Bone tissue and the underlying marrow are under strict regulation by lineage-specific transcription factors and factors involved in energy metabolism. Lineage allocation of the mesenchymal stem cells (MSCs), which give rise to bone forming osteoblasts or adipocytes, is dependent on pro-adipocytic transcription factor PPARγ2 and pro-osteoblastic Wnt signaling pathway. Activation of PPARγ2 in MSCs stimulates adipogenesis and acts as a dominant negative suppressor of osteoblastogenesis whereas upregulation of the Wnt pathway promotes bone accrual and reduces total body fat. This study examined the crosstalk between PPARγ2 and b-catenin, a key mediator of the Wnt signaling pathway, on MSC lineage determination.
Rosi-activated PPARγ2 induced rapid proteolytic degradation of b-catenin, which was prevented by LiCl, an antagonist of GSK3β. B-catenin degradation correlated with pro-adipocytic but not anti-osteoblastic activity of PPARγ2. As a result, stabilization of b-catenin with LiCl affected PPARγ2 mediated upregulation of adipocyte-specific markers without affecting osteoblast-specific markers. In addition, inhibition of pro-adipocytic activity of PPARγ2 by selective antagonist GW9662, protected b-catenin from degradation but did not affect anti-osteoblastic activity of PPARγ2. B-catenin physically interacts with PPARγ2 and its degradation depends on interaction with specific domains of PPARγ2 protein. Introducing a single nucleotide mutation within these domains of PPARγ2 protein protected b-catenin from dissociation from PPARγ2 and subsequent degradation and completely suppressed the transcriptional pro-adipocytic activity of PPARγ2, whereas lack of b-catenin binding to PPARγ2 failed to regulate PPARγ2 transcriptional activity, even in the absence of b-catenin degradation. Moreover, anti-osteoblastic activity of PPARγ2 was supported in conditions of PPARγ2 and b-catenin interaction but was inhibited in conditions of no interaction and no degradation of b-catenin. In conclusion, b-catenin sequesters PPARγ2 pro-adipocytic activity at the level of protein-protein interaction between these two transcriptional regulators and may resulate anti-osteoblastic activity through Wnt10b.
The crosstalk between marrow fat and osteoblasts raises questions regarding the regulation of bone by marrow fat. Fat occupies a significant portion of bone cavity however its function is largely unknown. Marrow fat expands during aging and in conditions, which affect energy metabolism, indicating that fat in bone is under similar regulatory mechanisms as other fat depots. On the other hand, its location may determine specific functions in the maintenance of an environment for bone remodeling and hematopoiesis. This study demonstrated that marrow fat has a distinctive phenotype, which resembles both white and brown adipose tissue (WAT and BAT respectively).
Marrow adipocytes express gene markers of brown adipocytes at levels characteristics of BAT including transcription factor Prdm16, and regulators of thermogenesis such as deiodinase 2 (Dio2) and PGC1α. The levels of expression of BAT-specific gene markers are decreased in bone of 24 mo old C57BL/6 and in diabetic yellow agouti Avy/a mice implicating marrow fat functional changes with aging and diabetes. Administration of anti-diabetic Rosi, which sensitizes cells to insulin and increases adipocyte metabolic functions, significantly increased both BAT (UCP1, PGC1, Dio2, 3AR, Prdm16, and FoxC2) and WAT (adiponectin and leptin) gene expression in marrow of normoglycemic C57BL/6 mice, but failed to increase the expression of BAT, but not WAT, gene markers in diabetic mice. Based on these data, the metabolic phenotype of marrow fat combines both BAT and WAT characteristics. Therefore, a decrease in BAT-like characteristics with aging and diabetes may contribute to the negative changes in the marrow environment supporting bone remodeling and hematopoiesis.
A relationship between the status of energy metabolism and bone mass has been actively debated. FoxC2 transcription factor regulates the expression of genes which determine the function of metabolically active brown fat, and these incude an adrenergic response, adaptive thermogenesis, mitochondrial biogenesis, and angiogenesis. Ectopic expression of FoxC2 in adipose tissues under the control of FABP4 promoter renders lean animals insulin sensitive, and protects them from high fat diet-induced obesity and insulin resistance. More importantly, these animals acquire significantly high bone mass. In general, bones of Tg-FoxC2 mice are larger in diameter with the same cortical thickness as compared to control mice. This results in an increase of the polar moment of inertia by 25%, which is an indicator of stronger bone. Both males and females have significantly more trabecular bone in proximal tibia, distal femur, and vertebra. Trabecular bone volume of Tg-FoxC2 mice is increased by approximately 2-fold due to increase in trabecular number, thickness, and connectivity. Remarkably, increased trabecular bnoe mass in tibia and femur is respectively associated with 4- and 65-fold increase in fat volume with corresponding regions, indicating a positive effect of fat on bone structure.
To investigate a possibility that marrow ADs regulate bone remodeling by modulating marrow environment, we expressed FoxC2 in AD2 in cell line representing committed marrow ADs. Ectopic expression of FoxC2 in ADs cells resulted in up-regulation of gene markers of brown ADs energy-producing activity, and included UCP-1, b3AR, Prdm16, and Dio2. Conditioned media from cultures of FoxC2-ADs increased osteoblast activity in osteoblasts indicating that these cells produce pro-osteoblastic paracrine factors. Furthermore, FoxC2 in ADs induced expression of Wnt10b, a member of Wnt signaling pathway, which has an anabolic effect on bone and positive effect on energy homeostasis. Taken together, these data suggest that FoxC2 induces BAT-like metabolic and/or paracrine activities in marrw ADs. These activities, which may include increased energy production and Wnt10b production, have beneficial effect on bone. The Tg-FoxC2 model provides a proof of concept that energy metabolism is integral for the maintenance of bone metabolism and allows for consideration of bone fat as therapeutic target to improve bone mass.
In conclusion, the intricate relationship between marrow fat and bone may determine the status of both skeletal and metabolic health. From the regulation of osteoblast and adipocyte phenotype by protei-protein interaction between lineage-specific transcription factors to the active changes in marrow fat with alterations in energy status to the modulation of bone formation by marrow fat via release of osteogenic factors, these studies provide proof of concept that the skeleton is an active organ altering and modulating overall energy metabolism. Understanding these underlying mechanisms would provide novel therapeutic targets for maintenance of skeletal health and, to some extent, treatment of metabolic disorders.