Progress 08/01/96 to 07/31/07
Outputs
1) Our observations revealed that some intracellular lipid-binding proteins enhance the transcriptional activities of particular nuclear receptors. 2) We found that vitamin D controls the nuclear import of both the transcription factor that mediates its biological activities, VDR, and the VDR dimerization partner RXR. These observations point at an interesting cross-talk between signalling by vitamin D and vitamin A.
Impacts
The identification of the bioplogical functions of the intracellular lipid binding proteins CRABP-II, FABP4, and FABP5 point at these proteins as novel tools in therapy and prevention of various diseases. For example, retinoic acid is currently used in treatment of several human cancers. Retinoic acid toxicity and the development of retinoic acid-resistance in tumors may be overcome by ectopic administration of the retinoic acid binding protein CRABP-II.
Publications
- Donato, L.J. and Noy, N. 2006. Quantitation of retinoic acid in biological samples using a fluorescence-based assay. Anal. Biochem. 357:249-256.
- Donato, L.J., Suh, J.H. and Noy, N. 2007. Suppression of mammary carcinoma cell growth by retinoic acid: the cell cycle control gene Btg2 is a direct target for RAR signaling. Cancer Research, 67: 609-615.
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Progress 01/01/05 to 12/31/05
Outputs
Work over the past year focused on the regulatory mechanisms that control the biological activities of three nuclear hormone receptors: the retinoid receptors RAR and RXR, and the vitamin D receptor (VDR). We also investigated the involvement of cellular retinoic acid binding protein, type II (CRABP-II) in enahncing the transcriptional activities of its ligand, retinoic acid, which are mediated by RAR. 1) The mechanisms by which RXR and VDR and their mutual heterodimer mobilize into the nucleus were examined. The results demonstrated that these two receptors move to the nucleus by distinct mechanisms: RXR is mobilized by importin beta, while VDR is imported by importin alpha. Surprisingly, we found that nuclear import of the active heterodimer RXR-VDR is under the control of VDR. This was demonstrated by the observations that the heterodimer is carried into the nucleus by the VDR moiety via its interactions with importin alpha, and that massive nuclear translocation
requires the presence of vitamin D. 2) We previously showed that CRABP-II delivers retinoic acid from the cytosol to RAR in the nucleus, thereby augmenting the transcriptional activity of the receptor. Recent work led to the identification of the nuclear localization signal of CRABP-II and to molecular insight into the structural features of the protein that underlie the response of this signal to ligand-binding. 3) Studies of the mechanisms that underlie the anti-carcinogenic activities of retinoic acid revealed several apro-apoptotic genes that are up-regulated by this hormone in mammary carcinoma cells, and identified caspase 9 as a novel direct target for RAR. These studies also showed that inhibition of growth of breast cancer cells by retinoic acid involves cell cycle arrest at the G1 phase, followed by induction of apoptosis. Finally, we demonstrated that CRABP-II enhances the RAR-mediated up-regulation of caspase 9, as well as augments RA-triggered apoptosis. The later
observations provide strong evidence for the critical role of the binding protein in regulating retinoic acid signalling.
Impacts
1) The observations that nuclear import, and thus the transcriptional activity, of the VDR-RXR heterodimer is controlled by vitamin D suggest that the partitioning of the 'master regulator' RXR between its multiple signalling pathways may be regulated by the specific conditions in particular cells. 2) The ligand-controlled nuclear localization signal of CRABP-II identified by our studies can be recognized in the three dimensional fold of the protein but not by examining its rimary sequence. This novel version of classical signals point at the importance of information emerging from structural genomics for understanding biological functions.
Publications
- Sessler, R.J. and Noy, N. 2005. A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II. Molecular Cell, 18:343-353.
- Donato, L.J. and Noy, N. 2005. Suppression of mammary carcinoma growth by retinoic acid: proapoptotic genes are targets for RAR and CRABP-II signalling in MCF-7 cells. Cancer Res. 65:8193-8199.
- Yasmin, R., Williams, R. M., Xu, M. and Noy N. 2005. Nuclear import of the retinoid X receptor, vitamin D receptor and their mutual heterodimer J Biol. Chem. 280:40152-60.
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Progress 01/01/04 to 12/31/04
Outputs
We previously showed that the ligand-inducible transcription factor termed RXR is unique among nuclear hormone receptors in that it self-associates into tetramers in the the absence of its activating hormone, the vitamin A metabolite 9-cis-retinoic acid. Our recent findings clarified the biological function of these tetramers. Specifically, we demonstrated that, in the context of particular target genes, tetrameric RXR acts as a DNA architectural factor in that it induces DNA-looping. DNA-looping enables transcription factors placed far from gene strat sites to regulate gene expression. As RXR tetramers dissociate upon binding of their ligand, thereby negating the loop, the ability of RXR to modulate DNA geometry reveals a novel mechanism for transcriptional regulation by vitamin A.
Impacts
The findings that retinoic acid can regulate transcription in an indirect fashion by altering the oligomeric properties of RXR reveal a completely new paradigm of transcriptional control by vitamin A.
Publications
- Yasmin, R., Yeung, K. T., Chung, R., and Noy, N. 2004. DNA-looping by RXR tetramers permits transcriptional regulation at a distance. J. Mol. Biol. 343:327-338
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Progress 01/01/03 to 12/31/03
Outputs
The vitamin A metabolite retinoic acid is an important modulator of cell differentiation and proliferation both in the embryo and in the adult. Retinoic acid and its synthetic derivatives (retinoids) are in current use in treatment and prevention and of multiple pathologies ranging from dermatological disorder to cancer. These compounds are also being tested in clinical trials for other disease states such as diabetes and arteriosclerosis. We continue to study the roles of nuclear hormone receptors and intracellular lipid-binding proteins in mediating the biological activities of retinoic acid. We previously showed that cellular retinoic acid binding protein-II (CRABP-II) enhances the transcriptional activity of retinoic acid by directly channeling this ligand to the nuclear receptor that is activated by it, namely, the retinoic acid receptor (RAR). We found further that the ability of CRABP-II to enhance RAR activity dramatically sensitizes carcinoma cells to the
growth inhibitory activities of retinoic acid in cultured cells. Work carried out this year revealed that, due to these activities, CRABP-II also acts as a tumor suppressor in vivo, a conclusion that was supported by studies of two independent mouse models of breast cancer. Interestingly, inhibition of tumor growth by CRABP-II was maximal at the level of retinoic acid endogenously present in the mice, and exogenous administration of this drug did not result in additional beneficial effects. A different set of experiments addressed possible new targets of retinoic acid action in cells. This work revealed that, in addition to RAR, retinoic acid functions as a ligand to another ligand-inducible transcription factor, namely, the orphan nuclear receptor PPARbeta/delta. These observations raise the possibility that retinoic acid functions by a dual pathway, resulting in opposing effects: it induces cell growth arrest through activating RAR, and it may have anti-proliferative activities
mediated by activating PPARbeta/delta. This working hypothesis is currently under investigation.
Impacts
The observations that CRABP-II and other intracellular lipid-binding proteins enhance the transcriptional activities of nuclear receptors point at these proteins as novel tools in therapy and prevention of various diseases. For example, retinoic acid is currently used in treatment of several human cancers. Retinoid therapy is however often confounded by the toxicity of these compounds at pharmacological doses, and by development of retinoic acid-resistance. Our findings demonstrate that forced expression of CRABP-II in carcinomas results in reversal of retinoic acid resistance, and in suppression of cell growth at physiological retinoic acid concentrations. Our data thus suggest that binding proteins may be utilized to significantly enhance the therapeutic efficacy of their cognate ligands by allowing utilization of lower doses. Our studies suggesting that retinoic acid functions by a dual pathway are of particular importance as various reports of opposing effects of
retinoic acid have bee published by the molecular basis for these conflicting observations remain obscure. Clarifying the mechanisms responsible for the multiple activities of this potent drug is expected to point at novel therapeutic targets.
Publications
- Manor, D., Shmidt, E. N., Budhu, A., Flesken-Nikitin, A., Zgola, M., Page, R., Nikitin, A.Yu., and Noy, N. 2003. Mammary carcinoma suppression by cellular retinoic acid binding protein-II. Cancer Research 63:4426-33.
- Shaw, N., Elholm, M., and Noy, N. 2003. Retinoic acid is a high-affinity selective ligand for PPARbeta/delta. J Biol Chem. 278:41589-41592
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Progress 01/01/02 to 12/31/02
Outputs
We continued to examine the mechanisms by which CRABP-II cooperates with the nuclear receptor RAR in regulating transcription. We found that the ability of CRABP-II to 'channel' retinoic acid (RA) to RAR sensitizes 2 mammary carcinoma cell lines to RA-induced growth arrest. Further, injection of an adenovirus that expresses CRABP-II into tumors that spontaneously arise in the cancer mouse model MMTV-neu significantly inhibited tumor progression. Our studies also revealed a novel nuclear localization signal in CRABP-II. We demonstrated that other lipid binding proteins operate similarly to CRABP-II in that they 'channel' ligands to nuclear receptors with which they share them. Specifically, we found that A-FABP and K-FABP directly interact with and enhance the activity of PPARg and PPARd, respectively. Another set of studies focused on the interactions of RXR with heterodimerization partners and with DNA. We found that nuclear localization of VDR requires the presence
of RXR as well as the presence of ligands for both receptors. Finally, we found that RXR tetramers, which we previously showed to form with a high affinity in the absence of a RXR ligand, are able to alter DNA geometry. Specifically, the data demonstrated that binding of RXR tetramers to oligonucleotides containing 2 complete response elements for this receptor, results in significant DNA looping. No such looping was observed in the presence of ligand (which leads to tetramer dissociation) or in the presence of A RXR mutant that is unable to form tetramers.
Impacts
The observations that CRABP-II funtions to suppress mammary tumor growth both in cultured cells and in two independent mouse models of cancer point at this protein as a novel target for therapy and perhaps prevention of breast cancer.
Publications
- Lee, W.-y., and Noy, N. 2002. Interactions of RXR with coactivators are differentially mediated by helix 11 of the receptor's ligand binding domain. Biochemistry 41:2500 -2508.
- Budhu, A. S., and Noy, N. 2002. Direct channeling of retinoic acid between CRABP-II and RAR sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest Mol. Cell. Biol. 22:2632-2641.
- Tan, N. S., Shaw, N. S., Vinckenbosch, N., Liu, P., Yasmin, R. Desvergne, B., Wahli, W. and Noy, N. 2002. Selective cooperation between fatty acid binding proteins and peroxisome proliferator activated receptors in regulating transcription. Mol. Cell. Biol. 22:5114-5227.
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Progress 01/01/01 to 12/31/01
Outputs
Studies during the last year focused on studying the role of CRABP-II in regulating the activity of retinoid receptors, and on structure/function relationships of RXR. Our findings showed that the cytosolic protein known as CRABP-II is critical for mediating the ability of its ligand, the vitamin A metabolite retinoic acid, to induce growth arrest in cancer cells. Specifically, we demonstrated that CRABP-II dramatically sensitizes two mammary carcinoma cell lines to the antiproliferative activity of its ligand. Additional work revealed that over-expression of CRABP-II reverses the transformation properties of SC115 cells and inhibits the tumorigenic potential of these cells in immunodeficient mice. Additionally, we delineated the molecular mechanism by which CRABP-II exerts its effects. We thus demonstrated that CRABP-II delivers retinoic acid to the nucleus where it 'channels' it to RAR, thereby enhancing the transcriptional activity of the receptor. Another set of
studies focused on the interactions of the 9-cis retinoic acid-responsive nuclear receptor RXR with transcriptional coactivators. The results of this work lead to the conclusion that the tetramerization region of RXR, which we previously localized to helix 11 of the ligand-binding domain of the protein, contributes to the interaction surface of this protein with p160 coactivators but not with TIF1. These data point at selectivity in coactivator-receptor interactions and at the structural basis for such selectivity.
Impacts
Our findings that CRABP-II sensitizes carcinoma cells to retinoic acid, and our identification of the molecular mechanisms by which this activity is mediated are important on two levels. On the level of basic research, these results identified a specific role of a protein that belongs to the large family of lipid-binding proteins. Proteins in this family are usually believed to act by storing and transporting their hydrophobic ligands through the aqueous space of the cytosol. In contrast, our data show that a member of this family acts by specifically interacting with the cellular target of its ligand (in this case, the nuclear receptor RAR). We showed further that these protein-protein interactions are important for facilitating the activation of the target and thus regulate the transcriptional activity of the receptor both in cultured cell models and in vivo. These conclusions point at a potential novel therapeutic strategy in treatment and prevention of cancer.
Publications
- Lee, W.-y., and Noy, N. 2002. Interactions of RXR with coactivators are differentially mediated by helix 11 of the receptor's ligand binding domain. Biochemistry, in press
- Budhu, A.S., Gillilan R., and Noy, N. 2001. Localization of the RAR interaction domain of CRABPII. J. Mol. Biol. 305:939-949
- Budhu, A. S., and Noy, N. 2002. Direct channeling of retinoic acid between CRABP-II and RAR sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest Mol. Cell. Biol. in press
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Progress 01/01/00 to 12/31/00
Outputs
During this year, we have investigated two aspects of the mechanisms by which the vitamin A metabolites retinoic acids regulate gene transcription. One project focused on the 9-cis-retinoic acid-responsive transcription factor known as RXR. We previously found that this receptor forms tetramers that serve to silence its transcriptional activity in the absence of its cognate ligand. We showed further that the receptor is silent under these conditions because the protein region that serves to connect RXR with transcriptional co-activators is inaccessible to some such proteins within the tetramer. However, we recently found that particular coactivators can interact with RXR tetramers in the absence of its ligand. These data suggest that RXR may be active in some cells even in the absence of 9-cis-retinoic acid, a hypothesis that is currently under investigation. These findings uncover, for the first time, a molecular mechanism by which selectivity of nuclear receptors
for transcriptional coactivators is determined. In another project, we continued our studies of the mechanisms of action of the cellular retinoic acid-binding proteins (CRABPs). This work resulted in the identification of a specific function for CRABP-II which is not shared by CRABP-I. We thus demonstrated that CRABP-II enhances the transcriptional activity of the retinoid receptor RAR. We showed further that CRABP-II acts through direct interactions with RAR, leading to facilitation of the formation of the active receptor. This work pointed at a novel mechanism by which the cellular activities of retinoic acid are regulated. Recently, our work led to the identification of the surface region of CRABP-II which mediates its interactions with RAR. Additional studies demonstrated that due to its ability to activate RAR, The presence of CRABP-II in mammalian carcinoma cells enhances their sensitivity to retinoic acid and allows this compound to induce cell growth arrest at lower
concentrations.
Impacts
The findings that CRABP-II acts to enhance the sensitivity of carcinoma cells to retinoic acid treatment suggest that this protein may be a target for devising strategies for enhancing the therapeutic/preventive properties of retinoic acid as an anti-cancer agent. This possibility is currently being pursued.
Publications
- Budhu, A.S., and Noy, N. (2000) On the role of the carboxyl-terminal helix of RXR in the interactions of the receptor with ligand , Biochemistry, 39:4090-4095
- Hoyos, B., Imam, A., Chua, R., Swenson, C., Tong, G.-X., Levi, E., Noy, N., and Hammerling, U. (2000) The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors. J. Exp. Med. 192:1-12
- Noy, N. Retinoid-binding proteins: mediators of retinoid action. Biochem. J. 2000 348:481-495
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Progress 01/01/99 to 12/31/99
Outputs
Over the past year we pursued studies of the following issues: 1. The role of the self-association of RXR into tetramers in regulator the transcriptional activity of this protein. Current work focuses on the ability of RXR tetramers to induce DNA-bending and on functional consequences of this effect. In addition, work on delineating the differential mode by which RXR interacts with different partner receptors, specifically, the peroxisome proliferators-activated receptor (PPAR) and the retinoic acid receptor (RAR), is on-going. 2. The role of cellular retinoic acid binding proteins (CRABPs) in regulating the transcriptional activity of RAR. We have found that CRABPII sensitizes cells to retinoic acid by directly delivering this ligand to RAR. 3. The nature of the physiological ligands for the nuclear receptor PPAR. PPAR is a transcription factor that plays critical roles in regulating lipid and sugar metabolism. This protein can be activated by synthetic compounds
that are widely used in treatment of hyperlipidemia. However, the identity of the physiological ligands for this receptor remains unclear. Our studies revealed that PPAR binds several naturally occuring long chain fatty acids with high affinity. These findings support the notion that this important nuclear receptor respond in vivo to these ligands.
Impacts
The findings that CRABPII is engaged in direct protein-protein interactions with RAR, and that this process facilitates the formation of the active form the receptor has several important implications. At the level of basic science, it provides insights into the functional differences between CRABPI and CRABPII, an issue that remained unresolved for over a decade. In addition, these observations point at CRABPII as a possible therapeutic agent that may allow for enhancing the therapeutic efficacy of retinoic acid in cancer treatment and prevention.
Publications
- Dong, D., Ruuska, S., Levinthal, D. J., and Noy, N. (1999) Distinct roles for cellular retinoic acid-binding proteins-I and II in regulating signalling by retinoic acid. J. Biol. Chem 274: 23695-23698
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Progress 01/01/98 to 12/31/98
Outputs
We continued to study the transcription factor known as the retinoid X receptor (RXR). Previously we proposed that, in the absence of its ligand, the vitamin A derivative 9-cis-retinoic acid, RXR exists as a stable, transcriptionally silent tetramer, and that ligand-binding activates the receptor by inducing dissociation of tetramers into active species, dimers and monomers. We found further that the interactions of RXR with two of its partner receptors - the retinoic acid- and the vitamin D-receptor - are regulated both by tetramer formation by RXR and by cross-talk between different hormones that bind to RXR and its different partners. Recently, we established that the initial events in the activation of RXR is constituted of two distinct ligand-dependent steps: 1) dissociation of receptor tetramers into dimers, and 2) a confomational change that stabilizes the active dimeric form. The two events could be dissected by utilizing a RXR-specific antagonist. In
addition, we began to explore the molecular basis underlying the silencing of the transcriptional activity of RXR by the receptor's self-association properties. Initial observations suggest that the protein domain which mediates tetramer formation also acts as an interface through which RXR interacts with transcriptional coactivators. Hence, self-association by RXR acts to mask a region of the protein that is critical for its communication with the general transcription machinery. We also began to examine the roles of the two isoforms of cellular retinoic acid binding proteins (CRABPI and CRABII) in delivering their ligand to the nucleus, where it serves to activate RAR. Our data suggest that CRABPII, but not CRABPI, directly interacts with RAR, resulting in facilitation of transfer of retinoic acid to the transcription factor. These observations are of particular interest because, although it is well document that the spatial and temporal expression profiles of the two proteins are
distinct, the details of their (assumed) different functions in the biology of vitamin A are currently unknown. Finally, we developed a new optical method to investigate the ligand selectivity of the peroxisome proliferator-activated receptor (PPARa), a transcription factor which is important in regulation of lipid metabolism and adipocyte differentiation. We found, in accordance with previous reports, that PPAR displays a broad ligand specificity but that its affinity for binding of these ligands is significantly stronger than previously proposed. These data imply that physiological fatty acids and some of their metabolites serve as the endogeneous activators of this receptors in vivo.
Impacts
(N/A)
Publications
- Chen, Z.-P., Iyer, J., Bourguet, W., Held, P., Mioskowski, C., Lebeau, L., Noy, N., Chambon, P., and Gronemeyer, H. (1998) Ligand and DNA-induced dissociation of RXR tetramers. J. Mol. Biol. 275:55-65.
- Dong, D., and Noy, N. (1998) Heterodimerization of the retinoid X receptor with the retinoic acid- and the vitamin D-receptors is regulated by ligands and by the self-association properties of RXR. Biochemistry, 37:10691-10700.
- Kersten, S., Dong, D., Lee, W.-y., Reczek, P. R., and Noy, N (1998) Auto-silencing by the retinoid X receptor. J. Mol. Biol. 284:21-32
- Lin, Q., Ruuska, S. E, Shaw, N. S., Dong, D., and Noy, N. (1998) Ligand selectivity of the peroxisome proliferator-activated receptor a. Biochemistry 38:185-190
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Progress 01/01/97 to 12/31/97
Outputs
We continued to study the transcription factor known as the retinoid X receptor (RXR). Previously we proposed that, in the absence of its ligand, the vitamin A derivative 9-cis-retinoic acid, RXR exists as a stable, transcriptionally silent tetramer, and that ligand-binding activates the receptor by inducing dissociation of tetramers into active species, dimers and monomers. This year we provided evidence for the validity of this model by examining the oligomeric state and the transcriptional activities of two point-mutants. One of these mutants was found to form tetramers that failed to dissociate upon-ligand binding, and, as predicted, was also transcriptionally inactive. The other mutant was impaired in its ability to form tetramers, and, also as predicted, was found to be constitutively active. We also explored the implications of tetrmer formation for the interactions of RXR with two of its heterodimerization partners, the retinoic acid- and the vitamin
D-receptors. We found that although heterodimers form with a significantly higher binding energy as compared to tetramers, a kinetic barrier prevents RXR tetramers from interacting with these partners. Our data further indicated that the RXR is directed to particular transcriptional pathways both by its oligomerization behavior and by cross-talk between different hormones that bind to RXR and to its different partners.
Impacts
(N/A)
Publications
- KERSTEN, S., GRONEMEYER, H., and NOY, N. 1997 The DNA-binding pattern of the retinoid X receptor is regulated by a ligand-dependent modulation of its oligomeric state. J. Biol. Chem. 272:12771-12777
- . KERSTEN, S., RECZEK, P. R., and NOY, N. 1997 The tetramerization region of the retinoid X receptor is important for transcriptional activation by the receptor. J. Biol. Chem. 272:29759-29768
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Progress 01/01/96 to 12/30/96
Outputs
We continued to explore the mechanisms by which the activity of the transcription factor known as the retinoid X receptor (RXR) is regulated by its ligand. We previously showed that RXR forms tetramers with a high affinity and that upon ligand-binding the tetramers dissociate to lower order species. This year, we undertook to locate the region in the protein that mediates tetramer formation and identified it to be located in the ligand-binding domain of RXR. Mutagenetic analysis mapped the region to 3 consecutive phenylalanine residues within this domain. In addition, we began to explore the interactions of RXR with other transcription factors such as the retinoic acid receptor. We found that, contrary to what is sometime believed, RXR within a complex comprised of the heterodimer RXR-RAR maintains its ability to interact with its ligand with a high affinity. Additional efforts were made to set up a system that will allow us to investigate the interactions of RXR with
the vitamin D receptor. This protein is now routinely expressed in this laboratory .
Impacts
(N/A)
Publications
- Kersten, S., Dawson, M. I., Lewis, B. A., and Noy, N. (1996) Individual subunits of heterodimers comprised of the retinoic acid- and retinoid X receptors interact with their ligands independently. Biochemistry 35, 3816-3824.
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Progress 01/01/95 to 12/30/95
Outputs
The transcription factor known as the retinoid X receptor (RXR) was studied. It was found that RXR forms tetramers with a high affinity and that these tetramers form stable complexes with cognate DNA. The oligomeric state of RXR was found to be tightly regulated by its ligand such that ligand-binding leads to a dissociation of receptor tetramers concomitantly with a significant increase in the population of receptor dimers. Studies of the DNA-binding pattern of RXR alone and in the presence of another retinoid nuclear receptor, RAR, led to the conclusions that ligand-binding by RXR results in activation of both the homodimeric and heterodimeric pathways which this receptor may be involved in. A model for understanding the mechanism by which RXR is activated by its ligand was developed.
Impacts
(N/A)
Publications
- KERSTEN, S., KELLEHER, D., CHAMBON, P., GRONEMEYER, H., and NOY, N. 1995. The retinoid X receptor forms tetramers in solution. Proc. Nat'l. Acad. Sci. USA 92:8645-8649.
- KERSTEN, S., PAN, L., CHAMBON, P., GRONEMEYER, H., and NOY, N. 1995. On the role of ligand in retinoid signaling: 9-cis retinoic acid modulates the oligomeric state of the retinoid X receptor. Biochemistry 34:13717-13721.
- KERSTEN, S., PAN, L. and NOY, N. 1995. On the role of ligand in retinoid signaling. Positive cooperativity in the interactions of 9- cis retinoic acid with tetramers of the retinoid X receptor. Biochemistry 34:14263-14269.
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