Deficiency of Insulin Receptor Substrate-1 Impairs Skeletal Growth Through Early Closure of Epiphyseal Cartilage

Morphological analyses in and around the epiphyseal cartilage of mice deficient in insulin receptor substrate-1 (IRS-1) showed IRS-1 signaling to be important for skeletal growth by preventing early closure of the epiphyseal cartilage and maintaining the subsequent bone turnover at the primary spongiosa. Introduction: IRS-1 is an essential molecule for intracellular signaling by IGF-I and insulin, both of which are potent anabolic regulators of cartilage and bone metabolism. To clarify the role of IRS-1 signaling in the skeletal growth, morphological analyses were performed in and around the epiphyseal cartilage of mice deficient in IRS-1 (IRS-1^-/-), whose limbs and trunk were 20-30% shorter than wildtype (WT) mice. Materials and Methods: The epiphyseal cartilage and the primary spongiosa at proximal tibias of homozygous IRS-1^-/- and WT male littermates were compared using histological, immunohistochemical, enzyme cytohistochemical, ultrastructural, and bone histomorphometrical analyses. Results: In and around the WT epiphyseal cartilage, IRS-1 and insulin-like growth factor (IGF)-I receptors were widely expressed, whereas IRS-2 was weakly localized in bone cells. Chronological observation revealed that height of the proliferative zone and the size of hypertrophic chondrocytes were decreased in WT mice as a function of age, and these decreases were accelerated in the IRS-1^-/- cartilage, whose findings at 12 weeks were similar to those of WT at 24 weeks. In the IRS-1 ^-/- cartilage, proliferating chondrocytes with positive proliferating cell nuclear antigen (PCNA) or parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor immunostaining had almost disappeared by 12 weeks. Contrarily, TUNEL⁺ apoptotic cells were increased in the hypertrophic zone, at the bottom of which most of the chondrocytes were surrounded by the calcified matrix, suggesting the closure of the cartilage. In the primary spongiosa, bone volume, alkaline phosphatase (ALP)⁺ osteoblasts, TRACP⁺ osteoclasts, and the osteopontin-positive cement line were markedly decreased. Bone histomorphometrical parameters for both bone formation and resorption were significantly lower in IRS-1^-/- mice, indicating the suppression of bone turnover. Conclusion: The IRS-1^-/- epiphyseal cartilage exhibited insufficient proliferation of chondrocytes, calcification of hypertrophic chondrocytes, acceleration of apoptosis, and early closure of the growth plate. Thus, the data strongly suggest that IRS-1 signaling is important for the skeletal growth by preventing early closure of the epiphyseal cartilage and by maintaining the subsequent bone turnover at the primary spongiosa.