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Departments of Medicine and Bioregulatory Science (M.S., K.G., A.T., T.Y., H.N.) and Geriatric Medicine (R.T.), Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and Center for Tsukuba Advanced Research Alliance (A.F.), Institute of Applied Biochemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan; and CREST (Core Research for Evolutional Science and Technology), JST (Japan Science and Technology) (R.T., K.G., T.Y., H.N.), Kawaguchi 332-0012 Japan
Address all correspondence and requests for reprints to: Hajime Nawata, M.D., Ph.D., Department of Medicine and Bioregulatory Science, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: nawata{at}intmed3.med.kyushu-u.ac.jp.
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-dihydrotestosterone (DHT)-bound AR-GFP in COS7 cells was abolished by the cotransfection of a CBP
(118–2393) fragment eliciting a dominant negative effect on the transactivation capacity of the AR. The N-terminal AR fragment (AR-AF-1-YFP), which has a strong constitutive transactivation function, formed foci without DHT, whereas the C-terminal AR fragment (AR-AF-2-CFP), which has a quite low transactivation function, was distributed homogeneously even in the presence of DHT. The reporter gene assay showed a synergism between the transactivation functions of AR-AF-1 and AR-AF-2. This synergism was not reflected by the above two-dimensional imaging. In contrast, a three-dimensional imaging method clearly showed a difference in the intranuclear spatial distribution. The DHT-bound wild-type AR-GFP alone or AR-AF-1-YFP plus DHT-bound AR-AF-2-CFP was distributed as approximately 300 discrete spots in one nucleus, whereas AR-AF-1-YFP alone was distributed as one volume in a reticular pattern. Furthermore, not only AR but also the glucocorticoid receptor-YFP, ER -GFP, and YFP-tagged SRC-1, TIF2, and CBP were found to be accumulated in identical spots in the presence of ligand. All of the above results indicate that CBP is one of the factors essential for foci formation of the AR, and may propose the hypothesis that transcriptionally activated steroid receptors, regardless of the type of receptor, are transferred to common compartments (foci) and form a complex with coactivators, and this process is essential to full transactivation. |
INTRODUCTION |
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(5, 6), vitamin D receptor (7), and AR (8, 9). Furthermore, in the MR and AR the foci formation of the steroid hormone receptor was shown to be closely linked to transcriptional activation by the receptor. As evidence, the MR and AR were also translocated from the cytoplasm to the nucleus by the addition of an antagonist that inhibited transcriptional activation; however, they were diffusely distributed in the nucleus without making any foci (4, 8, 9). In the MR, ER, and AR, the foci, distributed in a speckled pattern, were reported to be closely associated with the nuclear matrix (4, 6, 8). However, the mechanism and physiological significance of such foci formation have yet to be elucidated. The steroid hormone receptors have two major domains for transcriptional activation, activation function 1 (AF-1) in the N-terminal domain (NTD) and activation function 2 (AF-2) in the C-terminal ligand-binding domain (LBD). The transactivation function of the AF-1 region is constitutive, namely, ligand independent and autonomous, and that of the AF-2 is ligand dependent (10). It is known that the transcriptional activation function of the steroid receptor is enhanced by direct binding with coactivators (11) such as CREB-binding protein (CBP) (12), steroid receptor coactivator-1 (SRC-1) (13) and transcriptional intermediary factor 2 (TIF2) (14). The AR has been reported to be different from the other steroid hormone receptors such as the ER, GR, TR, and PR in that the AF-2 activity in the LBD is negligible or quite low in mammalian cells (15), whereas the intrinsic AF-1 activity is clearly detectable (16, 17, 18, 19). In the present study, based on analysis of a chimera of the AR and the fluorescent protein, CBP was found to be essential for the foci formation of the AR in the nucleus. Furthermore, a three-dimensional imaging method that has recently been developed (9) demonstrated that the intranuclear distribution pattern of the N-terminal fragment of the AR containing AF-1 was different than that of the full-length AR, although such differences in foci formation could not be detected by the previous two-dimensional imaging method. Three-dimensional imaging also revealed that in the presence of ligand, the AR, GR, and ER were all accumulated in identical compartments (foci) containing coactivators. These present findings may suggest that compartmentalization (complete foci formation) in the nucleus is one of the indispensable and common steps for steroid hormone receptor-mediated transactivation. |
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) but was translocated into the nucleus upon exposure to dihydrotestosterone (DHT). Moreover, the nuclear distribution of AR-CFP was not uniform, but showed a speckled pattern with the formation of foci (Fig. 1B). When pCMV-yellow fluorescent protein (YFP)-SRC-1 and pCMV-YFP-TIF2, in which YFP was fused to the N termini of SRC-1 and TIF2, respectively, were transfected into COS7 cells, these YFP-tagged coactivators of AR were distributed diffusely in the nucleus (Fig. 1, C and I). Cotransfection of pCMV-AR-CFP in the presence of DHT in addition to pCMV-YFP-SRC-1 or pCMV-YFP-TIF2 changed the subnuclear distribution of these coactivators from a uniform pattern to a foci-forming one (Fig. 1, F and J). The liganded AR-CFP was colocalized with these foci of the coactivators, indicating that the transcriptionally activated AR recruited YFP-SRC-1 and YFP-TIF2 and rearranged them in the nucleus (Fig. 1, G, H, K, and L). The cotransfection of pCMV-AR-CFP without DHT or the addition of DHT alone did not change the diffuse distribution pattern of the coactivators (Fig. 1, E and D). YFP-CBP, a general integrator for nuclear receptors, was distributed in a mixed pattern with fine foci formation in a diffuse background in the nucleus of COS7 cells (Fig. 2A), unlike YFP-SRC-1 and YFP-TIF2. Upon exposure to DHT, the expressed AR-CFP was translocated from the cytosol (Fig. 2A) to the nucleus, coinciding with CBP and making clear foci (Fig. 2B). Endogenous CBP, which was detected by immunostaining using commercially available anti-CBP antibodies, was found to be distributed in a finely speckled (microparticulate) pattern (Fig. 2C) in the nucleus of pCMV-AR-CFP-transfected cells in the absence of DHT. However, endogenous CBP was not stained immunologically in the AR foci in DHT-exposed cells (Fig. 2D). AR-CFP was diffusely distributed without forming any foci in the nucleus by exposure to the antiandrogenic agent hydroxyflutamide (OHF) instead of DHT as reported (8, 9) (Fig. 2E), i.e. the transcriptionally inactive AR does not form foci in the nucleus. Endogenous CBP was detectable immunostaining when this antiandrogen was added (Fig. 2F). These observations suggest that the AR, activated by the addition of DHT, bound to endogenous CBP and masked the anti-CBP antibody recognition site of CBP, and thus might suggest that the DHT-bound AR was immediately recruited by CBP and distributed in a foci-forming pattern. To confirm the essential role of CBP in foci formation, the effect of mutated CBP, which exerts a dominant negative effect on the transactivation function of the AR, on the foci formation of the AR was examined. A series of domain-deletion mutants of CBP were prepared. Among these CBP mutants, the CBP (118–2393) fragment, consisting of only the N-terminal and C-terminal regions, suppressed the AR-dependent transactivation in a dose-dependent manner, and this suppression was recovered by cotransfection of the expression vector for wild-type CBP (Fig. 3). When this CBP (118–2393) expression vector was cotransfected, the foci of AR-GFP were not formed (Fig. 2G), but the foci formation was recovered by further transfection of the wild-type CBP expression vector (Fig. 2H). The foci formation was not affected by cotransfection of the expression vector for CBP (738–2393) (Fig. 2I), CBP (313–468) or CBP (1570–1891) (data not shown), which did not show any dominant negative effect on transactivation (Fig. 3). The above findings show endogenous CBP to be indispensable for the foci formation of the AR. When only the CBP (118–2393) expression vector was transfected, this mutated CBP was found to be distributed diffusely in the nucleus (Fig. 2J), but the distribution of endogenous CBP was not affected by the forced expression of CBP (118–2393) and showed a microparticulate pattern (Fig. 2K) as seen in Fig. 2C. Similar results were obtained when pCMV-AR-CFP was cotransfected and exposed to DHT (Fig. 2, L and M). These observations suggest that CBP (118–2393) exerted its dominant negative effect by occupying a CBP-binding site of the AR but not by binding to a transactivation complex to which endogenous CBP binds. This explanation for the dominant negative effect of CBP (118–2393) is not inconsistent because the N-terminal 100 amino acid residues of CBP have been reported to contain a domain for nuclear receptor binding (12).
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, A and B). In contrast, AR-AF-2-CFP, in which CFP was fused to the C terminus of the AR-AF-2 fragment, consisting of the DBD and the AF-2-containing ligand-binding domain (LBD) of the AR, was distributed in a uniform pattern regardless of the presence of DHT (Fig. 4, G and H). The subnuclear distribution pattern was similar in the AR fragments in which fluorescent protein was fused to the N termini of the AR fragments (data not shown). When, in addition to pCMV-AR-AF-1-YFP, pCMV-AR-AF-2-CFP was cotransfected at an amount lower than that of pCMV-AR-AF-1-YFP on a molar basis, the foci formation of AR-AF-1-YFP collapsed (Fig. 4C). Cotransfection of pCMV-AR-AF-2-CFP at an amount higher than that of pCMV-AR-AF-1-YFP perfectly excluded AR-AF-1-YFP from the nucleus to the cytosol (Fig. 4D). However, AR-AF-1-YFP was translocated again into the nucleus with foci formation by the addition of DHT (Fig. 4E), and AR-AF-2-CFP was also distributed with foci formation coinciding with AR-AF-1-YFP (Fig. 4F). Both the AR-AF-1-YFP and AR-AF-2-CFP fragments contained the DBD. Therefore, there was a possibility that the overlapped DBDs affected the interaction between AR-AF-1 and AR-AF-2. The DBD-deleted NTD, (DBD)AR-AF-1-YFP, was distributed dominantly in the cytosol (Fig. 4I), probably because this fragment lacked the nuclear localization signal (NLS). (DBD)AR-AF-1-YFP was translocated into the nucleus with foci formation in the presence of DHT-bound AR-AF-2-CFP (Fig. 4J) as seen in cells transfected with both pCMV-AR-AF-1-YFP and pCMV-AR-AF-2-CFP (Fig. 4, E and F), indicating that the overlapped DBD did not hinder the function of each AR fragment. When pCMV-YFP-SRC-1 or pCMV-YFP-TIF2 was cotransfected with pCMV-AR-AF-1-CFP, these coactivators formed foci coinciding with AR-AF-1-CFP (Fig. 4, K and L). AR-AF-1-YFP activated the transcription of the luciferase reporter gene regardless of the presence of DHT, and the level of the transactivation function was about 70% of that of the AR-AF-1 not fused to YFP. Although AR-AF-2-CFP showed ligand-dependent transcription activation, it was less than 6% of that of AR-AF-1-YFP (Fig. 5). Ligand-independent transactivation by AR-AF-1-YFP was suppressed to about 17% of the original activity by cotransfection of equivalent amounts or twice as much of pCMV-AR-AF-2-CFP in the absence of DHT, but transactivation increased 3 to 4 times in the presence of DHT (Fig. 5). Essentially similar results were obtained with the AR fragments, which were not fused to fluorescent protein (Fig. 5) and in the experiments using CV-1 and LNCaP cells (data not shown).
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), the existence of foci formation did not reflect differences in the levels of the transcriptional activation function. The formation of foci was evenly observed by two-dimensional imaging among cells transfected with the expression vectors for the full-length AR-CFP, the AR-AF-1-YFP alone and the AR-AF-1-YFP in combination with the AR-AF-2-CFP (Figs. 1B and 4, B and E), although the levels of transactivation were different (Fig. 5). Therefore, to clarify whether any differences in the subnuclear localization exist among these AR fragments and the full-length AR, we analyzed the intranuclear distribution by a computer-assisted three-dimensional imaging method. Nuclear DNA in living cells was stained with Hoechst 33342, which has been used to discriminate the heterochromatin region from the euchromatin region. To obtain the heterochromatin image, the less stained areas (namely euchromatin region) were cut off and shown as blank images (Fig. 6E). Most of the liganded full-length AR-GFP was detected as discrete multiple spots (Fig. 6A, surface view), and the green spots were located in the euchromatin region (Fig. 6F, surface view). In contrast, AR-AF-2-CFP was homogenously distributed as a single large spot, except for the nucleoli, regardless of the presence of DHT (Fig. 6C, surface view), over the whole chromatin (Fig. 6, I, surface view, and J, tomographic view). AR-AF-1-YFP was distributed in an intermediate pattern between the patterns of the full-length AR and the AR-AF-2-CFP, i.e. a reticular pattern (Fig. 6B, surface), in which the spatial distribution of the YFP signal was not discontinuous and was calculated as one volume, mostly in the euchromatin region (Fig. 6, G, surface, and H, tomogram). However, in combination with DHT-bound AR-AF-2-CFP, the AR-AF-1-YFP formed isolated spots and showed distribution which was essentially identical with that of the full-length AR-GFP (Fig. 6, D, surface, and K, tomogram). The DHT-bound AR-AF-2-CFP in combination with the DBD-deleted NTD, (DBD)AR-AF-1, also formed isolated spots (Fig. 6L, surface), indicating that overlapped DBDs of the AR-AF-1 and AR-AF-2 fragments did not affect the interaction between the two fragments. Essentially the same results were obtained using CV-1 and LNCaP cells.
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by Three-Dimensional Imaging) were found to exist as a distinct volume in one nucleus (Fig. 7A). The nuclear volume occupied by these 300 spots was estimated to be approximately 10% of the total nuclear volume in the present experimental conditions. When the three-dimensional imaging method was applied to liganded GR-YFP and ER-GFP, in which YFP and GFP were fused to the C termini of GR and ER, respectively, a distribution of discrete spots in the nucleus was also observed (Fig. 7, B and C), and the numbers of spots (308 ± 23, n = 8) were similar to that of AR-CFP. The number of spots was calculated from the three-dimensional images processed under the same conditions. When pCMV-AR-CFP was cotransfected with pCMV-GR-YFP, they formed identical spots in the presence of their ligands (Fig. 7D), and naturally the number of spots was similar to that of AR-CFP or GR-YFP alone. Similar results were obtained by the cotransfection of AR-CFP and ER-GFP (Fig. 7E). The number of AR-CFP spots was not affected by cotransfection, i.e. overexpression, of coactivators such as YFP-SRC-1, YFP-TIF2 and YFP-CBP (see legend for Fig. 7). Furthermore, YFP-SRC-1 (Fig. 7F), YFP-TIF2 (Fig. 7G) and YFP-CBP (Fig. 7H) were all colocalized with AR-CFP, forming identical spots.
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). The endogenous CBP was also distributed in a speckled pattern, and it was found, although indirectly, that the liganded AR-CFP bound to the endogenous CBP (Fig. 2, D–F). The introduction of a CBP mutant with a dominant negative effect on the AR-dependent transactivation destroyed the foci formation of AR-GFP, and AR-GFP was distributed in a uniform pattern. The involvement of CBP in the AR foci formation is also supported by reports that the addition of 12-O-tetradecanoylphorbol-13-acetate (20) or transfection of the expression plasmid for NF-
B (21) repressed the AR-dependent transactivation, and competition for endogenous CBP was speculated as the mechanism of this repression (20, 21). A diffuse intranuclear distribution of GFP-SRC-1 in cells transfected with the GFP-SRC-1 expression vector alone and a distribution of endogenous CBP in a microparticulate (finely speckled) pattern in nontransfected cells have also been reported by other groups (6, 22). A physiological significance of the difference in the intranuclear distribution between CBP and p160 coactivators such as SRC-1 and TIF2 remains unknown, but one of the speculations is as follows. CBP is an integrator of multiple signal transductions and interacts with various transcription regulators (12). Therefore, endogenous or exogenous CBP may make discrete foci by forming complexes with transcription factors other than the steroid receptors as well.
The AR is unusual among steroid hormone receptors in its AF-1 and AF-2 functions. Most of the transactivation functions of the AR exist in AF-1 in the NTD (16, 17, 18, 19), and the ligand-dependent transactivation function of AF-2 in the LBD is extremely low in mammalian cells (15). In the absence of ligand, the LBD of the AR is thought to keep the AR retained in the cytosol (17) by masking the NLS, which was reported to exist within the DBD and the hinge region corresponding to amino acid residues 557–653 (23), 617–633 (17, 24), or 627–658 (25). The unliganded LBD has also been reported to suppress the constitutive transactivation function of the NTD (17, 23). The NTD of the AR has recently been shown to interact directly with TIF2 and SRC-1 (26, 27). The present study succeeded in perfectly visualizing these reports on AF-1 and AF-2 function in the AR (Fig. 4), as follows. Both the AR-AF-1-YFP and the AR-AF-2-CFP fragments prepared in the present study contained the exposed NLS, and therefore, each of them was translocated into the nucleus without ligand binding. The cotransfection of AR-AF-2-CFP and AR-AF-1-YFP suppressed the foci formation of AR-AF-1-YFP (Fig. 4C) and excluded the AR-AF-1-YFP to the cytosol (Fig. 4D). The transfection of AR-AF-1-YFP alone formed foci in the nucleus, reflecting its strong constitutive transactivation function, whereas the AR-AF-2-CFP alone did not produce foci even in the presence of ligand. YFP-SRC-1 and YFP-TIF2 were shown to be localized within the AR-AF-1-CFP foci (Fig. 4, K and L). The rapid recruitment of SRC-1 to the ER foci upon exposure to E2 was studied by Stenoien et al. (6, 28), who reported that helix 12 and the AF-2 domain in the LBD are essential for the agonist-induced recruitment of coactivators, but the AF-1 domain and DBD are dispensable. In contrast to the ER, AR-AF-1 can recruit SRC-1 and TIF2. Thus, the recruitment of coactivators is suggested to be an essential and common process for transactivation by the various steroid receptors, but the molecular mechanism is different among the receptors.
AR-AF-1 (NTD) and AR-AF-2 (LBD) showed a synergistic action. If the degree of transactivation of MMTV (mouse mammarian tumor virus) promoter induced by both AR-AF-1 (NTD) and liganded AR-AF-2 (LBD) is set at 100%, the transactivation induced by AR-AF-1 alone was 28% in COS7 cells, and that by liganded AR-AF-2 alone was less than 2% (Fig. 5). Similarly, the degree of transactivation by the AR NTD alone and by the liganded AR LBD alone have been reported to be 10–75% (18, 24, 29) and 0–3% (18, 19, 24, 25, 29), respectively, of that by the liganded wild-type AR in various types of cells. For an explanation of this synergistic mechanism, the following has been speculated. AR-AF-2, although its intrinsic activity is negligibly low, can associate with AR-AF-1 in the presence of ligand, and this intramolecular interaction results in the formation of a complete platform to recruit coactivators. This conformational change then elicits the full activity of the AR (19, 26, 27, 30, 31, 32). The above speculation has been deduced from overall consideration of the results obtained by a reporter gene assay, a GST pull-down assay and a two-hybrid assay. If intramolecular interplay between the AF-1 and AF-2 domains really proceeds in living cells, there must be a difference between the structures of the receptor/coactivator complex in the case with AR-AF-1 alone vs. the case with full-length AR or the coexistence of AR-AF-1 and AR-AF-2. However, that difference was not detectable by two-dimensional image analysis. Therefore, a three-dimensional imaging method, which has recently been developed by us to detect accurately any antiandrogenic effect (9), was applied to the analyses of the subnuclear distribution of the fluorescent protein-tagged receptors. Using this method, to our knowledge, we for the first time succeeded in obtaining direct evidence of structural differences between the receptor/coactivator complex of AR-AF-1 alone and that of the full-length AR (Fig. 6, A and B). Three-dimensional imaging also revealed that the native AR/coactivator complexes, i.e. the AR foci, are distributed inside euchromatin, where transcription is thought to be active. This three-dimensional imaging method allowed analysis of the accurate subnuclear spatial localization and quantitation of the foci formed by the steroid hormone receptor labeled with fluorescent protein. The following was thus revealed. The AR, GR, and ER were accumulated in identical subnuclear compartments in the presence of ligand. The maximum number of subnuclear compartments, which was considered to be observed with the overexpression of the steroid hormone receptors, i.e. with the transfection of their expression vectors, was approximately 300 in one COS7 cell nucleus. Coactivators such as SRC-1, TIF2, and CBP were also accumulated in the same subnuclear compartments with those of the steroid hormone receptors. An overexpression of these coactivators did not affect the maximum number of compartments. These findings might propose the hypothesis that transcriptionally activated steroid hormone receptors, regardless of the type of the receptor, are transferred to common compartments located in the euchromatin region and form a complex with coactivators. These receptor/coactivator complexes are then mobilized to the target genes of each receptor and are then rapidly returned to the compartment after use. This hypothesis would be supported by recent reports that liganded GFP-GR undergoes a rapid exchange between chromatin and the nucleoplasmic compartment (33) and that liganded GR and ER are retained in the nuclear matrix even after DNase-digestion of the nucleus (6, 34). Furthermore, the dynamics of the ER in the nucleus have recently been revealed by fluorescence recovery after photobleaching. It was shown that the E2-bound CFP-ER/YFP-SRC-1 complex associates with the nuclear matrix (i.e. formation of foci) but undergoes rapid exchange within seconds (35).
In conclusion, the present study on the AR and foci formation in the nucleus provides novel findings regarding the physiological significance of the subnuclear foci formation of the steroid hormone receptors. CBP is at least one of the factors essential to foci formation of the AR. Foci formation indicates the formation of a complete steric conformation of the AR essential for transactivation, for which both the NTD and LBD are necessary, and is also evidence of the intranuclear compartmentalization that may be common to other steroid receptors and essential for full transcriptional activation. In addition, the three-dimensional imaging method performed in the present study seems to be an effective modality for further investigating the mechanism of nuclear receptor-mediated transcription.
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(DBD)AR-AF-1), corresponding to AR 1–501, was prepared by excising the DBD between HindIII and KpnI sites of pCMX-AR-AF-1. The expression plasmids for the full-length mouse CBP 1–2443 (pcDNA/mCBP) and for the truncated mutants, pcDNA/mCBP (118–2393), pcDNA/mCBP (313–468), pcDNA/mCBP (738–2393), and pcDNA/mCBP (1570–1891), were prepared as previously described (37). Human cDNAs for the GR, SRC-1, and TIF2 were prepared as reported previously (29), and the human ER cDNA was provided by Dr. Shigeaki Kato (Tokyo University, Japan). The expression plasmids for AR-GFP (pCMV-AR-GFP) and AR-CFP (pCMV-AR-CFP) chimeras were constructed by inserting the full-length AR cDNA into the NheI-SmaI sites of pEGFP-N1 and pECFP-N1 (CLONTECH Laboratories, Inc., Palo Alto, CA), respectively. The expression plasmids for AR-AF-1-YFP (pCMV-AR-AF-1-YFP) and AR-AF-2-CFP (pCMV-AR-AF-2-CFP) chimeras, in which fluorescent proteins were fused to the C termini of the AR fragments, were prepared by inserting the AR-AF-1 and AR-AF-2 cDNAs into the NheI-XhoI sites of pEYFP-N1 (CLONTECH Laboratories, Inc.) and the NheI-KpnI sites of pECFP-N1, respectively, using PCR techniques. The expression plasmid for AR-AF-1-CFP (pCMV-AR-AF-1-CFP) was prepared by inserting the AR-AF-1 cDNA into the NheI-XhoI sites of pECFP-N1. Similarly, the expression plasmids for GR-YFP (pCMV-GR-YFP), ER-GFP (pCMV-ER-GFP), YFP-SRC-1 (pCMV-YFP-SRC-1), YFP-TIF2 (pCMV-YFP-TIF2) and (DBD)AR-AF-1-YFP were constructed. The plasmids for YFP-AR-AF-1 (pCMV-YFP-AR-AF-1) and CFP-AR-AF-2 (pCMV-CFP-AR-AF-2), in which fluorescent proteins were fused to the N termini of the AR fragments, were prepared by inserting the AR-AF-1 and AR-AF-2 cDNAs into the NheI-XhoI sites of pEYFP-C1 and the BglII-XhoI sites of pECFP-C1 (CLONTECH Laboratories, Inc.), respectively. The expression vector for YFP-CBP chimera (pCMV-YFP-CBP) was constructed by inserting the YFP cDNA into the HindIII site of pcDNA/mCBP, upstream of the CBP sequence.
Reporter Assay
Kidney-derived cell lines, COS7 and CV-1, and a human prostatic cancer cell-line, LNCaP, were obtained from American Type Culture Collection (Manassas, VA). COS7 and CV-1 cells were maintained in DMEM (Life Technologies, Inc.) supplemented with 10% FBS, 2 mM L-glutamine and 100 U/ml of penicillin-streptomycin. LNCaP cells were similarly maintained except for the use of Roswell Park Memorial Institute 1640 medium instead of DMEM. The cells, cultured in 6-well plates (3 x 105 cells per well), were transfected with 1 µg/well of pGL3-MMTV as the reporter, 2 ng/well of pRL-CMV (a Renilla luciferase vector, Promega Corp., Madison, WI) as the internal control, and 0.1 to 0.2 µg/well of the expression vector for the AR, the AR fragment, the chimeric receptor or the mutated CBP, using 7 µl/well of SuperFect reagent (QIAGEN, Hilden, Germany). For coexpression studies, the total amount of vector added to each well was equalized by the addition of empty vector, unless otherwise indicated. Starting 3 h after transfection, the cells were incubated for 48 h in DMEM with 10% charcoal-treated FBS in the presence or absence of 10-8 M 5-dihydrotestosterone, 10-8 M dexamethasone, 10-8 M E2, or 10-6 M OHF. The cells were then solubilized with 500 µl of lysis buffer (Promega Corp.) and the activities of the reporter gene were determined by the Dual-Luciferase Reporter Assay System (Promega Corp.). One-way analysis of variance followed by Scheffé’s test was used for multi-group comparisons.
Fluorescence Microscopy and Three-Dimensional Image Analysis
The cells were cultured in 35-mm glass-bottom dishes (MatTek) (3 x 105 cells/dish) and then transfected with various plasmids in a total amount of 0.5 µg/dish using 2.5 µl of SuperFect. For coexpression studies, the total amount of vector added to each dish was equalized by the addition of empty vector, unless otherwise indicated. Sixteen to 24 h after incubation in DMEM containing 10% charcoal-treated FBS, the culture media were replaced with fresh DMEM in the presence or absence of the hormones or chemicals, and then the cells were observed with a Leica Corp. TSP-SP invert confocal laser scanning microscope (Leica Corp. Microsystems, Heidelberg, Germany), using a 100x, 1.4 numerical aperture PL APO oil immersion objective. Imaging for GFP, YFP, and CFP was performed by excitation with the 488-nm, 514-nm, and 450-nm lines, respectively, from an argon laser, and the emissions were viewed through band passes ranging from 500 to 550 nm, from 530 to 590 nm, and from 470 to 500 nm, respectively, by band pass regulation with a Prism System (Leica Corp. Microsystems). For simultaneous imaging of multiple fluorescent proteins, the laser line was changed, and the band pass was further finely controlled so as not to overlap emissions. For example, for the simultaneous observation of AR-CFP and ER-GFP, the chimeras were imaged by excitation with 450-nm and 514-nm lines, respectively, and the emissions through band passes ranging from 460 to 480 nm and from 580 to 620 nm, respectively, were observed. The nuclei were stained with Hoechst 33342 (Molecular Probes, Inc., Eugene, OR) (2 µg/ml) and excited with the 350-nm line from a UV laser, and the emission was viewed through a band pass ranging from 400 to 450 nm. For immunostaining, the cells were washed using PBS and fixed in 4% (vol/vol) paraformaldehyde in PBS for 30 min at 25 C. After being blocked in 10% goat serum for 1 h, the cells were incubated with anti-CBP mouse monoclonal antibody [CBP (C-1) against the C terminus of CBP] or anti-CBP rabbit polyclonal antibodies [CBP (A-22) against the N terminus and CBP (451) against the CREB-binding domain] (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at 1:100-fold dilution for 1 h, followed by incubation with 0.33 µg/ml of Alexa Fluor 594-labeled antimouse or antirabbit IgG or Alexa Fluor 488-labeled antirabbit IgG (Molecular Probes, Inc.) for 1 h at 25 C. After being washed by PBS, the stained cells were observed with a confocal microscope by excitation with the 568-nm line from a krypton laser and emission from 600–640 nm for Alexa Fluor 594-labeled IgG or by excitation with the 514-nm line from an argon laser and emission from 520 to 560 nm for Alexa Fluor 488-labeled IgG. A three-dimensional imaging study was performed essentially in the same manner as previously reported (9). In brief, a series of 30–50 scanning images were collected for each single nucleus, and these two-dimensional tomograms were reconstructed using the three-dimensional analysis software of TRI Graphics Program (Ratoc System Engineering, Tokyo, Japan). Both the spatial distribution and calculations of the fluorescent proteins as a distinct volume were made possible by removing scattering background fluorescence and lens spherical aberrations and then by separating each particle.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Abbreviations: AF, Activation function; CBP, CREB-binding protein; CFP, cyan fluorescent protein; DBD, DNA-binding domain; DHT, dihydrotestosterone; GFP, green fluorescent protein; LBD, ligand-binding domain; MMTV, mouse mammarian tumor virus; NLS, nuclear localization signal; NTD, N-terminal domain; OHF, hydroxyflutamide; SRC-1, steroid receptor coactivator-1; TIF2, transcriptional intermediary factor 2; YFP, yellow fluorescent protein.
Received for publication July 17, 2001. Accepted for publication December 14, 2001.
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