Date of Award
Doctor of Philosophy (PhD)
Committee Member 1
Committee Member 2
Committee Member 3
Chondrocytes are a predominant cell type present in articular cartilage, whose integrity is jeopardized in joint degenerative diseases such as osteoarthritis (OA). In the chondrocytes of patients with OA, the elevated levels of inflammatory cytokines such as interleukin 1β (IL1β) and tumor necrosis factor α (TNFα) have been reported. These cytokines contribute to degradation of cartilage matrix by increasing activities of proteolytic enzymes. In addition to their contribution to proteolytic enzymes, these cytokines adversely affect anabolic activity of chondrocytes by inhibiting the production of proteoglycans and type II collagen. Therefore, blocking the action of these cytokines is a potential strategy to prevent cartilage degradation. Accumulating evidence suggests that mechanical loading contributes to the regulation of cartilage homeostasis. However, the underlying mechanisms are not clear as to how varying magnitudes of mechanical loading trigger differential intracellular signaling pathways at sub-cellular levels, which consequently lead to selective matrix synthesis and degradation. Furthermore, it is not known whether the loading-magnitude dependent responses are linked to degenerative diseases such as OA.
Tyrosine kinases such as Src and focal adhesion kinase (FAK) are known to play a crucial role in OA progression. We hypothesized that mechanical loading regulates the sub-cellular activation pattern of Src/FAK, and acts as a suppressor of the OA- or inflammatory cytokine-driven signaling activities. We used live cell imaging approach in conjunction with fluorescence resonance energy transfer (FRET)-based biosensors to investigate real-time molecular events at the sub-cellular level in live chondrocytes. Using two-dimensional (2D) cell culture and shear stress application, we found that Src is activated by inflammatory cytokines (i.e., IL1β and TNFα), and is regulated by shear stress in a magnitude-dependent manner. Importantly, the cytokine-induced Src activation can be suppressed by moderate shear stress (5 dynes /cm 2) and the ER stress inhibitor.
Next, to investigate the sub-cellular activation pattern of Src and FAK in response to inflammatory cytokines and mechanical loading, we used lipid raft-targeting (Lyn-FAK and Lyn-Src) and non-lipid raft-targeting (KRas-FAK and KRas-Src) biosensors. We also developed a three-dimensional (3D) cell culture system using collagen-coupled agarose gels to mimic the physiologically relevant cell microenvironment. The activities of Lyn-Src, KRas-Src and Lyn-FAK were up and down regulated by high (>10 μl/min) and moderate (5 μl/min) interstitial fluid flow, respectively, but KRas-FAK did not respond to the flow. We also found that Src activation by loading was blocked by inhibition of FAK, while inhibition of Src did not affect FAK activities, suggesting that FAK is necessary for interstitial fluid flow-induced Src activity. In contrast, Src was necessary for inflammatory cytokine-induced FAK activation. Furthermore, we developed a 3D ex vivo system that uses murine cartilage explants. This system in conjunction with 3D FRET imaging allowed us to visualize sub-cellular signaling activities of Src and FAK that closely mimic in vivo setting. We found that intermediate loading can inhibit inflammatory cytokine-induced activities of Lyn-Src, KRas-Src and Lyn-FAK, but not KRas-FAK.
AMP-activate kinase (AMPK) is a master regulator of cellular energy balance that activate when the ratio of (AMP+ADP)/ATP increases. Imbalance of AMPK regulation contributes to the development of diabetes, cardiovascular diseases, cancer, and found most recently, OA. In healthy cartilage, the treatment of IL1β and TNFα decreases AMPK activity, while how mechanical loading influences AMPK activities needs to be clarified. The recent design of FRET-based AMPK biosensors that can target various subcellular compartments (PM-AMPK targets plasma membrane, Cyto-AMPK target cytosol, Nuc-AMPK targets nucleus, ER-AMPK targets endoplasmic reticulum (ER), Golgi-AMPK targets Glogi apparatus, and Mito-AMPK targets mitochondria) enable the study about the regulation of AMPK compartmentalization by physical stimuli. The AMPK activities were upregulated by shear stress in 2D environment, while only Nuc- and PM-AMPK are responsive to loading in 3D environment, suggesting culture dimensionality alters the mechanosensitivity of chondrocytes. Moreover, to examine potential factors responsible for discrepancies between different culture models, we evaluated roles of cytoskeleton in mechanotransduction in 2D and 3D cultures and found that the differential cytoskeletal networks contribute to the different signaling in 2D versus 3D models.
Wan, Qiaoqiao, "The role of mechanical loading in chondrocyte signaling pathways" (2016). Open Access Dissertations. 877.