Differential Regulation of Vitamin D Receptor (VDR) by the p53 Family

Abstract

p63 and p73, members of the p53 family, have been shown to be functionally distinct from p53. Vitamin D receptor (VDR) is a ligand (vitamin D3)-dependent transcription factor, which is shown to play a major role in calcium homeostasis and keratinocyte differentiation. Vitamin D and its analogues in combination with DNA-damaging agents are extensively used for cancer chemotherapy. In this report, we examined whether p53 affects p63-mediated induction of VDR and studied the effect of DNA damage on VDR induction in p53 null cell lines. Our results demonstrate that p53 itself does not induce VDR expression, nor does it affect p63-mediated VDR induction in the cell lines tested in this study. Furthermore, we observed p53-independent activation of VDR upon DNA damage and associated the induction of VDR to p73. We have demonstrated that ectopic expression of various p73 isoforms can induce VDR expression. Inhibition of p73 in cells treated with DNA-damaging agents exhibited decreased VDR expression. Finally, we show that upon DNA damage, induction of VDR sensitizes the cells to vitamin D treatment. In conclusion, our results indicate that VDR is regulated by p63 and p73 and that the induction of VDR expression upon DNA damage is p73-dependent. p63 and p73, members of the p53 family, have been shown to be functionally distinct from p53. Vitamin D receptor (VDR) is a ligand (vitamin D3)-dependent transcription factor, which is shown to play a major role in calcium homeostasis and keratinocyte differentiation. Vitamin D and its analogues in combination with DNA-damaging agents are extensively used for cancer chemotherapy. In this report, we examined whether p53 affects p63-mediated induction of VDR and studied the effect of DNA damage on VDR induction in p53 null cell lines. Our results demonstrate that p53 itself does not induce VDR expression, nor does it affect p63-mediated VDR induction in the cell lines tested in this study. Furthermore, we observed p53-independent activation of VDR upon DNA damage and associated the induction of VDR to p73. We have demonstrated that ectopic expression of various p73 isoforms can induce VDR expression. Inhibition of p73 in cells treated with DNA-damaging agents exhibited decreased VDR expression. Finally, we show that upon DNA damage, induction of VDR sensitizes the cells to vitamin D treatment. In conclusion, our results indicate that VDR is regulated by p63 and p73 and that the induction of VDR expression upon DNA damage is p73-dependent. p53, the most frequently altered gene in human cancers, controls multiple signaling pathways by regulating the genes involved in cell cycle arrest, apoptosis, DNA repair, cellular senescence, and inhibition of angiogenesis (1Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1176) Google Scholar). Stabilization of p53 protein occurs in response to various stress stimuli including DNA damage, viral infection, or oncogenic activation (1Giaccia A.J. Kastan M.B. Genes Dev. 1998; 12: 2973-2983Crossref PubMed Scopus (1176) Google Scholar, 2Schuler M. Green D.R. Biochem. Soc. Trans. 2001; 29: 684-688Crossref PubMed Google Scholar). Although the functional significance of p53 and its interacting proteins have been studied extensively, it was not until 1997 that the functional homologues of p53, namely p63 and p73, were discovered. Despite being structurally similar to p53, p63 and p73 were shown to be functionally more diverse and distinct from p53 (3Yang A. Kaghad M. Wang Y. Gillett E. Fleming M.D. Dotsch V. Andrews N.C. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Abstract Full Text Full Text PDF PubMed Scopus (1840) Google Scholar, 4Kaghad M. Bonnet H. Yang A. Creancier L. Biscan J.C. Valent A. Minty A. Chalon P. Lelias J.M. Dumont X. Ferrara P. McKeon F. Caput D. Cell. 1997; 90: 809-819Abstract Full Text Full Text PDF PubMed Scopus (1536) Google Scholar). p63 knock-out mice were born with severe developmental defects, which included lack of skin and various epithelial tissues, whereas p73 knock-out mice were born with neuronal defects as well as changes in sexual behavior (5Mills A.A. Zheng B. Wang X.J. Vogel H. Roop D.R. Bradley A. Nature. 1999; 398: 708-713Crossref PubMed Scopus (1701) Google Scholar, 6Yang A. Walker N. Bronson R. Kaghad M. Oosterwegel M. Bonnin J. Vagner C. Bonnet H. Dikkes P. Sharpe A. McKeon F. Caput D. Nature. 2000; 404: 99-103Crossref PubMed Scopus (883) Google Scholar). Functional diversities associated with p63 and p73 are partly due to their ability to generate multiple transcripts. Differential promoter usage by both p63 and p73 results in the generation of either transactivation domain containing isoforms (TAp63 and TAp73) or NH2-terminally truncated isoforms (ΔNp63 and ΔNp73) (7Scoumanne A. Harms K.L. Chen X. Cancer Biol. Ther. 2005; 4: 1178-1185Crossref PubMed Scopus (48) Google Scholar). Additionally, multiple carboxyl termini variants, α, β, and γ of p63 and α, β, γ, ϵ, and δ of p73, are generated due to the differential splicing of COOH terminus. Due to their structural similarity with p53, TA isoforms of both p63 and p73 have been shown to activate various p53-responsive genes and promote cell cycle arrest and apoptosis (8Dietz S. Rother K. Bamberger C. Schmale H. Mossner J. Engeland K. FEBS Lett. 2002; 525: 93-99Crossref PubMed Scopus (59) Google Scholar, 9Harms K. Nozell S. Chen X. Cell Mol. Life Sci. 2004; 61: 822-842Crossref PubMed Scopus (255) Google Scholar). On the contrary, elevated levels of ΔNp63 and ΔNp73 isoforms have been reported in several human cancers, and these isoforms have also been associated with the induction of various genes involved in promoting cell proliferation (10Kang K.H. Lee J.H. Kim K.C. Ham S.W. Kim M.Y. Choi K.H. FEBS Lett. 2002; 522: 161-167Crossref PubMed Scopus (16) Google Scholar, 11Wu G. Osada M. Guo Z. Fomenkov A. Begum S. Zhao M. Upadhyay S. Xing M. Wu F. Moon C. Westra W.H. Koch W.M. Mantovani R. Califano J.A. Ratovitski E. Sidransky D. Trink B. Cancer Res. 2005; 65: 758-766PubMed Google Scholar, 12Senoo M. Matsumura Y. Habu S. Oncogene. 2002; 21: 2455-2465Crossref PubMed Scopus (67) Google Scholar, 13Sniezek J.C. Matheny K.E. Westfall M.D. Pietenpol J.A. Laryngoscope. 2004; 114: 2063-2072Crossref PubMed Scopus (103) Google Scholar, 14Casciano I. Mazzocco K. Boni L. Pagnan G. Banelli B. Allemanni G. Ponzoni M. Tonini G.P. Romani M. Cell Death Differ. 2002; 9: 246-251Crossref PubMed Scopus (185) Google Scholar). Additionally, ΔN isoforms of p63 and p73 have also been shown to act in a dominant negative manner and suppress the TAp63 and TAp73 isoforms and p53-mediated antiproliferative functions (15Westfall M.D. Mays D.J. Sniezek J.C. Pietenpol J.A. Mol. Cell Biol. 2003; 23: 2264-2276Crossref PubMed Scopus (289) Google Scholar). Several reports have implicated that, upon DNA damage, both TAp63 and TAp73 isoforms can lead to cell cycle arrest and apoptosis (16Moll U.M. Slade N. Mol. Cancer Res. 2004; 2: 371-386PubMed Google Scholar). DNA damage-induced expression of TAp63α promotes apoptosis in hepatocarcinoma cell lines (17Gressner O. Schilling T. Lorenz K. Schulze Schleithoff E. Koch A. Schulze-Bergkamen H. Maria Lena A. Candi E. Terrinoni A. Valeria Catani M. Oren M. Melino G. Krammer P.H. Stremmel W. Muller M. EMBO J. 2005; 24: 2458-2471Crossref PubMed Scopus (233) Google Scholar). TAp73 variants were also shown to be induced by a subset of DNA-damaging agents in various cancer cell lines (18Ozaki T. Nakagawara A. Cancer Sci. 2005; 96: 729-737Crossref PubMed Scopus (114) Google Scholar). In addition, TAp73α and TAp73β play a pivotal role in p53-independent chemosensitivity (19Irwin M.S. Kondo K. Marin M.C. Cheng L.S. Hahn W.C. Kaelin Jr., W.G. Cancer Cell. 2003; 3: 403-410Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). In particular, transcriptional activity of p73α is required for E2F1-mediated apoptosis of T-cells (20Lissy N.A. Davis P.K. Irwin M. Kaelin W.G. Dowdy S.F. Nature. 2000; 407: 642-645Crossref PubMed Scopus (288) Google Scholar). Although the precise role of p63 and p73 in p53-dependent apoptosis is still unclear, a significant down-regulation of p53-mediated apoptosis upon γ-irradiation in p63- and p73-ablated mouse embryo fibroblasts suggests that both p63 and p73 are required for the biological activity of p53 (21Flores E.R. Tsai K.Y. Crowley D. Sengupta S. Yang A. McKeon F. Jacks T. Nature. 2002; 416: 560-564Crossref PubMed Scopus (723) Google Scholar). 1α,25-Dihydroxyvitamin D3, the active form of secosteroid hormone vitamin D, exerts its biological actions through vitamin D receptor (VDR). 2The abbreviations used are: VDR, vitamin D receptor; VD3, 1α,25-dihydroxyvitamin D3; CYP27B1, 25-hydroxyvitamin D3 1α-hydroxylase; VDRE-Luc, vitamin D-responsive elements upstream of luciferase; shVDR, short hairpin VDR; siRNA, small interfering RNA; BES, 2-[bis(2-hydroxyethyl) amino]ethanesulfonic acid; PLB, passive lysis buffer; shNCS, short hairpin nonsilencing control. 2The abbreviations used are: VDR, vitamin D receptor; VD3, 1α,25-dihydroxyvitamin D3; CYP27B1, 25-hydroxyvitamin D3 1α-hydroxylase; VDRE-Luc, vitamin D-responsive elements upstream of luciferase; shVDR, short hairpin VDR; siRNA, small interfering RNA; BES, 2-[bis(2-hydroxyethyl) amino]ethanesulfonic acid; PLB, passive lysis buffer; shNCS, short hairpin nonsilencing control. VDR is a ligand-dependent transcription factor, which belongs to the nuclear receptor family of proteins (22Nezbedova P. Brtko J. Endocr. Regul. 2004; 38: 29-38PubMed Google Scholar). Activation of VDR by the active form of vitamin D results in heterodimerization of VDR with the retinoic acid receptor and subsequent transcriptional regulation of its responsive genes (22Nezbedova P. Brtko J. Endocr. Regul. 2004; 38: 29-38PubMed Google Scholar). Vitamin D receptor primarily regulates the genes involved in calcium homeostasis and bone formation (23Demay M.B. Ann. N. Y. Acad. Sci. 2006; 1068: 204-213Crossref PubMed Scopus (91) Google Scholar). Apart from exerting its classical functions, vitamin D receptor has been shown to exert antiproliferative actions in several different cancer cell lines (24Campbell M.J. Gombart A.F. Kwok S.H. Park S. Koeffler H.P. Oncogene. 2000; 19: 5091-5097Crossref PubMed Scopus (95) Google Scholar, 25Banerjee P. Chatterjee M. Mol. Cell. Biochem. 2003; 253: 247-254Crossref PubMed Scopus (133) Google Scholar). Recently, VDR has been implicated to play a broader role in the differentiation of epithelial keratinocytes and immunomodulatory activities (26Hawker N.P. Pennypacker S.D. Chang S.M. Bikle D.D. J. Invest Dermatol. 2007; 127: 874-880Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 27Gombart A.F. Luong Q.T. Koeffler H.P. Anticancer Res. 2006; 26: 2531-2542PubMed Google Scholar). Although extensive work has been performed on the biological function of vitamin D and its analogs, little is known about the transcriptional regulation of VDR. In this report, we studied the transcriptional regulation of VDR by p53 family members in both stressed and unstressed conditions. Our results demonstrate the differential regulation of VDR by p63 and p73 as well as the p53-independent activation of VDR upon DNA damage. Examination of the mechanism of VDR induction upon DNA damage demonstrated the requirement of p73 for DNA damage-induced expression of VDR. Cell Lines and Plasmids—Human non-small lung carcinoma cell line H1299 and human osteosarcoma cell line SaoS2 were obtained from ATCC and were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine calf serum, 250 units of penicillin, and 250 μg of streptomycin. Expression plasmids encoding TAp63γ, and TAp73α were in as R. Z. D.J. Mol. Cancer Res. 2006; 4: PubMed Scopus Google Scholar). VDR and promoter were as R. Oncogene. 2006; PubMed Scopus Google Scholar). p53 expression was a from was a from of and 1α,25-dihydroxyvitamin D3 were from VDR short hairpin expression was obtained from and the to VDR was the in the obtained from used for was as were to the of were performed with the plasmids in Dulbecco's modified Eagle's of the a of μg of expression encoding TAp63γ, p53, and were p53 and μg of p53 was a the medium was with Dulbecco's modified Eagle's medium with 10% fetal bovine calf and p73 by were in to the of cells were to calcium medium was and the cells were in with was with and BES, was for and to the negative nonsilencing was medium was and cells were to with of was performed as was the as the used for p73 were as μg of was used to by the transcription was performed in a on and on for VDR p73 and Life was performed by the as R. Z. D.J. Mol. Cancer Res. 2006; 4: PubMed Scopus Google Scholar, R. Oncogene. 2006; PubMed Scopus Google Scholar). and or protein was from cells were on 10% and and with were with to and were used to VDR, p53, and expression, was used to p53. were used as and the was used to the for of and cells were for protein and of protein was used for protein was with of for were and protein was with with μg of were with for by with to the with were on 10% and with the and cells were in cells were with of with the plasmids as of cells were with and in passive lysis were performed to both and activity the as the activity was by the of activity to the vitamin transcriptional activity of VDR, H1299 cells were in and with of with the plasmids as cells were treated with for and medium was with medium containing 1α,25-dihydroxyvitamin D3 of cells were to a as was performed by a Cell to H1299 cells were and cells were on a by for cells were and were to to the was protein for was to with or of the that was not to was used as both DNA and were and DNA was DNA was to Green as the for VDR and used for both VDR and were as of were of and p53 VDR Expression or p63-mediated of has demonstrated that p63 not p53 VDR expression R. Oncogene. 2006; PubMed Scopus Google Scholar). a has demonstrated that p53 can induce VDR expression R. F. M. Y. H. H. H. K. M. Y. H. K. Y. T. Cancer Res. 2006; PubMed Scopus Google Scholar). this we to the role of p53 family members in VDR the significant p53 and TAp63 we whether p53 effect on p63-mediated VDR we p53 and p63 or in H1299 and cell lines We demonstrated that by as VDR expression, of p53 with p63 not affect the ability of p63 to induce VDR expression and of was used as a for the transactivation of both p53 and results were obtained we tested the ability of p63 or with p53 to the activity of either the VDR or the VDR shown to be by p63 R. Oncogene. 2006; PubMed Scopus Google Scholar). Although p63 the activity of both of these p53 not affect p63-mediated transactivation of either of these VDR with our report, p53 was to induce expression of VDR or affect the VDR activity in both of the cell lines tested In to whether the effect of p53 on VDR was we tested the effect of of p53 on VDR expression We that p53 tested not in induction of VDR either the or protein in both of the cell lines tested and a of p53, was induced by p53 and as a control. In addition, p53 tested also not VDR or the VDR activity does not induce VDR expression. H1299 and cells were with either or of expression plasmids encoding p53 or p63 as was and to in VDR and levels to cell were to and H1299 and cells were with either VDR promoter or the VDR promoter with of expression plasmids encoding p53 or p63 as cells were in and to as the were to for to DNA Expression of VDR of p53 and with p73 Expression whether p53 is required for DNA damage-induced VDR expression, as demonstrated we examined the of DNA-damaging and on p53 null H1299 and SaoS2 DNA-damaging agents to induction of VDR with that DNA damage can lead to the transcriptional activation of VDR whether in VDR upon DNA damage to a in the transcriptional activity of VDR, we examined the levels of 25-hydroxyvitamin D3 a of VDR. in levels was also observed in the and cells in both the H1299 and SaoS2 cell lines reports have demonstrated that both of these cell lines have p73, and p73 is induced upon DNA damage in various cancer cell lines M. T. C. Genes Dev. 2004; PubMed Scopus Google Scholar, X. Zheng Y. J. J. Wang J. Oncogene. 2001; PubMed Scopus Google Scholar, A. P. N. M. V. G. M. L. G. C. M. Mol. Cell. 2002; 9: Full Text Full Text PDF PubMed Scopus Google we was a p73 and VDR expression we treated both H1299 and SaoS2 cell lines with or and levels of both VDR and p73 We demonstrated that both p73 and VDR are induced upon DNA damage. with the in the levels of both VDR and p73 upon DNA damage, we also observed in the protein levels of both VDR and p73 upon with DNA-damaging agents results were obtained in p53 null cell line not In to whether p73 the VDR induction upon DNA damage, we the p73 and VDR levels different the treatment. with a report, we observed a significant in p73 levels the DNA damage M. T. C. Genes Dev. 2004; PubMed Scopus Google Scholar). induction in VDR levels was observed the these results that VDR induction upon DNA damage p73. p73 for DNA Expression of whether p73 is required for the induction of VDR upon DNA damage, we tested whether VDR is by the p73 isoforms in a p53 null expression of TAp73β and TAp73α in H1299 cells to a significant in VDR and expression both and protein In to a in both VDR and expression levels with the TAp73 was used as a for both VDR and induction and We tested the ability of different p73 isoforms to the activity of the VDR shown to be by in VDR activity was observed with both TAp73β and TAp73α isoforms of p73 expression in cells to a in the levels of both VDR and CYP27B1, a of VDR Finally, with H1299 cells demonstrated that p73 protein to the VDR promoter of p73 to the a known of p73, was used as a control. these results demonstrate that VDR is by multiple p73 isoforms and that VDR is a of p73. demonstrate that the transcriptional activity of p73 is required for VDR induction upon DNA damage, we a dominant negative shown to the transcriptional activity of p73 C. C. Mol. Cell Biol. 1999; 19: PubMed Scopus Google Scholar). We tested whether p53 can the ectopic induction of VDR We observed that p53 induction of VDR protein levels p53 not affect induction of VDR. is with that p53 transcriptional activity C. C. Mol. Cell Biol. 1999; 19: PubMed Scopus Google Scholar). we studied the effect of this p53 on DNA damage-induced VDR induction to whether the p73α transcriptional activity affects the VDR induction upon DNA damage. We observed that inhibition of TAp73 transcriptional activity by p53 not p53 the VDR protein expression levels demonstrate that p73 is required for VDR expression, we examined whether p73 expression affects VDR induction upon DNA damage. shown in can lead to a significant in expression of both p73 and VDR in cells in VDR induction was in cells p73 in the or of treatment. Although VDR VDR expression in the or of it does not affect p73 in the of p73 siRNA, VDR protein levels were upon DNA damage results that VDR induction upon DNA damage p73 which occurs upstream of VDR. demonstrate that induction of VDR upon DNA damage in p53 null cells is induction of p73. DNA VDR Expression the to Vitamin D we have shown that VDR induction occurs upon DNA damage, and and its analogues are used extensively for cancer we to whether VDR induction upon DNA damage sensitizes cells to the of we tested the transcriptional activity a containing upstream of the gene in the or of VDR. of VDR was by a short hairpin to VDR, and as a negative we used a nonsilencing short hairpin that the can VDR, we H1299 cells with expression encoding with short hairpin to VDR or nonsilencing control. shown in not the in VDR levels induced by TAp63γ, the of the of DNA damage on a were studied in cells or Although not lead to a significant in to a significant in the which was in the of that to VDR as shown in the in activation is on activation of VDR by its we that VDR activity in the or of and Furthermore, to that induction of VDR is for the of VD3, we the activity in the and of in cells with p73 or We observed a significant in activity in cells treated with or in combination with in cells in which p73 was with cells with our results that upon DNA damage, induction of p73 upon DNA damage to VDR which can the cells to of transcriptional of p53 family members has significant the functional diversities of Oncogene. 2007; 26: PubMed Scopus Google Scholar). the functional p53 family on its regulation of and genes be in their in In this report, we demonstrate that p53 can induce VDR expression by itself nor affect the p63-mediated of VDR expression and the of or regulation of VDR by p53 in the cell lines tested in this study. Our results are to p53 was shown to induce VDR and VDR was shown to be induced upon DNA damage in in of VDR expression in multiple cancer cell lines stress has the of p53 in induction of VDR conditions. of VDR and protein expression levels upon DNA damage was observed in as well as in SaoS2 and in cells not a p53-independent VDR induction upon DNA damage. In addition, these results transcriptional activation of VDR protein of involved in the DNA damage response signaling which lead to and C. B. Nature. 1998; PubMed Scopus Google Scholar). p73 has been shown to be induced upon DNA damage in several different cancer cell lines and shown to be for chemosensitivity of these cells (10Kang K.H. Lee J.H. Kim K.C. Ham S.W. Kim M.Y. Choi K.H. FEBS Lett. 2002; 522: 161-167Crossref PubMed Scopus (16) Google Scholar, X. Zheng Y. J. J. Wang J. Oncogene. 2001; PubMed Scopus Google Scholar). we observed a p73 and VDR levels upon DNA damage in these cell lines expression of multiple p73 isoforms in transcriptional activation of VDR, and p73 expression to in VDR a role of p73 in DNA damage-induced VDR expression Recently, a subset of of p53 has been shown to p73 transcriptional activity through Y. C. Oncogene. 2007; 26: PubMed Scopus Google Scholar, C. M. J. T. C. Mol. Cell Biol. 2001; 21: PubMed Scopus Google Scholar). expression of p53 in down-regulation of VDR expression levels upon DNA damage, the requirement of p73 for DNA VDR induction In of this with we not of VDR and p53 as reported we observed p73 and p53 not lack of has been reported in cells p53 K. K. M. J. 1998; Google and of p53 in various cancer cell lines F. Lee M. M. K. Res. 2007; PubMed Scopus Google Scholar, M.J. K.E. S. M. S. Cancer Res. 2004; PubMed Scopus Google Scholar). Inhibition of DNA damage-induced p73 expression levels in a in VDR expression levels Furthermore, with our inhibition of p73 in down-regulation of VDR expression levels and its transcriptional a transcriptional activation of VDR by p73 both and stressed conditions. Our results demonstrate that DNA damage-induced VDR expression occurs p73, which be by p53 the of both p73 and p53 are in human cancer cell lines. is well that and its analogues exert their antiproliferative activities through VDR and are extensively used for cancer S.F. J.C. Gombart A.F. M.J. 2001; PubMed Scopus Google Scholar, R. M.J. Res. Cancer Res. 2003; PubMed Scopus Google Scholar, M.J. L. Ther. 2006; PubMed Scopus Google Scholar, P. M.J. N. O. J.M. A. 2005; PubMed Scopus Google Scholar, J.H. M.J. Mol. Cancer Ther. 2002; Google Scholar, R. C. P. F. Mol. Cell Biochem. 2000; PubMed Google Scholar). the of analogues on the levels of VDR J.M. J. I. J. M. A. M. A. J. Cell Biol. 2001; PubMed Scopus Google in VDR levels the antiproliferative from our a significant in vitamin VDR transcriptional activity with Additionally, we have demonstrated that transcriptional activation of VDR by p73 upon DNA damage is for the DNA of vitamin D. In conclusion, we demonstrated the differential regulation of VDR by p53 family members and a and p53-independent activation of VDR upon DNA damage. induction of VDR levels upon DNA damage in to in of DNA-damaging agents with vitamin D which in for cancer chemotherapy. We and for with and members and for and