Osteoporosis is a significant public health concern that affects an estimated 75 million people, accounting for 55% of the United States population over the age of fifty. The disease is characterized by low bone mass and a structural deterioration of bone tissue, and is associated with an elevated risk of bone fracture with resulting increased mortality rates. Bone is an inherently dynamic tissue and has long been demonstrated to adapt to meet its physical demands. Reduced mechanical loading during disuse contributes to decreased bone mass and the potential for osteoporotic fractures, while increased physical stimulation augments bone mass and structural strength. Thus, the ability of bone cells to sense and respond to mechanical stimuli, referred to as mechanotransduction, is a key component to maintaining healthy bone homeostasis.
Bone remodeling is a highly coordinated balance between the actions of bone-forming osteoblasts and bone-resorbing osteoclasts. These processes are likely regulated by mechanosensitive osteocytes, terminally differentiated bone cells that are embedded within the mineralized bone matrix. Despite broad efforts to identify the intracellular signaling pathways through which osteocytes transduce mechanical signals into biochemical responses, the molecular mechanisms that drive these processes have yet to be fully elucidated. The purpose of this dissertation is to broaden the current understanding of osteocyte mechanosensitivity, specifically by investigating the functional role of integrins, transmembrane cell adhesion proteins, in the transduction of a physical signal into altered cellular activity.
In this dissertation, both in vitro and in vivo techniques are utilized to examine the role of integrin signaling in bone mechanosensitivity. The first study employs in vitro cell culture experiments to probe particular intracellular signaling pathways that are mediated by integrins in response to a mechanical stimulus. In this study, it is shown that inhibiting integrin function by expression of a dominant negative β1 integrin attenuates both the anabolic and catabolic response of osteocyte-like cells to dynamic fluid flow. Cyclooxygenase-2 gene expression and extracellular prostaglandin E2 release are used as anabolic markers of the response of osteocyte-like cells to loading; the ratio of receptor activator of NF-κB ligand mRNA to osteoprotegerin mRNA is used to measure the catabolic response of cultured osteocytes to fluid flow. Interestingly, we found that intracellular calcium release is unaffected by expression of a function-perturbing β1 integrin fragment in an osteocyte-like cell line exposed to fluid flow. In this study, we show that integrin signaling is activated by dynamic fluid flow, leading to an upregulation in markers of bone formation and a downregulation in markers of bone resorption.
In the second study, the physiological relevance of integrin signaling in bone is examined by conducting in vivo ulnar loading experiments in conditional knock-out mice. In this study, we demonstrate that deletion of β1 integrins from cortical osteocytes using a Cre-lox strategy results in a reduction of mechanically-induced bone formation. This study demonstrates for the first time that β1 integrins are required for the anabolic response of bone tissue to physical loading.
Finally, in the third study, we consider a potential mechanism by which integrins may sense loading-induced stimuli at the cellular level. Using immunohistochemical analysis on non-decalcified, non-embedded bone, it is shown that integrins are distributed across the cell body and dendritic processes in embedded osteocytes. These data provide further evidence that, in situ, integrins are properly situated in the cell membrane to sense externally-applied forces and may serve as mechanosensors in bone.
Taken together, the data presented in this dissertation indicate that integrins play a critical role in bone mechanosensitivity. We provide in vitro and in vivo evidence that integrins are involved in the response of osteocytes to physical stimuli. This dissertation may serve as a platform from which future studies may continue to piece together mechanosensitive signaling pathways in bone. Further, these studies suggest that integrins may be a key target for the development of pharmaceutical interventions to treat bone pathologies such as disuse osteoporosis.
