Visualizing Chondrocyte Mechanotransduction in 3D

Abstract

Chondrocytes are the only cell type present in the articular cartilage and their response to mechanical stimuli influences the maintenance and remodeling of the cartilage. Numerous studies have shown that the balance between anabolic and catabolic responses of the chondrocytes to mechanical loading is dependent on the loading intensities (reviewed in ref. [1]). Moderate, physiological loading, for instance, increases synthetic activity of the extracellular matrix (ECM) such as collagen type II, aggrecan, and proteoglycan, while decreasing the catabolic activity of degradative enzymes such as matrix metalloproteinases (MMPs) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) [2,3]. In contrast to moderate loading, static or high-intensity loading has been shown to degrade the cartilage resulting from inhibition of matrix synthesis and up-regulation of catabolic activities [3,4]. Therefore, the importance of these load-dependent signaling pathways involved in the maintenance and remodeling of the cartilage is widely accepted. However, the underlying mechanisms as to how varying magnitudes of mechanical stimuli trigger differential signaling activities that consequently lead to selective gene expression are not clear. FAK and Src are considered to be the main mechanotransduction signaling proteins at the cell-ECM adhesion sites and their activities influence various structural and signaling changes within the cell, including cytoskeletal organization, migration, proliferation, differentiation, and survival [5]. Accumulating evidence has shown that Src and FAK play crucial roles in regulating cartilage maintenance and degeneration and their activation stimulates matrix catabolic genes and activity [6,7]. Rho family GTPases such as RhoA and Rac1 play critical roles in fundamental processes including morphogenesis, polarity, movement, and cell division [8]. They also contribute to cartilage development and degradation [9,10]. Despite these studies, much remains to be elucidated on how load-induced Src and FAK participate in chondrocyte functions, and how their interactions are linked and regulated in connection to the activities of RhoA and Rac1 under different loading conditions. In this study, we use fluorescence resonance energy transfer (FRET)-based biosensors to monitor activities of Src and FAK as well as individual GTPases and evaluate the potential linkage of a network of these signaling molecules under different loading conditions.