppression 9. Basically, two levels of inhibition by MDM2 are implemented. Firstly, MDM2 may ubiquitin-mark p53 for nuclear export or degradation by the 26S proteasome. Alternatively or in addition, MDM2 may be recruited to promoter-associated p53 and p73 to directly inhibit gene transactivation by coactivator inhibition, corepressor recruitment and suppression of the basal transcription machinery 11-16. Here we present data suggesting that MDM2 can regulate VDR in a manner that is analogous in several respects to the regulation of p53.

2

Results and Discussion Since both p53 and VDR can transactivate the MDM2 gene

17, 18

and p53 is regulated by

MDM2 9, we asked whether VDR might also be controlled by this protein. In a first set of experiments, human H1299 lung adenocarcinoma cells (p53-deficient) were transiently transfected with combinations of plasmids producing Flag-tagged human VDR and MDM2. Cell extracts were prepared for immunoprecipitation and incubated with either monoclonal anti-Flag antibody or irrelevant monoclonal IgG. Precipitated proteins were analyzed by standard western immunoblotting. MDM2 coprecipitated specifically with Flag-VDR (Fig. 1A). Conversely, when the cell extracts were incubated with monoclonal anti-MDM2 Downloaded by [University of Leeds] at 18:37 17 May 2015

antibody 3G9 or IgG instead of anti-Flag antibody, Flag-VDR coprecipitated with MDM2 (Fig. 1B). Note that the levels of endogenous MDM2 present in H1299 cells sufficed to bring down some Flag-VDR in immunoprecipitations with anti-MDM2

antibody.

The

coprecipitation of Flag-VDR with MDM2 was not inhibited by the presence of the ligand 1,25D (Supplementary Figure S1).

To obtain information on the protein domains involved in the interaction, H1299 cells were transfected with combinations of plasmids expressing MDM2 and either HA-tagged fulllength VDR, HA-VDR-N (aa 1-119) containing the DNA binding domain, or HA-VDR-C (aa 120-427) harbouring the ligand binding domain. Immunoprecipitation with anti-MDM2 antibody efficiently coprecipitated HA-VDR full-length and HA-VDR-C whereas HA-VDRN coprecipitated only weakly, suggesting that MDM2 is primarily contacted via the center and C-terminus of VDR (Fig. 1C). Next, cells were transfected to produce Flag-VDR and either full-length MDM2 (491 aa) or the MDM2 fragments 2-222 (containing the p53 binding domain), 6-339 (p53 binding and acidic/zinc finger domains) or D222-325 (lacking the acidic and zinc finger domain). When MDM2 was precipitated with anti-MDM2 antibody 3G9, Flag-VDR coprecipitated weakly with the N-terminal half of MDM2 but coprecipitated strongly with MDM2 D222-325 lacking the acidic and zinc finger domain and containing the C-terminus with the RING domain (Fig. 1D). Thus, the N-terminus and central domain of MDM2 are mostly dispensable for the interaction with VDR. To examine whether the interaction also occurs between endogenous VDR and endogenous MDM2, extracts of human p53-deficient Caco-2 colon adenocarcinoma cells which express both proteins to higher levels than H1299 cells, were prepared and were again incubated with anti-MDM2 antibody or irrelevant IgG. As is shown in Figure 1E, VDR coprecipitated with MDM2. Combined, these 3

data suggest that MDM2 can associate with VDR and that the interaction, although perhaps involving several domains on both proteins, is primarily mediated through the C-terminal portions of MDM2 and VDR.

p53 and MDM2 are ubiquitin-marked for degradation through the 26S proteasome and are therefore typically present only at low levels in many cell types. VDR, too, is frequently present only at low levels in cells

19

. Moreover, we had noticed that overproduced MDM2

often decreased the level of Flag-VDR in cells (see Fig. 1A,B and Supplementary Figure S1). To examine whether the low steady-state levels of VDR in cells could in part be due to proteasomal degradation, the effects of inhibition of the proteasome by MG132 on the levels Downloaded by [University of Leeds] at 18:37 17 May 2015

of transfected Flag-VDR and endogenous VDR were studied. Western blot analysis of protein expression in H1299 cells showed that both endogenous MDM2 and ectopic Flag-VDR levels increased following treatment with MG132 (Fig. 2A). Proteasome inhibition also elevated the level of the endogenous VDR protein (Fig. 2B). We therefore next asked whether VDR may be a substrate of MDM2. First, H1299 cells were transfected with combinations of plasmids producing either Flag-p53 or Flag-VDR, and in addition, MDM2 and HA-tagged ubiquitin. Proteasomal degradation was temporarily inhibited in order to accumulate ubiquitin-marked proteins in the cell. Either Flag-p53 or Flag-VDR was immunoprecipitated from cell extracts that had been denatured to preclude detection of coprecipitating ubiquitylated proteins rather than HA-ubiquitin covalently linked to Flag-p53 or Flag-VDR. As expected, Flag-p53 was only weakly HA-ubiquitylated in the presence of the low levels of endogenous MDM2 but was strongly HA-ubiquitylated in the presence of ectopic MDM2, consistent with MDM2 being a main ubiquitin ligase for p53 in these cells (Fig. 2C, left panel). In contrast, FlagVDR was strongly HA-ubiquitylated regardless of MDM2 transfection (Fig. 2C, right panel). However, Flag-VDR ubiquitylation was consistently slightly increased under ectopic MDM2, suggesting that VDR is targeted by several ubiquitin ligases including MDM2. We reasoned that if VDR is indeed ubiquitin-marked in part by MDM2, knockdown of endogenous MDM2 by siRNA might increase the level of endogenous VDR. Figure 2D summarizes the results of two independent siRNA transfections. While equal low levels of both MDM2 and VDR were detectable in untransfected cultures and cultures transfected with control siRNA, knockdown of MDM2 resulted in an increase of VDR level. Combined these findings suggest that the cellular steady-state VDR level is controlled in part by MDM2 and that MDM2 is one among perhaps several ubiquitin ligases that regulate VDR.

4

MDM2 was known to inhibit p53 not only through ubiquitylation but also through the suppression of transcription upon being recruited to p53-responsive promoters 16. To begin to examine if MDM2 can act similarly towards VDR, H1299 cells expressing low levels of endogenous VDR (Fig. 3A, inset) were transiently transfected either with a control reporter plasmid harbouring a minimal promoter driving the luciferase gene, or with a derivative of the reporter plasmid which in addition carried three VDRE in front of the minimal promoter. In the presence of 1,25D, the VDRE reporter gave rise to about 16-times higher luciferase activity than the control (Fig. 3A, white columns). Strikingly, when we cotransfected relative amounts of MDM2 plasmid that were much smaller than those required to elicit detectable HA-ubiquitylation in the study summarized in Figure 2C, a significant decrease in luciferase Downloaded by [University of Leeds] at 18:37 17 May 2015

expression was observed, suggesting that MDM2 is a potent inhibitor of VDR-mediated transactivation (Figure 3A, black columns). In contrast, similar quantities of MDM2 plasmid failed to suppress luciferase expression from the basal reporter plasmid that lacked the VDRE (not shown), indicating that the inhibitory effect of MDM2 on the VDR-mediated transactivation was not simply caused by the suppression of the basal transcription machinery 16

. Cotransfection of a plasmid producing an MDM2 mutant that is proficient for VDR

binding but defective for ubiquitylation yielded identical results and suggested that the suppressive effect does not require MDM2’s enzymatic activity (Figure 3A, grey columns). Since these transactivation studies have been done in the presence of 1,25D to stimulate VDR-induced gene expression, these findings are also consistent with our IP-western blot analysis suggesting that the MDM2:VDR interaction is not disrupted by 1,25D (Supplementary Figure S1). CYP24A1 encodes the 24-hydroxylase that is required for the degradation of vitamin D metabolites. It is a classical VDR target gene that is, in dependence of ligand, directly and specifically transactivated by VDR

20

. We therefore asked whether MDM2 knockdown by

siRNA would have an effect on the transcription of this gene, measured by quantitative RTPCR. MDM2 was efficiently knocked down in H1299 cells (Fig. 3B, inset). In the absence of 1,25D this knockdown produced no significant effect on the expression of the CYP24A1 gene (Fig. 3B, upper diagram). In contrast, MDM2 knockdown induced significantly higher levels of CYP24A1 transcripts at 4h, 8h and 12h after 1,25D treatment (Fig. 3B, lower diagram; note the difference in y-axis scale between the upper and lower diagrams). To corroborate this finding, similar experiments were performed with Caco-2 cells, which like H1299 cells are p53-deficient. Again, MDM2 was efficiently knocked down (Fig. 3C, inset), and this 5

knockdown resulted in significantly increased levels of CYP24A1 transcript (Fig. 3C). In both cell types, the most distinctive effect of MDM2 knockdown on CYP24A1 gene transactivation was observed at early times (2-4h) of 1,25D treatment. p21 (CDKN1A), a gene encoding a major inhibitor of cell cycle-dependent kinases that causes cell cycle arrest, is also a wellknown target of transactivation by liganded VDR

21

. As with CYP24A1, knockdown of

MDM2 failed to alter the expression of the p21 gene in the absence of 1,25D. In its presence, by contrast, MDM2 knockdown resulted in a 1.8-fold increase in p21 transcript as early as 2 h after ligand exposure (Fig. 3D). Collectively, these findings suggest that MDM2 can inhibit the VDR-mediated transactivation of genes. However, despite substantial effort, we were as yet not able to detect MDM2 at VDR-regulated genes by chromatin immunoprecipitation. We Downloaded by [University of Leeds] at 18:37 17 May 2015

also examined the effect of MDM2 knockdown on the expression of SOSTDC1 (Wise), a gene involved in the mammalian hair cycle that is suppressed, not transactivated, by liganded VDR in Caco-2 cells 22. MDM2 knockdown had no effect on the suppression of SOSTDC1 by VDR (not shown).

1,25D-liganded VDR displays potent antiproliferative action in many tumor cell types, as do activated members of the p53 family, through the induction of cell cycle arrest, senescence, differentiation and apoptosis 23. It is therefore perhaps no surprise to find crosstalk between these tumor suppressors (Fig. 4 and transactivate the VDR gene

25, 26

24

). p63 and p73, and less efficiently, p53 itself, can

. VDR, in turn, regulates several genes that are also targeted

by the p53 family, including p21 (CDKN1A), Bax, Bcl-2 and MDM2

18, 21, 23

. Transactivation

of the MDM2 gene by p53 and the subsequent repression of p53 by MDM2 establish a negative feedback loop

17

. The findings presented here suggest that VDR and MDM2 may

engage in negative feedback regulation as well (Fig. 4), at least in tissues in which the MDM2 gene is transactivated by VDR 18. Tumors frequently evolve to either loose or mechanistically inactivate their tumor suppressors. In the case of VDR, for example, functional incapacitation through the overexpression of CYP24A1 that breaks down 1,25D has been reported 27. In the light of the findings presented here, MDM2 overproduction may thus be another means by which VDR can be blunted in tumors. MDM2 overproduction is frequently observed, for instance, in sarcomas 28, leukemias and lymphomas 29, 30, breast cancers 31 and gliomas 32. Of interest in this context may also be the finding that 14-24 % of Caucasians are homozygous for a MDM2 promoter polymorphism, SNP309 (rs2279744), that causes elevated levels of MDM2 in response to stress or estrogen 33. Homozygosity appears to constitute a risk factor for cancer in humans and mice, presumably due to lower functional p53 levels 33, 34. It will be 6

interesting to examine whether VDR function is also compromised in humans and mice carrying SNP309. Materials and Methods Cell culture and transfection H1299 cells were maintained at 37°C, 5% CO2 in DMEM containing 10% FCS and antibiotics; Caco-2 cells were maintained under similar conditions but in MEM supplemented with 1% non-essential amino acids and 10% FCS. For transient transfection of DNA into cells, these were seeded into dishes to reach 60-70% confluency at the time of transfection. Downloaded by [University of Leeds] at 18:37 17 May 2015

DNA was transfected with jetPEI (Polyplus, Illkirch, France), according to the supplier’s instructions. siRNAs were transfected, at a final concentration of 5 nM, with HiPerFect transfection reagent (Qiagen), as specified by the manufacturer. The following siRNA were employed: control: MDM2: VDR- 8: VDR-9:

UUCUCCGAACGUGUCACGUTT

(sense)

ACGUGACACGUUCGGAGAATT

(antisense)

UCAUCGGACUCAGGUACAUTT

(sense)

AUGUACCUGAGUCCGAUGATT

(antisense)

CCACUGAUUUGGAGAUAUUTT

(sense)

AAUAUCUCCAAAUCAGUGGTA

(antisense)

AGACUCCAUUUGUAUUAUATT

(sense)

UAUAAUACAAAUGGAGUCUGA

(antisense)

Plasmids, reagents and antibodies Details on the construction and maps of pCMV-Flag-VDR expressing human Flag-tagged VDR, pCMV-HA-VDR-fl, pCMV-HA-VDR-N, pCMV-HA-VDR-C, pcmdm2 expressing human MDM2, pCMV-MDM2-2-222, pCMV-MDM2-6-339, pCMV-MDM2-D222-325, pCMV-Flag-p53 producing human Flag-tagged p53, and pcDNA3-HA-ubiquitin (Addgene) will be provided upon request. The VDRE reporter and control plasmids pDR3-luc and pCluc were purchased from Agilent Technologies, Santa Clara, CA, USA. MG132 was from Sigma, as were the b-actin monoclonal antibody AC-15, anti-Flag antibody M2 and the peroxidase-conjugated secondary anti-mouse antibody. The monoclonal anti-MDM2 antibody 3G9 was from Millipore. The irrelevant monoclonal anti-HRS3 IgG was kindly provided by 7

Michael Pfreundschuh (Internal Medicine I, Homburg, Germany). The monoclonal anti-HA antibody was from Covance, and the monoclonal anti-p53 antibody DO-1 was purchased from Santa Cruz Biotechnology. Anti-VDR antibody 9A7 was purchased from Abcam. The siRNAs were from Qiagen. The active vitamin D metabolite 1,25D was purchased from Sigma, dissolved at 1 mM in ethanol, and stored at -20°C. Immunoprecipitation and western blot analysis Coimmunoprecipitations were performed according to our recently published, detailed protocol

35

. For the study of in vivo ubiquitylation of Flag-p53 and Flag-VDR, the proteins

were immunoprecipitated from denatured cell extracts

35

. In brief, H1299 cells were

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transfected with the relevant plasmids as indicated in the figure legends. After 24 h, cells were treated with 10 µM MG132 for another 4 h. Cells were then washed in cold PBS; 1/10 of the cells was saved as input control. The rest was lysed in 400 µl TBS-lysis buffer (1 % SDS in TBS) per 10 cm-dish at 95°C for 5 min. Lysates were squeezed repeatedly through a 23 Gauge needle and vortexed vigorously for 10 seconds. 800 µl TBS-Triton buffer (1.5 % Triton X-100 in TBS) per 400 µl lysate was added and mixed prior to incubation with 100 µl of a 1:1 mix of protein G and protein A sepharose 4 Fast Flow (GE Healthcare), for 1 h on a rotating wheel at 4°C (pre-clearing). Samples were centrifuged for 5 min at max speed and supernatant was incubated with 100 µl of a 1:1 mix of protein G and protein A sepharose 4 Fast Flow pre-conjugated with 4 µg of the indicated antibody, for at least 4 h at 4°C on a rotating wheel. Samples were washed three times in 1 ml cold TBS mix (1 part TBS-lysis buffer plus 2 parts TBS-Triton buffer); beads were resuspended in 30 µl of 95°C SDS-sample buffer (100 mM Tris-HCl (pH 6.8), 100 mM DTT, 4 % SDS, and 20 % glycerol) and were boiled for 10 min. The proteins were separated by SDS-PAGE, immobilized on PVDF membrane (Immobilon P, Millipore) and detected by the indicated antibodies 35. For standard western blot analysis, cells were lysed in SDS lysis buffer (100 mM Tris-HCl pH 6.8, 4% SDS and 20% glycerol) at 100°C. Fifteen microgram of total cellular protein was run on an 8% SDS polyacrylamide gel and then transferred to a PVDF membrane (Immobilon-P, Millipore). The membrane was submersed in PBS supplemented with 5% (w/w) dry milk powder, and was incubated overnight with antibodies as indicated in the figure legends. The bound primary antibodies were detected by incubating the membranes for 1 h with a peroxidase-conjugated secondary anti-mouse antibody. Signals were detected by the Thermo Scientific ECL western blotting substrate, as recommended by the manufacturer.

8

Reporter gene assay H1299 cells were seeded into 24-well dishes at approximately 105 cells per dish. After 24 h, cells were transfected with jetPEI (Polyplus, Illkirch, France), as recommended by the manufacturer. At the time points indicated in the figure legends, cells were harvested, cell extracts were prepared and luciferase activity was measured in a luminometer with the Luciferase Assay System (Promega), as specified by the supplier. Quantitative Reverse Transcription-PCR Total cellular RNA was isolated with the RNeasy Mini Kit and QIAshredder from Qiagen as specified by the supplier. The RNA was digested with RQ1 RNase-Free DNase (Promega) as Downloaded by [University of Leeds] at 18:37 17 May 2015

additional step during RNA isolation with the RNeasy Mini Kit. 1 µg RNA was used for the first-strand cDNA synthesis with Omniscript RT Kit (Qiagen) and with random primers (Promega), as recommended by the manufacturer. 10 units per 20 µl volume of Recombinant RNasin® ribonuclease inhibitor (Promega) were added during cDNA synthesis to stabilize RNA. Quantitative RT-PCR analyses for CYP24A1, p21 and 18S rRNA were performed with QuantiTect SYBR® Green PCR Kit from Qiagen. The conditions were: CYP24A1 primers (from Qiagen, Cat.No. QT00015428); TA: 60°C; final concentration of primers and final MgCl2 concentration as specified by the supplier; p21 primers (see below); TA: 60°C; final concentration of primers: 0.5 μM; final MgCl2 concentration: 2 mM; 18S rRNA primers (from Qiagen, Cat.No. QT00199367); TA: 60°C; final concentration of primers and final MgCl2 concentration as specified by the supplier. qPCR analyses were performed on the Applied Biosystems® StepOnePlus™ Real-Time PCR System. p21:

GGCGGCAGACCAGCATGACAGATT

(forward)

ATGAAGCCGGCCCACCCAACCTC

(reverse)

Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Acknowledgements We thank Michael Pfreundschuh (Internal Medicine I, University Medical Center, Homburg, Germany) for materials. We are also grateful to Ms. Heike Palm for expert technical assistance.

9

Funding This work was supported by a University of Saarland grant to KR and by the Cancer Research Saar-Pfalz-Mosel Society. Author Contributions KH, T-CH and BB performed all lab experiments. JR helped with the conception of the work.

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KR conceived and supervised the study, and wrote the paper.

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19. Wang Y, Zhu J, DeLuca HF. Where is the vitamin D receptor? Arch Biochem Biophys 2012; 523:123-33. 20. Zou A, Elgort MG, Allegretto EA. Retinoid X receptor (RXR) ligands activate the human 25-hydroxyvitamin D3-24-hydroxylase promoter via RXR heterodimer binding to two vitamin D-responsive elements and elicit additive effects with 1,25-dihydroxyvitamin D3. J Biol Chem 1997; 272:19027-34. 21. Liu M, Lee MH, Cohen M, Bommakanti M, Freedman LP. Transcriptional activation of the Cdk inhibitor p21 by vitamin D3 leads to the induced differentiation of the myelomonocytic cell line U937. Genes Dev 1996; 10:142-53. 22. Haussler MR, Haussler CA, Bartik L, Whitfield GK, Hsieh JC, Slater S, et al. Vitamin D receptor: molecular signaling and actions of nutritional ligands in disease prevention. Nutr Rev 2008; 66:S98-112. 23. Feldman D, Krishnan AV, Swami S, Giovannucci E, Feldman BJ. The role of vitamin D in reducing cancer risk and progression. Nat Rev Cancer 2014; 14:342-57. 24. Reichrath J, Reichrath S, Heyne K, Vogt T, Roemer K. Tumor suppression in skin and other tissues via cross-talk between vitamin D- and p53-signaling. Front Physiol 2014; 5:166. 25. Kommagani R, Payal V, Kadakia MP. Differential regulation of vitamin D receptor (VDR) by the p53 Family: p73-dependent induction of VDR upon DNA damage. J Biol Chem 2007; 282:29847-54. 26. Maruyama R, Aoki F, Toyota M, Sasaki Y, Akashi H, Mita H, et al. Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation. Cancer Res 2006; 66:4574-83. 27. Skowronski RJ, Peehl DM, Feldman D. Vitamin D and prostate cancer: 1,25 dihydroxyvitamin D3 receptors and actions in human prostate cancer cell lines. Endocrinology 1993; 132:1952-60. 28. Oliner JD, Kinzler KW, Meltzer PS, George DL, Vogelstein B. Amplification of a gene encoding a p53-associated protein in human sarcomas. Nature 1992; 358:80-3. 29. Bueso-Ramos CE, Yang Y, deLeon E, McCown P, Stass SA, Albitar M. The human MDM-2 oncogene is overexpressed in leukemias. Blood 1993; 82:2617-23. 30. Watanabe T, Hotta T, Ichikawa A, Kinoshita T, Nagai H, Uchida T, et al. The MDM2 oncogene overexpression in chronic lymphocytic leukemia and low-grade lymphoma of Bcell origin. Blood 1994; 84:3158-65. 31. Sheikh MS, Shao ZM, Hussain A, Fontana JA. The p53-binding protein MDM2 gene is differentially expressed in human breast carcinoma. Cancer Res 1993; 53:3226-8. 32. Reifenberger G, Liu L, Ichimura K, Schmidt EE, Collins VP. Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations. Cancer Res 1993; 53:2736-9. 33. Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC, et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell 2004; 119:591-602. 34. Post SM, Quintas-Cardama A, Pant V, Iwakuma T, Hamir A, Jackson JG, et al. A high-frequency regulatory polymorphism in the p53 pathway accelerates tumor development. Cancer Cell 2010; 18:220-30. 35. Heyne K, Forster J, Schule R, Roemer K. Transcriptional repressor NIR interacts with the p53-inhibiting ubiquitin ligase MDM2. Nucleic Acids Res 2014; 42:3565-79.

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Figure 1. MDM2 binds to VDR in vivo. (A) Coimmunoprecipitation of MDM2 with Flag-VDR. H1299 cells were transfected with the indicated combinations of plasmids producing FlagVDR (4 mg) and MDM2 (4 mg). At 24 h after transfection, cells were treated with MG132 for another 4h. Cell extracts were prepared for standard coimmunoprecipitations and were incubated with anti-Flag antibody M2 (2 mg/IP) or irrelevant IgG (anti-HRS3, kindly provided by M. Pfreundschuh, Homburg, Germany). Immunoprecipitates (IP), coimmunoprecipitates (Co-IP) and the proteins in the total cell lysates (TCL) were analyzed by western blotting using the anti-Flag antibody M2 (1:10,000), monoclonal anti-MDM2 antibody 3G9 (1:2,000) or anti-b-actin antibody (1:10,000). (B) Coimmunoprecipitation of Flag-VDR with MDM2. Transfections, treatments and immunoprecipitations/western blot analyses were performed as specified in (A). (C) Coimmunoprecipitation of HA-tagged VDR fragments with MDM2. H1299 cells were transfected with combinations of plasmids expressing MDM2 (4 mg), HAVDR-fl (full-length; 4 mg), HA-VDR-N (aa 1-119; 4 mg) or HA-VDR-C (aa 120-427; 4 mg). After 24 h, the cultures were incubated with MG132 (10 mM) for another 4h. Standard coimmunoprecipitations with monoclonal anti-HA antibody (2 mg/IP) or irrelevant IgG were carried out as in (A). (D) Coimmunoprecipitation of Flag-VDR with fragments of MDM2. Cells were

transfected

and

treated

as

indicated

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and

specified

in

(C).

MDM2

was

immunoprecipitated anti-MDM2 antibody 3G9 that recognizes an epitope between aa 6 and 222. MDM2-fl, 2-222, 6-339 and D222-325 denote full-length MDM2 and the respective MDM2 fragments. Note that fragment 2-222, although expressed at low levels in the total cell lysate (TCL), was readily immunoprecipitable with 3G9. (E) Coimmunoprecipitation of endogenous VDR with endogenous MDM2. Caco-2 cells were treated with MG132 for 3 h in order to accumulate both proteins, and simultaneously, were either mock-treated or treated with active vitamin D ligand (1,25D; 100 nM). Cellular extracts were incubated with antiMDM2 antibody 3G9 (2 mg/IP) or irrelevant IgG, and the precipitating/coprecipitating proteins

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were analyzed by western blotting, as before.

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Figure 2. MDM2 affects steady-state level of VDR in cells. (A) Flag-VDR accumulates in response to proteasome inhibition. H1299 cells were treated with MG132 (10 mM). After the indicated times, total cell extracts were prepared and protein expression was analyzed by standard western blotting. Flag-VDR was detected with anti-Flag antibody M2 (1:10,000), MDM2 with anti-MDM2 antibody 3G9 (1:2,000) and b-actin with anti-b-actin antibody (1:10,000). (B) Levels of endogenous VDR increase under proteasome inhibition. H1299 cells were exposed to MG132 as before, and cell extracts were prepared after the indicated times of exposure. Western blot analysis was performed as in (A). VDR was detected with monoclonal anti-VDR antibody 9A7 (1:2000). unspec. = unspecific signal. (C) Flag-VDR is ubiquitylated in H1299 cells. H1299 cells were transfected for 24 h with the indicated combinations of plasmids to produce Flag-VDR (4 mg), Flag-p53 (0.5 mg), MDM2 (3 mg) and HA-ubiquitin (3 mg), and were then treated with MG132 (10 mM) for another 5 h. Flag-p53 (left panel) or Flag-VDR (right panel) were immunoprecipitated from denatured cell extracts with anti-Flag antibody M2 (2 mg/IP). Western blotting with anti-HA antibody (1:2000), anti-Flag antibody M2, anti-MDM2 antibody 3G9, and anti-b-actin antibody, was carried out as specified above. (D) Knockdown of MDM2 increases level of endogenous VDR. H1299 cells

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were mock-transfected (-) or transfected with scrambled control siRNA (C; 5 nM) or MDM2 siRNA (5 nM). At 24 h after transfection, total protein extracts were prepared and subjected

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to western blot analysis with the indicated antibodies as detailed in (A).

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Figure 3. MDM2 inhibits VDR-mediated gene transactivation. (A) Inhibition of VDR-induced reporter gene transactivation by MDM2. H1299 cells were transfected with 0.3 mg of either control plasmid harbouring a minimal promoter in front of the luciferase gene (pC-luc), or plasmid that in addition carries three copies of a VDR response element upstream of the minimal promoter (pDR3-luc). In addition, the cells received increasing amounts (0.01, 0.02, 0.04, 0.08 mg) of either empty control vector or vector producing MDM2 or the MDM2 RING domain mutant G448S/C449A (MDM2*) that is defective for E3 ligase activity. At 24 h after transfection, 1,25D (100 nM) was added for another 10 h. Luciferase assays were performed with the Luciferase Assay System. T-bars denote standard deviations from three experiments; P-values were calculated with student’s t-test (two-sided). Inset: Exponentially growing H1299 cells were transfected with either scrambled control siRNA (C) or VDR siRNA (5 nM) for 24 h. Standard western blot analysis was employed to detect endogenous VDR with the anti-VDR antibody 9A7 (1:2,000). unspec. = unspecific signals. (B) Knockdown of MDM2 increases transcript levels from the VDR-driven CYP24A1 gene in the presence of vitamin D. Exponentially growing H1299 cells were mock-transfected (grey bar; C) or transfected with either control siRNA (5 nM) or MDM2 siRNA (5 nM) for 24 h, and were then treated or not treated with 1,25D (100 nM). Inset: Knockdown of MDM2 was verified by

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western blot analysis with anti-MDM2 antibody 3G9 (1:2,000). Total RNA was prepared at the indicated time points after transfection/ligand treatment and the expression of CYP24A1 transcript relative to 18S rRNA transcript was determined in quantitative RT-PCRs on a StepOnePlus (Applied Biosystems). exp.1, exp.2; experiment 1, experiment 2. T-bars show standard deviations from three measurements. P-values were determined with student’s ttest (two-sided). (C) Knockdown of MDM2 increases the transcript levels from the VDRdriven CYP24A1 gene in a different cell type. Experiments and statistics were performed as specified in (B), except that the target cells were Caco-2 instead of H1299. (D) MDM2 knockdown increases the transcript levels from the p21 (CDKN1A) gene in Caco-2 cells. Experiments and statistics were performed as reported in (B). The diagram shows the result of a 2 h exposure of cells with 1,25D (100 nM). Similar results were obtained with H1299 Downloaded by [University of Leeds] at 18:37 17 May 2015

cells.

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Figure 4. Flow diagram depicting some of the relations between the p53 family proteins and VDR. Arrows denote activating, T-lines suppressing actions. Italic letters indicate genes; upright letters proteins. Dotted lines mark relatively weak interactions.

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MDM2 binds and inhibits vitamin D receptor.

The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Her...
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