Requirement of Retinoic Acid Receptor Isotypes {alpha}, ß, and {gamma} during the Initial Steps of Neural Differentiation of PCC7 Cells

Christina Zechel

Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, Medical School, 55099 Mainz, Germany

Address all correspondence and requests for reprints to: Christina Zechel, Institute of Physiological Chemistry and Pathobiochemistry, Johannes Gutenberg-University, Medical School, Duesberg Weg 6, 55099 Mainz, Germany. E-mail: zechel{at}uni-mainz.de.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Retinoic acid (RA) is indispensable for morphogenesis and differentiation of several tissues, including the nervous system. The requirement of the RA receptor (RAR) isotypes {alpha}, ß, and {gamma} and the putative role of retinoid X receptor-(RXR) signaling in RA-induced neural differentiation, was analyzed. For this compound-selective retinoids and the murine embryonal carcinoma cell line PCC7, a model system for RA-dependent neural differentiation was used. The present paper shows that proliferating PCC7 cells primarily express RXR{alpha} and RAR{alpha}, lower levels of RXRß, and barely detectable amounts of RARß, RAR{gamma}, and RXR{gamma}. At receptor-selective concentrations, only a RAR{alpha} or RAR{gamma} agonist induced the typical tissue-like differentiation pattern consisting of neuronal and nonneuronal cells. Differentiation-associated processes, such as the down-regulation of Oct4, up-regulation of certain nuclear receptors and proneuronal genes, and the induction of neuronal markers could be triggered by receptor-selective concentrations of a RAR{alpha}-, ß-, or {gamma}-selective agonist, although with distinct efficacy. The differences are only partially explained by the distinct RAR{alpha}, ß, and {gamma} expression levels and the dissociation constants for the bound retinoids, suggesting differential requirement of RAR isotypes during the initial stages of neural differentiation of PCC7 cells.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RETINOIC ACID (RA) is a biologically active derivative of vitamin A, which regulates proliferation, differentiation, apoptosis, and malignant transformation of cells. RA is indispensable for morphogenesis and differentiation of several tissues (1, 2, 3). In the developing nervous system, RA is necessary for primary neurogenesis and hindbrain development (4). Moreover, RA specifies motor neuron subsets and regulates the development of the spinal cord (5), and controls the expression of certain genes involved in neurotransmission processes (Refs. 3 and 6 and references therein). Postnatal vitamin A-deprived animals indicated that RA functions as an essential competence factor for long-term synaptic plasticity of the adult brain (7).

RA exerts its effects through two classes of ligand-dependent transactivators, the RA receptors (RARs) and the retinoid X receptors (RXRs), which are members of the nuclear receptor family (for overview, see Ref. 8). Owing to the structure of the ligand binding pocket and the flexure limits of the ligand, RXRs are bound and activated by 9-cis RA (9cRA), whereas RARs are bound and activated by both, all-trans RA (atRA) and 9cRA (9, 10). The three members (isotypes) of each class of retinoid receptors, designated RAR {alpha}, ß, and {gamma} and RXR {alpha}, ß, and {gamma} are encoded by different genes, each of which can generate multiple RNA splice variants encoding receptor isoforms with unique amino-terminal regions. RXRs can either act as homodimers, or as heterodimeric partners of RARs and several other members of the nuclear receptor family (11, 12). Gene targeting experiments in mice provided evidence that the RXR/RAR heterodimer transduces the retinoid signal during mouse development (13). RXRs enhance the RAR’s efficiency for binding to RA response elements (RAREs), the specificity of RARE recognition, and modulate RAR signaling (11, 12). The apo-RXR/RAR heterodimer provides surfaces for the interaction of nuclear corepressors, SMRT (silencing mediator of retinoid and thyroid receptors) and NCoR (nuclear receptor corepressor), both of which permit recruitment of corepressor complexes that repress transcription (14). RA induces conformational changes within the ligand binding domain of its cognate receptors, and involves the transconformation of the conserved core of the activation function 2 (Refs. 10 and 12 and references therein). This results in release of corepressor complexes and the concomitant binding of transcription-facilitating multiprotein complexes, the mechanistic basis of which has been well elaborated by means of molecular, biochemical, and structural analysis (14, 15, 16, 17, 18).

Both the RAR and RXR isotypes exhibit a high evolutionary conservation and spatiotemporally regulated expression, suggesting that each of them displays some specific function (11, 12). For example, RXR{alpha}/RAR{gamma} heterodimers are instrumental in patterning of the craniofacial skeletal elements, whereas RXR{alpha}/RAR{alpha} may be preferentially involved in the generation of neural crest-derived arterial smooth muscle cells, and both RXR{alpha}/RARß and RXR{alpha}/RAR{gamma} heterodimers function during development of the ocular mesenchyme (19). Moreover, RAR {alpha}, ß, and {gamma} take distinct roles in early embryonic morphogenesis and hindbrain patterning in particular (20, 21). Null mutation of RARß and RXR{gamma} resulted in locomotor defects and compromised learning in behavioral tests (22, 23). Specific and redundant functions of RXR and RAR isotypes became also evident from gene targeting experiments in the murine embryonal carcinoma cell line F9. In combination with assays using compound-selective synthetic retinoids, these experiments suggested the requirement of RXR{alpha} and RAR{gamma} for endodermal differentiation (24, 25, 26, 27).

Little is known about the potential of RXRs and RARs to induce the signal cascades involved in neural differentiation. In principle, the retinoid signal may be transduced by the RXR homodimer, the RXR/RAR heterodimer, or via RXRs that cooperate with distinct nuclear receptors known to modulate neurogenesis (Refs. 3 and 28 and references therein). To investigate the role of RXR and RAR isotypes in neural differentiation the murine embryonal carcinoma (EC) cell line PCC7, which resembles pluripotent stem cells of a neuroectodermal origin was used. This cell line reproducibly differentiates into a tissue-like pattern of neuronal and nonneuronal cells upon exposure to RA (29, 30, 31). Cell lineage determination starts within hours after retinoid addition, is completed within 3 d and accompanied by apoptotic death in a reproducible fraction of cells (30, 31, 32). The neuronal precursor cells develop neurites and express neuronal markers, and from d 4 on, the initial network of neuronal cells undergoes a cell death-associated restructuring. This involves the aggregation of the cell bodies of the immature neurons and results in the formation of neuron-aggregates that interconnect by fasciculated neurites (30, 31). In addition, the neuronal cells begin to develop polarity in a way that reflects the situation in the mouse brain and, which is completed in the second week by the acquirement of conductance properties (30). Like in brain tissue, nonneuronal cells support survival, as well as the sequentially ordered differentiation and structurally and functionally complete maturation of the neuronal derivatives (29, 30, 31, 32).

Proliferating PCC7 cells predominantly express RXR{alpha} and RAR{alpha}, lower levels of RXRß, and barely detectable amounts of RARß, RAR{gamma}, and RXR{gamma}. Because it has been reported that functional redundancies between retinoid receptor isotypes may be artifactually generated by compound knockouts (27, 33), I used compound-selective retinoids to delineate the role of the three RAR isotypes and of RAR-independent RXR signaling. Exposure of proliferating PCC7 cells to receptor selective agonists or antagonists indicated that neural differentiation requires RAR signaling. At isotype-selective concentrations the RAR agonists differed in their potential to induce neural differentiation and differentiation-associated gene expression. The paper shows that these differences are only partially explained by the distinct expression levels of RAR{alpha}, ß, and {gamma} and the dissociation constants (KDs) of the bound retinoids. The possible differential requirement of the RAR{alpha}, ß, and {gamma} during early stages of neural differentiation of PCC7 cells is discussed.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The EC cell line PCC7 is a useful model to study RA-dependent neural differentiation from the stage of fate choice decision until the development of functional neurons (29, 30, 31, 32). To determine the requirement of RXR and RAR signaling for the transduction of the RA-signal I treated PCC7 cultures with decreasing concentrations of receptor-selective retinoids and compared their morphology and gene expression pattern with those treated with RA. In this study, I did not discriminate between signaling through RXR{alpha}, ß, or {gamma} because RXR isotype-selective ligands are not yet available, due to the close similarities of the residues forming the ligand binding pockets (Refs. 10 and 34 and references therein).

Effects of Receptor-Selective Retinoids on Cofactor Binding and Transactivation
Numerous reports indicate that transcriptional regulation by RA involves ligand-regulated recruitment of the nuclear corepressor SMRT and the nuclear coactivators (NCoA)-1, -2, and -3, which constitute the p160 protein family; corepressor (CoR) and coactivator (CoA) binding is mutually exclusive (14, 16, 35). CoR and CoA binding, as well as the apo and holo state of the receptor’s ligand binding domain likely exist in a dynamic equilibrium and transcriptional activation is mechanistically linked to CoR release (36, 37, 38). In addition, the RXR/RAR heterodimer is only permissive for RXR-ligand signaling in cases where CoR binding to the heterodimer is largely impaired by e.g. the cofactor content of a given cell or RAR-selective ligands (39).

Glutathione-S-transferase (GST)-pull-down and mammalian two-hybrid analysis was performed to elucidate whether the retinoids used in this study permit CoR and/or CoA binding to RXRs (exemplary tested for RXR{alpha}) and the RAR isotypes {alpha}, ß, and {gamma}; atRA and the RAR pan-agonist CD367 served as controls. A 100-nM concentration of Am 580 induced NCoA-2 binding to RAR{alpha} and less efficiently to RARß, and 10 nM only to RAR{alpha} (Fig. 1CGo and data not shown). At 100 nM, CD2314 was not a potent inducer of NCoA-2 binding to RARß in vitro, albeit it could recruit NCoA2-VP16 to Gal4-RARß in human embryonic kidney (HEK) 293T cells (Fig. 1CGo and data not shown). At higher concentrations, CD2314 (1 µM) efficiently induced NCoA-2 binding to GST-RARß and only weakly to RAR{alpha} or RAR{gamma} (data not shown). At 100 nM, CD666 induced NCoA-2 binding to RAR{gamma}, weakly to RARß, but not to RAR{alpha}, whereas 10 nM of CD666 only triggered NCoA-2 binding to RAR{gamma} (Fig. 1CGo and data not shown). Thus, 10 nM of Am 580 and CD666 were fully receptor isotype-selective with respect to NCoA-2 binding in vivo and in vitro, and 1 µM of CD2314 was highly RARß selective. In addition, in transactivation assays with HEK293T cells, 10 nM Am 580, 1 µM CD2314, and 10 nM CD666 induced selective transactivation through Gal4-RAR{alpha}, Gal4 RARß, and Gal4-RAR{gamma}, respectively (Fig. 2Go), indicating a similar degree of RAR isotype selectivity in transcriptional activation. In this respect, it is important to note that the potential of ligands to mediate coactivator binding and transactivation may not only depend on ligand binding per se but also on their capability to efficiently induce the transconformation of the activation helix12 and its stabilization in the agonist position (18) that, in turn, could affect the formation of surfaces for the binding of distinct coactivators and corepressors.



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Fig. 1. Effect of Compound-Selective Retinoids on Cofactor Binding

A, Dissociation constants of the retinoids used in this study. Numbers in parentheses indicate references. KD values of atRA: RAR{alpha}, 0.2 nM; RARß, 0.36 nM; RAR{gamma}, 0.2 nM. For KD values of 9cRA for RARs and RXRs, see Ref. 9 . B and C, Pull-down analysis using GST-fused nuclear receptor (NR) ligand binding domains (LBD) and in vitro-translated coactivator NCoA-2. C, Similar analysis using GST-fused corepressors SMRT or NCoR and in vitro-translated full-length RAR{alpha} or RARß. The retinoid receptors analyzed are indicated on the left side of the corresponding set of data.

 


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Fig. 2. Ligand-Dependent CAT Expression in PCC7 and HEK293T Cells

Cells were transiently transfected with the indicated Gal4-retinoid receptor fusion constructs and the (17mer)5-TATA-CAT reporter plasmid. In case of PCC7 cells (A), pEGFP encoding the GFP was cotransfected, and the percentage of GFP+ cells expressing CAT was determined by immunocytochemistry and counting. In case of HEK293T cells (B–D), pCMV-ßGal was cotransfected and the relative CAT activity was determined by ELISA. Results represent three independent series of experiments (±SEM). A, Synthetic retinoids were used at receptor selective concentrations of 10 nM for Am 580 and CD666, and 1 µM for CD2314 in assays with PCC7 cells. For specificities of retinoids, see Fig. 1AGo; the concentrations used are indicated.

 
The RXR-selective ligand CD3640 efficiently induced NCoA-2 binding to RXR{alpha} in vivo and in vitro (Fig. 1BGo and data not shown). Interaction and transactivation assays with CD3640 and RAR isotypes, however, indicated that CD3640 only acts as a RXR-selective agonist (rexinoid) at concentrations below 1 µM. For example, at 1 µM but not at 100 nM CD3640 induced binding of NCoA-2 to GST-RARß, GST-RAR{gamma}, and less efficiently to GST-RAR{alpha} (Fig. 1CGo). Comparable results were obtained in a mammalian two-hybrid system using the Gal4-RAR isotypes and NCoA2-VP16 (data not shown). Moreover, 1 µM but not 100 nM of CD3640 enhanced transcription from the (17mer)5-TATA-chloramphenicol acetyltransferase (CAT) reporter in transiently transfected HEK293T cells 2-fold above the levels seen in the absence of retinoid for Gal4-RARß and Gal4-RAR{gamma}, and somewhat less efficiently Gal4-RAR{alpha} (Fig. 2Go). Notably, none of the RAR-selective retinoids induced NCoA-2 binding to RXR{alpha} even when applied at 1 µM (data not shown).

To analyze whether the receptor-selective concentrations of the RAR agonists or CD3640 were capable of inducing transactivation in PCC7 cells, I cotransfected Gal4-retinoid receptor fusions, the (17mer)5-TATA-CAT reporter and a plasmid coding for the green fluorescent protein (GFP), and determined the number of CAT expressing cells by immunocytochemistry. Note that the transfection rate of PCC7 cells was very low with any kind of technique (see Materials and Methods), excluding quantification of CAT activity in PCC7 cells. Figure 2AGo shows the number of CAT expressing GFP+ PCC7 cells that were obtained with receptor-selective concentrations of Am 580 (10 nM), CD2314 (1 µM), CD666 (10 nM), and CD3640 (100 nM). Keeping in mind that cotransfection of three separate plasmids bares a certain risk that one component might be missing or underrepresented in the transfectant, these data indicate that Am 580, CD2314, CD666, and CD3640 permit receptor-selective transcriptional activation in the PCC7 model at the respective concentrations.

Previous work showed that apo-RAR{alpha} is a potent repressor of transcription that efficiently interacts with SMRT, whereas RARß and RAR{gamma} are poor repressors, which less efficiently bind SMRT and may activate rather than repress target gene transcription in their apo-form (40, 41). SMRT, and its relative, NCoR are present in proliferating PCC7 cells and their expression does not significantly change during the first week of differentiation (my unpublished data).

GST-pull-down analysis was used to investigate the effects of the various agonists and antagonists on corepressor binding (examples in Fig. 1DGo). The results were verified by two-hybrid analysis using HEK293T cells, Gal4-cSMRT (or Gal4-NCoR), and VP16-fused RAR isotypes (data not shown). The strong interaction of SMRT with apo-RAR{alpha} was abrogated by receptor-selective concentrations of Am 580. Reminiscent to a situation observed for certain distinct antagonists (39, 40) RO 41-5253 impaired binding of SMRT and NCoR to RAR{alpha}. In contrast, AGN 193109 decreased the interaction of SMRT with RAR{alpha}, but increased binding of NCoR (Fig. 1DGo and data not shown). Thus, both antagonists might compromise SMRT-dependent repression by RAR{alpha} and render the RXR/RAR{alpha} heterodimer permissive for RXR signaling. The weak binding of SMRT to apo-RARß and apo-RAR{gamma} was impaired by AGN 193109, CD2665, but not by RO 41-5253, and receptor-selective concentrations of CD666 released SMRT from RAR{gamma}, whereas 1 µM of CD2314 had no detectable effect on the formation of the SMRT/RARß complex (Fig. 1DGo and data not shown). The latter suggests that the actual concentration of coactivators and corepressors present in a given cell determines how RARß will respond to CD2314.

Expression of Retinoid Receptors in Proliferating and Differentiating PCC7 Cells
To establish whether proliferating PCC7 cells could respond to the various RXR and/or RAR{alpha}-, ß-, or {gamma}-selective retinoids, I determined the expression of retinoid receptors by RT-PCR. The analysis revealed expression of mRNA for all three RXR and RAR isotypes when amplification was for 40 cycles and of all genes, except for rxr {gamma}, when amplification was for 35 cycles; at 30 cycles, only the rar {alpha}-, rxr {alpha}-, and to a lesser extent the rxr ß-specific primer pair amplified detectable amounts of PCR product (Fig. 3AGo and data not shown).



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Fig. 3. Expression of Retinoid Receptor Isoforms in Proliferating and Differentiating PCC7 Cells

A, RT-PCR analysis using random-primed cDNA derived from proliferating cells. B, RT-PCR analysis using random-primed cDNA derived from PCC7 cultures at the indicated days. Rounds of amplification used in second strand synthesis in A and B are indicated. C, Western blot analysis with specific antibodies and WCEs from PCC7 cells at the indicated stages. Actin served as a loading control. d0, Cells before exposure to retinoid; d1–3, Cells at d 1, 2, or 3 after exposure to retinoid. Retinoids were used at the indicated concentrations; for specificities and KD values, see Fig. 1AGo.

 
Next, I analyzed whether the expression of RXR and RAR isotypes changed during the stage fate choice, which is accomplished until d 3 after exposure to atRA (32). Note, in this respect, that RA-responsive elements have been found upstream of the RAR{alpha}2, RARß2, and RAR{gamma}2 gene (11, 12). I determined the expression of all RXR and RAR isotypes by RT-PCR but did not discriminate between the various RXR and RAR isoforms, except for RARß2. Expression of RAR{alpha}, RARß, and RAR{gamma} was also investigated by Western blot analysis.

After exposure to atRA, PCC7 cells showed a moderate up-regulation of RXR{alpha}, RXRß, and RAR{alpha} mRNA expression levels (data not shown). This resulted in an even less pronounced up-regulation of RAR{alpha} on the protein level (Fig. 3CGo). In contrast, expression of RAR{gamma}, and of RXR{gamma} and RARß in particular, was largely affected by atRA. Notably, RAR{gamma} mRNA expression was up-regulated during the first 24 h after atRA addition, doubled within the next day, and remained on this level for the next 24 h (Fig. 3BGo). However, the RAR{gamma} protein was not detectable by Western blot analysis with specific antibodies (data not shown). The very weak signal for RXR{gamma} expression obtained by RT-PCR with RNA from proliferating PCC7 cells turned into a strong band for RNA from d 3 cultures (Fig. 3BGo). In the case of RARß, three closely migrating bands of 51–56 kDa (most probably RARß2, RARß1, and RARß3) became detectable in Western blots with RARß-specific antibodies and protein extracts from d 1 cultures (Fig. 3CGo; fragment size determination was with sodium dodecyl sulfate (SDS) gels containing less polyacrylamide). Induction of RARß2 (51 kDa) expression, but not of the corresponding RARß1 (54 kDa) and RARß3 (56 kDa) isoforms, has also been reported from RA-treated P19 cells (42). RT-PCR analysis with primer pairs that either amplified all RARß isoforms, or solely RARß2, indicated that up-regulation was on the RNA level and included the RARß2 isoform (data not shown). The drastic up-regulation of RXR{gamma} and RARß during neural differentiation of PCC7 cells is particularly interesting in view of the finding that knockout mice indicated an essential role of RARß and RXR{gamma} in the acquirement of higher cognitive functions (22, 23), and that postnatal vitamin A-deprived mice showed a vitamin A deficiency-related cognitive effect and a reduction of 25–30% of RARß and RXRß/{gamma} expression in brain (43). Moreover, these results suggested that RARß is required for processes downstream of neural differentiation initiation, which is in agreement with the observation that RARß mRNA (various isoforms) was particularly abundant in the developing nervous system (11).

To investigate which RAR isotypes transduced the signal that up-regulated the retinoid receptors, I performed comparative RT-PCR and Western blot analysis with extracts from Am 580-, CD2314-, or CD666-treated cultures. Expression of RXR{alpha}, RXRß, and RAR{alpha} was only weakly modulated by these ligands, whereas expression of RAR{gamma}, and particularly RXR{gamma} and RARß, was strongly affected (Fig. 3Go and Table 1Go). Consistent with their potential to trigger neural differentiation (see below) and CoA binding, and reflecting their KD values and expression levels in proliferating PCC7 cells, 10 nM Am 580 more efficiently up-regulated RAR{gamma} expression than did 1 µM CD2314 or 100 nM CD666 (Fig. 3BGo and data not shown). In contrast, 100 nM (or 10 nM) of CD666 more efficiently up-regulated RXR{gamma} mRNA levels than 100 nM (or 10 nM) of Am 580, even though PCC7 cells express less RAR{gamma} than RAR{alpha}; 1 µM of CD2314 did not up-regulate RXR{gamma} (Fig. 3BGo and Table 1Go). Thus, up-regulation of RXR{gamma} expression may specifically involve RAR{gamma} signaling. When differentiation was induced with 100 nM (or 10 nM) of atRA and CD666, the strong induction of RARß expression was transient and confined to d 1. In contrast, Am 580 (>1 nM)- and CD2314 (≥1 µM)-mediated up-regulation of RARß persisted at least until d 3 (Fig. 3CGo), which might reflect differences in the transcription of the RARß-RARE by the RAR isotypes. Note that autoinduction of RARß expression has been reported from other cell lines (25, 27).


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Table 1. Effects of Synthetic Retinoids on Morphological Differentiation, Gene Expression, and Induction of Neuronal Differentiation Makers

 
Induction of Neural Differentiation by RAR-Selective Agonists
PCC7 cells were grown in monolayer cultures and treated with decreasing concentrations of RAR-selective agonists. The results are summarized in Table 2Go. Immunocytochemistry showed that 10 nM of CD367 or Am 580, and less efficiently CD666, induced formation of a tissue-like neural differentiation pattern with neuronal [GAP (growth-associated protein) 43+, MAP (microtubule-associated protein) 2+, SYP (synaptophysin) +, Tau+], fibroblast-like (Thy 1.2+), glial [GFAP (glial fibrillary acidic protein) +], and few endothelial (MESA+) cells, and triggered aggregation of the neuronal cells (Table 2Go and Fig. 4Go, c and o). This was similarly efficient as morphological differentiation triggered by 100 nM atRA or 9cRA (Fig. 4aGo, Table 2Go, and data not shown), and the pattern obtained with 100 nM atRA was in perfect agreement with previous work (30, 44). A 1-nM concentration of atRA or CD666 only triggered outgrowth of extensions and marker expression in a subgroup of cells, whereas 1 nM of CD367 and Am 580 efficiently induced expression of the neuronal markers GAP43, MAP2, Tau, and SYP, as well as partial aggregation of the neuronal cells (Table 2Go and Fig. 4kGo). The RARß agonist CD2314 only mediated a typical differentiation pattern consisting of neuron aggregates and nonneuronal cells when applied at 10 µM, when CD2314 might also transactivate through RAR{alpha} (Table 2Go and Figs. 2BGo and 4eGo). A 1-µM concentration of CD2314 induced outgrowth of extensions and induction of GAP43, MAP2, Tau, and SYP in a subgroup of cells that did not further differentiate or aggregate but remained in a loose network until d 6 and later (Fig. 4mGo). Finally, 100 nM of CD2314 only triggered the development of some crippled extensions (Table 2Go). Thus, receptor-selective concentrations of a RAR{alpha} and RAR{gamma} but not a RARß agonist triggered typical neural differentiation of PCC7 cells.


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Table 2. Effects of Synthetic Retinoids on Morphological Differentiation

 


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Fig. 4. Effect of Compound-Selective Retinoids on Morphological Differentiation of PCC7 Cells

Immunocytochemistry analysis with the neuron-specific antibody {alpha}GAP43 and corresponding Dapi nuclear stain. {alpha}Gap43 was revealed with a Texas Red (TxR)-coupled secondary antibody. Open arrows exemplary point to aggregates of neuronal cells, filled arrows to fasciculated, interconnecting neurites. Areas without fibroblast-like cells are exemplary highlighted by circles, neuron aggregates with incomplete aggregation by rectangles. Bars, 50 µM. Retinoids were used at the indicated concentrations; for specificities and KD values, see Fig. 1AGo.

 
Next, I compared the ratio of neuronal to nonneuronal cell types and the time when they became first detectable by immunocytochemistry. When exposed to 10 nM of RA, CD367, Am 580, and CD666, respectively, subpopulations of cells coexpressing the neuronal markers GAP43, MAP2, and SYP were first seen at d 1; Thy1.2+ cells became detectable at d 3/4, GFAP+ cells at >d 6 and MESA+ cells at >d 6 (data not shown). Thus, the time course of the appearance of the various cell types was not affected. In contrast, the relative number of neuronal to nonneuronal cells was modulated. In particular, 100 nM of CD666, and 10 nM of Am 580 or CD367 increased the number of neuronal cells and neuron aggregates above those seen with 100 nM RA, whereas the relative number of fibroblast-like cells was reduced, resulting in differentiation pattern with large fibroblast-like cell-free areas [Fig. 4Go: compare {alpha}GAP43 stain in panels a, c, and g with the corresponding Dapi (4',6-diamidino-2-phenylindole) nuclear stain in panels b, d, and h].

Together, the above experiments showed that CD666, and Am 580 and CD367 in particular, are more potent inducers of neural differentiation and neuronal fate than atRA. This is unexpected considering their KD values (Fig. 1AGo) but may be related to a more rapid metabolization of atRA as compared with the synthetic retinoids. Alternatively, atRA but not the synthetic retinoids might have affected RXR signaling, owing to traces of the 9cis isomer and/or isomerization in the cells (11).

Considering that the KD value of Am 580 is 8 nM for RAR{alpha}, whereas that one of CD666 is 68 nM for RAR{gamma} (Fig. 1AGo), and that proliferating PCC7 cells express RAR{alpha} at much higher levels than RAR{gamma} (Fig. 3Go), this study suggests that the retinoid signal initiating differentiation may be transduced by both, RAR{alpha} and RAR{gamma}. This was further supported by the observation that coapplication of atRA (or CD367) and a RAR{alpha}- (RO 41-5253) or RARß/{gamma}- (CD2665) selective antagonist did not abrogate differentiation (Table 2Go and Fig. 4Go and see Fig. 7AGo). Involvement of RAR{alpha} and RAR{gamma} has been also reported for morphological differentiation of the EC cell line P19, and is in contrast to that of the EC cell line F9, which appeared to involve RAR{gamma} (Refs. 27 and 45 and references therein).



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Fig. 7. Expression of Neuronal Markers in Retinoid-Treated PCC7 Cultures

A, Immunochemistry analysis of neuron aggregates in differentiating PCC7 cultures. {alpha}MAP2 was detected with a FITC-coupled {alpha}SYP with a Texas Red (TxR)-coupled secondary antibody. Dapi nuclear stain revealed the neuronal and nonneuronal cells. Polar localization of nuclei in neuronal cells is exemplary indicated by filled arrowheads. Bars, 16 µM. B, Western blot analysis of the somato-dendritic marker MAP2. C, Expression of the axonal markers GAP43 and SYP. Actin served as a loading control in B and C. d0, Cells before exposure to retinoid; d1, d2, d3, cells at d 1, 2, or 3 after exposure to retinoid. Ligands were used at the indicated concentrations; for specificities and KD values, see Fig. 1AGo.

 
Influence of RAR-Selective Agonists on Proliferation and Apoptosis
Stepwise reduction of the retinoid concentration to 10 nM for atRA and CD666, or 1 nM for Am 580 resulted in a differentiation pattern that consisted of increasing numbers of both, neuronal and nonneuronal cells (Fig. 4Go and data not shown). Further reduction to 1 nM for atRA and CD666, or 0.1 nM for Am 580 resulted in loss of typical neural differentiation (Fig. 4mGo and data not shown).

To determine the percentage of proliferating cells present during the stage of fate choice, the antigen Ki67 was used. All cells in retinoid-free cultures appeared Ki67-positive in immunocytochemistry, reflecting the fact that Ki67 is specifically expressed in proliferating cell (data not shown). Until d 3, 100 nM of atRA, Am 580 and CD666 reduced the percentage of Ki67+ cells to 18, 12, and 17%, respectively, whereas CD2314 had no detectable effect (Fig. 5AGo and data not shown). Reduction of the CD666 but not the Am 580 concentration from 100 nM to 1 nM enhanced the number of Ki67+ cells (Fig. 5AGo and data not shown). This indicated that Am 580 was a more potent inhibitor of proliferation than CD666, consistent with the binding KDs and the distinct expression level of RAR{alpha} and RAR{gamma} in the responding cells (Figs. 1AGo and 3AGo). Thus, the potential of the RAR-isotype selective retinoids to inhibit proliferation correlated with their potential to induce neural differentiation.



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Fig. 5. Effects of atRA and RAR Isotype-Selective Retinoids on Proliferation and Cell Death

A, Percentage of proliferating cells at 68 h after exposure to retinoid as determined by the number of Ki67+ cells. Results are expressed as percentage of total cell number and represent a minimum of three independent experiments (±SEM). Note that all cells stained positive for the antigen Ki67 in retinoid-free cultures. B, Viability at 24 h after exposure to retinoids (WST-1 test). Results are expressed as percentage of viability of an untreated control and represent a minimum of three independent experiments (±SEM). Retinoids were used at the indicated concentrations; for specificities and KD values, see Fig. 1AGo.

 
RA-induced differentiation of PCC7 is associated with apoptosis (31). The degree of cell death at 24 and 48 h after exposure to RAR-selective agonists was determined by 1) specific staining and counting of apoptotic and/or necrotic cells, and 2) measurement of the percentage of living cells by WST-1 test. With Am 580, the WST-1 values at 24 h and 48 h after retinoid application were very similar to those measured for the identical concentration of atRA (Fig. 5BGo), and the percentage of Annexin-V+ and Propidium Iodide+ cells, a measure for apoptotic and necrotic cells, respectively, was not significantly different for 100 nM (or 10 nM) Am 580 and atRA (data not shown). With CD666, I observed less cell death and a higher percentage of living cells than with the identical concentrations of Am 580 (Fig. 5BGo), which again reflects the binding KDs and the expression levels of RAR{gamma} and RAR{alpha}. In agreement with its poor potential to induce differentiation, CD2314 was not a potent inducer of differentiation-associated cell death. At 1 µM CD2314, the percentage of living cells was not significantly different from that of the untreated control, and the percentage of dead cells did not significantly differ from that observed in retinoid-free cultures (Fig. 5BGo and data not shown).

Together, these data showed that log-wise reduction of the atRA, Am 580, or CD666 concentration resulted in progressive reduction in the levels of both cell death and inhibition of proliferation. This explains the increased cell numbers present in the corresponding cultures during the stage of fate choice and why a more dense network of neuronal and nonneuronal cells could develop in cultures that were treated with lower retinoid concentrations.

RAR-Dependent and -Independent RXR Signaling in Neural Differentiation
RXRs do not only heterodimerize with RARs but are promiscuous partners of several nuclear receptors, enhancing their binding to cognate response elements and modulating their transcriptional activities (11, 12). Although it has been shown that it is the RXR/RAR heterodimer that preferentially transduces the retinoid signal in endodermal differentiation of F9 cells and mouse embryonic development (13, 26, 27, 46), RA-induced neural differentiation might involve signaling through RAR-independent pathways. To elucidate this possibility, I used a RXR-selective agonist (rexinoid) and/or a pan RAR antagonist, either alone or in the presence of atRA, 9cRA, or RAR-selective agonists.

In agreement with the observation that rexinoids failed to trigger differentiation in other EC cells (46), RXR-selective concentrations of CD3640 (100 nM) also did not induce neural differentiation of PCC7 cells. Instead the culture continued to proliferate and, after 1 wk, the cells had developed only some crippled extensions (example in Fig. 4uGo). To further investigate the significance of RXR vs. RAR signaling, cells were treated with atRA (or 9cRA) and the pan RAR antagonist AGN193109. AGN193109 has been shown to completely abrogate RA function in human ectocervical epithelial cells at a molar ratio of 10:1 of AGN193109 to RA (47), and to efficiently counteract RA-induced alleviation of age-related relational memory deficits in mice (48). In the presence of 1 µM AGN193109, 100 nM atRA (or 9cRA) could no longer trigger neural differentiation of PCC7 cells (Fig. 4qGo and Table 2Go), indicating that RAR signaling is required for neural differentiation.

As has been observed in other models (27, 46), concentrations of a rexinoid and a RAR-selective agonist that did not induce differentiation when applied alone synergistically triggered neural differentiation of PCC7 cells (Table 2Go). For example, 100 nM CD3640 plus 1 nM CD666 (or <1 nM Am 580) induced the typical pattern of neuron aggregates and nonneuronal cells seen with 100 nM atRA (Table 2Go and data not shown). The presence of CD3640 (100 nM, but no lower) and CD2314 (100 nM, but no lower) also induced the typical differentiation pattern consisting of neuron aggregates and nonneuronal cells (Fig. 4sGo and data not shown), indicating that the RARß/RXR heterodimer may initiate neural differentiation. Based on the expression level of RAR{alpha}, ß, and {gamma} in proliferating cells (Fig. 3AGo), the binding KDs of Am 580, CD2314, and CD666, as well as the efficacy of the differentiation process in the absence and presence of CD3640 (Table 2Go), these results suggest that induction of neural differentiation was initiated by the RXR/RAR{alpha} and/or RXR/RAR{gamma} rather than the RXR/RARß heterodimer.

Expression of Proneuronal Genes
Neurogenesis requires basic helix-loop-helix (bHLH) transcription factors (2). Moreover, overexpression of neuro D2, mash-1, and ngn-1 cDNA in P19 cells resulted in neuron formation, indicating that these bHLH proteins were sufficient to confer a neuronal fate in uncommitted mammalian cells (49), and, together with the homeobox protein Prox-1, Mash-1 delineate early steps in differentiation of neural stem cells in the developing central nervous system (50).

RT-PCR analysis (35 cycles) revealed expression of mash-1, ngn-1, and prox-1, and neuro-D mRNA in proliferating PCC7 cells (data not shown), which provides a molecular explanation for their strict commitment to neural differentiation. Expression of these proneuronal genes was efficiently up-regulated by atRA, Am 580, and CD666 (Fig. 6AGo and Table 1Go). A 1-µM concentration of the RARß agonist CD2314 also mediated up-regulation of these proneuronal genes, although it could not induce the typical differentiation pattern of PCC7 cells. This suggests that neural differentiation in PCC7 cells requires processes in addition to the up-regulation of mash-1, ngn-1 and prox-1, and neuro-D.



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Fig. 6. Expression of Selected Genes and Oct4 in Retinoid-Treated PCC7 Cultures

A, RT-PCR analysis using random-primed cDNA derived from proliferating or neurally differentiating PCC7 cultures. Rounds of amplification are indicated. I, Proneuronal genes; II, gene encoding the nuclear receptor GCNF; III, internal control gapdh (glyceraldehyde-3-phosphate dehydrogenase). For details, see text. B, Western blot analysis with an antibody specific for the key regulator of differentiation Oct4. Actin served as loading control. d0, Cells before exposure to retinoid; d 1–3, cells at d 1, 2, or 3 after exposure to retinoid. Retinoids were used at the concentrations indicated; for specificities and KD values; see Fig. 1AGo.

 
RT-PCR and Western blot analysis suggests certain preferences for RAR{alpha} and RAR{gamma} in the up-regulation of the neuro-D, ngn-1, and mash-1 mRNA. Firstly, the maximal expression level and the time course of the up-regulation of Neuro D mRNA and protein was not significantly different for 100 nM (or 10 nM) of Am 580 or CD666 (Fig. 6AGo, Table 1Go, and data not shown). Considering the KD values of these retinoids (Fig. 1AGo) and the higher expression level of RAR{alpha} (Fig. 3Go), this proposes a preferential role of RAR{gamma} in the induction of the signal cascades that lead to Neuro D up-regulation. Secondly, weak ngn-1 expression in proliferating PCC7 cells was up-regulated and peaked at d 3 with 1 nM Am 580 and 10 nM CD666 (Table 1Go). Increasing the concentration of Am 580 to 10 nM or 100 nM shifted the expression peak to d 2 and d 1, respectively, and 100 nM CD666 induced a maximal expression at d 2/3 (Fig. 6AGo and Table 1Go). This suggested that RAR{alpha} was the predominant inducer of ngn-1 expression, in keeping with its higher expression level (Fig. 3Go). Finally, 10 nM Am 580, as well as 100 nM atRA triggered an up-regulation of mash-1 that persisted from d 1–3 (Fig. 6AGo and Table 1Go). Increasing the concentrations of Am 580 to 100 nM resulted in a mash-1 expression peak at d 1, whereas an expression peak at d 3 was observed with 1 nM Am 580; the expression profile was also affected by variation of the concentration of CD666 or atRA (Table 1Go). This suggests that mash-1 expression is persistent at a certain retinoid level and transient with higher or lower concentrations. Although it is not known how retinoids affect expression of Neuro D, Mash-1 or Ngn-1, these results suggest a functional redundancy of RAR{alpha} and RAR{gamma} for the up-regulation of proneuronal bHLH factors, with a possible preference of RAR{gamma} for Neuro D induction.

Work by Torii et al. (50) revealed the loss of Prox-1 expression in the developing brain of Mash-1 mutants and suggested that Mash-1 expression did not directly specify neuronal lineage differentiation, but converted multipotent stem cells into Mash-1+/Prox-1+ secondary precursors. In the present analysis, 1 nM (or 10 nM) Am 580 and 10 nM (or 100 nM) CD666 comparably triggered up-regulation of prox-1 mRNA (Table 1Go and examples in Fig. 6AGo). Considering the different binding KDs and lower RAR{gamma} expression level, this suggests that RAR{alpha} and RAR{gamma} may well equally induce the signal cascades that up-regulate prox-1 expression. Importantly, both, the much lower expression of prox-1 than mash-1 in proliferating PCC7 cells and the similar expression level of these genes during fate choice (Fig. 6AGo), are consistent with the suggestion by Torii et al. (50) that Mash-1 is upstream of Prox-1. In addition, the data obtained with Am 580 and CD666 suggests that mash-1 expression may already decline when prox-1 levels are still high (compare prox-1 and mash-1 signals in panels Am 580 and CD666 in Fig. 6AGo).

Expression of GCNF and Oct4
The orphan nuclear receptor GCNF (NR6A1; also known as RTR) is essential for embryonic development and survival, as well as for RA signaling in hindbrain development and its expression is spatiotemporally regulated in the developing central nervous system (Refs. 51 and 52 and references therein). In PCC7 cells, the repressor GCNF affected fate choice decision and promoted differentiation of the neuronal cells (44). The RT-PCR analysis in Fig. 6AGo shows the atRA-triggered up-regulation of gcnf during fate choice (Fig. 6AGo). Gcnf mRNA up-regulation with 1 nM Am 580 was as efficient as with 10 nM CD666, and peaked at d 3 (Table 1Go). Stepwise increase of the concentration of Am 580 shifted the peak to d 2 (10 nM) and d 1 (100 nM), respectively (Fig. 6AGo and Table 1Go). Although it is not yet known which signal cascades are involved in gcnf up-regulation, these results suggest a preferential induction via RAR{alpha}, in accordance with its higher expression level (Fig. 3AGo).

Retinoid-triggered up-regulation of gcnf mRNA levels is important in view of data showing that expression of the transcription factor Oct4, a key regulator of differentiation (53), is regulated by atRA and that oct4 is a target gene of the repressor GCNF (54). A 1-nM concentration (10 nM) Am 580 more efficiently down-regulated Oct4 than 10 nM (or 100 nM) CD666, which paralleled their potential to up-regulate gcnf expression, (Fig. 6BGo and Table 1Go).

In agreement with its capability to up-regulate expression of proneuronal genes, and in contrast to its failure to trigger typical neural differentiation, 1 µM CD2314 up-regulated gcnf expression and down-regulated Oct4 (Fig. 6Go, A and B, and Table 1Go).

Expression of the Neuronal Markers MAP2, GAP43, and SYP
The immunocytochemistry analysis in the present study was in agreement with previous work. (30, 31) showing that the somato-dendritic marker MAP2 and the axonal markers GAP43 and SYP are induced in a subpopulation of differentiating cells within 1 d after exposure to 100 nM atRA. RAR isotype-selective concentrations of Am 580 and CD666 and CD2314 also induced MAP2, GAP43, and SYP, although with distinct efficacy (Table 1Go). The typical differentiation-associated changes in the subcellular localization of MAP2, GAP43, and SYP, as well as polarization of the nucleus was only observed with receptor-selective concentrations of Am 580 and CD666, but not CD2314 (Fig. 7AGo and data not shown). Moreover, compared with cultures treated with 10 nM (or 100 nM) of atRA or Am 580, the strength of the MAP2 signal appeared stronger in cultures exposed to 10 nM (or 100 nM) of CD666, or a combination of atRA (100 nM) and the RAR{alpha} antagonist RO 41-5253 (1 µM) (Fig. 7AGo and data not shown). This suggested that RAR{alpha} and RAR{gamma} might differentially influence MAP2 expression. To confirm on quantitative changes in MAP2, GAP43, or SYP expression, Western blot analysis was used.

MAP2 expression levels at d 2 were at least 2-fold stronger with CD666 than with identical concentrations of Am 580, as was revealed by quantification. Moreover, 100 nM CD666 but not Am 580 induced a MAP2 signal at d 1, irrespective of the lower expression level of RAR{gamma} in the responding cells (Figs. 3AGo and 7BGo, and Table 1Go). This confirmed the immunocytochemistry data on the stronger MAP2 induction by CD666 and additionally indicated that the RAR{gamma} agonist shortened the time course of MAP2 induction. Because neuron-specific MAPs, such as MAP2 affect neurite initiation through several mechanisms (55), this could explain why CD666 efficiently triggered neural differentiation despite its less favorable KD and the lower expression level of RAR{gamma} compared with the KD of Am 580 and the expression level of RAR{alpha} (Tables 1Go and 2Go). MAP2 expression in the presence of 1 µM CD2314 resembled the expression seen with 1 nM Am 580 (Table 1Go). Together this suggests that the signal cascades responsible for the induction and up-regulation of MAP2 expression are preferentially initiated by RAR{gamma}, although all three RAR isotypes could exert this function.

Expression of GAP43 is crucial for differentiation of neural precursor cells and axonal outgrowth during initiation and remodeling of neural connections in the developing vertebrate nervous system (Ref. 56 and references therein). Western blot analysis with PCC7 whole cell extracts showed that the concentrations of RAR{alpha}-, ß-, and {gamma}-selective agonists, which efficiently triggered neurite outgrowths (Table 2Go), also efficiently induced GAP43 expression (Fig. 7CGo and Table 1Go). Induction of the synaptic vesicle protein SYP (for recent overview, see Ref. 57) was more efficient by receptor-selective concentrations of Am 580 than CD666 (or RA), and delayed by 1 µM of CD2314 (Table 1Go). Overall, the potential of Am 580, CD666 but not CD2314 to induce the axonal proteins GAP43 and SYP reflects their binding KDs and the expression levels of the RAR isotypes in the responding cells, suggesting that RAR{alpha} and RAR{gamma}, rather than RARß are involved in this process.

Conclusion
I used the PCC7 model and compound-selective retinoids to investigate the requirement of RXR and RAR{alpha}, RARß, and RAR{gamma} signaling for the transduction of the retinoid signal in RA-dependent neural differentiation. The concentrations at which the respective retinoids specifically acted through RAR{alpha}, RARß, RAR{gamma}, or RXR{alpha} were determined by in vitro and in vivo interaction assays with cofactors (NCoA2, and the corepressors SMRT and NCoR), as well as transactivation assays in HEK293T and PCC7 cells.

Proliferating PCC7 cells predominantly express RXR{alpha} and RAR{alpha}, less RXRß, and very little RXR{gamma}, RARß, and RAR{gamma}. During the stage of fate choice decision, which is accomplished until d 3 after onset of neural differentiation (32), the expression of RAR{gamma} was increased, but remained largely below that of RAR{alpha} at all times. The RARß and RXR{gamma} levels were drastically up-regulated at specific stages during fate choice, suggesting that these receptors have important roles down-stream of the initial events. Up-regulation of RARß was transient with atRA and the RAR{gamma} agonist CD666, but persisted, at least until d 3, with receptor-selective concentrations of the RAR{alpha} agonist Am 580 and the RARß agonist CD2314.

In view of the many similarities between neural differentiation of PCC7 cells and neural stem cells of higher vertebrates, this paper might predict the significance of retinoid receptor isotypes in RA-dependent differentiation of neural stem cells. The results show that initiation of RA-dependent neural differentiation requires RAR-signaling. The signals that initiate the cascades that ultimately lead to 1) inhibition of proliferation, 2) differentiation-associated cell death, 3) outgrowth of extension, 4) induction of neuronal markers, 5) development of neuronal and nonneuronal cells, 6) formation of neuron-aggregates and polarization of the nucleus within the neuronal cells, 7) up-regulation of pro-neuronal bHLH genes and the homeobox gene prox-1, 8) up-regulation of the transcriptional repressor GCNF, and 9) down-regulation of the GCNF target gene and key regulator of differentiation Oct4 may be transduced by the RAR{alpha} or RAR{gamma}, and perhaps to a much lesser extent, by RARß.

In the majority of cases, the potential of Am 580 (RAR{alpha} agonist) or CD666 (RAR{gamma} agonist) to induce these processes paralleled their binding KDs and the expression levels of RAR{alpha} and RAR{gamma} in the responding cells, indicating that RAR{alpha} and RAR{gamma} are functionally redundant in most aspects. However, although the higher expression level of RAR{alpha} suggests that most signaling will occur through RAR{alpha}, the results propose that up-regulation of RXR{gamma}, Neuro D, and MAP2 may preferentially occur via RAR{gamma}. Receptor-selective concentrations of the RARß agonist CD2314 were less efficient in the initiation of several of the above processes, and receptor-selective concentrations of CD2314 could not induce the tissue like differentiation pattern consisting of neuronal and nonneuronal cells. This is only partially explained by the binding KD of CD2314 and the expression level of RARß, proposing that RARß is not involved in the initiation of neural differentiation.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmids and Fusion Proteins
Bacterial expression vectors for GST-fusion proteins were constructed in pGEX-2TK (Amersham Biosciences, San Francisco, CA). Expression and purification of GST-murine (m) RXR{alpha}(DE) (residues Met205-end), GST-mRAR{alpha}(DEF) (residues Met153-end), GST-mRARß(DEF) (residues Met146-end), GST-mRAR{gamma}(DEF) (residues Met155-end), GST-cSMRT (residues 982-end), and GST-NCoR (residues 1629-end) was as described previously (40). Eukaryotic expression vectors for full-length mRAR{alpha}, mRARß, mRAR{gamma}, and mRXR{alpha} were gifts by Dr. P. Chambon (Strasbourg, France). Gal4- (residues 1–147 of the yeast activator Gal4) and VP16-fusion proteins contained the identical RAR, RXR, SMRT, or NCoR sequences as the corresponding GST constructs. pSG5-based expression vectors encoded residues 982-1492 of mouse cSMRT and residues 624-1287 of mouse NCoA-2. The (17mer)5-TATA-CAT plasmid with five copies of the Gal4 response element in front of a simple TATA motif and the CAT gene served as reporter gene. Sequences of oligonucleotides used for RT-PCR, cloning, and sequencing are available on request.

Neural Differentiation of PCC7 Cells and Quantitative Assessment of Cell Numbers
Properties of the embryonal carcinoma cell line PCC7 (29), as well as culture conditions and induction of neural differentiation with atRA or 9cRA (Sigma, St. Louis, MO) were as described elsewhere (30, 31, 32). In differentiation assays DMEM (Sigma) containing 12.5% of charcoal-treated fetal calf serum (FCS) was used (FCS: Invitrogen Life Technologies, Carlsbad, CA). The compound-selective synthetic retinoids were provided by Galderma laboratories (Sophia, France).

The potential to induce neural differentiation was analyzed for: atRA (or 9cRA) at 1 µM–1 nM; the pan RAR agonist CD367 (58) at 1 µM–1 nM; the RAR{alpha} agonist Am 580/CD336 (59) at 1 µM–0.1 nM; the RARß agonist CD2314 (60) at 10 µM–10 nM; the RAR{gamma} agonist CD666 (59) at 1 µM–1 nM, and the RXR pan agonist CD3640 at 10 µM–10 nM. The following compounds served as inhibitors of retinoid-induced neural differentiation: pan RAR antagonist AGN193109 (47); RAR{alpha} antagonist RO 41-5253/CD2503 (60); RARß/{gamma} antagonist CD2665 (59).

To investigate the relative number of proliferating cells, the percentage of Ki67 expressing cells was determined. Cells were grown in 24-well tissue culture plates at low density (0.5 x 104 cells/cm2). At 68 h after exposure to retinoids cells were stained with {alpha}Ki67/fluorescein isothiocyanate (FITC), followed by counting of antigen-positive cells. The following strategies were applied in parallel assays: 1) Cells were stained with {alpha}Ki67/FITC, followed by counting in situ. 2) Cells were harvested and replated on adhesive coverslips before immunological staining and counting.

The number of living vs. apoptotic cells was determined at 24 h and 48 h after exposure to ligand using the following strategies: 1) Cells were harvested followed by staining with trypan blue and counting; 2) Cells were stained with the Annexin-V-FLUOS staining Kit (Roche Molecular Biochemicals, Mannheim, Germany), which allowed for discrimination of both, apoptotic and necrotic cells from living cells [combined Annexin-V and DNA (propidium iodide) stain], followed by counting. 3) Cells were grown in 96-well plates and subjected to WST-1 tests (Roche) as described by Esdar et al. (31).

Transient Transfection and Reporter Gene Activity
Cells were grown in DMEM containing charcoal-treated FCS (PCC7, 12.5%; HEK293T, 10%). Transfection of PCC7 cells with the calcium-phosphate technique, electroporation or chemical transfection reagents was very inefficient, as became obvious by cotransfection of the GFP-encoding plasmid pEGFP (BD Biosciences, San Diego, CA). At best, one cell in 1000 was transfected, whereas transfection rates exceeded 80% in parallel cultures of HEK293T cells. Therefore, induction of CAT expression in PCC7 cells was determined in situ using immunocytochemistry. The best results were obtained with 0.5 µg Gal4-fusion plasmid, 0.25 µg reporter plasmid [(17mer)5-TATA-CAT], 0.25 µg pEGFP and 4 µl SuperFect transfection reagent (QIAGEN, Valencia, CA) per fibronectin-coated coverslip (2 cm2). After replacement of the medium, cells were maintained for 16 h in culture before exposure to ligands. 24 h after application of retinoids (or carrier) cells were fixed and stained with a CAT-specific antibody (Sigma) according to the manufacturer’s instruction. The percentage of CAT-expressing GFP-positive cells was determined by counting. Three independent series of experiments using six coverslips per retinoid (or carrier) were performed. To quantify the potential of Am 580, CD2314, CD666 and CD3640 to induce transactivation and to mediate coactivator binding in vivo HEK293T cells were transiently transfected using the calcium-phosphate technique (61). The conditions applied in transactivation assays and mammalian two-hybrid analysis were as described previously (Refs. 39 and 40 and references therein). CAT expression was determined using a CAT ELISA Kit (Roche) and normalized to the ß-galactosidase concentration resulting from cotransfection of 1 µg of pCMV-ßGal (gift from Dr. P. Chambon) per 58-cm2 dish.

Preparation of RNA and Semiquantitative RT-PCR
For RT-PCR analysis I used total RNA from cells treated with atRA (100 nM, 10 nM), CD367 (100 nM), Am 580 (100 nM, 10 nM, 1 nM), CD2314 (1 µM, 100 nM), and CD666 (100 nM, 10 nM). Cells were washed twice with ice-cold PBS and harvested in 1x TEN [10 mM Tris-HCl (pH 7.5), 1 mM EDTA (pH 8.0), 150 mM NaCl]. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Valencia, CA). Characterization of nucleic acids and proteins was according to standard protocols (61).

Semiquantitative RT-PCR was performed with deoxyribonuclease I-treated RNA from a minimum of two independent series of cells. cDNA synthesis was with the avian myeloblastosis virus-first-strand cDNA synthesis Kit (Roche) using 500 ng of total RNA in random-primed cDNA synthesis. Second-strand synthesis was performed with 1/40 of the first-strand reaction and comprised at least three independent experiments with varying numbers of cycles: cycling was for 26, 28, 30, 32, 35, and/or 40 rounds, except for the internal control glyceraldehyde-3-phosphate dehydrogenase (20, 23, or 25 cycles). One fifth of the individual second-strand reaction was run on agarose gels. Signals were quantified using the GS250 Molecular Imaging System (Bio-Rad, Hercules, CA).

Primary Antibodies
The following monoclonal (mAb) or polyclonal (pclAb) primary antibodies were used: mouse pan-Actin mAb (Chemicon International, Temecula, CA); rabbit {alpha}CAT pclAb (Sigma); rabbit affinity-purified serum against GAP-43 (gift from Dr. T. Herget, Darmstadt, Germany); mouse {alpha}GFAP mAb (Chemicon); rabbit anti-Ki67 mAb (Dianova, Hamburg, Germany); rat {alpha}MESA mAb (gift from Dr. C. Goridis, Paris, France); mouse {alpha}MAP2 mAb (Chemicon); rabbit pclAb against Neuro D (Chemicon); rabbit {alpha}Oct4 pclAb (Active Motif, Carlsbad, CA); rabbit pclAb against RAR{alpha}, RARß or RAR{gamma} [Biomol (Butler Pike, PA) and gifts by Drs. P. Chambon and C. Rochette-Egly (Strasbourg, France)]; rabbit pclAb against SYP (gift from Dr. R. Jahn, Göttingen, Germany); rabbit anti-Tau (Sigma); rat {alpha}Thy1.2 mAb (BD Biosciences).

Whole Cell Protein Extracts (WCEs) and Western Blot Analysis
WCEs were prepared as described previously (44), using proliferating PCC7 cells and cultures treated with atRA (100 nM–1 nM), Am 580 (100 nM–1 nM), CD2314 (10 µM–10 nM), or CD666 (100 nM–1 nM). Proteins were characterized and quantified according to standard protocols (61).

If not indicated otherwise, Western blot analysis with antibodies specific for the RARs, Oct4, synaptophysin, Neuro D, or GAP43 was with 20 µg (or 40 µg) of WCE. Proteins were resolved by SDS-PAGE (10% resolving gel, 4% stacking gel; Minigel system Protean II; Bio-Rad, Hercules, CA) and transferred onto nitrocellulose membrane (Bio-Rad) using the semidry transfer system TransBlot SD (Bio-Rad). For analyses with {alpha}MAP2, 80 µg WCE were used. Electrophoresis was at 35–55 V for 12–16 h at room temperature [run buffer: 25 mM Tris base (pH 8.3), 250 mM glycine, 0.1% (wt/vol) SDS], using the Hoefer electrophoresis system (SE 600 series) and SDS-PAGE (4–10% resolving gel; 4% stacking gel). Transfer of proteins was in buffer containing 25 mM Tris-base (pH 8.3), 250 mM glycine, 0.3% (wt/vol) SDS, 20% ethanol. In any case, bound antibodies were revealed by chemiluminescence using peroxidase-conjugated secondary antibodies (Beckman-Coulter, Fullerton, CA; or Sigma), SuperSignal West Pico (Pierce, Rockford, IL) and Kodak (Rochester, NY) BioMax x-ray films.

Immunocytochemistry
For immunocytochemistry studies, cells were differentiated on glass coverslips in 24-well plates. Fixation was with ethanol/acetic acid (95:5 vol/vol), except for Thy1.2 (no fixation before antibody treatment). Cells were treated with Triton X-100 before {alpha}Ki67 stain. Dapi nuclear staining was with Hoechst 33258 according to the manufacturer instruction. The following secondary antibodies were used: rabbit antimouse FITC (Dako, Carpinteria, CA), rabbit antirat FITC (Dako), goat antimouse FITC M4 (Caltag Laboratories, Burlingame, CA), goat antirabbit Texas Red (Jackson ImmunoResearch, West Grove, PA); goat antimouse Texas Red (Jackson ImmunoResearch), goat antirabbit Cy3 (Jackson ImmunoResearch). Immunofluorescence was recorded using Leica (Solms, Germany) DM IBRE and accompanying software IM50.

Pull-Down Analysis
GST pull-down was essentially as described (40). The nuclear receptor or cofactor encoding plasmids were in vitro translated using the TNT-T7 Quick Coupled Transcription/Translation System (Promega Biosciences Inc., San Luis Obispo, CA). For the pull down assays 1.5 µl (35S-labeled NCoA-2, RAR{alpha}, RARß, RAR{gamma}, RXR{alpha}) or 2 µl (35S-labeled cSMRT) lysate were incubated with a suspension of glutathione beads (Amersham Biosciences) loaded with 4 µg of GST, 8 µg of GST-NR(LBD), 20 µg of GST-cSMRT and 20 µg of GST-cNCoR, respectively.


    ACKNOWLEDGMENTS
 
I am grateful to Drs. P. Chambon (Strasbourg, France), C. Goridis (Paris, France), T. Herget (Darmstadt, Germany), R. Jahn (Göttingen, Germany), and C. Rochette-Egly (Strasbourg, France) for plasmids or antibodies, to Dr. V. Lallemand-Mezger (Paris, France) for PCC7 and Dr. C. Pietrzik (Mainz, Germany) for HEK293T cells, and to Galderma laboratories (Sophia, France) for CD synthetic retinoids. I thank Drs. C. Behl and B. Lutz (both Mainz, Germany) for support and E. Waldron for reading the manuscript. I am indebted to I. Koziollek-Drechsler for contributions to immunocytochemistry studies and acknowledge excellent technical assistance D. Dormann.


    FOOTNOTES
 
This research was supported by grants of the Johannes Gutenberg-University of Mainz.

First Published Online April 14, 2005

Abbreviations: atRA, All-trans RA; bHLH, basic helix-loop-helix; CAT, chloramphenicol acetyltransferase; CoA, coactivator; 9cRA, 9-cis RA; Dapi, 4',6-diamidino-2-phenylindole; EC, embryonal carcinoma; FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GAP, growth-associated protein; GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein; GST, glutathione-S-transferase; HEK, human embryonic kidney; KD, dissociation constant; m, murine; mAb, monoclonal antibody; MAP, microtubule-associated protein; NCoA, nuclear CoA; NCoR, nuclear receptor corepressor; pclAb, polyclonal antibody; RA, retinoic acid; RAR, RA receptor; RARE, RA response element; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SMRT, silencing mediator of retinoid and thyroid receptors; SYP, synaptophysin; WCE, whole cell protein extract.

Received for publication December 29, 2004. Accepted for publication April 5, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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