The cellular identity and precise nature of osteoclast progenitors are longstanding and important biological questions. Based on our cellular, molecular, genetic, pathological, and pharmacological evidence, in vivo and ex vivo, we conclude that the osteoclast lineage resides in the PPARγ-expressing hematopoietic bone marrow cell population, and we have identified the quiescent PPARγ+ bone marrow cells as the osteoclast progenitors. Importantly, we have established PPARγ-tTA TRE-H2BGFP reporter mice as an unprecedented tool to visualize, isolate, quantify, and trace the lineage of osteoclast progenitors. As a complement, we have also established PPARγ-tTA TRE-Cre mice as a genetic tool to interrogate the function and regulation of osteoclast progenitors in vivo by inducing flox-mediated gene deletion or activation. Using these tools, we have uncovered previously unrecognized effects of ovariectomy and rosiglitazone, two resorption-enhancing stimuli, on the early osteoclast lineage. Mechanistically, we have identified GATA2 as a novel yet critical PPARγ target gene in osteoclast progenitors. Therefore, both conceptually and technically, this study opens an exciting new path to the fundamental understanding of both osteoclast lineage specification and PPARγ function.
In the PPARγ-tTA TRE-H2BGFP reporter mice, GFP+
cells also label adipocyte progenitors and mature adipocytes (46
). Intriguingly, several reports show that mammalian cells of the adipocyte lineage and the macrophage lineage share numerous functional and antigenic properties. Gene expression profiling revealed that preadipocytes share a surprisingly closer signature with macrophages than with adipocytes, and preadipocytes can be effectively converted to macrophages in a macrophage environment (10
). This appears to be an evolutionarily conserved phenomenon, because in invertebrates, such as Drosophila
, hemocytes (blood cells) and fat bodies also share the expression of fate-determining genes (14
). Our findings that PPARγ+
cells label both adipocyte progenitors and macrophage/osteoclast progenitors provided mechanistic evidence for convergence and/or plasticity in the adipocyte and macrophage lineage specification. Together with the dual roles of PPARγ ligand in stimulating both adipogenesis and osteoclastogenesis, our findings illuminate a potential molecular basis for the close correlation between insulin-sensitizing effects and bone loss effects, as well as the emerging connections between fat and bone.
Stem/progenitor cells are defined as multipotent; hence, in order to target osteoclast progenitors experimentally, it is impossible to completely rule out other differentiation outcomes, and specificity is only relative. For example, Tie2 labels not only osteoclast progenitors, but also all other hematopoietic progenitors, as well as endothelial cells (13
), whereas PPARγ labels osteoclast and adipocyte progenitors but not other hematopoietic lineages ( and ). The advantages of the PPARγ-tTA-based mouse models include the following: (i) they distinguish macrophages/osteoclasts from other hematopoietic lineages; (ii) they target the entire osteoclast lineage, including osteoclast progenitors and mature osteoclasts; and (iii) they permit temporal control of inducible cell labeling and genetic manipulations in the osteoclast lineage. Our results show that osteoclast progenitors are 140-fold enriched in the PPARγ+
bone marrow cell population, and thus, only <1% of osteoclast progenitors may be derived from PPARγlow
cells. Moreover, previous studies have documented that Notch activation in lymphoid progenitors causes T-cell lymphoblastic leukemia in humans and mice (16
). In our study, PTNICD mice did not develop lymphoma, which further supports the notion that PPARγ specifically directs hematopoiesis toward the monocyte/macrophage lineage and that the PPARγ-expressing bone marrow population does not contain lymphoid progenitors. Since PPARγ also labels adipocyte progenitors (46
), it is possible that Notch constitutive activation in PTNICD mice may also affect other PPARγ+
cells, such as adipocytes, in addition to osteoclast progenitors.
Several drivers targeting macrophage precursors or mature osteoclasts have been elegantly described; nonetheless, because they do not target osteoclast progenitors, they are not suitable for in vivo
study of early osteoclast lineage specification. For example, CD11b or lysozyme drivers are useful to target macrophage precursors because they are upregulated only upon macrophage differentiation (12
). In addition, Ctsk or TRAP drivers are useful to target preosteoclasts and mature osteoclasts because they are upregulated only upon osteoclast differentiation (11
). Therefore, the PPARγ-tTA-based models represent a novel osteoclast progenitor-targeting strategy that is complementary to other existing models for the comprehensive investigation of osteoclast lineage specification and differentiation.
Indeed, the PTNICD and PTDTA genetic models provide compelling in vivo
evidence that the osteoclast lineage resides in the PPARγ+
bone marrow population under physiological conditions; in particular, the PTNICD model supports the notion that PPARγ+
cells represent osteoclast progenitors. In the PTDTA model, the DTA was an “attenuated” version of diphtheria toxin, thus explaining the relatively mild bone phenotype, which was supported by the survival of the PTDTA mice in contrast to the embryonic lethality in the global PPARγ knockout (KO) mice (7
). Furthermore, both OVX and BRL, representing pathological and pharmacological resorption-enhancing stimuli, triggered the PPARγ+
cells to proliferate and differentiate, further supporting the notion that osteoclast progenitors reside in the PPARγ+
bone marrow population in vivo
. The rapid increase (1 to 2 weeks) in the percentage of GFP+
bone marrow cells in response to BRL treatment in vivo
indicates that this effect was not likely secondary to any BRL alteration of adipocytes followed by changes in hematopoiesis, which takes at least 4 weeks (1
The Ets family transcription factor PU.1 is essential for the development of both myeloid and B-lymphoid cells (42
). This suggests that additional transcription factors are required to function in combination with PU.1 and confer lineage specificity (22
). Since osteoclasts are of myeloid lineage, PU.1 is also essential for the generation of osteoclast progenitors. Indeed, PU.1 deletion in mice precludes osteoclast development, leading to arrested bone resorption and osteopetrosis (47
). A recent study revealed that, in macrophages, PPARγ colocalizes with PU.1 in areas of open chromatin and histone acetylation near a distinct set of hematopoietic genes (31
). Our results suggest that in the PPARγ+
cells, PPARγ cooperates with PU.1 to activate the transcription of a subset of genes, including GATA2, thereby directing macrophage/osteoclast lineage commitment. In contrast, in the PPARγ−
cells, the absence of PPARγ prevents GATA2 transcription and alters the subset of genes regulated by PU.1, thereby directing B-lymphoid lineage commitment. Therefore, our identification of PPARγ+
bone marrow cells as osteoclast progenitors provides in vivo
evidence for the notion that the collaborative interaction between PPARγ and PU.1 on a subset of promoters is essential to activate the transcriptional program required for macrophage/osteoclast lineage commitment. Furthermore, our results suggest that the expression of PPARγ, rather than the ligand activation of PPARγ, promotes osteoclast progenitor specification by enhancing GATA2 expression, which is downregulated during the quiescence-to-proliferation switch and thus is absent in osteoclast precursors (53a
) (G). Together, our current and previous studies reveal that PPARγ plays dual roles in osteoclastogenesis that involve multiple mechanisms and target genes (G): PPARγ expression promotes osteoclast progenitors by inducing GATA2, and PPARγ ligand activation stimulates osteoclast differentiation by inducing c-fos.