pRb, dialing between bone and brown fat

Abstract

Calo et al. report in a recent issue of Nature how the absence of the transcriptional regulator pRb enhances differentiation of mesenchymal precursors towards the brown adipocyte lineage in detriment of the osteoblast and white fat populations. This work could be therapeutically relevant for the fields of cancer, bone and metabolic disease.

pRb, which constitutes together with p107 and p130 the pocket protein family of transcriptional regulators, was identified as a tumor suppressor that inhibits cell cycle progression through binding and inhibition of the transcription factor E2F1. As pRb is phosphorylated by the G1-S CDK/cyclin complexes, it becomes unable to interact and inhibit E2F1, allowing the cell to enter S phase. However, the list of the functions of pRb has expanded beyond cell cycle control and it is now established as a relevant player in the differentiation of a wide array of cell types. Although the importance of pRb in cell differentiation was initially associated to its ability to induce cell cycle exit, more recently pRb has also been shown to regulate differentiation through specific binding and modulation of the activity of master transcription factors that specify cell fate (Korenjak and Brehm, 2005).

pRb has in this context been extensively studied for its roles during adipocyte and osteoblast differentiation. During adipocyte differentiation, pRb has a dual function. In confluent preadipocytes, the repressive E2F4/pRb complex inhibits transcription of E2F-responsive genes including PPARγ, the master regulator of adipocyte differentiation (Fajas et al., 2002b). This involves the recruitment of histone deacetylase 3 to PPARγ-binding sites (Fajas et al., 2002a). When these quiescent preadipocytes are then stimulated to differentiate they enter the so-called clonal expansion phase, coinciding with the loss of the repressive E2F4 complex and the induction of E2F1, which triggers PPARγ expression and the adipogenic program (Fajas et al., 2002b). Perhaps even more relevant is that during the final stages of adipocyte differentiation, pRb activation determines the characteristics of the prevailing adipocyte lineage, pushing the process towards white adipocytes, in detriment of brown fat cells (Hansen et al., 2004). Coherently, adipocyte-specific deletion of pRb protects mice from obesity, with reduced white fat mass and increased amounts of energy-spending brown fat (Dali-Youcef et al., 2007). During osteoblast differentiation, pRb enhances the transcription of core binding factor A1, also called RUNX2, a transcription factor indispensable for osteoblast differentiation (Thomas et al., 2001) and blocks E2F1-mediated proliferation of precursor cells, thus allowing proper osteoblast differentiation (Berman et al., 2008). As a result, ossification is impaired in pRb^−/− mice due to defective osteoblast differentiation (Berman et al., 2008).

Although the above evidence indicates that pRb regulates adipocyte and osteoblast differentiation, Calo et al. now unite these scattered data by proving that pRb blocks differentiation of common mesenchymal precursors to adipocytes and favors their osteoblasts fate. In preosteoblasts, the presence of pRb commits them towards the osteoblast lineage. In the absence of pRb, the preosteoblasts de-differentiate into multipotent precursors, that without pRb will preferentially commit towards the adipocyte lineage. Only those preadipocytes that start to express pRb during the final stages of differentiation will become white adipocytes, whereas the continuous absence of pRb favors the brown adipocyte fate, which fits with the robust increase in brown fat in pRb^−/− mice (Calo et al., 2010; Dali-Youcef et al., 2007), and the predominance of hibernomas in p53/pRb double-deficient mice (Calo et al.).

pRb is subject to many posttranslational modifications, which opens up the possibility to modulate its function through these upstream pathways. Within this context, it is well known that pRb is inactivated via phosphorylation by CDK/cyclin complexes in the G1-S phase of the cell cycle. This led to the identification of specific CDK inhibitors that have been successfully used in mouse cancer models (Puyol et al., 2010). Exploring the effects of such compounds on the developmental switches in question should be interesting.

The absence of pRb promotes the formation of more aggressive tumors, a feature mainly attributed to inhibitory effects of pRb on cell cycle progression. The pluripotent nature of pRb deficient cells, resembling cancer stem cells, may also contribute to tumor aggressiveness. A dilemma to be solved, however, is why is pRb so commonly mutated in osteosarcomas, when its genetical ablation pushes mesenchymal cells away from the osteoblastic state. The authors suggest that osteosarcomas derive from already committed osteoblasts, where pRb loss constitutes a proliferative advantage, without altering their lineage commitment. Another consideration is that most tumors express a mutated pRb protein that still retains some activity, unlike the situation in genetically engineered mouse models, where gene and protein are lost.

pRb deficient mice have clear deficiencies in bone formation, whereas at the same time there is a noticeable expansion of brown fat mass. From these results it is not clear whether common mesenchymal bone/fat precursors, in the absence of pRb, develop into brown adipocytes instead of into osteoblasts in an adult individual; or whether there are two separate precursor populations, pre-osteoblasts and pre-adipocytes, without the possibility of in vivo interchange between them. The development of additional spatiotemporally controlled genetically engineered mouse models to define the function of pRb during the different sites and stages of differentiation, including adulthood, will therefore be highly relevant.

In the light of this work, one can speculate that modulation of pRb activity, as achieved by CDK inhibitors (Puyol et al., 2010), could be exploited to select osteoblast or adipocyte lineages at will. The possibilities of pRb-modulating compounds are multiple. In the iPS era, modulating pRb activity during in vitro differentiation of pluripotent stem cells could specify the generation of osteoblasts or adipocytes. In a clinical setting, selective enhancers of pRb function (e.g. CDK inhibitors) could be used to promote osteogenesis, a feature of interest in conditions such as osteoporosis, complex fractures and bone degenerating processes. Also inhibitors of pRb could have therapeutic relevance. Recently, brown fat was recognized as an important contributor to energy dissipation in adult humans (Kajimura et al., 2010). Interestingly, not all brown adipocytes are alike. The brown adipocytes in the interscapular region are of dermomyotomal origin, and it is the zinc-finger protein PRDM16 which specifies the fate of these precursor cells towards either skeletal myoblasts or brown adipocytes (Kajimura et al., 2010). Interestingly, white adipose tissue, that is of mesodermal origin, also contains an expendable pool of “brown-adipocyte-like” or “brown-in-white” cells (Kajimura et al., 2010). Given that these cells are of mesodermal origin, inhibition of pRb could stimulate their expansion. The fact that adipocyte-specific pRb^−/− mice are protected against obesity, as a consequence of the expansion of brown adipocytes and the induction of energy expenditure, validates such an approach already in vivo (Dali-Youcef et al., 2007). Characterization of the endogenous signaling pathways and identification of pharmacological compounds that modify pRb activity may therefore hold also value to treat obesity and associated disorders. It is needless to remind, however, that modulating pRb’s activity can also have undesired effects, most notably tumor promotion.

Altogether, the piece of work by Calo et al. unequivocally defines the important in vivo contributions of pRb to mesenchymal lineage differentiation. It not only sets an impressive example of the potential of mouse genetics to understand basic biological processes, but also brings up a number of interesting clinical perspectives that should stimulate further research aimed at specifically modulating the many functions coordinated by pRb. If successful, such strategies could have ramifications in fields like development/stem cells, cancer, bone health, and metabolism.

Figure 1.

pRb determines the lineage commitment of mesodermal precursor cells. The presence of pRb enhances differentiation of mesodermal precursors into osteobasts, while its absence shifts this process towards preadipocytes. In turn, preadipocytes in which pRb is reexpressed will develop into white fat cells, while constant absence of pRb will lead to brown adipocytes. This last cell type can also derive from dermomyotomal precursors that, upon expression of PRDM16, will differentiate into brown fat, whereas the absence of PRDM16 will lead them to become skeletal myoblasts.