Deficiency of interleukin-9 inhibits osteogenic potential in mice
Wang Yi, Chen Chichi, Zhou Xichao, Shi Qin
Department of Orthopedics, the First Affiliated Hospital of Soochow University, Institute of Orthopedics of Soochow University, Suzhou 215006, Jiangsu Province, China
Abstract: BACKGROUND: Mouse osteogenic potential is regulated by the JAK-STAT signaling pathway, and interleukin-9 can regulate multiple cellular functions through the JAK-STAT pathway, which has the potential to be a novel cytokine that regulates osteogenic potential.
OBJECTIVE: To investigate the effect of interleukin-9 deficiency on osteogenic potential in mice
METHODS: The femurs collected from 2-month-old wild-type and interleukin-9 knockout mice were subjected to Micro-CT scanning to analyze the changes in bone mass. Then, hematoxylin-eosin staining, Masson staining, and immunohistochemical staining of type I collagen were performed on the slices of the femurs of mice. Bone marrow cells from 2-month-old wild-type and interleukin-9 knockout mice were extracted for colony-forming assay and detection of osteogenic gene expression in bone marrow mesenchymal stem cells. To further verify whether interleukin-9 worked through the JAK-STAT pathway, the expression of STAT3 protein was detected by western blot.
RESULTS AND CONCLUSION: Micro-CT results showed the bone mass of interleukin-9 knockout mice decreased significantly compared with that of wild-type mice. In addition, the bone mineral density, bone volume fraction, trabecular number significantly decreased and trabecular separation markedly escalated in interleukin-9 knockout mice. The findings of hematoxylin-eosin staining were consistent with Micro-CT results. Interleukin-9 knockout mice had lower bone trabecular density. Type I collagen immunohistochemistry staining and Masson staining indicated the number of type I collagen positive osteoblasts was significantly reduced and the capacity of collagen formation was damaged in interleukin-9 knockout mice. The results of colony-forming assay indicated that the mineralization capacity of osteoblast in interleukin-9 knockout mice were significantly lower than that in wild-type mice. Western blot results showed that osteogenesis induction activated STAT3 signaling, and the pSTAT3 level in wild-type mice with osteogenic induction was significantly higher than that in interleukin-9 knockout mice with osteogenic induction. These findings suggest that interleukin-9 regulates osteogenesis through the JAK-STAT3 pathway and interleukin-9 deficiency inhibits osteoblast differentiation and function, which may lead to reduced bone mass in interleukin-9 knockout mice.
Key words: interleukin-9, osteoporosis, mesenchymal stem cell, osteogenic capability, osteogenic induction, osteogenic mineralization