Abstract
C57BL/6J (B6), but not C3H/HeJ (C3H), mice responded to mechanical loading with an increase in bone formation. A 30-min steady fluid shear of 20 dynes/cm(2) increased [(3)H]thymidine incorporation and alkaline phosphatase activity and up-regulated the expression of early mechanoresponsive genes (integrin beta1 (Igtb1) and cyclooxygenase-2 (Cox-2)) in B6 but not C3H osteoblasts, indicating that the differential mechanosensitivity was intrinsic to osteoblasts. In-house microarray analysis with 5,500 gene fragments revealed that the expression of 669 genes in B6 osteoblasts and 474 genes in C3H osteoblasts was altered 4 h after the fluid shear. Several genes associated with the insulin-like growth factor (IGF)-I, the estrogen receptor (ER), the bone morphogenetic protein (BMP)/transforming growth factor-beta, and Wnt pathways were differentially up-regulated in B6 osteoblasts. In vitro mechanical loading also led to up-regulation of these genes in the bones of B6 but not C3H mice. Pretreatment of B6 osteoblasts with inhibitors of the Wnt pathway (endostatin), the BMP pathway (Noggin), or the ER pathway (ICI182780) blocked the fluid shear-induced proliferation. Inhibition of integrin and Cox-2 activation by echistatin and indomethacin, respectively, each blocked the fluid shear-induced up-regulation of genes associated with these four pathways. In summary, up-regulation of the IGF-I, ER, BMP, and Wnt pathways is involved in mechanotransduction. These four pathways are downstream to the early mechanoresponsive genes, i.e. Igtb1 and Cox-2. In conclusion, differential up-regulation of these anabolic pathways may in part contribute to the good and poor response, respectively, in the B6 and C3H mice to mechanical loading.
Highlights
Mechanical loading is essential for maintenance of skeletal architectural integrity
We postulate that the differential osteogenic response to mechanical stress in B6 and C3H inbred strains of mice is intrinsic to bone cells and that comparative global gene expression profiling studies in osteoblasts derived from this pair of inbred mouse strains in response to fluid shear could provide information concerning potential signaling pathways involved in the mechanical stimulation of bone formation
The objectives of this study were 4-fold and are as follows: 1) to confirm that the differential anabolic response to mechanical loading in B6 and C3H strains of mice is intrinsic to osteoblasts, using an in vitro fluid flow shear stress model as a surrogate of mechanical loading [4]; 2) to perform in-house microarray analyses in isolated B6 and C3H osteoblasts to identify potential signaling pathways that in part contribute to the differential osteogenic response; 3) to confirm that the pathwaysof-interest are essential for fluid shear-induced cell proliferation; and 4) to determine the relationship between the pathways-of-interest and the early mechanoresponsive gene products, such as integrins and cyclooxygenase-2 (Cox-2)
Summary
Mechanical loading is essential for maintenance of skeletal architectural integrity. Loading stimulates bone formation and suppresses bone resorption, leading to an overall increase in bone mass [1], whereas unloading results in an overall decrease in bone mass, because of an inhibition of formation along with an increase in resorption [2]. We postulate that the differential osteogenic response to mechanical stress in B6 and C3H inbred strains of mice is intrinsic to bone cells and that comparative global gene expression profiling studies in osteoblasts derived from this pair of inbred mouse strains in response to fluid shear could provide information concerning potential signaling pathways involved in the mechanical stimulation of bone formation. This would yield important information about the identity of mechanosensitivity genes that determine the good and poor mechanical response in bone formation, respectively, in B6 and C3H mice. The objectives of this study were 4-fold and are as follows: 1) to confirm that the differential anabolic response to mechanical loading in B6 and C3H strains of mice is intrinsic to osteoblasts, using an in vitro fluid flow shear stress model as a surrogate of mechanical loading [4]; 2) to perform in-house microarray analyses in isolated B6 and C3H osteoblasts to identify potential signaling pathways that in part contribute to the differential osteogenic response; 3) to confirm that the pathwaysof-interest are essential for fluid shear-induced cell proliferation; and 4) to determine the relationship between the pathways-of-interest and the early mechanoresponsive gene products, such as integrins and cyclooxygenase-2 (Cox-2)
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