It is widely believed that osteoprogenitors and adipocyte progenitors originate from common mesenchymal stem cells located in the bone marrow (40
). In addition, several lines of evidence have suggested that differentiation of osteoblasts and adipocytes is regulated reciprocally (4
). 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
). Conversely, we have reported an osteosclerotic phenotype accompanied by decreased adipogenesis in transgenic mice that overexpress the AP-1 transcription factor ΔFosB under the control of the NSE promoter (25
In our initial study of NSE-ΔFosB 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 ΔFosB 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 ΔFosB 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 ΔFosB induces the changes in osteoblast and adipocyte differentiation, and especially to explore the possible role of indirect effects, we generated transgenic mice that express ΔFosB 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
), without any expression in osteoclasts (65
). The phenotype of the bitransgenic OG2-tTA × TetOp-ΔFosB mice clearly showed that the effects of ΔFosB on osteoblast and adipocyte differentiation are independent of each other.
Complete histomorphometric analysis showed that the restricted expression of ΔFosB 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-ΔFosB transgenic mice. Thus, these results confirm our previous conclusion that ΔFosB-induced osteosclerosis is a direct effect of ΔFosB overexpression in osteoblasts (25
). However, in sharp contrast to the clear inhibition of adipogenesis in NSE-ΔFosB mice, no differences were observed in either the abdominal fat or the adipogenic capacity of the bone marrow cells of OG2-ΔFosB transgenic mice. This study therefore also indicates that the decreased adipogenesis in the NSE-ΔFosB mice is not simply a consequence of the increased osteoblast differentiation, but rather is probably the direct result of ΔFosB overexpression in preadipocytes or adipocytes. While we cannot rule out the possibility that the absence of the adipocyte phenotype in the OG2-ΔFosB mice is due in part to a lower level of expression of ΔFosB in the osteoblasts of these mice than in the osteoblasts of the NSE-ΔFosB 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-ΔFosB mice, while the additional bone formation differed by more than 10-fold between those lines (47
), and also that when the NSE-ΔFosB 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 ΔFosB expression than the bone phenotype. Thus, ΔFosB most likely affects both osteoblast and adipocyte differentiation directly and independently. Whether ΔFosB 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 ΔFosB, however, Fra-1 overexpression had no effect on adipogenesis in vivo, much like the phenotype seen in the OG2-ΔFosB 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/EBPβ and C/EBPδ. These transcription factors in turn activate the expression of C/EBPα and PPARγ, which are essential for the stimulation of several adipocyte-specific genes (7
). In fact, knocking out either the C/EBPα gene or the PPARγ gene results in severely diminished adipogenesis (44
We found that C/EBPα expression was downregulated in primary bone marrow stromal cell cultures from NSE-ΔFosB mice but not in those from OG2-ΔFosB mice (Fig. ), suggesting that ΔFosB 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 ΔFosB on adipocyte differentiation by overexpressing the ΔFosB isoforms in the preadipocytic 3T3-L1 cell line and the less committed ST2 bone marrow stromal cell line. Overexpression of either of the ΔFosB isoforms in ST2 cells inhibited the induction of C/EBPα and PPARγ, 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-ΔFosB mice. However, in contrast to the results obtained in ST2 cells, overexpression of ΔFosB in the more committed 3T3-L1 cells had little effect on the induction of C/EBPα or PPARγ expression. Consistent with this failure of ΔFosB 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 ΔFosB 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 ΔFosB 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
; our unpublished observations). This indicates that none of the FosB isoforms (FosB, ΔFosB, and Δ2ΔFosB) are strictly required for osteoblast and adipocyte differentiation to occur and suggests that the effects of overexpressed ΔFosB are not due to the displacement of full-length FosB from some critically important complex or to an increased amount of a required ΔFosB-containing complex.
ΔFosB and the further N-terminally truncated Δ2ΔFosB 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 ΔFosB and Δ2ΔFosB 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 Δ2ΔFosB isoform, which lacks ΔFosB's transcriptionally active N-terminal Fos homology domain [13
], 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 ΔFosB.) Thus, the identification of transcription factors that interact with overexpressed ΔFosB during osteoblast or adipocyte differentiation will contribute to understanding the mechanisms that are critically important for the differentiation of these cells.
The ability of ΔFosB to prevent the increased expression of C/EBPα that normally occurred in primary bone marrow stromal cells and ST2 cells in response to adipogenic conditions suggested that ΔFosB interacts with a factor that promotes the expression of C/EBPα. We therefore examined whether ΔFosB interacts with or affects the function of C/EBPβ, another basic leucine zipper transcription factor that promotes adipocyte differentiation by upregulating the expression of several adipogenic genes, including C/EBPα (27
), and which has also recently been implicated in osteoblast differentiation (18
). We found that ΔFosB indeed bound to C/EBPβ but did not bind to other C/EBP proteins, indicating the specificity of the interaction. Furthermore, the presence of the ΔFosB isoforms altered the binding of C/EBPβ-containing protein complexes to a consensus C/EBPβ response element.
Interestingly, the full-length ΔFosB isoform, like FosB itself, caused a small reduction in C/EBPβ DNA binding, while the Δ2ΔFosB isoform strongly increased the binding of C/EBPβ to the DNA. The strong potentiation of the C/EBPβ-DNA interaction by the N-terminally truncated Δ2ΔFosB isoform, in contrast to the markedly weak effects of FosB and ΔFosB, suggests that the Δ2ΔFosB isoform might be the actual mediator of changes in osteoblast and adipocyte differentiation observed in the NSE-ΔFosB transgenic mice. Indeed, recent data show that overexpressing the Δ2ΔFosB 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 ΔFosB isoforms to C/EBPβ, the alteration of C/EBPβ-DNA interactions by ΔFosB isoforms, and the ΔFosB-induced changes in the amount of the small C/EBPβ isoform in the ST2 cells provide a strong indication of the involvement of C/EBPβ in the mechanism by which ΔFosB inhibits adipocyte formation. C/EBPβ biology is complex, however, presenting challenges to elucidating its possible role in the ΔFosB-dependent mechanisms. Differential usage of initiation methionines in a single C/EBPβ mRNA generates isoforms that activate (LAP) and inhibit (LIP) gene expression (12
). 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/EBPβ both positively (12
) and negatively (56
) regulates the expression of the albumin gene, where it interacts with the same C/EBPβ-binding site that we used in these experiments.
The challenge of elucidating the ΔFosB mechanism is further increased by the existence of the full-length and Δ2ΔFosB isoforms of ΔFosB and the different effects of those isoforms on the C/EBPβ-DNA interaction and probably on the transcriptional activity of the resulting complexes. While elucidation of the combined effect of ΔFosB and the C/EBPβ isoforms on the C/EBPα promoter is thus beyond the scope of this study, such an analysis might reveal that interactions between C/EBPβ and ΔFosB contribute to both the increase in osteoblastogenesis and the inhibition of adipocyte differentiation seen in the NSE-ΔFosB mice.
In conclusion, this study establishes that ΔFosB-induced osteosclerosis is a direct effect of the overexpression of ΔFosB in osteoblasts. In addition, under these conditions, osteosclerosis is induced independently of decreased adipogenesis, suggesting that ΔFosB isoforms exert direct effects on both osteoblast and adipocyte differentiation processes. Finally, the inhibitory effect of ΔFosB on adipogenesis is also cell autonomous and appears to occur at an early stage of stem cell commitment, possibly via an interaction with C/EBPβ.