Retinoid X Receptor and c-erbA/Thyroid Hormone Receptor Regulate Erythroid Cell Growth and Differentiation

Petr Bartunk and Martin Zenke

Max-Delbrück-Center for Molecular Medicine D-13122 Berlin, Germany


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Nuclear receptors are important regulators of erythroid cell development. Here we investigated the impact of retinoid X receptor (RXR), retinoic acid receptor (RAR), and of the c-erbA/thyroid hormone (T3) receptor (c-erbA/TR) on growth and differentiation of erythroid cells using an in vitro culture system of stem cell factor-dependent erythroid progenitors. RXR, RAR, and c-erbA/TR-specific ligands were found to induce erythroid-specific gene expression and to accelerate erythroid differentiation in culture, with T3 being most effective. Furthermore, while ligand-activated c-erbA/TR accelerated differentiation, unliganded c-erbA/TR effectively blocked differentiation and supported sustained progenitor growth in culture. Thus, c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. Additionally, to determine the impact of RXR for erythroid cell development, dominant interfering mutant RXRs, lacking the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire DNA-binding domain, were introduced into erythroid progenitor cells via recombinant retrovirus vectors and analyzed for RXR-specific effects. It was found that expression of wild-type RXR and of the RXR mutants devoid of AF-1 and/or AF-2 supported a transient outgrowth of erythroid cells. In marked contrast, expression of the dominant interfering {Delta}DNA-binding domain RXR, containing a deletion of the entire DNA-binding domain, was incompatible with erythroid cell growth in vitro, suggesting a pivotal role of RXR for erythroid cell development.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The c-erbA thyroid hormone (T3) receptor (c-erbA/TR), retinoic acid (RA) receptor (RAR), and the retinoid X receptor (RXR) are members of the large family of nuclear receptors (NRs) that effectively induce gene transcription when activated by cognate ligand (1, 2, 3, 4, 5, 6). Both c-erbA/TR and RAR require heterodimerization with RXR for high-affinity binding to their cognate response elements (4, 5). Furthermore, in addition to ligand-dependent activation of transcription, selected NRs including the c-erbA/TR and RAR repress basal transcription in the absence of ligand (see Refs. 5, 6, 7, 8 for references). Most significantly, both gene activation and repression by these factors occur through recruitment of specific coactivators and corepressors, respectively (Refs. 8, 9, 10, 11, 12, 13 and reviews of 5–7, 14, 15). This way, the overall transcriptional activity of a given gene depends on multiple signals determined by factors that either directly bind to DNA and act as activators or repressors, or alternatively, by ligand-dependent DNA-binding proteins, like the NRs, that in a ligand-dependent fashion recruit specific coactivators or repressors to activate or repress transcription. Additionally, more recent studies demonstrated that NR coactivators and corepressors are components of multimeric enzymatic complexes, containing histone acetyltransferases and histone deacetylases, respectively, that might cause chromatin remodeling at the promoter site (reviewed in Refs. 6, 16).

NRs regulate complex biological processes like cell growth and differentiation, morphogenesis, and organogenesis by specifically modulating gene transcription. Additionally, abnormal and/or mutated NRs were found to lead to aberrant gene expression and oncogenic transformation (17, 18, 19, 20). The v-erbA oncogene, a retrovirus-transduced version of c-erbA/TR contained in the avian erythroblastosis virus AEV, represents such an oncogenic version. V-erbA lacks hormone binding activity and has suffered multiple point mutations and short N- and C-terminal deletions (19, 20). Most significantly, v-erbA lost the ligand-dependent transcriptional activator function AF-2, but still contains functional silencing domains that bind corepressors (11, 12, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28). In erythroid cells v-erbA effectively suppresses expression of erythroid specific genes [like band 3 and carbonic anhydrase II (CAII)] and inhibits terminal differentiation (18, 19, 20, 29, 30, 31). In marked contrast, ligand-activated c-erbA/TR was found to effectively induce gene transcription and to accelerate erythroid cell differentiation (19, 32, 33, 34).

Erythroid progenitor cells can be isolated from chicken and human bone marrow, human cord blood preparations, or human CD34+ peripheral blood stem cells, and amplified in vitro in the presence of stem cell factor (SCF) or transforming growth factor type {alpha} (TGF{alpha}), erythropoietin, and steroid hormones as homogenous cell populations to large cell numbers (35, 36, 37, 38, 39, 40, 41, 42, 43). Additionally, cells can be induced to undergo normal terminal differentiation in the presence of specific differentiation factor, like erythropoietin and insulin, and yield fully mature erythrocytes. Such in vitro systems allow the study of NR function and of the activity of NR-specific ligands within an ongoing differentiation program under well defined culture conditions. Furthermore, a number of recombinant retrovirus vectors are available for introduction and expression in chicken erythroid cells of NRs or mutated versions thereof, to determine their impact on erythroid cell development (19, 30, 31, 32, 33, 37, 41, 44, 45). These experimental systems therefore complement and extend previously developed in vitro growth and differentiation systems based on erythroid progenitor cells that were conditionally transformed by temperature-sensitive (ts) tyrosine kinase oncogenes (18, 19, 38, 39).

Previous studies addressed the question of the impact of hormone-activated c-erbA/TR and RAR for induction of erythroid cell differentiation (19, 32, 33, 34). It was found that ligand-activated c-erbA/TR effectively induced erythroid gene transcription (like CAII) and accelerated erythroid cell differentiation in vitro. Here we show that additionally unliganded c-erbA/TR affects erythroid cell development. In the absence of hormone, c-erbA/TR inhibits differentiation and supports sustained growth of normal untransformed SCF- dependent erythroid progenitor cells in culture and therefore exerts an activity similar to v-erbA. Furthermore, we addressed the question of the impact of RXR, the obligate heterodimeric partner of c-erbA/TR, for erythroid cell growth and differentiation by ectopic expression of dominant interfering RXR mutants. Our results support the idea that RXR is a pivotal factor for erythroid cell growth and differentiation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Unliganded c-erbA/TR Supports Sustained Growth of Erythroid Progenitor Cells in Vitro
Ligand-activated c-erbA/TR effectively induces erythroid cell-specific gene transcription and accelerates erythroid differentiation in vitro (19, 32, 33, 34). A number of more recent studies demonstrated that unliganded c-erbA/TR affects gene transcription by binding transcriptional corepressors, like N-CoR/SMRT (nuclear receptor corepressor/silencing mediator of retinoic acid and thyroid hormone receptor), that silence expression of c-erbA/TR responsive promoters (8, 9, 11–13, 28 and reviewed in Refs. 5, 6, 7, 14). Thus, additionally unliganded c-erbA/TR can be expected to contain a specific activity and to affect growth and/or differentiation of erythroid cells. Such an idea is conceivable in light of the fact that N-CoR/SMRT corepressors are widely expressed in various tissues and cell lines (14).

To test this hypothesis, SCF-dependent erythroid progenitor cells were prepared from chicken bone marrow and infected with a c-erbA/TR containing recombinant retrovirus to augment c-erbA/TR levels in these cells and thereby achieve a more pronounced biological effect. As a control, cells were infected with v-erbA virus, or with neo virus only or left uninfected. Cells were then cultured in medium (that was extensively deprived of T3 and retinoids; see Materials and Methods) and SCF, and cell numbers were determined in regular time intervals (Fig. 1AGo). An outgrowth of c-erbA/TR and v-erbA retrovirus-infected cells was readily obtained. Additionally, by Western blot analysis we show that cells expressed the c-erbA/TR and v-erbA proteins, respectively (Fig. 1BGo). Cells also expressed the erythroid-specific transcription factor GATA-1, demonstrating that they were of erythroid origin. As expected, control cells infected with neo virus only or left uninfected exhibited a restricted growth potential and ceased proliferation after 8–10 days in culture (Fig. 1AGo and data not shown). However, most significantly c-erbA/TR in the absence of T3 blocked erythroid differentiation and effectively supported sustained growth of erythroid progenitors (Fig. 1Go, A and C) yielding at day 8 of culture up to 10-fold higher cumulative cell numbers than control; v-erbA cells grew, as expected, faster and reached about 30-fold higher cumulative cell numbers than control (day 8). Cell numbers further increased with time in culture resulting at day 12 in a more than 500-fold (c-erbA/TR) or 3500-fold (v-erbA) amplification of erythroid progenitor cells over control; c-erbA/TR cells grew for at least 30 days without signs of senescence similar to v-erbA control. In addition, td359 v-erbA that contains a specific point mutation in the N-CoR/SMRT binding site (11, 27, 46) was essentially inactive in supporting erythroid progenitor cell growth (Fig. 1AGo). Interestingly, unliganded RAR and vitamin D3 receptor, which belong to the same NR family as c-erbA/TR and also bind corepressors, were deficient in inducing sustained progenitor cell growth, thus pointing to a specific role of c-erbA/TR in erythroid cells (P. Bartunek and M. Zenke, unpublished).



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Figure 1. Unliganded c-erbA/TR Supports Sustained Growth of SCF-Dependent Erythroid Progenitors

A, Growth of c-erbA/TR, wild-type v-erbA, and td359 mutant v-erbA expressing SCF-dependent erythroid progenitor cells (c-erbA/TR, v-erbA, and td359, respectively) was analyzed by counting cells in regular time intervals. Cumulative cell numbers are shown. Control, Cells expressing neo virus only. B, SCF-dependent erythroid progenitor cells expressing c-erbA/TR or v-erbA (same cells as in panel A, day 7) were analyzed by Western blotting using an erbA-specific antibody. GATA-1 protein expression is also shown. C, Cells at day 10 of culture (c-erbA/TR and v-erbA cells of panel A, as indicated) were centrifuged onto slides and stained with neutral benzidine and histological dyes.

 
Finally, when aliquots of the cultures were seeded in colony assays, unliganded c-erbA/TR clearly supported colony formation, and the number of colonies obtained was, as expected from the results shown above, lower than that for v-erbA control cells (data not shown). We also note that in some experiments (like the one shown in Fig. 1AGo) growth of c-erbA/TR cells was somewhat delayed at day 8–10 of culture as compared with v-erbA cells, which is probably due to selection of cells with higher proliferative potential and/or higher c-erbA/TR levels. Additionally, c-erbA/TR cells were fully competent in differentiating terminally into mature erythrocytes and differentiation was, as expected, effectively accelerated by T3 treatment (data not shown).

In summary, it appears that unliganded c-erbA/TR behaves very similarly to v-erbA in supporting sustained proliferation of SCF-dependent erythroid progenitor cells in vitro and was only slightly less efficient than v-erbA. This might be because the differentiation block imposed by unliganded c-erbA/TR is less effective as compared with that of v-erbA (Fig. 1CGo; see also Discussion). Alternatively, some residual T3 that could not be removed from serum might compromise c-erbA/TR repressor function.

Ligand-Activated RXR, RAR, and c-erbA/TR Accelerate Erythroid Cell Differentiation and Induce Erythroid Gene Transcription
RXR subserves the role of an obligate heterodimeric partner to several NRs including c-erbA/TR and RAR and can also function as a homodimer (4, 5). RXR expression is found in both tyrosine kinase oncogene-transformed and also normal untransformed erythroid progenitor cells of chicken (data not shown). Furthermore, while previous studies demonstrated the importance of c-erbA/TR and RAR for erythroid cell-specific gene expression and differentiation (19, 32, 33, 34), the impact of RXR for proper erythroid cell development has so far not been studied. To address this question, erythroid cells were, first, treated with specific ligands that activate RXR, RAR, or c-erbA/TR and then analyzed for ligand-specific effects on differentiation and erythroid-specific gene expression. Second, since erythroid progenitor cells express at least two RXR isoforms (RXR{alpha} and RXR{gamma}, Ref. 47 ; N. Koritschoner, P. Bartunek, and M. Zenke, in preparation) dominant-negative RXR versions were introduced and expressed in erythroid cells via recombinant retroviral vectors to interfere with RXR function; cells were then analyzed for RXR-specific effects.

First we determined the individual contribution of activated RXR, RAR, and c-erbA/TR on erythroid cell differentiation by administration of specific ligands. SCF-dependent erythroid progenitor cells were prepared and treated with T3, all-trans-RA (atRA), 9-cis-RA (9cRA), and RXR agonist SR11237, and various combinations thereof. Such progenitor cells can be induced to undergo normal terminal differentiation in vitro in the presence of anemic chicken serum (as a source of erythropoietin) and insulin (36, 39, 40, 41). After induction of differentiation, cells begin to accumulate hemoglobin and gradually acquire the morphology of normal avian erythrocytes (Fig. 2AGo). During differentiation, cells undergo four to five cell divisions which are accompanied by a decrease in cell size and followed by cell cycle arrest (Fig. 2BGo and data not shown). Under these conditions such erythroid progenitors differentiate into fully mature erythrocytes after 4 days in culture (40). Interestingly, administration of T3, atRA, and 9cRA, or various combinations thereof effectively accelerated the differentiation process. While untreated cells at day 2 of differentiation were only partially hemoglobinized, T3-, atRA-, and 9cRA-treated cells had already further progressed in differentiation, T3 being the most effective factor (Fig. 2CGo). The RXR agonist SR11237 was as effective as atRA (data not shown). Additionally, simultaneous administration of T3 plus atRA, 9cRA, or SR11237 was more efficient in accelerating differentiation than any treatment with single factor (Fig. 2CGo and data not shown). For example, at day 2 of culture, T3 plus 9cRA-treated cells had reached a differentiation stage that was equivalent to day 3 of differentiation of untreated cells. The ligand-specific effects on differentiation were also evident when analyzing reduction in cell size in response to factor and loss of proliferative potential in [3H]thymidine incorporation assays (Fig. 2DGo and data not shown).



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Figure 2. T3, atRA, and 9cRA Accelerate Erythroid Cell Differentiation

A, Cytospin preparation of SCF-dependent erythroid progenitors before (d0) and 2 and 3 days after differentiation (2d and 3d, respectively) were stained with neutral benzidine and histological dyes as in Fig. 1CGo. B, Cell profiles of the cells shown in panel A demonstrate the reduction in cell size during differentiation; undifferentiated cells (dark), differentiated cells at day 2 (gray) and at day 3 (white). C, SCF-dependent erythroid progenitor cells were differentiated in the presence of T3, atRA, 9cRA, T3+atRA, or T3+9cRA or left untreated (no) as indicated. Cytospin preparations at day 2 of differentiation stained with neutral benzidine and histological dyes are shown. D, Cell profiles of the cells shown in panel C demonstrate the effect of T3+9cRA on reduction of cell size (gray); untreated cells (white).

 
In summary, these results demonstrate that, when individual compounds were administered, T3 was most effective in accelerating erythroid cell differentiation as judged 1) by morphological changes and accumulation of hemoglobin that were assessed histologically and 2) by reduction in cell size and proliferative potential. Simultaneous administration of T3 and 9cRA, which activate multiple receptors, was the most effective factor combination. Essentially the same result was obtained when clonal cell populations of ts-v-myb + epidermal growth factor receptor-transformed erythroblasts (48) were induced to differentiate by temperature shift to 42 C minus EGF (to inactivate ts-v-myb and epidermal growth factor receptor, respectively) in the presence of T3, atRA, 9cRA, or T3 plus 9cRA (data not shown).

Second, to assess T3 and RA-specific effects on erythroid cell differentiation quantitatively, RNA was prepared and analyzed for expression of the erbA target gene CAII (19, 32). SCF-dependent erythroid progenitor cells were treated with ligand (Fig. 2Go, C and D) and 24 h later analyzed for CAII expression in Northern blots (Fig. 3Go, A and B). CAII mRNA levels were low in untreated cells and induced by T3, atRA, 9cRA, and SR11237, with T3 and 9cRA being most effective. Simultaneous treatment with multiple ligands (to activate multiple receptor complexes) further increased CAII expression which was highest for T3 plus atRA and T3 plus 9cRA (~6- to 7.3-fold higher than in untreated control; Fig. 3Go, A and B, and data not shown). Interestingly, RXR agonist SR11237 exhibited some activity on its own (Fig. 3Go, A and B), which is surprising in light of the concept that RXR complexed with unliganded c-erbA/TR or RAR should be transcriptionally silent (Ref. 49 and references therein). Some residual T3 that could not be removed from serum and that synergizes with SR11237 might account for the activity observed.



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Figure 3. T3, atRA, 9cRA, and SR11237 (SR) induce CAII Transcription in SCF-Dependent Erythroid Progenitor Cells

A, SCF-dependent erythroid progenitor cells were differentiated in the presence of various ligands and combinations thereof, as indicated, or left untreated (no). After 24 h, RNA was isolated and analyzed for CAII expression by Northern blotting (10 µg total RNA/lane). The blot was subsequently hybridized with a 18S rRNA-specific probe to demonstrate equal RNA loading per lane. B, CAII mRNA levels of panel A were quantified by PhosphorImager and are plotted as fold induction of control (untreated cells, no) set arbitrarily at 1. Average values of three independent experiments are shown. Standard deviations are indicated.

 
Finally, the ligand-specific effects measured on CAII transcription were also observed in undifferentiated proliferating cells. SCF/TGF{alpha}-dependent erythroid progenitor cells were grown in the presence of SCF and TGF{alpha} (36, 37), treated with ligand for 3 h, and analyzed for specific effects on CAII transcription by Northern blotting. Again, when single compounds were administered T3 was found to be the most effective factor; simultaneous treatment with T3 and 9cRA yielded maximum CAII transcription (Fig. 4AGo). We also emphasize that in these experiments activation of endogenous nuclear receptors rapidly induced transcription of an endogenous gene (CAII) within 3 h of ligand treatment which, most importantly, occurred uncoupled from differentiation. While these results are in line with our previous studies, which identified CAII as a direct target gene of hormone-activated c-erbA/TR, so far only an erbA-binding site has been identified within this gene (32); the identity of additional NR response elements remains to be determined.



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Figure 4. T3, atRA, and 9cRA Induce CAII Transcription in Proliferating Erythroid Cells

A, Proliferating erythroid progenitor cells (grown in the presence of SCF, TGF{alpha}, and steroid hormones; see Materials and Methods) were treated with T3, atRA, 9cRA, or T3+9cRA for 3 h or left untreated (no) as indicated. Total RNA (10 µg per lane) was analyzed for CAII and c-myb mRNA levels by Northern blot hybridization (CAII and c-myb, respectively). 18S rRNA stained with methylene blue is shown (18S). B, HD3 erythroblasts overexpressing c-erbA/TR (32 ) were treated with T3 for 2, 4, and 6 h or left untreated (0). RNA was analyzed for CAII and c-myb expression by Northern blotting. 18S rRNA stained with methylene blue shows equal RNA loading per lane (18S).

 
Interestingly, in such cells there was an inverse relationship between the induction of CAII transcription and c-myb mRNA levels. Expression of c-myb was highest in untreated cells and reduced by 2-fold within 3 h of treatment with T3, atRA, 9cRA, and T3 plus 9cRA (Fig. 4AGo). Essentially the same result was obtained with HD3 erythroblasts that ectopically expressed c-erbA/TR (32); since these cells contained higher c-erbA/TR levels, the magnitude of c-myb down-regulation after 2, 4, and 6 h T3 treatment was even more pronounced (Fig. 4BGo). This observation is interesting since in erythroid progenitor cells c-myb expression correlates with their proliferative potential and declines when cells differentiate (19, 31, 32, 40, 45). Most importantly, in these experiments hormone treatment led to c-myb down-regulation uncoupled from differentiation, suggesting that ligand-activated NRs might cause and/or contribute to growth arrest by down-regulating c-myb expression. Whether ligand-activated RXR, RAR, or c-erbA/TR directly effect c-myb transcription by binding to c-myb regulatory elements still remains to be determined. Our results also indicate that ligand-activated NRs are dominant over c-kit/SCF or TGF{alpha} receptor in regulating c-myb expression. In summary, all these data point to an important function of ligand-activated RXR, RAR, and c-erbA/TR in induction of erythroid cell differentiation and gene transcription, with, among the NRs analyzed, T3-activated c-erbA/TR being most effective.

Dominant-Negative RXR Receptors Affect Erythroid Cell Growth
While the experiments described above assessed RXR activity on differentiation, we were also interested in determining whether RXR in the absence of ligand would, similar to unliganded c-erbA/TR, affect growth of erythroid progenitor cells. Since erythroid progenitor cells express at least two RXR isoforms (N. Koritschoner, P. Bartunek, and M. Zenke, in preparation) we sought to subvert endogenous RXR activity in such cells by ectopic expression of dominant interfering RXR versions.

To this end, retroviral vectors were constructed that contained mutated RXRs devoid of AF-1 and AF-2, or of AF-2 only, or that had the DNA-binding domain (DBD) deleted (140–448 RXR, 1–445 RXR and {Delta}DBD RXR, respectively; Fig. 5AGo). 1–445 RXR and 140–448 RXR were expected to target RXR/RXR homodimers and also heterodimeric RXR complexes where RXR is the transcriptionally active partner and to render them transcriptionally inactive. Additionally, both 1–445 RXR and 140–448 RXR were still able to form heterodimers with c-erbA/TR since such heterodimers did respond to T3 and 9cRA in tyrosine kinase oncogene-transformed erythroblasts expressing these mutants (P. Bartunek and M. Zenke, unpublished). Furthermore, both RXR mutants effectively heterodim-erized with RAR and supported RAR-specific transactivation in response to ligand (50). The {Delta}DBD RXR, however, should target both homodimeric and heterodimeric RXR complexes and render them nonfunctional for DNA binding and thus should generate a functional knock-out of all RXR complexes within the respective cell.



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Figure 5. Transcriptional Activity and Expression of Wild-Type and Mutant RXR

A, Schematic representation of human RXR{alpha}, human 1–445 RXR{alpha}, mouse 140–448 RXR{alpha}, and human {Delta}DBD RXR{alpha} (RXR, 1–445, 140–448, and {Delta}DBD, respectively). The DBD is shown in black. B, RXR, 1–445 RXR, 140–448 RXR, and {Delta}DBD RXR contained in recombinant retrovirus vectors were analyzed for transcriptional activity in response to 9cRA in transient cotransfection experiments in CEF. The luciferase reporter construct contains the rat CRBPII RXRE (RXRE). Control, No RXR expression plasmid transfected. C, CEF stably expressing RXR, 1–445 RXR, 140–448 RXR, and {Delta}DBD RXR were analyzed by Western blotting using an RXR-specific antibody as indicated. Control, CEF containing empty vector only.

 
As expected 1–445 RXR, 140–448 RXR, and {Delta}DBD RXR were transcriptionally inactive in inducing 9cRA-dependent expression of a RXRE reporter plasmid in transient cotransfection experiments in chicken embryo fibroblasts (CEF); {Delta}DBD RXR also suppressed endogenous RXR activity (Fig. 5BGo). Additionally, {Delta}DBD RXR effectively silenced c-erbA/TR- and T3-dependent transcription from a TRE-driven reporter construct (data not shown). Activities of 1–445 RXR and 140–448 RXR were described previously (50, 51). Recombinant retroviral vectors containing wild-type or mutant RXRs were then transfected into CEF together with RCAN helper virus DNA and virus producing neomycin-resistant cells were selected. Cells effectively expressed wild-type and mutant RXR proteins as determined by Western blot analysis (Fig. 5CGo).

The effect of wild-type and mutant RXRs on the proliferative potential of erythroid progenitor cells was then tested, using a strategy employed before for studying c-erbA/TR function (see above). SCF-dependent erythroid progenitor cells were prepared from chicken bone marrow and infected with recombinant retroviruses containing 1–445 RXR, 140–448 RXR, {Delta}DBD RXR, and wild-type RXR. As controls, cells were infected with v-erbA virus or neo control virus only or left untreated. Growth of retrovirus-infected cells was then monitored by determining cell numbers in regular time intervals (Fig. 6Go, A and C). At day 7 post infection, expression of the various RXR proteins was readily detectable in Western blots (Fig. 6BGo and data not shown). Control cells infected with neo virus only or uninfected cells showed the expected growth potential and ceased proliferation at day 7–8 of culture (Fig. 6Go, A and C, and data not shown). Additionally, cells containing either wild-type RXR, 1–445 RXR, or 140–448 RXR behaved very similarly and grew only marginally faster than control, achieving 2- to 3-fold higher cumulative cell numbers at day 7–8 of culture (Fig. 6Go, A and C). Thus, both wild-type and the 1–445 or 140–448 mutant RXRs were found to support a transient outgrowth of erythroid cells. In marked contrast, {Delta}DBD RXR cells were severely compromised in growth, and there was no outgrowth of erythroid progenitor cells in such cultures (Fig. 6Go, A and C). In some experiments, nonerythroid cells were eventually obtained that expressed {Delta}DBD RXR and myeloid- specific markers (data not shown). v-erbA control cells showed, as expected, maximal growth rates (Fig. 6Go, A and C).



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Figure 6. RXR, 1–445 RXR, and 140–448 RXR Transiently Enhance Growth Rates of SCF-Dependent Erythroid Progenitor Cells in Vitro, while {Delta}DBD RXR is Deleterious to Growth

A, Growth of erythroid progenitor cells expressing RXR, {Delta}DBD RXR or, as a positive control, v-erbA was monitored as in Fig. 1AGo. Control, Cells containing empty vector only. B, Progenitor cells express RXR and {Delta}DBD RXR (day 7) as demonstrated by Western blotting using a RXR{alpha}-specific antibody. Control, Progenitor cells containing empty vector only. C, Growth properties of erythroid progenitor cells expressing RXR, 1–445 RXR, 140–448 RXR, {Delta}DBD RXR, c-erbA/TR, and v-erbA. Cumulative cell numbers at day 7 of culture (average values of three independent experiments) are shown. Control, Progenitor cells containing empty vector only.

 
Taken together, these studies demonstrate that a functional knock-out of RXR is incompatible with erythroid cell growth in vitro. We emphasize, however, that {Delta}DBD RXR is not cytotoxic per se, since the myeloid cells eventually obtained expressed {Delta}DBD RXR protein (data not shown). {Delta}DBD RXR was also efficiently expressed in CEF (Fig. 5CGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The activities of the c-erbA/TR, RAR, and RXR NRs and their functional domains have been extensively studied in transient transfection systems and more recently in whole animals by targeted gene inactivation experiments (1, 2, 3, 4, 5, 6, 7). However, there is clearly a paucity of in vitro differentiation systems with primary cells, where the activities of NR-specific ligands and by ectopic and/or overexpression the impact of individual NRs on growth and differentiation can be faithfully investigated. In vitro systems of primary erythroid progenitor cells, like the one described in this paper, provide such opportunities. Obviously NRs in such culture systems will affect the expression of multiple genes, both directly and indirectly, as well as positively and negatively. While it is well established that ligand-activated c-erbA/TR induces red cell-specific gene expression and promotes erythroid differentiation, we demonstrate in this study that additionally unliganded c-erbA/TR affects erythroid cell development. Furthermore, we have addressed the question of the impact of RXR, the obligate heterodimeric partner of c-erbA/TR, on erythroid cell growth and differentiation.

Here we show that unliganded c-erbA/TR represses erythroid cell differentiation and supports sustained growth of SCF-dependent erythroid progenitor cells in vitro. This finding extends our previous observation in tyrosine kinase oncogene-transformed erythroblasts where overexpressed gag-c-erbA/TR and chimeric v-erbA/c-erbA/TRs in the absence of ligand suppressed gene expression and differentiation (19). Thus, c-erbA/TR appears to act as a binary switch affecting erythroid cell fate: unliganded c-erbA/TR supports growth while ligand-activated c-erbA/TR induces differentiation. This dual activity of c-erbA/TR appears to be due to its ability to repress and activate gene expression, such as CAII, by recruitment of specific corepressors and coactivators, respectively, in response to hormone (Fig. 7Go; Refs. 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 19).



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Figure 7. Schematic Representation of c-erbA/TR and RXR Action during Erythroid Cell Growth and Differentiation

In the absence of hormone c-erbA/TR blocks differentiation and supports growth of erythroid progenitors (A) while a hormone-activated c-erbA/TR promotes differentiation (B). In the absence of hormone, c-erbA/TR recruits corepressors that silence gene transcription of gene X (e.g. CAII). Alternatively and depending on the target gene (Y) corepressor binding might also lead to gene activation (54 ). Ligand-activated c-erbA/TR induces transcription of gene X (e.g. CAII) via coactivators and simultaneously might suppress transcription of other genes Y (e.g. c-myb).

 
Furthermore, in its unliganded state c-erbA/TR exhibits an activity very similar to its oncogenic variant v-erbA. This observation is most interesting, since both unliganded c-erbA/TR and v-erbA appear to recruit a similar or identical set of transcriptional corepressors through the N-CoR/SMRT-binding site residing in the hinge region of the erbA molecules (9, 11, 12, 13, 28). Thus, the experiments described in this paper would be in accord with a model where the v-erbA oncoprotein is locked in an inactive receptor conformation resembling unliganded c-erbA/TR and due to its association with corepressor proteins acts as a constitutive repressor of transcription. Such a concept would also be in line with studies in which a specific point mutation contained in the N-CoR/SMRT binding site present in td359 v-erbA, which abrogates corepressor binding if put in the context of c-erbA/TR, affects the silencing function of v-erbA and renders it nontransforming (11, 27, 28, 46). There are, however, differences between unliganded c-erbA/TR and v-erbA, which are also revealed in the present study; v-erbA is, for example, more effective in supporting sustained progenitor cell growth than unliganded c-erbA/TR, which might be due to the specific point mutations both within and outside of the DBD that affect sequence-specific DNA binding (52, 53). Thus, gene regulation in erythroid cells by v-erbA might comprise a more broad set of target genes including those regulated by other NRs and/or transcription factors. Our recent finding that erythroid cells express a number of other NRs and orphan receptors, which might be important for red blood cell development (N. Koritschoner, P. Bartunek, and M. Zenke, unpublished), would be in line with such an idea. Alternatively, v-erbA might be more restricted in its DNA-binding specificity than c-erbA/TR, as suggested before (47, 52, 53), or even have a specific set of v-erbA-specific sites that are not shared with other transcription factors. Furthermore, other studies proposed that v-erbA mimics activities of hormone-activated estrogen receptor and glucocorticoid receptor (37, 42, 45) indicating that v-erbA might act through more than one mechanism.

Both c-erbA/TR and v-erbA associate with RXR, an obligate heterodimeric partner of a number of NRs (4, 5, 47). Erythroid cells express RXR{alpha} and RXR{gamma} (N. Koritschoner, P. Bartunek, and M. Zenke, in preparation) and in the present study we have addressed the question of the impact of RXR for erythroid cell growth and differentiation. It was found that selective ligands, which activate RXR or the c-erbA/TR and RAR proteins contained in the heterodimeric RXR/c-erbA/TR and RXR/RAR complex, accelerate erythroid differentiation and induce gene transcription (like CAII; Fig. 7Go). When individual compounds were administered T3 was most effective, supporting the view that c-erbA/TR is one of the major ligand-dependent transcription factors in erythroid cells. Additionally, at the same time ligand-activated NRs, such as c-erbA/TR, down-regulate the expression of other genes like c-myb (Fig. 7Go). Whether this involves a direct interaction of NRs with c-myb regulatory sequences remains to be determined. Furthermore, GATA-1 and GATA-2, which play decisive roles in red blood cell development (Refs. 41, 44 and references therein) are not directly regulated by c-erbA/TR, RAR, and RXR (K. Briegel, P. Bartunek, and M. Zenke, unpublished).

Finally to determine the impact of RXR on erythroid cell growth, dominant interfering mutant RXRs lacking the transcriptional activator functions AF-1 and AF-2, or AF-2 only, or the entire DBD (140–448 RXR, 1–445 RXR, {Delta}DBD RXR, respectively) were ectopically expressed in erythroid cells. Interestingly, expression of wild-type RXR, 140–448 RXR, or 1–445 RXR exhibited activities that supported a transient outgrowth of erythroid cell, probably by enhancing the transcriptional repressor function of unliganded endogenous c-erbA/TR. In support of this idea, both wild-type and 140–448 and 1–445 mutant RXRs contain heterodimerization and DNA-binding functions (50) and bind c-erbA/TR and thereby might target such hereodimeric complexes to specific genes that are silenced. Alternatively, unliganded RXR might have, similar to unliganded c-erbA/TR, some activity on its own in supporting erythroid progenitor cell growth, potentially through N-CoR/SRMT binding. The observation that RXR binds N-CoR/SMRT less efficiently than c-erbA/TR (11) might explain why only a transient outgrowth is observed. Most importantly, forced expression of {Delta}DBD RXR severely compromised erythroid cell growth, while its expression was not deleterious for myeloid cells and CEF. {Delta}DBD RXR targets both homodimeric and heterodimeric RXR complexes and renders them nonfunctional for DNA binding and thus generates a functional knock-out of all RXR complexes within the cell. Thus, our studies demonstrate an important role of RXR expression in erythroid cell development.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Tissue Culture
SCF and TGF{alpha}-dependent erythroid progenitor cells were prepared from bone marrow of 3- to 10-day-old SPAFAS chicks essentially as described (36, 39, 41). Briefly, after Ficoll-Hypaque centrifugation (density 1.077 g/cm3; Eurobio, Paris, France) bone marrow cells (2–4 x 106 cells/ml) were cultured in modified CFU-E medium (18, 39) containing 100 ng/ml avian SCF (55). Tested batches of FCS (Boehringer Mannheim, Mannheim, Germany), chicken serum (ChS, Sigma, St. Louis, MO), and detoxified, delipidated BSA (fraction V, Sigma) were used. Recombinant chicken myelomonocytic growth factor [cMGF, 40 ng/ml (56, 57)] was present during the initial phase of culture (days 1 and 2) to induce differentiation and adherence of macrophages and macrophage precursor cells. At day 2, nonadherent cells were recovered and grown in CFU-E medium plus 100 ng/ml avian SCF. Routinely, homogenous cultures of SCF-dependent erythroid progenitor cells were obtained after 3–4 days of culture at 37 C in 5% CO2 atmosphere and high humidity (95%).

To prepare TGF{alpha}-dependent progenitors, chicken bone marrow cells were prepared and cultured in modified CFU-E medium essentially as described above, but containing 100 ng/ml SCF, 5 ng/ml TGF{alpha} (Promega, Madison, WI), 10-6 M estrogen (Sigma), and 10-6 M dexamethasone (Sigma; Refs. 36, 37, 42). Homogenous populations of TGF{alpha}-dependent erythroid progenitor cells were obtained at days 12–14. Cell growth was monitored by measuring cell numbers in regular time intervals by the CASY-1 Cell Counter and Analyser System (Schàrfe, Reutlingen, Germany).

For transformation of CEF with recombinant retroviral vectors (see below), cells were grown in DMEM supplemented with 8% FCS (Boehringer Mannheim), 2% ChS (Sigma), 10 mM HEPES, pH 7.0, and 100 U/ml penicillin and streptomycin (in the following referred to as standard growth medium). CEF were transfected with 10 µg retroviral vector DNA and 1 µg RCAN helper virus DNA as described previously (41, 44). After G418 selection, these virus-producing CEF were then used for infection of chicken bone marrow progenitor cells in modified CFU-E medium [containing FCS and ChS that were extensively deprived of T3 and retinoids (58, 59)]. Briefly, chicken bone marrow was prepared (see above) and cocultured with virus-releasing, mitomycin C-treated CEF in the presence of SCF for 2 days. At day 2, nonadherent cells infected with v-erbA, td359 v-erbA, c-erbA/TR, RXR, 1–445 RXR, 140–448 RXR, or {Delta}DBD RXR viruses were collected and cultured in modified CFU-E medium (deprived of T3 and retinoids; see above) plus 100 ng/ml SCF and 40 ng/ml recombinant human insulin-like growth factor type 1 (long-R3 IGF-I; Sigma). CEF producing empty neo vector pSFCV-LE (60) served as control and were used for infection of bone marrow cells accordingly. Cell growth was assessed by counting cells in regular time intervals (see above).

Construction of Retroviral Vectors
Retroviral vectors containing RXR, 1–445 RXR, 140–448 RXR, or {Delta}DBD RXR were constructed by subcloning wild-type human RXR{alpha} cDNA (61), human 1–445 RXR{alpha} (51), mouse 140–448 RXR{alpha} (dnRXR{alpha}{Delta}AB in 50), and human {Delta}DBD RXR{alpha} into the EcoRI site of pSFCV-LE (60); in {Delta}DBD RXR amino acid positions 46–222 comprising the entire DBD of human RXR{alpha} are deleted. Recombinant retrovirus vectors containing v-erbA and c-erbA/TR, respectively, were described previously (23, 32).

Differentiation Assays
To induce erythroid differentiation, cultures of SCF-dependent erythroid progenitors were incubated in CFU-E medium without chicken serum (2 x 106 cells/ml; Ref. 18) supplemented with 3% anemic ChS (as a source for erythropoietin) plus 10 ng/ml recombinant human insulin (Novo Nordisk). Cells were treated with receptor-specific ligands [1 x 10-7 M T3, Sigma; 1 x 10-6 M atRA, Sigma; 1 x 10-6 M 9cRA, Sigma; 1 x 10-6 M SR11237 (62)] that were applied individually or in various combinations thereof. Erythroid differentiation was assessed by determining hemoglobin accumulation in cytospin preparations stained with neutral benzidine and Diff-Quik [Baxter, Switzerland (39, 44)], by measuring reduction in cell size with the CASY-1 Cell Counter and Analyser System (40, 41) and loss of proliferative potential in [3H]thymidine incorporation assays (39, 44). Photographs were taken with Axiophot II microscope (Carl Zeiss, Jena, Germany) and Kontron ProgRes 3012 CCD camera followed by image processing with Adobe Photoshop software.

RNA Analysis and Northern Blotting
Total RNA was prepared and analyzed by Northern blotting as described previously (41, 44). Blots were stained with methylene blue to demonstrate equal RNA loading per lane. Probes corresponding to cDNA of human RXR{alpha} (61), chicken v-myb [EcoRI-XbaI fragment (63)], CAII (19, 32), and to 18S rRNA were prepared by random priming and used for hybridization. Blots were hybridized, washed, and exposed to film and/or evaluated by PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Transient Transfections
Calcium phosphate coprecipitation was used to transiently transfect CEF with retroviral expression vectors containing RXR, 1–445 RXR, 140–448 RXR, or {Delta}DBD RXR (see above) together with a luciferase reporter containing the rat CRBPII RXRE (5'-GCTGTCACAGGTCACAGGTCACAGGTCACAGT TCA-3') between HindIII and XhoI at position -109 of the thymidine kinase promoter (32, 61). RSV-ß-gal was cotransfected to normalize for transfection efficiencies. Transfected cells were grown in standard growth medium containing FCS and ChS that were extensively deprived of T3 and retinoids (see above). 9cRA (10-6 M) was added, and 2 days later cell extracts were prepared and analyzed for luciferase and ß-galactosidase activity as described (32). Luciferase values were normalized for ß-galactosidase activity.

Western Blot Analysis
Total cell extracts were separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (BA85, Schleicher & Schuell, Dassel, Germany). After blocking membranes overnight in TBS (25 mM Tris-HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, 0.7 mM CaCl2, 0.5 mM MgCl2, 0.6 mM Na2HPO4) containing 3% BSA, 1 mM EDTA, 0.05% Tween-20, and two washes with wash buffer (50 mM Tris-HCl, pH 8.0, 0.1 M NaCl, 0.1% Tween-20), antibody was added and incubated in blocking buffer for 1 h. The primary antibodies used were erbA-specific rabbit polyclonal antibody (a kind gift of J. Ghysdael, Orsay, France), RXR{alpha}-specific mouse monoclonal antibody 4RX-3A2(DE) (Ref. 64 ; a kind gift of M.-P. Gaub and P. Chambon, Strasbourg, France), and GATA-1-specific rabbit polyclonal antipeptide antibody (41). Subsequently, blots were washed three times with wash buffer and incubated with the appropriate secondary antibodies [enhanced chemiluminescence (ECL) kit, Amersham, Little Chalfont, UK] in TBS supplemented with 5% nonfat milk powder for 45 min. All reactions and incubations were performed at room temperature. Membranes were washed another five times with wash buffer, developed in ECL reagents, and exposed to film.


    ACKNOWLEDGMENTS
 
We would like to thank P. Chambon, H. Gronemeyer, and H. Stunnenberg for recombinant plasmids, RXR-specific antibodies, and SR11237; J. Ghysdael for anti-erbA-specific antiserum; and P. Chambon, H. Gronemeyer, and N. Koritschoner for critical review of the manuscript; G. Blendinger for expert technical assistance; and I. Gallagher for typing the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Martin Zenke, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, D-13122 Berlin, Germany. E-mail: zenke{at}mdc-berlin.de

Received for publication February 18, 1998. Revision received May 21, 1998. Accepted for publication June 15, 1998.


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