Macro-molecular complexes in the cell membrane, including caveolae, play a central role in receiving both biochemical and mechanical cues. Through protein-protein interactions with signaling molecules, these structures are poised to control the process of mechanotransduction and convert information from outside the cell into altered cellular function. Caveolae are comprised of caveolin-1 and other proteins to form a subtype of lipid raft that deform the cell membrane to generate flask-shaped pits. These membrane invaginations are dynamic and disassemble to provide strain relief for cells under excessive membrane tension, which releases proteins that were bound to caveolae into the cytosol. Similarly, the binding of matrix fragments to integrins located within caveolae stimulates internalization of the complex. We hypothesize that the demands placed on the caveolae system in chondrocytes under conditions that reflect osteoarthritis (excessive loading requiring membrane tension relief and high levels of matrix degradation products) compromise chondrocyte function. Specifically, chondrocytes will have a reduced capacity to respond to oxidative stress due to the release of caveolae-associated proteins that interfere with antioxidant pathways, and excessive internalization of fibronectin fragments will amplify catabolic signaling. The proposed work tests this hypothesis by modulating the density of caveolae through caveolin-1 plasmid overexpression and CRISPR/Cas9 genetic knockout of caveolin- 1.
In aim 1, we will use an innovative microfluidics approach to apply membrane tension to chondrocytes embedded within a hydrogel to mimic excessive compressive loading. We will apply an oxidative challenge and measure the cytosolic levels of reactive oxygen species, with assessment of peroxiredoxin hyperoxidation as a readout of oxidative stress.
In aim 2, we will apply fibronectin fragments to recapitulate matrix turnover. We will quantify the effect of caveolae density on internalization through imaging of fluorescently-labeled fragment and will use analysis of the MAP kinase pathway to determine the effect on catabolic cell signaling. The proposed work will yield much needed insight into caveolae function in a cell type exposed to frequent loading and matrix turnover demands. This work will also establish a high-throughput microfluidics platform for exposing cells to physiologically relevant strains. Ultimately, successfully completion of this work will catalyze therapeutic approaches that seek to delay the progression of osteoarthritis. Combined with a personalized training plan that prioritizes career development, this training period will enable the applicant to continue on a trajectory towards running an innovative research lab as an independent investigator.
The proposed research is relevant to public health because of the role that caveolae may play in mediating the response of chondrocytes to external stimuli, which can be critical in the development of osteoarthritis. Caveolae can unfurl/disassemble in response to biochemical or mechanical cues by fulfilling roles in endocytosis and membrane protections respectively. Elucidating how the modification of cellular signaling by caveolae impacts the response to cytosolic oxidative challenge is important for understanding the process of chondrocyte dysfunction and may support the development of new therapies for osteoarthritis.
