The long-term objective of the proposed project is to determine the effects of mechanical loading on osteoarthritic (OA) cartilage. Integrin-mediated Src/FAK signaling as well as Rho family GTPase such as RhoA is known to play an important role in cartilage maintenance and degradation. Accumulating evidence suggests that in OA cartilage inflammatory cytokines such as interleukin 1? (IL1?) and tumor necrosis factor ? (TNF?) elevate catabolic activities, whereas mechanical loading may act as a beneficial regulator depending on the loading magnitude. Despite previous studies, it remains unclear whether mechanical loading could attenuate cytokine-induced activation of Src/FAK and Rho GTPases that is linked to cartilage degradation. Furthermore, little has been known about spatiotemporal dynamics of intracellular signaling of Src/FAK and GTPases at the sub-cellular level in response to cytokines and interstitial fluid flow. We address a question whether normal and OA chondrocytes have differential baseline activities of Src, FAK, and Rho family GTPases. We hypothesize that interstitial fluid flow generates spatiotemporal patterns of these signaling molecules distinctively different in normal and OA/cytokine-treated chondrocytes, and it suppresses cytokine-induced and/or OA-induced signaling activities. To test our hypotheses, we propose three specific aims using normal chondrocytes, cytokine-treated chondrocytes, and chondrocytes isolated from OA patients under varying magnitudes of interstitial fluid flow. " Aim 1: Determine the spatiotemporal patterns of activities of Src and FAK " Aim 2: Determine the spatiotemporal patterns of activities of Rho family GTPases (RhoA, Rac1, etc.)" Aim 3: Examine interactions of Src and FAK to Rho family GTPases In this proposal, fluorescence resonance energy transfer (FRET)-based biosensors together with a three- dimensional (3D) culture model will allow us to visualize the activities of signaling molecules at the sub- cellular level and evaluate the responses to fluid flow in the presence and absence of inflammatory cytokines. We expect that this project will advance our understanding of the molecular mechanism of mechanotransduction involved in cartilage degradation, and it will contribute to treatment of degenerative joint diseases such as osteoarthritis.
Chondrocytes, the only cell type present in the cartilage, are subjected to complex mechanical stimuli during common daily activities such as cell and matrix deformation and interstitial fluid flow. These individual mechanical factors play different, but crucial roles in regulating chondrocytes, and subsequently degenerative disease such as osteoarthritis. Using fluorescence resonance energy transfer (FRET)-based biosensors together with a novel three-dimensional cell culture model, this proposal will visualize molecular activitie of living chondrocytes at a sub-cellular level under dynamic interstitial fluid flow.
|Wan, Qiaoqiao; Xu, Wenxiao; Yan, Jing-long et al. (2014) Distinctive subcellular inhibition of cytokine-induced SRC by salubrinal and fluid flow. PLoS One 9:e105699|