Abstract
Insulin receptor substrate-1 (IRS-1) is an essential molecule for intracellular signaling of insulin-like growth factor (IGF)-I and insulin, both of which are potent anabolic regulators of bone and cartilage metabolism. To investigate the role of IRS-1 in bone regeneration, fracture was introduced in the tibia, and its healing was compared between wild-type (WT) mice and mice lacking the IRS-1 gene (IRS-1(-/-) mice). Among 15 IRS-1(-/-) mice, 12 remained in a non-union state even at 10 weeks after the operation, whereas all 15 WT mice showed a rigid bone union at 3 weeks. This impairment was because of the suppression of callus formation with a decrease in chondrocyte proliferation and increases in hypertrophic differentiation and apoptosis. Reintroduction of IRS-1 to the IRS-1(-/-) fractured site using an adenovirus vector significantly restored the callus formation. In the culture of chondrocytes isolated from the mouse growth plate, IRS-1(-/-) chondrocytes showed less mitogenic ability and Akt phosphorylation than WT chondrocytes. An Akt inhibitor decreased the IGF-I-stimulated DNA synthesis of chondrocytes more potently in the WT culture than in the IRS-1(-/-) culture. We therefore conclude that IRS-1 deficiency impairs bone healing at least partly by inhibiting chondrocyte proliferation through the phosphatidylinositol 3-kinase/Akt pathway, and we propose that IRS-1 can be a target molecule for bone regenerative medicine.
Highlights
In efforts to develop more advanced skeletal regenerative medicine through genetic manipulation, we have been attempting to identify genes implicated in bone and cartilage formation in vivo
Insulin receptor substrates (IRSs)-1 and IRS-2 are known to be essential for intracellular signaling of Insulin-like growth factor-I (IGF-I)/insulin, these two adaptor molecules have distinct biological roles and are differentially expressed in a variety of cells
Insulin receptor substrate-1 (IRS-1) plays an important role in the metabolic actions of insulin mainly in skeletal muscle and adipose tissue, whereas IRS-2 does so in the liver [29, 30]
Summary
In efforts to develop more advanced skeletal regenerative medicine through genetic manipulation, we have been attempting to identify genes implicated in bone and cartilage formation in vivo. For the former assay, primary chondrocytes from WT or IRS-1Ϫ/Ϫ mice were inoculated at a density of 5 ϫ 104 cells/well in a 24-multiwell plate and cultured to confluency in DMEM, 10% FBS for 2 days.
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