Interaction of BAG-1 with Retinoic Acid Receptor and Its Inhibition of Retinoic Acid-induced Apoptosis in Cancer Cells

Abstract

BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone. We show here that BAG-1 also interacts with retinoic acid receptor (RAR). Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs). A glutathioneS-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR. Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays. In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers. When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis. In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1. These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells. BAG-1 (also known as RAP46) is an anti-apoptotic protein, which has been shown previously to interact with a number of nuclear hormone receptors, including receptors for glucocorticoid, estrogen, and thyroid hormone. We show here that BAG-1 also interacts with retinoic acid receptor (RAR). Gel retardation assays demonstrated that in vitro translated BAG-1 protein could effectively inhibit the binding of RAR but not retinoid X receptor (RXR) to a number of retinoic acid (RA) response elements (RAREs). A glutathioneS-transferase-BAG-1 fusion protein also specifically bound RAR but not RXR. Interaction of BAG-1 and RAR could also be demonstrated by yeast two-hybrid assays. In transient transfection assays, co-transfection of BAG-1 expression plasmid inhibited the transactivation activity of RAR/RXR heterodimers but not RXR/RXR homodimers. When stably expressed in breast cancer cell lines, BAG-1 inhibited binding of RAR/RXR heterodimer to a number of RAREs and suppressed RA-induced growth inhibition and apoptosis. In addition, RA-induced suppression of Bcl-2 expression was abrogated by overexpression of BAG-1. These results demonstrate that BAG-1 can regulate retinoid activities through its interaction with RAR and suggest that elevated levels of BAG-1 protein could potentially contribute to retinoid resistance in cancer cells. Development of a multicellular organism requires tightly regulated cellular processes, such as proliferation, differentiation, and cell death. Failure to maintain the balance among these fundamental and mechanistically related processes may result in abnormal cell growth, as seen in cancer cells where cell death is often inhibited (1White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1325) Google Scholar, 2Reed J.C. J. Cell. Biol. 1994; 124: 1-6Crossref PubMed Scopus (2390) Google Scholar). Retinoids, a group of natural and synthetic vitamin A derivatives, are currently used to treat epithelial cancer and promyelocytic leukemia and are being evaluated for prevention and therapy of other human cancers (3Gudas L.J. Sporn M.B. Roberts A.B. Sporn M.B. Roberts A.B. 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The effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) 1The abbreviations used are: RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response elements; TR, thyroid hormone receptor; MTT, 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide; GST, glutathioneS-transferase; tk, thymidine kinase; MHC, myosin heavy chain; CAT, chloramphenicol acetyl- transferase. and retinoid X receptors (RXRs). RARs and RXRs are encoded by three distinct genes (α, β, and γ) and are members of the steroid/thyroid/retinoid hormone receptor superfamily that function as ligand-activated transcription factors (12Zhang X.-k. Pfahl M. TEM. 1993; 4: 156-162Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 13Kastner P. Mark M. Chambon P. Cell. 1995; 83: 859-869Abstract Full Text PDF PubMed Scopus (939) Google Scholar, 14Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2837) Google Scholar). 9-cis RA is a high affinity ligand for both RARs and RXRs, whereas all-trans-RA (trans-RA) is a ligand for only RARs. RARs and RXRs primarily function as RXR/RAR heterodimers that bind to a variety of RA response elements (RAREs) and regulate their transactivation activities. Regulation of gene expression either positively or negatively by nuclear hormone receptors is modulated by additional factors. Some of them appear to provide a direct link to the core transcriptional machinery and to modulate chromatin structure (15Horwitz K.B. Jackson T.A. Bain D.L. Richer J.K. Takimoto G.S. Tung L. Mol. Endocrinol. 1996; 10: 1167-1177Crossref PubMed Scopus (837) Google Scholar), such as SRC-1 (16Onate S.A. Tsai S.Y. Tsai M.-J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2058) Google Scholar), SUG-1 (17Lee J.W. Ryan F. Swaffield J.C. Johnston S.A. Moore D.D. 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Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar), whereas a number of other cellular proteins, such as AP-1, have been implicated in the regulation of nuclear hormone receptor activity, probably through their interaction with receptors (27Pfahl M. Endocr. Rev. 1993; 14: 651-658Crossref PubMed Scopus (447) Google Scholar). The involvement of retinoid receptors in retinoid-induced apoptosis has been demonstrated in several studies. Expression of RARβ may be involved in the apoptosis of mesenchyme of the interdigital regions during mouse limb development (28Kochhar D.M. Jiang H. Harnish D.C. Soprano D.R. Prog. Clin. Biol. Res. 1993; 383: 815-825Google Scholar). RARβ is required for RA-induced apoptosis of breast cancer (5Liu Y. Lee M.-O. Wang H.-G. Li Y. Hashimoto Y. Klaus M. Reed J.C. Zhang X.-k. Mol. Cell. Biol. 1996; 16: 1138-1149Crossref PubMed Scopus (330) Google Scholar) and lung cancer (7Li Y. Dawson M.I. Agadir A. Lee M.-O. Jong L. 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In interacts with a number of nuclear hormone receptors, including receptor and thyroid hormone receptor M. U. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). are known to an in the activity of members of the steroid/thyroid/retinoid receptor D.O. Endocr. Rev. 1997; PubMed Scopus Google Scholar), it is that BAG-1 could the function of these transcriptional it was unknown BAG-1 the activities of the nuclear hormone receptors and BAG-1 interacts with retinoid demonstrate that BAG-1 interacts with the RAR but not the both in vitro and in and the yeast two-hybrid assays show that BAG-1 interacts with RAR but not RXR. BAG-1 RAR/RXR heterodimer binding and RA-induced transactivation activity of RARs of BAG-1 in and breast cancer cells the of to inhibit the growth and induce as as its of Bcl-2 results demonstrate that BAG-1 can interact with RARs and is an in the retinoid response suggest that interaction may a in the regulation of retinoid-induced growth inhibition and processes, potentially to retinoid resistance in cells and breast cancer cells in with and breast cancer cells in with in and with of for cell number was the Q. Li Y. Liu R. Agadir A. Lee M.-O. Liu Y. Zhang X.-k. EMBO J. 1997; 16: PubMed Scopus Google Scholar). the (5Liu Y. Lee M.-O. Wang H.-G. Li Y. Hashimoto Y. Klaus M. Reed J.C. Zhang X.-k. Mol. Cell. Biol. 1996; 16: 1138-1149Crossref PubMed Scopus (330) Google Scholar), cells with or cells with in in with in and with by and with cells a are in and and of in by and the in The three with and for in three in by with an cells in a transfection as previously X.-k. B. G. Pfahl M. 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When of in vitro BAG-1 protein with RAR and RXR, the binding of RAR/RXR heterodimers was inhibited in a BAG-1 a of BAG-1 protein to the binding was The of BAG-1 RAR/RXR binding was of receptor not show BAG-1 also effectively inhibited the binding of to the with a that BAG-1 can interact with M. U. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the of BAG-1 RAR/RXR heterodimer binding is to the used RA which is the RARβ B. J.M. Zhang X.-k. T. G. Pfahl M. Mol. Endocrinol. 4: Scopus Google Scholar). shown in binding of RAR/RXR the was also inhibited by the of BAG-1 results with other including and not inhibition of RAR/RXR binding to their by BAG-1 is of We inhibition of RAR/RXR heterodimer binding by BAG-1 is due to interaction of BAG-1 with RAR or RXR. in vitro RAR or alone not bind to RARE, used expressed RAR or When a of BAG-1 protein was the binding of was whereas binding of was not These results suggest that inhibition of RAR/RXR or heterodimer binding by BAG-1 is due to its interaction with RAR or but not with RXR. the interaction BAG-1 and RAR, used an in A fusion protein was expressed in and The with in vitro RAR or the a In to the of RAR but not by but not by a known BAG-1 protein S. T. Krajewski S. K. S. Reed J.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar), bound to We also a BAG-1 protein in which the acid are its S. S. C. Reed J.C. EMBO J. 1997; 16: PubMed Scopus Google Scholar). protein can interact with Bcl-2 but not with RAR but not was also by the that the interaction of BAG-1 with RAR is of its binding to Interaction RAR and BAG-1 was also evaluated by the two-hybrid in that of BAG-1 and the in whereas of BAG-1 and Interaction BAG-1 and was of either BAG-1 or with the not the RAR and BAG-1 also interact in cells. the the effects of the BAG-1 RAR transactivation activity a number of RAREs by transient transfection When cells with expression with either or gene activity was inhibited by co-transfection of BAG-1 expression plasmid in a The is to BAG-1 co-transfection of of expression not inhibit gene BAG-1 also RARβ activity the However, RARβ activity was not that RAR activity is to the of BAG-1. A of BAG-1 was also expression was used not We also the effects of BAG-1 thyroid hormone activity, and a of BAG-1 with the of BAG-1 to bind the protein M. U. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). the of BAG-1 activity, plasmid and expression However, the activity was not by co-transfection of BAG-1 with that BAG-1 not interact with in vitro and The suggest that BAG-1 may function as a of We previously that effectively the growth and apoptosis of human breast cancer cell (5Liu Y. Lee M.-O. Wang H.-G. Li Y. Hashimoto Y. Klaus M. Reed J.C. Zhang X.-k. Mol. Cell. Biol. 1996; 16: 1138-1149Crossref PubMed Scopus (330) Google Scholar). stably expressed BAG-1 in human breast cancer cell and and that stably expressed high levels of the BAG-1 plasmid not to the of BAG-1 overexpression RA activities. shown in effectively inhibited the growth of and cells. However, the resistance to the growth effects of The growth of the was by not the growth of and its the growth of these cells was as with its cells The activity was stably with the and to as that with the cell BAG-1 the growth effects of human breast cancer cells. We the effects of BAG-1 apoptosis of and cells the was by in and cells. In the shown in and of the and cells apoptosis in response However, and cells the with only and the of cells was by BAG-1 overexpression overexpression of BAG-1 apoptosis in breast cancer cells. Retinoids have been shown to the expression of Bcl-2 in leukemia E. Linker-Israeli M. Umiel T. Le J. Grillier I. Said J. Shintaku I.P. Krajewski S. Reed J.C. Binderup L. Koeffler H.P. Cancer Res. 1996; 56: 3570-3576PubMed Google Scholar). We BAG-1 expression of Bcl-2 in cells. Bcl-2 was expressed in and its expression was by as by In of or Bcl-2 expression These suggest that BAG-1 may inhibit apoptosis in through its effects such as the of BAG-1 gene nuclear and cells and their binding to including RARE, and shown in and binding to these whereas binding of the in and cells was inhibited in cells. the of the cells was with either or or to binding that either or but not inhibited the of the that the RAR and RXR. These demonstrate that overexpression of BAG-1 RAR/RXR binding and suggest that the of RAR transcriptional activity may contribute to the of BAG-1 RA it was that BAG-1 can interact with several hormone receptors M. U. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). However, the effects of the interaction are In show that the BAG-1 that is known to be expressed in G. M. J. 1997; PubMed Scopus Google Scholar) can RAR activity through its direct interaction with retardation assays, that BAG-1 binding of RAR/RXR heterodimers to several RAREs In that BAG-1 interacts with RAR in yeast two-hybrid assays that the interaction could in a interaction was demonstrated by that co-transfection of BAG-1 RAR transactivation activities several RAREs RAR/RXR binding was abrogated in cells that BAG-1 the interaction of BAG-1 with hormone receptors can be to We also here that BAG-1 can binding and transactivation However, BAG-1 not interact with nuclear hormone receptors, as shown here by the of BAG-1 to bind to and modulate the activity of or of gene transcription by nuclear hormone receptors requires their interaction with cellular factors. These receptor which their receptor transactivation activity by and chromatin structure (15Horwitz K.B. Jackson T.A. Bain D.L. Richer J.K. Takimoto G.S. Tung L. Mol. 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EMBO J. 1997; 16: PubMed Scopus Google Scholar, S. S. C. Reed J.C. EMBO J. 1997; 16: PubMed Scopus Google Scholar, M. M. U. EMBO J. 1997; 16: PubMed Scopus Google Scholar). the of in transcriptional by retinoid receptors remains L.J. M. Chambon P. PubMed Scopus Google Scholar, Mol. Cell. Biol. 1995; Scopus Google Scholar), and other are known to in the regulation of several other hormone receptors D.O. Endocr. Rev. 1997; PubMed Scopus Google Scholar). is to that BAG-1 may also RAR activity through that it binding and a of BAG-1 its which not bind to was of binding to RAR in vitro the in BAG-1 required for with RAR and appear to be expressed in the BAG-1 protein was of its effects gene the of BAG-1 with to its function as of RAR remains to be of the of BAG-1 is its to cell S. T. Krajewski S. K. S. Reed J.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, K. S. Reed J.C. Mol. Endocrinol. 1997; PubMed Google Scholar, A. M. H. S. A. Y. S. Reed J.C. K. 1997; 14: PubMed Scopus Google Scholar). The was previously to its interaction with Bcl-2 S. T. Krajewski S. K. S. Reed J.C. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). In addition, BAG-1 interacts with growth and growth receptors, their to that cell A. P. D. S. C. C. EMBO J. 1996; 15: PubMed Scopus Google Scholar). BAG-1 can also bind to and its activity H.-G. S. Reed J.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: PubMed Scopus Google Scholar). that overexpression of BAG-1 apoptosis and of Bcl-2 expression suggest that interaction with RAR may by which BAG-1 cell These also suggest that overexpression of BAG-1 may contribute to retinoid resistance in with that BAG-1 protein levels are elevated in breast and cancers S. Krajewski S. M. S. J. K. D. G. D. G. E. Reed J.C. Cancer 1998; Scholar), BAG-1 may an of cell and growth, which may contribute in to and resistance to The of has been to receptor that BAG-1 may either or the of nuclear receptors B. and J. C. J. Biol. We S. for of the