Role Of Leptin For Human Oa Chondrocytes Rhoa/limk1/cofilin Cytoskeletal Regulatory Pathway

Background:Osteoarthritis (OA) is a multifactorial disease that leads to a complete alteration of articular cartilage and other articular tissues such as subchondral bone. Over the last decade, the impact of certain risk factors such as aging and overweight in OA has been actively studied. Since obesity has become a public health problem, various laboratories have set up in vitro models in order to study mechanical stresses involved in the weight-associated cartilage degradative process, as well as the role of mediators involved in obesity. Leptin is a small polypeptide excreted by white adipose tissue, whose circulating concentrations are proportional to the degree of adiposity. In 2003, Dumond et al. found that leptin was strongly overexpressed in human OA cartilage and synovial fluid, which indicates leptin may play a key role in the pathophysiology of OA. However, the mechanism(s) by which leptin affects the cartilage cell in OA patient is not well understood. We were particularly interested in the role of the RhoA/LIMK1/Cofilin pathway because this system seems to play a critical role in leptin-induced cardiomyocyte hypertrophy and vascular smooth muscle hypertrophy, although its role in cartilage tissue has not been studied. Accordingly, in this work, our aims were to (1) to elucidate whether leptin-mediated cartilage cell reorganization occurs via the RhoA/LIMK1/Cofilin pathway, (2) to identify the downstream mediators that participate in this process, and (3) to elucidate accompanying cytoskeletal remodeling. These investigations would provide novel insights into the physical and morphological properties of chondrocytes and how they may interact with the pericellular matrix in healthy and diseased tissue.Methods:pRaichu-1237x vector, encoding YFP-RhoA-PKN-CFP fusion protein, was transfected into normal chondrocytes via Human Chondrocyte NucleofectorTM Kit. Chondrocytes were incubated with lOng/ml leptin and the FRET signal of RhoA was recorded during the incubation. Besides, we also used western blot method to detect the activities of the key proteins in the RhoA/LIMK1/Cofilin pathway and elucidate accompanying cytoskeletal remodeling.Results:During the incubation, we observed a pseudopodium contraction and a significant increase of RhoA activation appeared from 2 min to 30 min post-stimulation with peak values attained 10 min after leptin addition. The results of Western blot showed leptin induced a time-dependent stimulation in both LIMK1 and cofilin-2 phosphorylation with peak values attained 60 min and 24 h after leptin addition, respectively.Fluorescence microscopy showed that the cytoskeletal arrangement in chondrocytes was modified in response leptin treatment. Staining for the cytoskeletal F-actin showed that enhanced F-actin polymerization with the actin fibers localized in the peripheral region of the cellsConclusions:Low-dose leptin can mediate cartilage cell reorganization via the RhoA/LIMK1/Cofilin pathway.