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Molecular and Cellular Biology, April 2004, p. 2820-2830, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2820-2830.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

{Delta}FosB Induces Osteosclerosis and Decreases Adipogenesis by Two Independent Cell-Autonomous Mechanisms

Marie Kveiborg,1,{dagger} George Sabatakos,1,{ddagger} Riccardo Chiusaroli,1 Meilin Wu,1 William M. Philbrick,2 William C. Horne,1 and Roland Baron1*

Departments of Cell Biology and Orthopaedics,1 Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 065102

Received 1 May 2003/ Returned for modification 27 June 2003/ Accepted 15 December 2003


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ABSTRACT
 
Osteoblasts and adipocytes may develop from common bone marrow mesenchymal precursors. Transgenic mice overexpressing {Delta}FosB, an AP-1 transcription factor, under the control of the neuron-specific enolase (NSE) promoter show both markedly increased bone formation and decreased adipogenesis. To determine whether the two phenotypes were linked, we targeted overexpression of {Delta}FosB in mice to the osteoblast by using the osteocalcin (OG2) promoter. OG2-{Delta}FosB mice demonstrated increased osteoblast numbers and an osteosclerotic phenotype but normal adipocyte differentiation. This result firmly establishes that the skeletal phenotype is cell autonomous to the osteoblast lineage and independent of adipocyte formation. It also strongly suggests that the decreased fat phenotype of NSE-{Delta}FosB mice is independent of the changes in the osteoblast lineage. In vitro, overexpression of {Delta}FosB in the preadipocytic 3T3-L1 cell line had little effect on adipocyte differentiation, whereas it prevented the induction of adipogenic transcription factors in the multipotential stromal cell line ST2. Also, {Delta}FosB isoforms bound to and altered the DNA-binding capacity of C/EBPß. Thus, the inhibitory effect of {Delta}FosB on adipocyte differentiation appears to occur at early stages of stem cell commitment, affecting C/EBPß functions. It is concluded that the changes in osteoblast and adipocyte differentiation in {Delta}FosB transgenic mice result from independent cell-autonomous mechanisms.


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INTRODUCTION
 
Although osteoblasts and adipocytes represent two morphologically and functionally distinct cell types, it has been proposed that they may develop from a common mesenchymal precursor in the bone marrow (36, 40, 43). Indeed, an inverse relationship between adipocyte and osteoblast differentiation has been suggested, as exemplified by increased bone marrow adipocytes in age-related bone loss (4, 6, 29, 32, 60) or after treatment with glucocorticoids (57). Several regulatory factors involved in osteoblast and adipocyte differentiation have been identified (27, 42, 45). However, the identities of the factors that control commitment at the branching point between the osteoblast and adipocyte lineages and the degree of plasticity between the two cell types are still uncertain (33, 38).

We have recently reported that transgenic mice overexpressing {Delta}FosB, a member of the activator protein 1 (AP-1) family of transcription factors, under the control of the neuron-specific enolase (NSE) promoter develop not only a severe and progressive osteosclerotic phenotype, characterized as increased bone formation, but also a pronounced decrease in adipogenesis and fat levels (25, 47, 53). The AP-1 family of basic leucine zipper transcription factors comprises various combinations of Jun (c-Jun, JunB, and JunD) and Fos (c-Fos, FosB, Fra-1, and Fra-2) proteins, which upon dimer formation regulate gene transcription by binding to consensus response elements present in the promoter region of target genes (23). Several studies have demonstrated an important regulatory role of AP-1 factors, especially the Fos-related proteins, in bone formation and osteoblast function (16, 19, 20, 26, 59). {Delta}FosB is a C-terminally truncated splice variant of FosB that lacks the proline-rich transactivation domain but has maintained the ability to bind DNA and heterodimerize with other AP-1 factors (30, 31, 64).

Although the increased bone formation in the NSE-{Delta}FosB mice was shown to be due, at least in part, to a cell-autonomous effect on cells of the osteoblast lineage, the dramatic decrease in adipogenesis observed in these mice, as revealed by decreased abdominal fat, low leptin levels in serum, and reduced number of adipocytes in the bone marrow, could be independently cell autonomous to the adipocyte lineage or secondary to the alteration in osteoblast differentiation. This uncertainty is due to the fact that the NSE promoter directs {Delta}FosB transgene expression in several tissues in addition to the brain, including bone and white adipose tissue. We therefore generated transgenic mice overexpressing {Delta}FosB under the control of the mouse osteocalcin promoter, OG2, thereby directing the transgene specifically to cells of the osteoblastic lineage, and determined their bone and fat phenotypes. OG2-{Delta}FosB mice demonstrated increased osteoblast function and an osteosclerotic phenotype, firmly establishing that the skeletal phenotype is cell autonomous to the osteoblast lineage, independent of the fate of adipocytes. In contrast, no change in adipocyte formation was observed, indicating that the decreased adipogenesis phenotype of the NSE-{Delta}FosB mice is independent of the osteosclerotic phenotype. Furthermore, in vitro studies with cells transfected with {Delta}FosB indicated that the inhibitory effects of {Delta}FosB on adipocyte differentiation occur mainly at early stages of stem cell commitment, possibly by affecting C/EBPß functions. It is concluded that the bone and fat phenotypes of {Delta}FosB transgenic mice result from independent and cell-autonomous mechanisms.


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MATERIALS AND METHODS
 
Construction of plasmids. The pII1.3Luc construct containing 1.3 kb of the murine osteocalcin promoter OG2 (15) was provided by G. Karsenty, the promoter was subcloned into pBluescript SK (Stratagene) for subsequent sequencing, and the construct was designated pBS-OG2. The 1.3-kb OG2 promoter was released from pBS-OG2 by digestion with KpnI and HindIII restriction endonucleases and subcloned into the ptTAk vector (51), provided by Eric Nestler (8), to create pOG2-tTA. {Delta}FosB cDNA in the pTetOp vector was provided by Eric Nestler (8), cut out by digestion with KpnI and XbaI, and subcloned into the pcDNA3.1 expression vector (Invitrogen). A construct encoding only the truncated isoform of {Delta}FosB, termed {Delta}2{Delta}FosB, which arises from translational initiation from the methionine at position 79 was generated by replacing the EcoRV-BstEII fragment with the HindIII-BamHI fragment encoding {Delta}2{Delta}FosB. A mutant form of {Delta}FosB that expresses only the full-length isoform, named 2i3i, was generated by mutating the second and third methionines at positions 50 and 79 to isoleucines by two rounds of PCR-induced mutation of {Delta}FosB with the Quickchange kit (Stratagene), with primers M2F, M2R, M3F, and M3R (Table 1).


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  		TABLE 1. Sequences of oligonucleotides used for PCR and electrophoretic mobility shift assay

Glutathione S-transferase (GST) fusion constructs were generated by PCR either from {Delta}2{Delta}FosB cDNA with primers D2GSTF and DFGSTR (Table 1) or from 2i3i cDNA with primers DFGSTF and DFGSTR (Table 1), containing EcoRI and XhoI restriction sites. PCR products were digested and cloned into pGEX4T2 (Amersham). EcoRI/XhoI fragments were excised from 2i3i-pGEX4T2 and {Delta}2-pGEX4T2 and subcloned into pCMV-Myc (Clontech). C/EBP{alpha}, C/EBPß, peroxisomal proliferator-activated receptor {gamma} (PPAR{gamma}), adipsin, and lipoprotein lipase cDNA, used as probes in Northern blot analysis, were kindly provided by J. Gimble, and C/EBPß cDNA in pBlueskript SK, used for transfections, was received from D. P. Ramji.

Transgenic mice. A DNA fragment containing the promoter, open reading frame, simian virus 40 intron, and polyadenylation signal in pOG2-tTA was gel purified and microinjected into the pronuclei of oocytes from (SJL x C57BL6)F2 mouse ova. Viable embryos were implanted into pseudopregnant recipients and allowed to develop to term. Founders were identified by isolating tail DNA with the Tissue DNeasy kit (Qiagen) and analyzing for the transgene by PCR with primers TTAF and TTAR (Table 1), which were also subsequently used for routine genotyping of the transgenic mice. The {Delta}FosB transgene was identified with primers FosBF2 and FosBB2 (Table 1).

As previously described (8, 24, 25, 47, 53), NSE-{Delta}FosB mice were generated by crossing mice expressing the tetracycline transactivator (tTA) under the control of the NSE promoter with mice carrying the {Delta}FosB gene under the control of the tetracycline-responsive promoter TetOp. Similarly, OG2-{Delta}FosB mice were generated by cross-breeding the OG2-tTA transgenic mice with the TetOp-{Delta}FosB mice. For all experiments, the bitransgenic (NSE-{Delta}FosB or OG2-{Delta}FosB) mice were bred as heterozygotes, and monotransgenic (NSE-tTA or OG2-tTA) littermates were used as controls. All animal protocols were approved by the Yale University Institutional Animal Care and Use Committee.

Histomorphometric analysis. All mice used for analysis were injected with calcein (20 mg/kg; Sigma) and demeclocycline (20 mg/ml; Sigma) at 10 and 3 days before sacrifice, respectively, to label bone mineralization fronts (52). Bone sections were fixed in 3.7% formaldehyde-phosphate buffered saline and embedded by standard procedures in methylmethacrylate resin (52). Five-micrometer toluidine blue-stained and 10-µm unstained sections were analyzed for bone histomorphometry by standard procedures (37) with the Osteomeasure system (Osteometrics, Atlanta, Ga.). All measurements were performed in a blinded fashion. Von Kossa staining of proximal tibia to illustrate increased trabecular bone volume was done according to standard procedures (2).

Quantification of abdominal fat and serum leptin levels. Ten-week-old OG2-{Delta}FosB mice and control littermates were anesthetized with metofane (Medical Developments) and bled by cardiac puncture, and serum samples were used immediately for measurement of leptin levels with the QuantikineM radioimmunoassay (R&D Systems, Inc.) Abdominal adipose tissue was removed and weighed.

Cell cultures. Primary calvarial cell cultures were prepared from 10-week-old control, OG2-{Delta}FosB, and NSE-{Delta}FosB mice by standard techniques (3) and used immediately for expression analysis. Primary bone marrow cells were established from 10-week-old control, OG2-{Delta}FosB, and NSE-{Delta}FosB mice by plating the marrow flushed from tibia and femur in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin (complete medium). Nonadherent cells were removed after 5 days, and the adherent cells were trypsinized, replated, and allowed to reach confluency. Complete medium supplemented with 50 µg of ascorbic acid per ml, 5 mM ß-glycerophosphate, and 10 nM dexamethasone (all from Sigma) was added (day 0) to induce differentiation, and the cultures were maintained under these conditions for the indicated number of days.

3T3-L1 cells, obtained from the American Type Culture Collection, were cultured in complete medium. Differentiation was induced in confluent preadipocytes (designated day 0) by adding 10 µg of insulin per ml, 1 µM dexamethasone, and 0.5 mM 3-isobutyl-1-methylxanthine (all from Sigma) in complete medium. After 48 h, the medium was replaced with complete medium containing 1 µg of insulin per ml for an additional 48 h. The cells were fully differentiated by day 8, and the adipocyte phenotype was examined at the time points indicated. The murine stromal cell-line ST2, provided by Aventis Pharma, Inc., was cultured in RPMI 1640 medium supplemented with 10% sodium bicarbonate, 10% fetal bovine serum, and 1% penicillin-streptomycin. Adipocyte differentiation was induced by culturing the cells for 2 days in medium supplemented with 0.5 µM hydrocortisone (Sigma), 500 µM 3-isobutyl-1-methylxanthine, and 60 µM indomethacin (Sigma). 3T3-L1 and ST2 cells were transfected with the FuGene6 transfection reagent as recommended by the manufacturer (Roche), and lines stably expressing the transgene were established by geneticin (G-418) (Invitrogen) selection.

Staining. Bone marrow cultures were fixed in 10% formalin, and osteoblastic cells were visualized by the alkaline phosphatase leukocyte staining kit (Sigma). To stain lipid-filled vacuoles, fixed cells were incubated with Oil Red O (Sigma) for 30 min, followed by three washes with phosphate-buffered saline, as described (50). The degree of adipocyte differentiation was estimated by eluting the stain in isopropanol and measuring the absorbance at 540 nm.

RNA purification and real-time RT-PCR. Total RNA was extracted from various tissues isolated from 10-week-old OG2-{Delta}FosB mice and control littermates with the Trizol (Gibco) method (9). Quantitative {Delta}FosB mRNA expression in tissues was determined by real-time reverse transcription-PCR (RT-PCR) analysis on the iCycler (Bio-Rad), with the one-step QuantiTect SYBR Green RT-PCR kit (Qiagen) as described by the manufacturer. Primers {Delta}FosBF, {Delta}FosBB, ACTINF, and ACTINR (Table 1) were designed with the Primer Express V1.0 software (Perkin-Elmer Applied Biosystems Inc.).

Northern blotting. Twenty micrograms of total RNA was resolved in 1% denaturing agarose-formaldehyde gels and transferred onto Hybond-N nylon membranes (Amersham) as described (48). Randomly 32P-labeled cDNA probes were purified on Wizard minicolumns (Promega), and membranes were incubated at 42°C overnight in hybridization buffer (50% formamide, 5x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA, pH 7.7], 5x Denhardt's solution, 0.1% sodium dodecyl sulfate [SDS]). Membranes were washed for 1 h at 42°C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate)-0.1% SDS and 1 h at 42°C in 1x SSC-0.1% SDS before being exposed to X-ray film. 18S rRNA was used as an internal control.

Immunoprecipitation and Western blot analysis. Cells were lysed in modified radioimmunoprecipitation assay (mRIPA) buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA) supplemented with 1 mM NaF, 1 mM Na3VO4, 10 µg of leupeptin per ml, 10 µg of aprotinin per ml, 10 µg of pepstatin per ml, and 1 mM phenylmethylsulfonyl fluoride. The lysates were cleared by centrifugation, and the protein concentration was determined by the BCA assay (Pierce); 500 µg of total cell lysate protein was incubated with 3 to 5 µg of polyclonal anti-C/EBPß antibody (Santa Cruz) in 500 µl of mRIPA buffer at 4°C with shaking for 2 h. The samples were then incubated with 50 µl of a 50% protein G-Sepharose slurry (Amersham Pharmacia Biotech) for 1 h at 4°C with shaking. The beads were then washed three times in mRIPA buffer and boiled in 30 µl of loading buffer. Proteins were electrophoresed through an SDS-12% polyacrylamide gel and transferred at 35 V overnight to Protran nitrocellulose membranes (Schleicher & Schleicher Inc.). Total cell lysates (50 µg) were electrophoresed on 10 to 12% gradient SDS-PAGE gels and transferred to nitrocellulose membranes. Western blotting was performed with specific antibodies by standard techniques. Immunoreactive bands were detected by the ECL method (Amersham Pharmacia Biotech). All antibodies were purchased from Santa Cruz Biotechnology.

GST pull-down. GST fusion proteins were expressed and purified with the B-PER bacterial protein extraction reagent (Pierce) according to the manufacturer's instructions. In short, transformed Escherichia coli BL21 cells expressing the GST fusion proteins were harvested and lysed in B-PER reagent. The cellular extracts were cleared by centrifugation and incubated with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 20 min at room temperature with gentle agitation. The suspensions were washed and resuspended in mRIPA buffer to make a 50% slurry. The purified GST fusion proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. For interaction studies, GST fusion proteins were incubated with 500 µg of total cell lysate protein in mRIPA buffer for 2 h at 4°C. The samples were washed three times in mRIPA buffer, the beads were sedimented and boiled in 10 µl of loading buffer, and the complexes were analyzed by Western blotting.

Electrophoretic mobility shift assay. Nuclear extracts from cells were prepared as described previously (49). The electrophoretic mobility shift assay and supershift analysis were performed as described (46), with a 32P-labeled double-stranded oligonucleotide corresponding to a consensus C/EBPß binding site (D-site, Table 1) (56). For supershift analysis, anti-C/EBPß antibody (Santa Cruz) was incubated with nuclear extracts for 30 min on ice before addition of the labeled probe. Following electrophoresis, gels were dried, and the protein-DNA complexes were visualized by autoradiography.

Statistical analysis. The data are represented as means ± standard error of the mean, and statistical analysis was performed with Student's t test. A P value of less than 0.05 was considered statistically significant.


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RESULTS
 
Bone-specific overexpression of {Delta}FosB. With the same tetracycline-regulated system as used for the previously described NSE-{Delta}FosB transgenic mice (8, 47), bone-specific overexpression of the {Delta}FosB transgene was obtained by driving transgene expression with the osteoblast-specific osteocalcin promoter OG2. Transgenic mice expressing the tetracycline transactivator (tTA) under the control of the OG2 promoter were generated and crossed with transgenic mice carrying the {Delta}FosB cDNA under the control of the TetOp promoter, which requires the binding of tTA in order to drive transcription. In the absence of tetracycline, tTA binds to the promoter and activates transcription of {Delta}FosB cDNA, whereas in the presence of tetracycline, tTA undergoes a conformational change and cannot bind to the TetOp promoter, preventing transcription of the transgene.

As shown in Fig. 1, the use of the OG2 promoter resulted in bone-specific overexpression of {Delta}FosB. Real-time RT-PCR analysis of the {Delta}FosB transcript in a number of tissues revealed an approximately threefold overexpression of {Delta}FosB in bone, whereas no statistically significant differences in {Delta}FosB expression levels were observed in liver, lung, brain, or adipose tissues (Fig. 1A). In addition, Western blot analysis of {Delta}FosB expression in osteoblasts isolated from OG2-{Delta}FosB transgenic mice further demonstrated overexpression of the transgene, although at lower levels than in the NSE-{Delta}FosB transgenic animals (Fig. 1B). Since osteocalcin is known to be upregulated towards the end of osteoblast differentiation, it is, however, likely that transgene expression would be higher in more mature cells.



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  		FIG. 1. Bone-specific overexpression of the {Delta}FosB transgene in OG2-{Delta}FosB mice. (A) Real time RT-PCR analysis of {Delta}FosB mRNA levels in fat, brain, liver, bone, and lung tissues in 10-week-old OG2-{Delta}FosB transgenic mice compared to control littermates. (B) Western blot analysis of {Delta}FosB protein levels in primary calvarial osteoblasts established from 10-week-old OG2-{Delta}FosB transgenic mice compared to control littermates and a low-expressing founder line of the NSE-{Delta}FosB transgenic mice. The full-length {Delta}FosB and truncated {Delta}2{Delta}FosB isoforms are indicated. Actin levels were used as an internal control.

OG2-{Delta}FosB transgenic mice are osteosclerotic. Having established that bone-specific overexpression of {Delta}FosB occurs in the OG2-{Delta}FosB transgenic mice, the bone phenotype was examined. As in the NSE-{Delta}FosB transgenic mice, Von Kossa staining of mineralized tissue in the proximal tibia from 10-week-old animals demonstrated an increased trabecular bone volume in the bone marrow of OG2-{Delta}FosB mice compared to control littermates (Fig. 2A). Detailed histomorphometric analysis showed a more than twofold increase in trabecular bone volume by this age (Fig. 2B). The increased trabecular bone volume was associated with statistically significant increases in trabecular thickness and trabecular number (Fig. 2C and D). The dynamic bone formation parameters (mineralizing surface, mineral apposition rate, and bone formation rate) were all significantly higher in the OG2-{Delta}FosB mice (Fig. 2E, F, and G), and the number of osteoblasts per total area of bone tissue was more than doubled (Fig. 2H). However, the number of osteoblasts relative to the bone perimeter was unchanged (not shown), indicating that osteoblast differentiation was increased while the amount of bone formed per osteoblast was unchanged. Also, neither the osteoclast surface nor osteoclast number was significantly different between the OG2-{Delta}FosB transgenic animals and control littermates (Fig. 2I and J), suggesting that, as in the NSE-{Delta}FosB mice, bone resorption was unchanged.



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  		FIG. 2. Osteosclerotic phenotype in OG2-{Delta}FosB transgenic mice. (A) Von Kossa-stained proximal tibia from 10-week-old control and OG2-{Delta}FosB transgenic mice. (B to J) Histomorphometric analysis of toluidine blue-stained sections from OG2-{Delta}FosB transgenic mice and control littermates at 10 weeks of age. (B) Trabecular bone volume (BV/TV); (C) trabecular thickness (TbTh); (D) trabecular number (TbN); (E) mineralizing surface (MS/BS); (F) mineral apposition rate (MAR); (G) bone formation rate (BFR/BS); (H) osteoblast number (ObN/TAr); (I) osteoclast surface (OcS/BS); (J) osteoclast number (OcN/TAr). Data presented are means ± standard error of the mean; *, P < 0.05; **, P < 0.005, Student's t test.

Unchanged adipogenesis in OG2-{Delta}FosB transgenic mice. In contrast to the recurrence of the osteosclerotic phenotype, there was no apparent change in adipogenesis when {Delta}FosB expression was targeted exclusively to cells of the osteoblast lineage. Similar amounts of abdominal fat were found in the transgenic mice and control littermates (Fig. 3A). Consistent with this finding, and in contrast to the NSE-{Delta}FosB mice, the level of the adipocyte-secreted hormone leptin in serum was unaltered (Fig. 3B).



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  		FIG. 3. Unchanged adipogenesis in OG2-{Delta}FosB transgenic mice. (A) Abdominal fat weight in 10-week-old OG2-{Delta}FosB and control female and male animals. Data are shown as means ± standard error of the mean. There was no significant difference between gender-matched OG2-{Delta}FosB and control animals. (B) Immunoassay quantification of leptin levels in serum in 10-week-old OG2-{Delta}FosB and control mice. Data are shown as means ± standard error of the mean. There was no significant difference between gender-matched OG2-{Delta}FosB and control animals. (C) Long-term primary bone marrow mesenchymal cell cultures established from control, OG2-{Delta}FosB, and NSE-{Delta}FosB transgenic mice and maintained under osteogenic conditions were stained for alkaline phosphatase to visualize osteoblasts and with Oil Red O to visualize lipid-filled vacuoles in mature adipocytes, as described in Materials and Methods. (D) Western blot analysis of C/EBP{alpha} and C/EBPß protein levels in total cell extracts from cultures of primary bone marrow cells from control, OG2-{Delta}FosB, and NSE-{Delta}FosB transgenic mice at days 0, 8, and 16 of differentiation. Mainly the 42-kDa (p42) and 30-kDa (p30) C/EBP{alpha} isoforms and the 32- to 35-kDa (LAP) and 20-kDa (LIP) C/EBPß isoforms were detected. Actin was used as an internal control.

The adipogenic capacity of bone marrow mesenchymal progenitor cells was also studied. Long-term culture of wild-type primary bone marrow stromal cells with ascorbic acid and ß-glycerophosphate to induce osteoblast differentiation typically produces both osteoblasts and clusters of mature lipid-filled adipocytes. We had previously found, however, that marrow cells from the NSE-{Delta}FosB mice formed markedly fewer adipocytes in culture than did those from control animals (47). When bone marrow stromal cells from control, OG2-{Delta}FosB, and NSE-{Delta}FosB mice were cultured with ascorbic acid and ß-glycerophosphate, the adipogenic capacity of bone marrow cultures from OG2-{Delta}FosB transgenic mice, shown by staining with Oil Red O, was comparable to that of cultures established from control mice, while adipocyte formation was dramatically reduced in cultures from NSE-{Delta}FosB transgenic mice, as reported previously (25, 47) (Fig. 3C).

To examine adipogenesis at the molecular level, total cell lysates were isolated from control, OG2-{Delta}FosB, and NSE-{Delta}FosB bone marrow cultures at days 0, 8, and 16 of differentiation, and the adipocytic transcription factors C/EBP{alpha} and C/EBPß were quantified by Western blotting (Fig. 3D). The major isoforms of C/EBP{alpha} are p42 (42-kDa) and p30 (30-kDa) proteins, whereas C/EBPß mRNA produces mainly a 32- to 35-kDa isoform, termed LAP, and a 20-kDa isoform, termed LIP. The expression of both the 42-kDa and 30-kDa isoforms of C/EBP{alpha} was clearly increased with progressive adipocyte differentiation in extracts from control and OG2-{Delta}FosB mouse cell cultures but not in NSE-{Delta}FosB mouse cell cultures, further confirming the lack of an adipocytic effect in the osteoblast-specific transgenic mice. In contrast, there was little change in the expression of C/EBPß isoforms in any of the cultures.

{Delta}FosB inhibits the expression of adipogenic transcription factors at an early stage of stem cell commitment. The unchanged adipogenesis in the OG2-{Delta}FosB transgenic mice suggested that the inhibitory effects on adipocyte differentiation in NSE-{Delta}FosB transgenic mice were the result of direct effects of the transgene product in adipocytes or their precursors. In order to test this hypothesis and to elucidate at which stage of adipocyte differentiation the inhibition occurs, we examined the effect of {Delta}FosB expression on differentiation of the preadipocytic cell line 3T3-L1, which undergoes a well-characterized adipocytic differentiation program (27, 42), as well as in the multipotential mesenchymal cell line ST2, which has the potential to differentiate along both the osteoblastic and adipocytic lineages (35, 39, 63) and thus represents an earlier stage of mesenchymal cell differentiation.

3T3-L1 cells stably transfected with {Delta}FosB or an empty vector control were grown to confluence and then induced to undergo differentiation by treatment with adipogenic agents. Total cell lysates were isolated from preadipocytes (day 0), differentiating adipocytes (days 1 and 4), and fully mature adipocytes (day 8), and the expression levels of several adipocyte transcription factors were examined by Western blotting. As shown in Fig. 4A, the presence of {Delta}FosB had little or no effect on the clear increase in the protein level of C/EBP{alpha} isoforms, PPAR{gamma}, and sterol regulatory element binding protein 1 (SREBP-1) transcription factors that occurred in 3T3-L1 cells cultured in adipogenic medium. The early adipogenic transcription factor C/EBPß remained essentially unchanged in both control-transfected and {Delta}FosB-expressing cells throughout the experiment. We also examined steady-state mRNA levels in undifferentiated and differentiated 3T3-L1 cells by Northern blot analysis (Fig. 4B). In these already committed preadipocytic cells, {Delta}FosB had no effect on the levels of C/EBP{alpha} and C/EBPß RNA transcripts in either the preadipocytes or the mature adipocytes, consistent with the results obtained by Western blotting.



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  		FIG. 4. {Delta}FosB has little effect on 3T3-L1 adipocyte differentiation. (A) Western blot analysis of the adipogenic transcription factors C/EBP{alpha}, C/EBPß, PPAR{gamma}, and SREBP-1 in cell extracts from 3T3-L1 adipocytes stably transfected with {Delta}FosB or the empty vector control at days 0, 1, 4, and 8 of adipocyte differentiation. Mainly the 42-kDa (p42) and 30-kDa (p30) C/EBP{alpha} isoforms and the 32- to 35-kDa (LAP) and 20-kDa (LIP) C/EBPß isoforms were detected. Actin served as an internal control. (B) Northern blot analysis of C/EBP{alpha}, C/EBPß, and PPAR{gamma} in preadipocytes (pre) and mature adipocytes (adip). (C) Northern blot analysis of two markers of mature adipocytes, lipoprotein lipase (LPL) and adipsin. 18S rRNA served as an internal control. (D) Secreted leptin in conditioned medium from control- and {Delta}FosB-transfected cells at day 8 of adipocyte differentiation was quantified as described in Materials and Methods (*, P < 0.05). (E) 3T3-L1 cells stably transfected with {Delta}FosB or empty vector were stained with Oil Red O at day 8 of differentiation, and the degree of staining was quantified as described in Materials and Methods. Data are presented as means ± standard error of the mean. *, P < 0.05, Student's t test.

We also examined adipocyte markers expressed at later stages of adipocyte differentiation. Both adipsin and lipoprotein lipase mRNA levels were strongly increased in mature adipocytes compared to preadipocytes in both control and {Delta}FosB-expressing 3T3-L1 cells (Fig. 4C). Finally, at day 8 of differentiation, at which point more than 80% of the cells had acquired the phenotype of mature adipocytes, secreted leptin (Fig. 4D) and lipid accumulation (Fig. 4E) were measured as described in Materials and Methods. Although overexpression of {Delta}FosB reduced leptin secretion by about 30% (Fig. 4D), there were no statistically significant differences in lipid accumulation by the {Delta}FosB and control cultures and the morphologies of the {Delta}FosB and control cultures were similar, supporting the finding that {Delta}FosB overexpression has little effect on adipocyte differentiation in 3T3-L1 preadipocytes.

The findings that {Delta}FosB had little effect on the development of fully mature adipocytes from the cell-line 3T3-L1, which is considered already committed to the adipocytic lineage, but repressed adipogenesis from the primary bone marrow progenitor cells from NSE-{Delta}FosB mice suggested that {Delta}FosB exerts its inhibitory effects at early stages of stem cell commitment. This possibility was tested by overexpressing {Delta}FosB in ST2 cells, which have the potential to differentiate along both the osteoblastic and adipocytic lineages and thus represent an earlier stage of mesenchymal cell differentiation. Western blot analysis of adipocyte transcription factors at days 0, 2, 3, and 7 of adipocyte differentiation (Fig. 5) demonstrated that stable overexpression of {Delta}FosB repressed the increase in C/EBP{alpha} and PPAR{gamma} protein levels that occurred in control cells. No clear change in expression levels of the high-molecular-weight isoform of C/EBPß (LAP) was observed, but expression of the short C/EBPß isoform (LIP) as well as a band of slightly higher molecular weight that most likely represents a posttranslationally modified form of LIP was reduced in the {Delta}FosB-transfected cells. In contrast, the expression pattern of the adipocyte transcription factor SREBP-1 was unchanged.



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  		FIG. 5. {Delta}FosB isoforms inhibit the expression of adipogenic transcription factors in the ST2 bone marrow stromal cell line. Western blot analysis of C/EBP{alpha}, C/EBPß, PPAR{gamma}, and SREBP-1 in cell extracts from ST2 cells stably transfected with {Delta}FosB, the full-length 2i3i isoform, the truncated {Delta}2{Delta}FosB isoform, or empty vector at days 0, 2, 3, and 7 of adipocyte differentiation. Mainly two C/EBP{alpha} isoforms at 42 to 45 kDa (probably p42 and a posttranslationally modified form) were detected, whereas less of the 30-kDa (p30) isoform was seen. For C/EBPß, mostly the 32- to 35-kDa (LAP) isoform and less of the 20-kDa (LIP) isoform were detected. At higher expression levels, a third band, most likely representing a posttranslationally modified LIP form, was observed. Actin served as an internal control.

The {Delta}FosB mRNA transcript gives rise to both the {Delta}FosB protein and a further truncated isoform, termed {Delta}2{Delta}FosB, that is lacking the N-terminal Fos homology domain of {Delta}FosB. In order to elucidate the relative importance of these two isoforms in regulating adipocyte differentiation, ST2 cells were transfected with a {Delta}2{Delta}FosB cDNA construct or with a mutated {Delta}FosB construct, termed 2i3i, that cannot generate the {Delta}2{Delta}FosB protein. Transfection with either 2i3i or {Delta}2{Delta}FosB caused a decrease in PPAR{gamma}, C/EBP{alpha}, and the LIP isoform of C/EBPß but had no effect on SREBP-1, similar to the pattern seen when the cells were transfected with the native {Delta}FosB cDNA (Fig. 5). Thus, the Fos homology domain, which is present in {Delta}FosB and 2i3i but not in the further truncated {Delta}2{Delta}FosB isoform, is not required for the {Delta}FosB-induced changes in the expression of the adipogenic transcription factors.

{Delta}FosB interacts with transcription factor C/EBPß. Since the inhibitory effect of {Delta}FosB on adipogenesis appeared to occur at an early stage of adipocyte differentiation, {Delta}FosB could interact with one or more transcription factors known to be involved in the early commitment to adipogenesis. C/EBPß is one of the earliest inducers of adipogenesis and, like {Delta}FosB, is a basic leucine zipper transcription factor. We therefore investigated the possibility that the two proteins interact. For this purpose, we generated chimeric proteins of GST fused to the full-length 2i3i isoform or to {Delta}2{Delta}FosB and used them in pull-down experiments with total cell lysates from ST2 cells that overexpressed a C/EBPß construct (Fig. 6A). Both GST-{Delta}2{Delta}FosB and GST-2i3i fusion proteins bound C/EBPß in the cell extracts, whereas GST by itself did not, demonstrating in vitro an interaction between {Delta}FosB and C/EBPß.



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  		FIG. 6. {Delta}FosB interacts with the early adipogenic transcription factor C/EBPß. (A) Pull-down experiments were performed with GST-{Delta}FosB and GST-{Delta}2{Delta}FosB and cellular extracts from ST2 cells transfected with a C/EBPß construct. Bound proteins were analyzed by Western blotting with an anti-C/EBPß antibody. (B) C/EBPß was immunoprecipitated (IP) from ST2 cells transfected with C/EBPß and either the Myc-{Delta}2{Delta}FosB or Myc-2i3i isoform, and the immune complexes were blotted for Myc to detect the Myc-tagged {Delta}FosB isoforms. Western blot analysis of c-Myc and C/EBPß in total cell lysates (TCL) from transfected cells verified equal expression levels of the proteins.

To confirm that this interaction also occurs in a cellular environment, ST2 cells were transfected with C/EBPß and either Myc-tagged {Delta}2{Delta}FosB or Myc-tagged 2i3i. Immunoprecipitation of C/EBPß followed by immunoblotting of the immune complexes with an anti-Myc antibody identified both {Delta}FosB isoforms (Fig. 6B), confirming that the C/EBPß and {Delta}FosB transcription factors were present in the same complex. The endogenous level of C/EBPß was sufficient to precipitate some of the Myc-tagged {Delta}FosB proteins, although not as much as when C/EBPß was overexpressed. No interactions between {Delta}FosB and C/EBP{alpha} or C/EBP{delta} were detected by either GST pull-down or coimmunoprecipitation (data not shown).

{Delta}2{Delta}FosB enhances binding of C/EBPß to its consensus DNA-binding site. The interaction of {Delta}FosB isoforms with C/EBPß suggests that the interaction might affect the ability of C/EBPß to heterodimerize with other transcription factors and to bind DNA response elements, thereby affecting the regulation of the transcription of downstream target genes (11, 27). We therefore examined the effect of {Delta}FosB isoforms on C/EBPß binding to its consensus response element (11, 41) in an electrophoretic mobility shift assay. As shown in Fig. 7A, incubation of the C/EBPß response element with nuclear extracts from control-transfected ST2 cells produced several complexes. The complexes observed were shown to be specific by competition with unlabeled probe. When nuclear extracts from cells overexpressing FosB or the full-length {Delta}FosB isoform were used, the levels of the complexes were slightly reduced from the levels seen with the control nuclear extracts. In contrast, overexpression of the truncated {Delta}2{Delta}FosB isoform dramatically enhanced complex formation. Supershift analysis with an antibody against C/EBPß indicated that C/EBPß was part of all the complexes formed on the response element. The expression of FosB isoforms in the nuclear extracts used for the electrophoretic mobility shift assay was verified by Western blotting (Fig. 7B).



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  		FIG. 7. Truncated {Delta}2{Delta}FosB isoform enhances binding of C/EBPß to its consensus DNA response element. (A) Electrophoretic mobility shift assay of nuclear extracts from ST2 cells transfected with empty vector (control) or cells overexpressing FosB, the truncated {Delta}2{Delta}FosB isoform, or the full-length 2i3i isoform. Complex formation was inhibited with excess unlabeled oligonucleotide (comp), or protein complexes were supershifted with an anti-C/EBPß antibody. (B) Western blot analysis of nuclear extracts to verify overexpression of {Delta}FosB isoforms.


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DISCUSSION
 
It is widely believed that osteoprogenitors and adipocyte progenitors originate from common mesenchymal stem cells located in the bone marrow (40, 43). In addition, several lines of evidence have suggested that differentiation of osteoblasts and adipocytes is regulated reciprocally (4, 11, 32). Park et al. reported that differentiated adipocytes from human bone marrow could dedifferentiate and then redifferentiate into osteoblasts (38), indicating a high degree of plasticity in the osteoblast and adipocyte lineages. In vivo, an inverse relationship between the number of osteoblasts and bone marrow adipocytes has been demonstrated in several forms of osteopenia, where decreased bone mass is often associated with increased adipogenesis (6, 29, 57, 60). Conversely, we have reported an osteosclerotic phenotype accompanied by decreased adipogenesis in transgenic mice that overexpress the AP-1 transcription factor {Delta}FosB under the control of the NSE promoter (25, 47, 53).

In our initial study of NSE-{Delta}FosB mice, we found that the NSE promoter directs transgene expression to both bone and fat as well as to several other tissues (47). Thus, the concurrence of increased osteoblast formation and function and decreased adipocyte formation could result from the independent action of {Delta}FosB in the two cell types. On the other hand, given the postulated reciprocity in the development of the two cell lineages, the change in one cell type could be a consequence of the action of {Delta}FosB in the other. We previously examined the possibility that the decreased circulating leptin levels in these mice contributed to the bone phenotype, since it has been suggested that the adipocyte-secreted hormone leptin acts as a mediator coupling adipocyte differentiation to osteoblast function (14). While we found that correcting the level of circulating leptin had little effect on the excessive bone formation (25), this did not completely exclude the possibility of interdependence between the increased osteoblast differentiation and decreased adipocyte differentiation observed in the transgenic animals.

To further understand how {Delta}FosB induces the changes in osteoblast and adipocyte differentiation, and especially to explore the possible role of indirect effects, we generated transgenic mice that express {Delta}FosB in a bone-specific manner, with the mouse OG2 (osteocalcin) promoter used to drive the expression of the tetracycline transactivator. Osteocalcin is considered a late marker of differentiating osteoblasts (54), and while the OG2 promoter produces a lower level of expression than the collagen 1a1 promoter (22), it has been used successfully to direct transgene expression specifically to mature osteoblastic cells (10, 22, 28), without any expression in osteoclasts (65, 66). The phenotype of the bitransgenic OG2-tTA x TetOp-{Delta}FosB mice clearly showed that the effects of {Delta}FosB on osteoblast and adipocyte differentiation are independent of each other.

Complete histomorphometric analysis showed that the restricted expression of {Delta}FosB in osteoblasts was sufficient to cause a significant increase in all bone formation parameters without affecting bone resorption, as had been seen in the NSE-{Delta}FosB transgenic mice. Thus, these results confirm our previous conclusion that {Delta}FosB-induced osteosclerosis is a direct effect of {Delta}FosB overexpression in osteoblasts (25, 47). However, in sharp contrast to the clear inhibition of adipogenesis in NSE-{Delta}FosB mice, no differences were observed in either the abdominal fat or the adipogenic capacity of the bone marrow cells of OG2-{Delta}FosB transgenic mice. This study therefore also indicates that the decreased adipogenesis in the NSE-{Delta}FosB mice is not simply a consequence of the increased osteoblast differentiation, but rather is probably the direct result of {Delta}FosB overexpression in preadipocytes or adipocytes. While we cannot rule out the possibility that the absence of the adipocyte phenotype in the OG2-{Delta}FosB mice is due in part to a lower level of expression of {Delta}FosB in the osteoblasts of these mice than in the osteoblasts of the NSE-{Delta}FosB mice, we think that this is unlikely, given our earlier findings that the abdominal fat weights were essentially identical in the high-expressing and low-expressing lines of NSE-{Delta}FosB mice, while the additional bone formation differed by more than 10-fold between those lines (47), and also that when the NSE-{Delta}FosB mice are given doxycycline to prevent transgene expression, the low weight of the fat pad is essentially unchanged at a time (2 weeks of treatment) when bone formation and osteoblast number have been reduced to levels well below those of the control littermates (53).

These results suggest that, if anything, the fat phenotype is more likely to be induced by low levels of {Delta}FosB expression than the bone phenotype. Thus, {Delta}FosB most likely affects both osteoblast and adipocyte differentiation directly and independently. Whether {Delta}FosB regulates the two events by similar mechanisms is unknown. Interestingly, a similar osteosclerotic phenotype has been described in mice that overexpress another Fos family member, Fra-1 (19). In contrast to {Delta}FosB, however, Fra-1 overexpression had no effect on adipogenesis in vivo, much like the phenotype seen in the OG2-{Delta}FosB mice, but it enhanced osteoclastogenesis in vitro.

Differentiation of mesenchymal cells into adipocytes involves a cascade of transcriptional events, including the induction of the early adipocyte transcription factors C/EBPß and C/EBP{delta}. These transcription factors in turn activate the expression of C/EBP{alpha} and PPAR{gamma}, which are essential for the stimulation of several adipocyte-specific genes (7, 61, 62). In fact, knocking out either the C/EBP{alpha} gene or the PPAR{gamma} gene results in severely diminished adipogenesis (44, 55, 58).

We found that C/EBP{alpha} expression was downregulated in primary bone marrow stromal cell cultures from NSE-{Delta}FosB mice but not in those from OG2-{Delta}FosB mice (Fig. 3), suggesting that {Delta}FosB might be altering this key adipogenic regulatory mechanism when expressed in adipocytes or their precursors. We therefore explored the possible mechanism of a direct effect of {Delta}FosB on adipocyte differentiation by overexpressing the {Delta}FosB isoforms in the preadipocytic 3T3-L1 cell line and the less committed ST2 bone marrow stromal cell line. Overexpression of either of the {Delta}FosB isoforms in ST2 cells inhibited the induction of C/EBP{alpha} and PPAR{gamma}, further supporting the possibility that downregulation of the expression of these transcription factors could account, at least in part, for the low-fat phenotype of the NSE-{Delta}FosB mice. However, in contrast to the results obtained in ST2 cells, overexpression of {Delta}FosB in the more committed 3T3-L1 cells had little effect on the induction of C/EBP{alpha} or PPAR{gamma} expression. Consistent with this failure of {Delta}FosB to inhibit expression of the adipocyte master regulators in 3T3-L1 cells, its presence had no effect on the development of adipocyte morphology, lipid accumulation, or the expression of the late adipocyte markers adipsin and lipoprotein lipase, although the amount of secreted leptin decreased by about 30%. Collectively, these data suggest that {Delta}FosB exerts its antiadipogenic effect at an early stage of adipogenesis yet beyond the presumed branching point of the osteoblast and adipocyte lineages. The partial downregulation of leptin secretion by the 3T3-L1 cells suggests that it may also act in a more limited way during later stages of differentiation.

Since {Delta}FosB is a splicing variant of the AP-1 transcription factor FosB, it is likely to exert its effects by altering transcriptional regulation. No changes in bone or fat formation have been observed in mice that lack the fosB gene (5, 17; our unpublished observations). This indicates that none of the FosB isoforms (FosB, {Delta}FosB, and {Delta}2{Delta}FosB) are strictly required for osteoblast and adipocyte differentiation to occur and suggests that the effects of overexpressed {Delta}FosB are not due to the displacement of full-length FosB from some critically important complex or to an increased amount of a required {Delta}FosB-containing complex.

{Delta}FosB and the further N-terminally truncated {Delta}2{Delta}FosB lack one and both of FosB's transactivation domains, respectively, but contain both the DNA-binding and heterodimerization domains (31). Based on these structural considerations, it is likely that the truncated isoforms bind transcription factors and DNA response elements that are similar or identical to those bound by full-length FosB but that they alter the transcriptional activity of the complexes. Overexpressing the {Delta}FosB and {Delta}2{Delta}FosB proteins could decrease transcriptional activity by reducing the DNA-binding affinity or altering the DNA-binding specificity of the protein complex or by preventing the interaction with transcriptional coactivators. Alternatively, transcription could be increased due to the displacement of transcriptional repressors. (Since the {Delta}2{Delta}FosB isoform, which lacks {Delta}FosB's transcriptionally active N-terminal Fos homology domain [13, 21], appears to be sufficient to induce the observed effects in the ST2 cells, the phenotype is unlikely to be a consequence of direct transcriptional activity of {Delta}FosB.) Thus, the identification of transcription factors that interact with overexpressed {Delta}FosB during osteoblast or adipocyte differentiation will contribute to understanding the mechanisms that are critically important for the differentiation of these cells.

The ability of {Delta}FosB to prevent the increased expression of C/EBP{alpha} that normally occurred in primary bone marrow stromal cells and ST2 cells in response to adipogenic conditions suggested that {Delta}FosB interacts with a factor that promotes the expression of C/EBP{alpha}. We therefore examined whether {Delta}FosB interacts with or affects the function of C/EBPß, another basic leucine zipper transcription factor that promotes adipocyte differentiation by upregulating the expression of several adipogenic genes, including C/EBP{alpha} (27, 42, 45), and which has also recently been implicated in osteoblast differentiation (18). We found that {Delta}FosB indeed bound to C/EBPß but did not bind to other C/EBP proteins, indicating the specificity of the interaction. Furthermore, the presence of the {Delta}FosB isoforms altered the binding of C/EBPß-containing protein complexes to a consensus C/EBPß response element.

Interestingly, the full-length {Delta}FosB isoform, like FosB itself, caused a small reduction in C/EBPß DNA binding, while the {Delta}2{Delta}FosB isoform strongly increased the binding of C/EBPß to the DNA. The strong potentiation of the C/EBPß-DNA interaction by the N-terminally truncated {Delta}2{Delta}FosB isoform, in contrast to the markedly weak effects of FosB and {Delta}FosB, suggests that the {Delta}2{Delta}FosB isoform might be the actual mediator of changes in osteoblast and adipocyte differentiation observed in the NSE-{Delta}FosB transgenic mice. Indeed, recent data show that overexpressing the {Delta}2{Delta}FosB isoform by itself under control of the NSE promoter is sufficient to cause both the osteoblast and the adipocyte phenotypes in mice (unpublished data).

Together, the binding of {Delta}FosB isoforms to C/EBPß, the alteration of C/EBPß-DNA interactions by {Delta}FosB isoforms, and the {Delta}FosB-induced changes in the amount of the small C/EBPß isoform in the ST2 cells provide a strong indication of the involvement of C/EBPß in the mechanism by which {Delta}FosB inhibits adipocyte formation. C/EBPß biology is complex, however, presenting challenges to elucidating its possible role in the {Delta}FosB-dependent mechanisms. Differential usage of initiation methionines in a single C/EBPß mRNA generates isoforms that activate (LAP) and inhibit (LIP) gene expression (12, 34). The levels of these isoforms are differentially regulated (1), possibly in a species- and cell type-specific manner, and the specific response of target genes depends on the relative amounts of the different isoforms (12). This may explain the apparently conflicting reports that C/EBPß both positively (12) and negatively (56) regulates the expression of the albumin gene, where it interacts with the same C/EBPß-binding site that we used in these experiments.

The challenge of elucidating the {Delta}FosB mechanism is further increased by the existence of the full-length and {Delta}2{Delta}FosB isoforms of {Delta}FosB and the different effects of those isoforms on the C/EBPß-DNA interaction and probably on the transcriptional activity of the resulting complexes. While elucidation of the combined effect of {Delta}FosB and the C/EBPß isoforms on the C/EBP{alpha} promoter is thus beyond the scope of this study, such an analysis might reveal that interactions between C/EBPß and {Delta}FosB contribute to both the increase in osteoblastogenesis and the inhibition of adipocyte differentiation seen in the NSE-{Delta}FosB mice.

In conclusion, this study establishes that {Delta}FosB-induced osteosclerosis is a direct effect of the overexpression of {Delta}FosB in osteoblasts. In addition, under these conditions, osteosclerosis is induced independently of decreased adipogenesis, suggesting that {Delta}FosB isoforms exert direct effects on both osteoblast and adipocyte differentiation processes. Finally, the inhibitory effect of {Delta}FosB on adipogenesis is also cell autonomous and appears to occur at an early stage of stem cell commitment, possibly via an interaction with C/EBPß.


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ACKNOWLEDGMENTS
 
We thank Karen Ford and Wayne Grant for excellent technical assistance. We are grateful to J. Gimble and D. P. Ramji for supplying various cDNA constructs.

This work was supported by a postdoctoral fellowship from the Danish Research Council to M.K. and for the most part by a grant from the National Institutes of Health (AR48218) to R.B. Other support included grants from the NIH to W.M.P. (DE12616) and to the Yale Core Center for Musculoskeletal Diseases (AR46032) and from Aventis Pharma and Proskelia to R.B.


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FOOTNOTES
 
* Corresponding author. Mailing address: Departments of Orthopedics and Cell Biology, Yale University School of Medicine, 333 Cedar St., SHM IE-55, New Haven, CT 06510. Phone: (203) 785-5986. Fax: (203) 785-5986. E-mail: roland.baron{at}yale.edu. Back

{dagger} Present address: Institute of Molecular Pathology, University of Copenhagen, 2100 Copenhagen, Denmark. Back

{ddagger} Present address: Procter & Gamble Pharmaceuticals, Mason, OH 45040. Back


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Molecular and Cellular Biology, April 2004, p. 2820-2830, Vol. 24, No. 7
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.7.2820-2830.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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