2021 Theses Doctoral
The osteocyte primary cilium is a mechanoresponsive organelle that regulates cytoskeletal adaptation and coordinates mechanotransduction with adenylyl cyclases.
Osteoporosis and low bone mass are devastating and costly diseases affecting over half the US population over 50-years old. Peak bone mass is reached around age 30 and begins to decline in the following years, leading to a multi-decade disease. While there are several treatment options, including both ant-resorptive agents -- preventing bone loss -- and anabolic agents -- promoting bone formation -- they are inadequate due to either the limited scope of approval or decreased patient compliance from the perceived high risk of complications. Fracture risk increases significantly in osteopenic individuals with vertebral, hip, wrist, and pelvic fractures being among the most common. While there is a significant annual cost of osteoporotic fractures – on the order of $25 billion annually – the increased rate of mortality after osteoporotic fracture is halting. Risk of mortality one year after hip fracture ranges from 20% to 40%. Therefore, there is a clinical and economic need to develop the next-generation of bone saving therapeutics.
Bone has the innate ability to respond to mechanical loading -- forming new bone to accommodate increased mechanical loads, and resorbing bone when there is a period of low mechanical loading or disuse. If we can determine how mechanical loads are detected on the cellular level, we can open up a new avenue for drug development. The osteocyte, a terminally differentiated cell embedded in the bone mineral matrix, is accepted as a key bone mechanotransducer -- detecting mechanical loading and translating it into biochemical signals promoting bone formation. There are several hypothesized models of how the osteocyte detects mechanically loading – fluid flow through the canalicular environment stimulates the osteocyte's dendritic processes; fluid flow results in deformation of the primary cilium within the lacunar cavity; or matrix deformation is directly transduced through integrin attachments at the lacunar wall. The primary cilium is a solitary antenna-like organelle that forms a distinct signaling domain with a unique protein pool, which makes it an attractive therapeutic target.
In this thesis, we seek to unravel the role of the primary cilium in bone mechanotransduction in order to open new avenues for drug development. We examine if the osteocyte primary cilium contributes to load-induced bone formation, determine if adenylyl cyclase 3 (AC3) -- a cAMP catalyzing enzyme that localizes to the primary cilium -- contributes to osteocyte mechanotransduction, and investigate if the primary cilium coordinates actin adaptation in response to mechanical loading. Using a Cre-lox system to knockout Ift88, a gene encoding a critical protein for cilia formation, we find that load-induced bone formation is dependent on whether one or two alleles of Ift88 are present globally, but not if they are only deleted in the osteocyte cell population. We also find that knocking down AC3 mRNA expression leads to an increased response to mechanical stimulation and altered primary cilia length, likely through decreased cAMP production. Finally, we determine that inhibiting primary cilia formation dysregulates actin adaptation to mechanical loading and prevents the actin-dependent mechanoresponse of Taz, a transcriptional co-regulator. This action is likely through alterations in the expression of actomyosin components and in the activation of focal adhesion kinases. Together, this work demonstrates that the primary cilium plays a role in load-induced bone formation, but this effect is not localized to the osteocyte cell population. We also show that adenylyl cyclases play a role in osteocyte mechanobiology, and that whole cell mechanosensitivity may be determined through the primary cilium in its function regulating the actin cytoskeleton.
- Duffy_columbia_0054D_16632.pdf application/pdf 2.19 MB Download File
More About This Work
- Academic Units
- Biomedical Engineering
- Thesis Advisors
- Guo, X. Edward
- Ph.D., Columbia University
- Published Here
- June 14, 2021