The Studies On Function Of Pten And Gab1 In Bone Homeostasis Using Osteoblast-specific Gene Knockout Mice

A rigid skeleton makes it possible to support weight and ensures protection for the organs. Bone strength is determined by bone mass and structure. Bone is a dynamic structure with a continuous tissue renewal called remodeling in which osteoclasts responsible for bone resorption and osteoblasts responsible for bone formation, and the maintenance of bone mass is determined by the coupling of bone resorption to bone formation. Bone remodeling is a danymic process performed by the coordinated activities of osteoclasts, osteoblasts and osteocytes, and tightly controlled by the interaction of these cells. The activation of osteoblasts and osteoclasts is regulated by growth factors, cytokines, hormones and mechanical forces. Further understanding of osteoblastic bone formation is critical for the development of bone anabolic agents and the treatment for osteoporosis.PI3K (phosphoinositide 3 kinase)/AKT signaling regulate a number of biological processes such as proliferation, differentiation, migration and survival. However, the function of PI3K/AKT signaling on osteoblasts and bone homeostasis in vivo is poorly understood. In the current study, we explored the function of Pten and Gab1 genes which are involved in the regulation of PI3K/AKT signaling in osteoblasts and bone metabolism,.Pten (phosphatase and tensin homolog deleted from chromosome 10) is an important tumor suppressor, participating in the regulation of diverse cellular biological process by suppressing PI3K/AKT signaling. In this study, we created osteoblast-specific Pten knockout mice (Ptenflox/flox;OC-Cre) using the Cre–LoxP system. We found that Pten mutant mice exhibited osteopetrosis phynotype. We studied the function of Pten in osteoblasts and bone homeostasis and explored the underlying molecular mechanism by combining radiological, histological, cellular and molecular methods.Osteoblast-specific Pten mutant mice were obtained by breeding OC-Cre transgenic mice created by ourselves previously with Ptenflox/flox mice. Femurs from Pten mutant mice of both sexes displayed significant increases in bone mineral density (BMD) relative to their wild-type controls at 4 month of age. We performed three-dimensional micro computed tomography (μCT) analysis on femurs from 4-month-old mice. Pten mutant femurs showed pronounced increases in trabecular bone volume in longitudinal section and distal femur region. Trabeculae from mutant femurs were increased and became thicker compared with those of controls. Cortical thickness in the midshaft femurs was increased while bone cavity was decreased significantly. Furthermore, we performed bone histomorphometric analyses of the proximal tibias. Von Kossa staining showed a significantly increased bone volume in the mutant mice. The trabecular bone volume/tissue volume ratio (BV/TV) in tibias of Pten deficient mice was increased relative to that of controls at the age of 4 months. In addition, trabecular thickness (Tb.th) and number of osteoblasts/bone perimeter (N.Ob/B.Pm) were significantly increased in Pten mutants. Double calcein labeling revealed a dramatically increased bone formation rate (BFR) and mineral appositional rate (MAR) in 4-month-old mutant mice compared with controls. These results showed that disruption of Pten in osteoblasts leads to increased bone formation. Additionally, primary calvarial osteoblasts culture exhibited increased bone nodules in Pten mutants compared with controls. We also found that interference of Pten in preosteoblasts increased activation of AKT and accelerated osteoblast differentiation. We next analyzed the osteoclasts using tartrate-resistant acid phosphatase (TRAP) staining. Bone histomorphometric analyses revealed that the number of mature osteoclasts (OcN/BPm) was significantly increased in Pten mutant mice. Real-time PCR analyses displayed that the expression of TRAP and cathepsin K (CathK), which are lysosomal enzymes essential for osteoclastic bone resorption, were markedly increased in femoral bones of Pten mutant mice. These results indicated that disruption of Pten in osteoblasts leads to increased bone resorption. However, the expression levels of osteoprotegerin (OPG) and the ligand receptor activator of NF-κB (RANKL) in serum were comparable between control and Pten mutant mice, indicating that increased osteoclast numbers in Pten mutant mice is driven by other regulator but not by RANKL or OPG. Real time PCR showed that Pten muant bone enhanced expression of the monocyte chemoattractants (MCP-2, MCP-5, SDF-1), which may recruit osteoclast precursors to bone and influence osteoclastogenesis.The Grb2-associated binder 1 (Gab1), which serves as a scaffolding adaptor protein, is phosphorylated by diverse receptor tyrosine kinases, and subsequently activate PI3K/AKT and Ras/MAPK (mitogen activated protein kinase) pathways transmitting key signals from cytokine and growth factor. We generated osteoblast-specific Gab1 mutant mice (Gab1flox/flox;OC-Cre) and provided the first evidence that Gab1mutant mice developed osteoporosis. In this study, we showed that the molecular scaffold Gab1 was critical for normal postnatal bone homeostasis verified by combined radiological, histological, cellular and molecular methods. Three-point bending test demonstrated that femurs from Gab1 deficiency mice exhibited decreased biomechanical properties. Bone mineral density (BMD) from femurs of mutant mice is significant decreases relative to that of controls at 2 months and 6.5 months old. Soft x-ray analyses of vertebrae and femora revealed that Gab1 mutant mice exhibited lower bone mineral density than their wild-type littermates at 6.5 months of age.μCT analysis revealed that decreased trabecular bone volume and cortical thickness from Gab1 mutant femurs compared with controls. We also performed analyses about bone formation and bone resorption at 2-month-old proximal tibias. Bone histomorphometric analyses revealed that both bone formation and bone resorption parameters, the trabecular bone volume/tissue volume ratio, the number of osteoblasts/bone perimeter (N.Ob/B.Pm and the percentage of bone surface covered by mature osteoclasts (OcS/BS), were significantly decreased in in Gab1 mutant mice compared with controls. Double calcein labeling revealed a dramatically declined bone formation rate/bone surface (BFR/BS) and mineral appositional rate (MAR) in 2-month-old mutant mice compared with controls. These data showed that Gab1 mutant mice developed low turnover osteoporosis. In vitro primary osteoblasts culture systems showed that Gab1 deficiency in osteoblasts caused three cell autonomous abnormalities: suppressed osteoblast mineralization, increased susceptibility to apoptosis and decreased RANKL expression to support osteoclastogenesis. We also found that interference of Gab1 in preosteoblasts increased susceptibility to apoptosis and decreased osteoblast mineralization. In addition, we found that phosphorylation of AKT and ERK was significantly decreased in mutant osteoblasts under stimulation by IGF-1 and insulin, suggesting that Gab1 has a critical role in mediating insulin and IGF-1 stimulated activation of AKT and ERK in osteoblasts.In conclusion, we revealed the physiological functions of Pten and Gab1 genes, which are able to regulate PI3K/AKT pathway by a negative or positive way, in the maintenance of bone homeostasis. The osteoblast-specific Pten knockout mice which developed osteopetrosis and osteoblast-specific Gab1 knockout mice which exhibited osteoporosis will serve as new animal models for further investigation on molecular mechanisms underlying the maintenance of bone homeostasis.