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Cell Growth & Differentiation Vol. 12, 563-572, November 2001
© 2001 American Association for Cancer Research

The Nuclear Orphan Receptor TR4 Promotes Proliferation of Myeloid Progenitor Cells1

Nicolás P. Koritschoner2, Jaime Madruga, Signe Knespel, Gitta Blendinger, Birgit Anzinger, Albrecht Otto, Martin Zenke3 and Petr Bartunek

Max-Delbrück-Center for Molecular Medicine, D-13122 Berlin, Germany [N. P. K., J. M., S. K., G. B., B. A., A. O., M. Z., P. B.], and Institute of Molecular Genetics, 166 37 Prague 6, Czech Republic [P. B.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Nuclear receptors represent key regulators in cell proliferation, differentiation, and development. Here we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues and have analyzed the impact of TR4 in this cell compartment. We show that TR4, when ectopically expressed in bone marrow cells via retrovirus vector, promotes proliferation of myeloid progenitor cells. Cells represent promyelocytes as judged by morphological features, expression of cell surface molecules, and specific markers like Mim-1 and CAAT/enhancer binding protein ß. We also demonstrate that the growth promoting activity of TR4 is not exclusively dependent on its association with DNA, because expression of a mutated TR4 version devoid of its DNA binding domain exhibits a similar proliferative potential as wild-type TR4. In conclusion, these data position the orphan receptor TR4 as an important regulator of myeloid progenitor cell proliferation and development.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
NRs4 comprise a large family of transcription factors that are involved in the regulation of diverse biological processes, such as cell growth, differentiation, development, and homeostasis (1, 2, 3) . In many cases, NRs exert their regulatory functions on binding of known ligands, such as retinoids, steroids, thyroid hormone, eicosanoids, and vitamin D. In addition, there is an even larger set of NRs for which no regulatory ligands exist or have not been identified thus far, which is designated the orphan receptor subfamily (4 , 5) . NRs are composed of functionally distinct domains which show a variable degree of amino acid sequence homology between different receptors. The highly conserved DNA binding domains have been studied extensively, and their respective target DNA sequences have been defined for many receptors. The mechanism of NR action on their target genes involves the recruitment of several auxiliary proteins known as corepressors and coactivators, which in turn modify the chromatin structure by changing the acetylation status of histones (6) .

A number of studies demonstrated that NRs have a profound impact on development of hematopoietic cells, e.g., RXR and TR/c-erbA effectively regulate erythroid cell growth and differentiation (Refs. 7 and 8 and references therein). Also in this context, an oncogenic version of TR/c-erbA, the v-erbA oncoprotein, arrests differentiation of erythroblasts and cooperates with v-erbB in producing a fatal erythroleukemia in chickens (9 , 10) . Additionally, activation of both glucocorticoid receptor and ER is required for an efficient induction of self renewal of erythroid progenitor cells (11, 12, 13, 14) . In other cell systems, activation of RAR{alpha} by retinoic acid directly mediates a growth arrest of human HL60 leukemia cells and promotes their differentiation into neutrophils (15) . Furthermore, a chromosomal translocation that characterizes most of acute PMLs results in a fusion protein containing the NH2-terminal part of the PML gene and the DNA binding and ligand binding domains of RAR{alpha} (16 , 17) . Interestingly, introduction of a dominant-negative RAR in bone marrow cells immortalized a lymphohematopoietic progenitor, which can be induced to differentiate into lymphoid, myeloid, and erythroid lineages (18) .

In view of the central role of NRs as key factors in regulating molecular mechanisms of hematopoietic cell development, we focused on the identification of potential new members of the NR gene family by applying a reverse transcriptase-PCR approach. This strategy used degenerate primers encompassing the region encoding the DNA binding domain of most NR genes (19) . During the course of this study, we consistently found the TR4 gene as the most abundant cDNA in primary cultures of chicken bone marrow cells.

TR4 (TAK1 and NR2C2; Ref. 20 ) is an orphan receptor that is closely related to TR2, and both TR2 and TR4 define a subclass within the steroid/thyroid receptor family (21, 22, 23) . TR4 and TR2 form homodimers as well as TR4/TR2 heterodimers that synergize in repressing gene expression (24) . However, TR4 and TR2 do not heterodimerize with RXR, which is an obligate partner for most NRs that are able to form heterodimers. Several lines of evidence suggest that TR4 is an important regulator of distinct NR signaling pathways, e.g., it has been shown that TR4 effectively represses RAR, RXR, peroxisome proliferator-activated receptor, vitamin D3 receptor, and TR-mediated gene expression (Refs. 22 , 24, 25, 26 , and references therein). TR4 is able to repress transcription by interaction with DR1-HREs and DR2 elements (27) . TR4 also appears to repress ER-mediated transactivation specifically in bone cells but not in kidney or breast cancer cells (28) . In contrast, some genes containing DR4-HRE motifs or nonclassical thyroid HREs were described to be activated by TR4 (27) . Such a positive transcriptional activity of TR4 was observed for the ciliary neurotrophic factor receptor gene (29) , and a cooperation of TR4 with COUP-TF and TR{alpha}1 in activation of the HIV-long terminal repeat promoter was also reported (30) . More recently, it was demonstrated that TR4 interacts with the androgen receptor, and as a consequence, both TR4 and androgen receptor mediated pathways were influenced (31) . The current mode of TR4 action is still a subject of debate and may involve distinct mechanisms, such as: (a) occupation of different HREs by TR4 homodimers or TR4/TR2 heterodimers; (b) sequestration of (a) coactivator(s); (c) association of TR4 with (an) as yet undefined corepressor(s); (d) modulation of TR4 activity by thus far unknown ligand(s); or (e) other posttranslational events.

In this work, we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells. We additionally analyzed the impact of TR4 on primary bone marrow cells by ectopic expression of TR4 via retrovirus vector. Additionally, we show that a DNA binding-deficient TR4 protein exerts a similar effect as its natural counterpart and provide information of the potential role of such proteins on hematopoietic cell growth and differentiation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Nuclear Orphan Receptor TR4 Is Highly Expressed in Hematopoietic Tissues and Cell Types.
To determine the expression profile of NRs in hematopoietic cells, we applied a reverse transcriptase-PCR-based strategy that used degenerate primers encompassing the highly conserved DNA binding domain of NRs (19) , thereby following an approach that was successfully used before (32) . Several members of the NR gene family were found to be expressed in various types of progenitors derived from chicken bone marrow (data not shown). Interestingly, one of the most abundant cDNA clones found corresponded to the chicken homologue of nuclear orphan receptor TR4. To additionally explore the expression pattern of TR4, human and chicken TR4 cDNA were used as probes in Northern blots containing a panel of RNA from hematopoietic and nonhematopoietic tissues and cell types (Fig. 1, A–C)Citation .



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Fig. 1. Hematopoietic cells and tissues express TR4. Total RNA of various cell types and tissues from human (A), mouse (B), and chicken (C) was analyzed by Northern blotting for TR4 expression. TR4-specific transcripts are indicated (arrowhead and star). 28S rRNA stained with Methylene Blue is shown to demonstrate RNA loading. Erbls, erythroblasts; DC, dendritic cell; DCprog, DC progenitors; SCF and STED, SCF- and SCF/transforming growth factor{alpha}-dependent erythroblasts, respectively.

 
Prominent TR4 expression was seen in human myeloid cell lines (U937 and HL60) and primary monocytes and dendritic cells (Fig. 1ACitation and data not shown). Similarly, TR4 was highly expressed in murine myeloid cells (Fig. 1B)Citation . TR4 expression was moderate in the T-cell compartment and low in B cells. In addition, TR4 was readily detectable in erythroid cell lines of human, mouse, and chicken (K562, MEL, and HD3, respectively) and also in primary red blood cell progenitors (Fig. 1, A–CCitation and data not shown). As expected from previous studies (21 , 33) , TR4 expression was high in brain. TR4 levels were high in thymus and somewhat lower in bursa and spleen (Fig. 1C)Citation . Accordingly, TR4 transcripts were found in T cells, B cells, and dendritic cells from chicken. Thus, TR4 is expressed in a variety of cell types of the hematopoietic system.

TR4 Effectively Induces Proliferation of Myeloid Progenitor Cells.
Whereas previous studies demonstrated the importance of TR4 in repressing several NR pathways, the impact of TR4 in primary cells and particularly in those of the hematopoietic system has thus far not been studied. Therefore, to get insight into its potential role in hematopoietic cells, TR4 was ectopically expressed in chicken bone marrow cells, and its effects on proliferation and/or development of such cells were investigated. To this end, a retrovirus vector harboring TR4 cDNA was constructed and transfected into CEF, and neomycin-resistant cells were selected. Cells effectively expressed TR4 protein (see below, Fig. 3Citation ) and were used as a source of virus to infect bone marrow cells. Pilot experiments using different culture conditions with various combinations of growth factors, hormones, sera, and conditioned media allowed us to optimize the growth of TR4-infected precursor cells. Most significantly, TR4 caused an outgrowth of cells within 4–5 days, whereas this was not observed in control cultures infected with empty vector (Fig. 2A)Citation . Cells grew in large aggregates containing >100 cells (Fig. 2BCitation , left panel) and ceased proliferation after 14–16 days.



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Fig. 3. Expression and subcellular localization of HA-TR4 and HA-{Delta}TR4. A, schematic representation of TR4 constructs in retrovirus vectors. Wild-type human TR4 cDNA (hTR4), the HA-tagged TR4 (HA-TR4), and the DNA binding domain-deficient version (HA-{Delta}TR4) are shown. B, Western blot analysis of HA-TR4 and HA-{Delta}TR4 expression in retrovirus-transduced CEF with HA-specific antibody. Control, neo-resistant CEF with empty vector. C, subcellular localization of TR4 proteins. CEF-expressing HA-TR4 and HA-{Delta}TR4 proteins were analyzed by indirect immunofluorescence with an anti-HA-specific antibody ({alpha}-HA). DAPI and tetramethylrhodamine isothiocyanate-phalloidin detected DNA and F-actin, respectively. Please note that the weak cytoplasmic/perinuclear staining in the HA-{Delta}TR4 DAPI panel is attributable to spillover of the bright anti-HA staining into the DAPI channel.

 


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Fig. 2. TR4 promotes proliferation of bone marrow cells in vitro. A, growth curve of bone marrow cells infected with TR4 retrovirus (•) or empty vector (control, {square}). Cells were grown in medium containing 100 ng/ml SCF, and cumulative cell numbers were determined at regular time intervals. A representative experiment is shown. B, photomicrograph of TR4 cells at day 8 of culture (left panel). Cells were also centrifuged onto slides and stained with histological dyes (DiffQuik; right panel). C, expression of cell type-specific markers on TR4 cells. Cells were obtained as above and analyzed for cell surface markers by flow cytometry at day 8. Monoclonal antibodies were: MC-47/83 (myeloid), JS4 (erythroid), JS8 (transferrin receptor), and CD3 (T cell). Control, cells incubated with FITC-labeled secondary antibody only (open area).

 
Histological examination of TR4 cells revealed that they resemble myeloid progenitors (Fig. 2BCitation , right panel). Cells had a mean diameter of 9 µm and were additionally characterized for cell surface marker expression by flow cytometry using a panel of monoclonal antibodies (Fig. 2CCitation and data not shown). TR4 cells highly expressed the myeloid markers MC47–83, MC51–2, and MC22–3 and also MHC class II. K1, a macrophage marker, was negative. Transferrin receptor (detected by JS8 antibody) was present, which was expected because cells were in the exponential phase of growth. TR4 cells were, however, devoid of T-cell-specific markers (CD3, CD4, and CD8), erythroid-specific markers (JS4), and the eosinophilic marker (EOS47; Fig. 2CCitation and data not shown). HEM-CAM, an adhesion molecule of early hematopoietic progenitors, was also absent, whereas CD44, another adhesion molecule expressed by several hematopoietic cell types, was expressed. MEP17/{alpha}2/ß1 integrin was also present in TR4 cells, whereas MEP21/thrombomucin, which is highly expressed on thrombocytes and multipotent progenitors of myeloid, thrombocytic, and erythroid cells (34, 35, 36) , was absent.

In conclusion, morphological features and the cell surface marker profile clearly indicate that TR4 cells are myeloid progenitors. This is in accordance with expression in these cells of myeloid proteins, such as C/EBPß (see below Fig. 7Citation ).



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Fig. 7. TR4 cells highly express the myb-induced myeloid protein 1 (Mim-1) and the transcription factor C/EBPß. In A, protein extracts from cells shown in Fig. 5Citation were analyzed by 12% SDS-PAGE and stained with Coomassie blue. The protein corresponding to the prominent Mr 35,000 band (arrowhead) was sequenced and shown to be Mim-1. A degradation product of Mim-1 (Mr 18,000, *) is also more abundant in HA-TR and HA-{Delta}TR4 cells than in RXR and {Delta}RXR cells. In B, expression of Mim-1 protein of the samples in A was revealed by Western blotting and Mim-1-specific antibody. In C, Western blot of the same extracts as in A reacted with an anti-C/EBPß antibody.

 
TR4 Growth Promoting Activity Is Compromised but not Abolished on Deletion of Its DNA Binding Domain.
TR4 has been described as a transcription factor involved in repressing and activating gene expression. Such a dual activity may largely depend on TR4-DNA contacts, TR4-cofactor(s) binding, or both. Therefore, to investigate which activity of TR4 might be involved in promoting growth of bone marrow cells as observed here, retroviruses containing HA-tagged versions of full-length TR4 or a mutant TR4 devoid of its DNA binding domain were constructed and analyzed (Fig. 3A)Citation . Efficient expression of the HA-TR4 and HA-{Delta}TR4 proteins was detected by Western blotting using an anti-HA-specific antibody (Fig. 3B)Citation . Additionally, the subcellular localization of TR4 proteins was determined by indirect immunofluorescence (Fig. 3C)Citation . In CEF, HA-TR4 was clearly confined to the nucleus, whereas HA-{Delta}TR4 protein was found in both the nucleus and cytoplasm. The latter observation is not surprising because TR4 and its closest relative, TR2, share a highly homologous nuclear localization signal in their DNA binding domain that has been shown to be functionally important in TR2 (37) , and this motif is deleted in HA-{Delta}TR4. As expected, CEFs containing empty vector were negative for staining with the HA-specific antibody (data not shown).

Bandshift assays demonstrated that HA-TR4 effectively bound to the TR4 binding site contained in DR1-HRE (AGGTCAAAGGTCA; Ref. 26 ), whereas HA-{Delta}TR4 did not (Fig. 4A)Citation . The band corresponding to HA-TR4 migrated slightly higher than untagged TR4. HA-TR4 binding was clearly sequence specific because it was efficiently competed by unlabeled DR1-HRE but not by a mutated oligonucleotide (Fig. 4ACitation and data not shown). Moreover, the anti-HA-specific monoclonal antibody was able to supershift the band of HA-TR4, whereas it did not affect the pattern of HA-{Delta}TR4. These data were additionally confirmed by Shift-Western experiments (38) where the proteins in the bandshift gel were transferred to a membrane and probed with the anti-HA antibody; as expected, the antibody detected HA-TR4, which corresponds to the shifted band (data not shown).



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Fig. 4. DNA binding and transcriptional activities of HA-TR4 and HA-{Delta}TR4. In A, DNA binding activity of TR4 proteins was analyzed in bandshift assays using whole cell extracts from stably transfected CEF shown in Fig. 3BCitation . A 32P-labeled oligonucleotide containing a DR1 motif was used. CEF (control) and neo-resistant CEF with empty vector (neoR) showed some endogenous binding activity (Lanes 2 and 3, respectively). HA-TR4 extracts exhibited a predominant band (arrowhead in left panel, Lanes 4 and 6), which was absent in HA-{Delta}TR4 samples (Lane 7). TR4 gave rise to a complex migrating slightly faster than HA-TR4 (Lanes 8 and 11). The addition of anti-HA-specific antibody ({alpha}-HA) supershifted the HA-TR4 band (marked by *), whereas HA-{Delta}TR4 and TR4 remained unaffected. Lane 1, no extract (no). Lane 5, competition with 100-fold excess of unlabeled oligonucleotide. Open arrowheads, background bands. In B, HA-TR4 and HA-{Delta}TR4 were analyzed for transcriptional activity in transient cotransfection experiments in CEF. The luciferase reporter construct contains the rat CRBPII-RXR responsive element. Transfection reactions contained 500 ng of both reporter and pSG5-RXR plasmids (all samples) and increasing amounts (5, 10, 50, 100, and 500 ng) of retrovirus vectors expressing HA-TR4 or HA-{Delta}TR4 as indicated. Luciferase activity was normalized by cotransfection of a ß-galactosidase reporter plasmid. 9cRA was added to a final concentration of 10-6 M as indicated (+).

 
To analyze the transcriptional activity of TR4 constructs, transient transfection experiments were performed. It was found that HA-TR4 repressed RXR- and TR-dependent transcriptional activation, whereas HA-{Delta}TR4 did not (Fig. 4BCitation and data not shown, respectively), which is in accord with previous observations (39) . Similar results were obtained in hematopoietic cells (HD3 erythroblasts, data not shown).

Having determined that HA-{Delta}TR4 does not bind DNA nor represses transcription, we next sought to examine whether the DNA binding activity of TR4 was responsible for the TR4-induced phenotype observed. Therefore, HA-TR4 and HA-{Delta}TR4 retroviruses were used to infect bone marrow cells as above. HA-TR4 behaved identically to unmodified TR4, and growing myeloid cells were readily obtained (Fig. 5Citation and data not shown). Surprisingly, an outgrowth of myeloid cells was also observed for HA-{Delta}TR4, though the total cell numbers were somewhat lower than for HA-TR4 (Fig. 5)Citation . As expected, control cells infected with neo-virus exhibited a restricted growth potential and ceased proliferation after 4–6 days in culture. In addition, retrovirus-transduced cells efficiently expressed HA-tagged TR4 proteins as revealed by Western blot analysis using the anti-HA-specific antibody (Fig. 5)Citation . Finally, both by morphology and cell surface marker expression, TR4, HA-TR4, and HA-{Delta}TR4 cells were identical (data not shown).



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Fig. 5. Activity of HA-TR4, HA-{Delta}TR4, RXR, and {Delta}RXR in promoting proliferation of bone marrow cells. Growth curves of bone marrow cells infected with HA-TR4, HA-{Delta}TR4, RXR, and {Delta}RXR as indicated. Control, cells with empty neo-virus. Cells were grown in medium with 100 ng/ml SCF, and cumulative cell numbers were determined daily. Inset: HA-TR4 and HA-{Delta}TR4 cells effectively express TR4 proteins as revealed by Western blotting and anti-HA-specific antibody.

 
In summary, TR4 and, to a lesser extent, also HA-{Delta}TR4 effectively supported an outgrowth of myeloid cells from bone marrow, indicating that the growth-promoting activity of TR4 is not lost by deletion of its DNA binding domain.

RXR and Its DNA Binding-deficient Variant {Delta}RXR Do Not Fully Recapitulate TR4 Effects on Bone Marrow Cells.
TR4 functions as a strong repressor of RXR-mediated transcription as published elsewhere and also shown here. Therefore, to test whether a dominant-interfering RXR would display a similar phenotype as TR4, {Delta}RXR lacking the DNA binding domain (7) was introduced into bone marrow cells via retrovirus. {Delta}RXR was demonstrated before to act as a dominant-interfering RXR allele in transient transactivation experiments (7) . Full-length RXR was used as a control. While there was an initial outgrowth of {Delta}RXR and RXR cells, at day 9 of culture, cells ceased proliferation, and cell numbers remained constant or started to decrease (Fig. 5)Citation . All these data indicate that while RXR promotes growth of myeloid cells, extended proliferation is specifically accomplished only by TR4 and TR4 variants. This difference in their proliferative potential is likely not to be due to different expression levels of TR4 and RXR, because both are expressed from the very same retroviral vector. Additionally, {Delta}RXR had a similar effect on bone marrow cells as full-length RXR, indicating that as seen for TR4, the DNA binding of RXR is dispensable for this activity.

To examine the differentiation potential of TR4 cells, inducers of myeloid differentiation, such as 9cRA or TPA, were added, and cells were investigated for morphological changes and adhesive properties (Fig. 6, A and B)Citation . As a control, cells were treated with chicken myelomonocytic growth factor, which in previous experiments showed growth-promoting but no differentiation-inducing activity (data not shown). On 9cRA treatment, only 5–10% of the HA-TR4 and HA-{Delta}TR4 cells became adherent (Fig. 6B)Citation . However, the majority of RXR and {Delta}RXR cells adhered to the dish after the addition of 9cRA and differentiated into macrophage-like cells (Fig. 6, A and BCitation and data not shown). This might indicate that TR4 and RXR transformants might represent different cell types, at least in response to 9cRA, and this might account for the differences observed. TPA rapidly induced adhesion of the entire cell population, and no obvious differences were observed for TR4, {Delta}TR4 or RXR, {Delta}RXR cells.



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Fig. 6. Differentiation of TR4 and RXR cells in response to different stimuli. In A, differentiation of HA-TR4, HA-{Delta}TR4, RXR, and {Delta}RXR cells at day 12 of culture was induced by 9cRA (1 x 10-6 M) or TPA treatment (1 µg/ml) for 48 h, and differentiated adherent cells were stained with histological dye (DiffQuik). Chicken myelomonocytic growth factor (CMGF, 40 ng/ml) was added as a control. Because there was no outgrowth of empty-neo virus-infected bone marrow cells (Fig. 5)Citation , empty vector-infected cells could not be included as control. In B, the density of adherent cells in A was determined by NIH image software, and values for TPA-treated cells were arbitrarily set to 100.

 
TR4 and {Delta}TR4 Cells Express High Levels of C/EBPß and the Promyelocyte Marker Mim-1.
Next, protein expression in TR4 cells was analyzed. Interestingly, both HA-TR4 and HA-{Delta}TR4 cells abundantly expressed a protein with a molecular mass of Mr 35,000 (Fig. 7A)Citation . This band was also seen in RXR and {Delta}RXR cells but to a much lesser extent than in TR4 cells. This observation prompted us to determine the identity of this protein. Therefore, the band was isolated from a gel, and peptide sequencing was performed. The analysis of two peptide sequences allowed us to identify the protein as the myb-induced myeloid protein 1, referred to as Mim-1 (40) . Western blotting with an anti-Mim-1-specific antibody additionally confirmed the identity of this protein (Fig. 7B)Citation . The Mr 18,000 band seen in Coomassie-blue stained gels is most probably a Mim-1 degradation product because it was also stained with anti-Mim-1 antibody (data not shown). Mim-1 is found in cytoplasmic granules in normal and transformed promyelocytes (Ref. 41 and references therein).

To further extend the analysis of TR4 cells, the presence of C/EBPß, another protein that is highly expressed in myeloid progenitors and required for efficient Mim-1 expression (42 , 43) , was tested by immunoblotting. It was found that TR4- and HA-{Delta}TR4-containing cells displayed high levels of C/EBPß (Fig. 7C)Citation . C/EBPß was also expressed in RXR and {Delta}RXR cells, although its level was lower.

Taken together, all these data support a role of TR4 in inducing outgrowth of committed granulocyte progenitors and/or in preventing terminal differentiation of such cells.


    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
NRs have a central role in development and differentiation of various hematopoietic lineages. It is demonstrated here that nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues of vertebrate organisms and particularly abundant in myeloid cells. Additionally, the potential impact of TR4 in hematopoiesis was revealed by ectopic expression of TR4 in bone marrow cells, thereby following a strategy that was successfully used before for other NRs (7, 8, 9 , 13 , 44 , 45) . It was found that TR4 led to an effective outgrowth of promyelocytes, thereby implicating TR4 in proliferation and differentiation of myeloid progenitor cells. Moreover, we also show that TR4, as well as {Delta}TR4 devoid of its DNA binding domain, exhibited essentially the same biological activities. Thus, it appears that for TR4 to exert some of its functions in hematopoietic cells, its DNA binding activity is not required.

As shown here, TR4 is expressed in several hematopoietic lineages, but after ectopic expression in bone marrow cells, an effective outgrowth was only observed in the myeloid compartment. Interestingly, whereas erythroid cells showed a prominent expression of TR4 in all species analyzed, an outgrowth of erythroid progenitor cells was not observed. In addition, if introduced into established erythroid progenitors (STED progenitors; Ref. 7 ), TR4 had only a marginal growth promoting activity (data not shown). This might be because ectopic expression represents one approach to assess gene function but does not necessarily recapitulate all activities of the respective protein in its physiological environment.

TR4 is believed to exert its transcriptional activity as a repressor by several mechanisms, including target site competition (24 , 26 , 27 , and references therein). Accordingly, TR4-mediated repression on certain DNA elements requires an intact DNA binding domain as also demonstrated here. Alternatively, transcriptional repression might be mediated by sequestration of cofactors (transrepression) that, e.g., bind to AF-2, and, therefore, both TR4 and {Delta}TR4 might exert transrepression. Interestingly, in vivo TR4 and {Delta}TR4 exhibit similar biological activities in promoting promyelocyte proliferation. It is therefore tempting to speculate that most, if not all, of the activity of ectopically expressed TR4 in myeloid cells results from the sequestration of (a) cofactor(s) that is/are involved in proper myeloid development. Evidence coming from experiments in other hematopoietic cell types would also be in line with this idea. For example, in erythroid cells, the ER can arrest differentiation via a mechanism that requires an intact AF-2 domain but is independent of AF-1 and DNA binding domains (45) .

Recently, it has become evident that NRs are capable of modulating target gene expression not only as DNA-bound factors but also by mechanisms that do not require direct DNA interactions (6 , 12) . Such DNA independent activities of NRs are in most situations executed by protein-protein interactions. The most likely candidates for such function are corepressors and coactivators that are known to interact with the AF-2 domain of NRs (reviewed in Ref. 6 ). The observations reported here demonstrating a similar activity for TR4 and {Delta}TR4 in promyelocyte proliferation would be in accordance with such a DNA-independent mechanism of TR4 action. A candidate for such a function is RIP-140, a coactivator that has been shown to interact with TR4 (25 , 26) . Additionally, transrepression by TR4 attributable to sequestration of RIP-140 is not without precedence and was observed, e.g., in peroxisome proliferator-activated receptor/RXR-controlled gene expression (26) .

Investigation of the subcellular localization of the TR4 proteins showed that TR4 was nuclear, whereas {Delta}TR4 was localized mostly in the nucleus but also present in the cytoplasm of TR4-transduced CEF and myeloid cells. This might be because the putative nuclear localization signal of TR4 (corresponding to the respective nuclear localization signal in the DNA binding region of TR2; Ref. 37 ) is deleted in {Delta}TR4. Thus, the reduced nuclear expression of {Delta}TR4 might well explain the somewhat diminished proliferative potential of {Delta}TR4 cells as compared with TR4 cells.

There is a large body of evidence demonstrating that different RAR alleles, such as PML-RAR or full-length and truncated RARs, effectively block myeloid cell development at the promyelocyte stage (Refs. 17 , 18 , 46, 47, 48 , and references therein) and thus exhibit a phenotype similar to the TR4 promyelocytes as reported here. However, RAR, RAR variants, and TR4 might mediate their effects on myeloid cells by different mechanisms that do or do not require DNA binding, respectively. In addition, thus far no ligand has been identified that regulates TR4 activity. In this study, the experiments with TR4 cells involved serum-containing media, and, therefore, we cannot exclude the presence of a serum-borne ligand (or a metabolite) that might influence TR4 activity. In this respect, we note though that both TR4 and {Delta}TR4 proteins would be affected similarly by ligand because the putative ligand-binding domain is present in both constructs.

Finally, TR4 as a regulator of myeloid development might represent a pharmacological target and thus might open new possibilities for treatment of myeloid leukemia. In particular, synthetic agonists and/or antagonists that would modulate TR4 activity might provide valuable agents in combination with established treatments for differentiation therapy.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cells and Cell Culture.
Chicken bone marrow cells were prepared from 3–10-day-old SPAFAS chicks maintained at Max-Delbrück-Center for Molecular Medicine, and cells were cultured as described before (7 , 32) . Briefly, bone marrow cells were plated for 2–4 h in DMEM (Life Technologies, Inc.), 8% FCS (Boehringer Mannheim), 2% ChS (Sigma Chemical Co., St. Louis, MO), 20 mM HEPES (pH 7.3), and 100 units of penicillin/streptomycin (Life Technologies, Inc.), referred to as standard growth medium. Nonadherent cells were recovered and used for infection with recombinant retrovirus, and infected cells were cultured in DMEM, 8% FCS, 5% ChS, 0.128 mg/ml chicken transferrin (conalbumin; Sigma Chemical Co.), 100 ng/ml recombinant chicken SCF (49) , 20 mM HEPES (pH 7.3), and 100 units of penicillin/streptomycin with 2–3 x 106 cells/ml cell density. Cell number and cell size were assessed in regular time intervals with the CASY-1 Cell Counter and Analyzer System (Schärfe Systems, Reutlingen, Germany).

CEFs were grown in standard growth medium. Production of retrovirus-producing CEFs and infection of bone marrow cells were as described before (7) . Infections were performed in the presence of 1 µg/ml recombinant human insulin (Actrapid HM40; Novo Nordisk Pharma) and 100 ng/ml SCF.

Construction of Retroviral Vectors.
A retroviral vector containing the full-length TR4 cDNA downstream of the cytomegalovirus promoter was constructed by subcloning wild-type human TR4 (21) , kindly provided by J. P. H. Burbach (Utrecht, the Netherlands), into the EcoRI site of pSFCV-LE (50) . The HA-tagged versions of TR4 were first engineered in a shuttle vector containing coding sequences for three HA epitopes upstream of the cloning sites.5 In HA-{Delta}TR4, amino acid positions 64–232, comprising the entire DNA binding domain of human TR4, are deleted. The HA-TR4 and HA-{Delta}TR4 were then subcloned into the EcoRV-EcoRI sites of pSFCV-LE. Recombinant retrovirus vectors containing RXR and {Delta}RXR (that is devoid of its DNA binding domain; {Delta}DBD RXR) were described (7) .

cDNA Synthesis and PCR.
For RNA preparation, cDNA synthesis, and PCR amplification, see Ref. 32 . Briefly, first-strand cDNA was synthesized from 2–5 µg of total RNA using 100 units of Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and random hexamers in a total volume of 20–50 µl. For PCR amplification, a set of degenerate primers for the region encoding the DNA binding domain of NRs was designed as described previously (19) . The sequences of these primers were as follows:

NHR1: 5'-GAYRARDCNWSNGGNTDYCAYT-3';

NHR2: 5'-GAYMGNDSNWSNGGNAARCAYT-3';

NHR3: 5'-CAYTTNYKNWRNCKRCANKMNKGRCA-3';

NHR4: 5'-TGYAARNNNTTYTTYMRNMG-3'.

First-strand cDNA was subjected to 40 cycles of PCR amplification using Taq DNA polymerase (Boehringer Mannheim) and applying the following cycle profile: 94°C for 30 s, 37°C for 90 s, and 72°C for 20 s. The primer combinations used were as follows: in the first reaction, NHR1 plus NHR3 or NHR2 plus NHR3. The product of this reaction was diluted 1:300 and used as a template for a hemi-nested reaction with NHR3 and NHR4. PCR products were purified from agarose gels and cloned into pCRII vector (Invitrogen, San Diego, CA), and clones were subjected to DNA sequencing using an Applied Biosystems DNA sequencing device. Sequences were then analyzed using the BLAST alignment tool. GenBank accession no. of chicken TR4: AF133054.

RNA Analysis.
RNA was isolated by standard procedures from tissues and the following cells. Human: K562 (erythroid); U937 and HL60 (myeloid); Namalwa and Raji (B-lymphoid); and CEM, Jurkat (T-lymphoid), and HepG2 (hepatocytes). Mouse: MEL and E31 (erythroid); WEHI-3 and RAW (myeloid); A20 (B-lymphoid); and NIH3T3 (fibroblast). Chicken: DT40 (B-lymphoid); MSB-1 (T-lymphoid); and v-relER-transformed progenitors and dendritic cells (51) . Primary cells were as follows. Human: erythroid progenitors (52) , peripheral blood T cells, and monocytes (53) . Mouse: dendritic cells (53) . Chicken: SCF and STED erythroblasts (7 , 32) . A total of 15 µg of RNA/lane was analyzed by Northern blotting (GeneScreen membranes; DuPont) as described (7) . Filters were hybridized with 32P-labeled human (21) or chicken TR4-specific probes, washed at moderate stringency, and exposed to film.

Flow Cytometry.
For flow cytometry, cells were recovered, washed with 1% BSA (fraction V; Sigma Chemical Co.) in PBS, and incubated with primary antibody (1 h). The following monoclonal antibodies were used: MC47–83, MC51–2, and MC22–3 (54) ; K1 (55) and MHC class II ß chain [2G11, specific for the nonpolymorphic region of chicken BL (56) ; JS4, JS8 (57 , 58) , CD3, CD4, and CD8 (all Southern Biotechnology Associates); MEP17, MEP21, MEP26, and EOS47 (34) ; DM-GRASP (BEN1; Ref. 59 ); HEM-CAM (60) ; and CD44 (AV6, kindly provided by T. F. Davison, Compton, United Kingdom). Cells were then reacted with FITC-conjugated antimouse IgG (1 h; Sigma Chemical Co.), washed, and resuspended in PBS containing 1% BSA and propidium iodide (2 µg/ml; Sigma Chemical Co.) for gating on viable cells. Cells were analyzed by flow cytometry using a FACS Calibur device with CellQuest software (Becton Dickinson).

Western Blotting.
Total cell extracts were separated on a 10% SDS-PAGE gel and blotted onto nitrocellulose membranes (BA85; Schleicher & Schuell) using a semidry blotting system (Pharmacia). Antibody reactions were performed as described previously (49 , 51) . The following antibodies were used: anti-HA 12CA5, anti-C/EBPß (kindly provided by K. H. Klempnauer, Münster, Germany), and anti-Mim-1 (Ref. 40 ; kindly provided by A. Leutz and E. Kowenz-Leutz, Berlin, Germany). Subsequently, blots were washed and incubated with the respective secondary antibody (enhanced chemiluminescence kit; Amersham) in Tris-buffered saline supplemented with 5% nonfat milk powder for 45 min (49) . All incubations were done at room temperature. Blots were developed with enhanced chemiluminescence reagents and exposed to film.

Immunofluorescence.
CEF containing TR4 versions were cultured as above, trypsinized, and allowed to adhere to glass slides (1 h, 37°C). Cells were washed and fixed with 3.7% paraformaldehyde in PBS (15 min). After fixation, cells were permeabilized with 0.5% NP-40 in PBS (15 min), incubated with 0.5% heat-inactivated FCS in PBS (30 min) to block unspecific binding, and reacted with anti-HA monoclonal antibody (12CA5) for 1 h, followed by reaction with FITC-conjugated antimouse IgG (1 h; Sigma Chemical Co.). Samples were also stained for actin with tetramethylrhodamine isothiocyanate-labeled phalloidin (Sigma Chemical Co.). DAPI (0.5 µg/ml; Sigma Chemical Co.) was used to stain nuclei. Samples were washed with PBS and mounted in Mowiol 4.88 (Hoechst) containing 50 mg/ml DABCO (Sigma Chemical Co.) as antibleaching agent. All incubations were done at room temperature. Photographs were taken with an Axioplan 2 fluorescence microscope (Zeiss) equipped with a Photometrics Quantix CCD camera. Images were processed with IPLab Spectrum and Adobe PhotoShop software.

Protein Purification and Sequencing.
Coomassie blue-stained protein bands were excised and digested in the gel with sequencing-grade modified trypsin (Promega; Ref. 61 ). Tryptic peptide maps were obtained by reversed-phase high-performance liquid chromatography on a µRPC C2/C18 SC 2.1/10 column using the Smart system (Pharmacia Biotech). The flow rate was 100 µl/min at 25°C using a linear gradient of acetonitrile in 0.1% trifluoroacetic acid. Peptides of interest were loaded onto a Biobrene-coated glass filter fiber of a Procise sequencer (Perkin-Elmer/Applied Biosystems), and sequencing was carried out using standard protocols.

Bandshift Assays.
CEFs were lysed in a buffer containing 20 mM Tris (pH 7.5), 2 mM DTT, 20% glycerol, and 400 mM KCl (1 h on ice). Whole cell extracts (5–10 µg) were incubated for 15 min on ice with a 32P-labeled oligonucleotide containing a DR1-HRE binding site: (5'-GGGGATCCGGCAGAGGTCAAAGGTCAAACGT-3'; Ref. 26 ). Poly(dI-dC) (1 µg/reaction) was added as a nonspecific competitor. For competition experiments, a mutated version of the DR1-HRE site was used: (5'-GGGGATCCGGCAGTGGACAATGGACAAACGT-3'). Binding reactions were separated on 5% polyacrylamide gels in 0.25 x Tris-borate EDTA (44) .

Transient Transfections.
Calcium phosphate coprecipitation was used to transiently transfect CEFs with retroviral expression vectors containing TR4, HA-TR4, and HA-{Delta}TR4 in combination with eukaryotic expression vectors carrying RXR in pSG5 vector and a luciferase reporter with the rat CRBPII-RXR responsive element (7) . Rous sarcoma virus-ß-gal or pCH110 plasmids were cotransfected to normalize for transfection efficiencies. Transfected cells were grown in standard growth medium containing FCS and ChS that were extensively deprived of retinoids (7) . 9cRA (10-6 M final concentration) was added, and 48 h later, cell extracts were prepared and analyzed for luciferase and ß-galactosidase activity as described (44) . Luciferase values were normalized for ß-galactosidase activity.


    Acknowledgments
 
We thank H. Beug, J. P. H. Burbach, C-L. Chen, M. D. Cooper, T. F. Davison, T. Graf, J. Kaufman, K. H. Klempnauer, E. Kowenz-Leutz, J. Young, and O. Vainio for providing cDNA probes and monoclonal antibodies. We also thank H. Gronemeyer and G. Schütz for critical reading of the manuscript, M. Meyer for her initial contribution to this work, and I. Gallagher for expert secretarial assistance.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported in part by Deutsche Forschungsgemeinschaft Grant Ze432/1 to M. Z. N. P. K. was a recipient of a Max-Delbrück postdoctoral fellowship and is an Investigator of CONICET (Argentina). Back

2 Present address: Departamento de Bioquímica Clínica, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, Ciudad Universitaria, 5000 Córdoba, Argentina. Back

3 To whom requests for reprints should be addressed, at Max-Delbrück-Center for Molecular Medicine, Robert-Rössle Str. 10, D-13122 Berlin, Germany. Phone: 49-30-9406 3343; Fax: 49-30-9406 3329; E-mail: zenke{at}mdc-berlin.de Back

4 The abbreviations used are: NR, nuclear receptor; 9cRA, 9-cis retinoic acid; CEF, chicken embryo fibroblast; DR, direct repeat; HA, hemagglutinin; HRE, hormone responsive element; RAR, retinoic acid receptor; RXR, retinoid X receptor; TR, thyroid hormone receptor; ER, estrogen receptor; PML, promyelocytic leukemia; C/EBP, CAAT/enhancer binding protein; TPA, 12-O-tetradecanoylphorbol-13-acetate; ChS, chicken serum; SCF, stem cell factor; DAPI, 4',6-diamidino-2-phenylindole. Back

5 N. P. Koritschoner, P. Bartunek, and M. Zenke, unpublished data. Back

Received for publication 7/25/01. Revision received 9/20/01. Accepted for publication 9/20/01.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
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