Foxo1, a Novel Regulator of Osteoblast Differentiation and Skeletogenesis

Abstract

Skeletogenesis depends on the activity of bone-forming cells derived from mesenchymal cells. The pathways that control mesenchymal cell differentiation are not well understood. We propose that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblasts. In mouse embryos, Foxo1 expression is higher in skeletal tissues, while Foxo1 silencing has a drastic impact on skeletogenesis and craniofacial development, specially affecting pre-maxilla, nasal bone, mandible, tibia, and clavicle. Similarly, Foxo1 activity and expression increase in mouse mesenchymal cells under the influence of osteogenic stimulants. In addition, silencing Foxo1 blocks the expression of osteogenic markers such as Runx2, alkaline phosphatase, and osteocalcin and results in decreased culture calcification even in the presence of strong osteogenic stimulants. Conversely, the expression of these markers increases significantly in response to Foxo1 overexpression. One mechanism through which Foxo1 affects mesenchymal cell differentiation into osteoblasts is through regulation of a key osteogenic transcription factor, Runx2. Indeed, our results show that Foxo1 directly interacts with the promoter of Runx2 and regulates its expression. Using a tibia organ culture model, we confirmed that silencing Foxo1 decreases the expression of Runx2 and impairs bone formation. Furthermore, our data reveals that Runx2 and Foxo1 interact with each other and cooperate in the transcriptional regulation of osteoblast markers. In conclusion, our in vitro, ex vivo, and in vivo results strongly support the notion that Foxo1 is an early molecular regulator in the differentiation of mesenchymal cells into osteoblast.Undifferentiated mesenchymal cells can differentiate into osteoblasts (bone-forming cells), adipocytes (fat cells), chondrocytes (cartilage cells), and myocytes (muscle cells) under the influence of various hormones and growth factors (1). Commitment and differentiation of mesenchymal cells into osteoblasts is crucial during skeletal development and bone growth.Whether mesenchymal cells differentiate along the osteogenic or other pathway depends on the activation of specific transcription factors. The importance of transcription factors in controlling skeletal development can be appreciated in the human skeletal disorder cleidocranial dysplasia. In this condition, deregulation of an important osteogenic transcription factor, Runx2, produces a striking phenotype with anterior fontanelle, hypoplasia or aplasia of the clavicle, wide pubic symphysis, and short stature (2).Although some of the transcription factors that control osteoblast differentiation are well characterized, the role of others remains unclear. One such factor is Foxo1 (forkhead box class O). Foxo1 belongs to the winged helix/forkhead family of transcription factors that is characterized by a 100-amino acid monomeric DNA-binding domain called the FOX domain. Other portions of the forkhead proteins, such as the DNA transactivation or DNA transrepression domains, a...

miR-182 is a negative regulator of osteoblast proliferation, differentiation, and skeletogenesis through targeting FoxO1

“…The expression of FoxO1 was increased during osteoblast differentiation. (30) miR-182, a negative regulator of FoxO1, was also increased along with osteogenesis in the present study and we speculated that it might be a feedback response to prevent further increases in expression of the target gene.…”

“…First, FoxO1 inhibited apoptosis and promoted differentiation of osteoblast lineage cells, in direct contrast to the effects of miR-182 overexpression in osteo-lineage cells. (8,9,30) The expression of Runx2 and Osterix has also been shown to be suppressed in FoxO1 knockout mice. (8,9) Second, miR-182 has been demonstrated to regulate FoxO1 in a breast cancer cell line.…”

“…(8) Moreover, FoxO1 silencing in C3H10T1/2 cells suppressed osteogenic differentiation by repressing the expressions of Runx2 and Osterix, and impaired bone formation drastically with some increase in apoptosis. (30) Although the roles and effects of FoxO1 differ somewhat depending on the stage of differentiation of development, it is clear that FoxO1 is an important positive regulator of bone formation. However, prior to this study, little was known about the regulators of FoxO1 in bone.…”

“…It promotes osteoblast proliferation by maintaining protein synthesis and the redox balance, and also stimulates osteoblast differentiation. (9,30) An osteoblast-specific FoxO1 knockout mice model generated using a1(1) Collagen-Cre showed decreased bone mineral density (BMD), a decreased bone formation rate, and a decrease in viable osteoblast numbers. (9) Another specific FoxO1 mouse model that was generated using Mx-Cre showed increased apoptosis and decreased osteogenic differentiation.…”

Transcriptional Control of Osteogenesis

“…Expression of Runx2 is controlled by Foxo1 which can also directly interact with this transcription factors. Teixeira and coworkers suggested that Foxo1 is an early molecular regulator during mesenchymal cell differentiation into osteoblast exerting its effects through regulation and interaction with Runx2 (Teixeira et al, 2010). Several studies have indicated that Runx2 is a context-dependent transcriptional activator and repressor and may interact with other regulatory proteins, suggesting a complex mechanism of osteoblastogenesis control by this factor.…”