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.
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ABSTRACT |
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INTRODUCTION |
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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 , ß, and
and RXR
, ß, and
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 RARs 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/RAR
heterodimers are instrumental in patterning of the craniofacial skeletal elements, whereas RXR
/RAR
may be preferentially involved in the generation of neural crest-derived arterial smooth muscle cells, and both RXR
/RARß and RXR
/RAR
heterodimers function during development of the ocular mesenchyme (19). Moreover, RAR
, ß, and
take distinct roles in early embryonic morphogenesis and hindbrain patterning in particular (20, 21). Null mutation of RARß and RXR
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
and RAR
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 and RAR
, lower levels of RXRß, and barely detectable amounts of RARß, RAR
, and RXR
. 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
, ß, and
and the dissociation constants (KDs) of the bound retinoids. The possible differential requirement of the RAR
, ß, and
during early stages of neural differentiation of PCC7 cells is discussed.
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RESULTS AND DISCUSSION |
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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 receptors 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) and the RAR isotypes
, ß, and
; atRA and the RAR pan-agonist CD367 served as controls. A 100-nM concentration of Am 580 induced NCoA-2 binding to RAR
and less efficiently to RARß, and 10 nM only to RAR
(Fig. 1C
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. 1C
and data not shown). At higher concentrations, CD2314 (1 µM) efficiently induced NCoA-2 binding to GST-RARß and only weakly to RAR
or RAR
(data not shown). At 100 nM, CD666 induced NCoA-2 binding to RAR
, weakly to RARß, but not to RAR
, whereas 10 nM of CD666 only triggered NCoA-2 binding to RAR
(Fig. 1C
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
, Gal4 RARß, and Gal4-RAR
, respectively (Fig. 2
), 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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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 2A 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 is a potent repressor of transcription that efficiently interacts with SMRT, whereas RARß and RAR
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. 1D). 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
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
. In contrast, AGN 193109 decreased the interaction of SMRT with RAR
, but increased binding of NCoR (Fig. 1D
and data not shown). Thus, both antagonists might compromise SMRT-dependent repression by RAR
and render the RXR/RAR
heterodimer permissive for RXR signaling. The weak binding of SMRT to apo-RARß and apo-RAR
was impaired by AGN 193109, CD2665, but not by RO 41-5253, and receptor-selective concentrations of CD666 released SMRT from RAR
, whereas 1 µM of CD2314 had no detectable effect on the formation of the SMRT/RARß complex (Fig. 1D
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-, ß-, or
-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
, when amplification was for 35 cycles; at 30 cycles, only the rar
-, rxr
-, and to a lesser extent the rxr ß-specific primer pair amplified detectable amounts of PCR product (Fig. 3A
and data not shown).
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After exposure to atRA, PCC7 cells showed a moderate up-regulation of RXR, RXRß, and RAR
mRNA expression levels (data not shown). This resulted in an even less pronounced up-regulation of RAR
on the protein level (Fig. 3C
). In contrast, expression of RAR
, and of RXR
and RARß in particular, was largely affected by atRA. Notably, RAR
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. 3B
). However, the RAR
protein was not detectable by Western blot analysis with specific antibodies (data not shown). The very weak signal for RXR
expression obtained by RT-PCR with RNA from proliferating PCC7 cells turned into a strong band for RNA from d 3 cultures (Fig. 3B
). In the case of RARß, three closely migrating bands of 5156 kDa (most probably RARß2, RARß1, and RARß3) became detectable in Western blots with RARß-specific antibodies and protein extracts from d 1 cultures (Fig. 3C
; 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
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
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 2530% of RARß and RXRß/
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, RXRß, and RAR
was only weakly modulated by these ligands, whereas expression of RAR
, and particularly RXR
and RARß, was strongly affected (Fig. 3
and Table 1
). 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
expression than did 1 µM CD2314 or 100 nM CD666 (Fig. 3B
and data not shown). In contrast, 100 nM (or 10 nM) of CD666 more efficiently up-regulated RXR
mRNA levels than 100 nM (or 10 nM) of Am 580, even though PCC7 cells express less RAR
than RAR
; 1 µM of CD2314 did not up-regulate RXR
(Fig. 3B
and Table 1
). Thus, up-regulation of RXR
expression may specifically involve RAR
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. 3C
), 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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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. 1A) 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, whereas that one of CD666 is 68 nM for RAR
(Fig. 1A
), and that proliferating PCC7 cells express RAR
at much higher levels than RAR
(Fig. 3
), this study suggests that the retinoid signal initiating differentiation may be transduced by both, RAR
and RAR
. This was further supported by the observation that coapplication of atRA (or CD367) and a RAR
- (RO 41-5253) or RARß/
- (CD2665) selective antagonist did not abrogate differentiation (Table 2
and Fig. 4
and see Fig. 7A
). Involvement of RAR
and RAR
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
(Refs. 27 and 45 and references therein).
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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. 5A 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. 5A
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
and RAR
in the responding cells (Figs. 1A
and 3A
). Thus, the potential of the RAR-isotype selective retinoids to inhibit proliferation correlated with their potential to induce neural differentiation.
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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. 4u). 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. 4q
and Table 2
), 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 2). 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 2
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. 4s
and data not shown), indicating that the RARß/RXR heterodimer may initiate neural differentiation. Based on the expression level of RAR
, ß, and
in proliferating cells (Fig. 3A
), 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 2
), these results suggest that induction of neural differentiation was initiated by the RXR/RAR
and/or RXR/RAR
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. 6A and Table 1
). 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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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 1 and examples in Fig. 6A
). Considering the different binding KDs and lower RAR
expression level, this suggests that RAR
and RAR
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. 6A
), 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. 6A
).
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. 6A shows the atRA-triggered up-regulation of gcnf during fate choice (Fig. 6A
). Gcnf mRNA up-regulation with 1 nM Am 580 was as efficient as with 10 nM CD666, and peaked at d 3 (Table 1
). Stepwise increase of the concentration of Am 580 shifted the peak to d 2 (10 nM) and d 1 (100 nM), respectively (Fig. 6A
and Table 1
). Although it is not yet known which signal cascades are involved in gcnf up-regulation, these results suggest a preferential induction via RAR
, in accordance with its higher expression level (Fig. 3A
).
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. 6B and Table 1
).
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. 6, A and B, and Table 1
).
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 1). 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. 7A
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
antagonist RO 41-5253 (1 µM) (Fig. 7A
and data not shown). This suggested that RAR
and RAR
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 in the responding cells (Figs. 3A
and 7B
, and Table 1
). This confirmed the immunocytochemistry data on the stronger MAP2 induction by CD666 and additionally indicated that the RAR
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
compared with the KD of Am 580 and the expression level of RAR
(Tables 1
and 2
). MAP2 expression in the presence of 1 µM CD2314 resembled the expression seen with 1 nM Am 580 (Table 1
). Together this suggests that the signal cascades responsible for the induction and up-regulation of MAP2 expression are preferentially initiated by RAR
, 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-, ß-, and
-selective agonists, which efficiently triggered neurite outgrowths (Table 2
), also efficiently induced GAP43 expression (Fig. 7C
and Table 1
). 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 1
). 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
and RAR
, 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, RARß, and RAR
signaling for the transduction of the retinoid signal in RA-dependent neural differentiation. The concentrations at which the respective retinoids specifically acted through RAR
, RARß, RAR
, or RXR
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 and RAR
, less RXRß, and very little RXR
, RARß, and RAR
. During the stage of fate choice decision, which is accomplished until d 3 after onset of neural differentiation (32), the expression of RAR
was increased, but remained largely below that of RAR
at all times. The RARß and RXR
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
agonist CD666, but persisted, at least until d 3, with receptor-selective concentrations of the RAR
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 or RAR
, and perhaps to a much lesser extent, by RARß.
In the majority of cases, the potential of Am 580 (RAR agonist) or CD666 (RAR
agonist) to induce these processes paralleled their binding KDs and the expression levels of RAR
and RAR
in the responding cells, indicating that RAR
and RAR
are functionally redundant in most aspects. However, although the higher expression level of RAR
suggests that most signaling will occur through RAR
, the results propose that up-regulation of RXR
, Neuro D, and MAP2 may preferentially occur via RAR
. 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.
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MATERIALS AND METHODS |
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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 µM1 nM; the pan RAR agonist CD367 (58) at 1 µM1 nM; the RAR agonist Am 580/CD336 (59) at 1 µM0.1 nM; the RARß agonist CD2314 (60) at 10 µM10 nM; the RAR
agonist CD666 (59) at 1 µM1 nM, and the RXR pan agonist CD3640 at 10 µM10 nM. The following compounds served as inhibitors of retinoid-induced neural differentiation: pan RAR antagonist AGN193109 (47); RAR
antagonist RO 41-5253/CD2503 (60); RARß/
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 Ki67/fluorescein isothiocyanate (FITC), followed by counting of antigen-positive cells. The following strategies were applied in parallel assays: 1) Cells were stained with
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 manufacturers 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 CAT pclAb (Sigma); rabbit affinity-purified serum against GAP-43 (gift from Dr. T. Herget, Darmstadt, Germany); mouse
GFAP mAb (Chemicon); rabbit anti-Ki67 mAb (Dianova, Hamburg, Germany); rat
MESA mAb (gift from Dr. C. Goridis, Paris, France); mouse
MAP2 mAb (Chemicon); rabbit pclAb against Neuro D (Chemicon); rabbit
Oct4 pclAb (Active Motif, Carlsbad, CA); rabbit pclAb against RAR
, RARß or RAR
[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
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 nM1 nM), Am 580 (100 nM1 nM), CD2314 (10 µM10 nM), or CD666 (100 nM1 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 MAP2, 80 µg WCE were used. Electrophoresis was at 3555 V for 1216 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 (410% 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 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, RARß, RAR
, RXR
) 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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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