Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction

Abstract

Background/Aims: Osteocytes can sense and respond to extracellular stimuli, including biochemical factors throughout the cell body, dendritic processes, and cilia bending. However, further exploration is required of osteocyte function in response to substrate stiffness, an important passive mechanical cue at the interface between osteocytes and the extracellular matrix, and the deep bio-mechanism in osteocytes involving mechanosensing of cell behavior. Methods: We fabricated silicon-based elastomer polydimethylsiloxane substrates with different stiffnesses but with the same surface topologies. We then seeded osteocytes onto the substrates to examine their responses. Methodologies used included scanning electron microscopy (SEM) for cell morphology, confocal laser scanning microscopy (CLSM) for protein distribution, western blot for protein levels, co-immunoprecipitation for protein interactions, and quantitative real-time polymerase chain reaction for gene expression. Results: SEM images revealed that substrate stiffness induced a change in osteocyte morphology, and CLSM of F-actin staining revealed that substrate stiffness can alter the cytoskeleton. These results were accompanied by changes in focal adhesion capacity in osteocytes, determined via characterization of vinculin expression and distribution. Furthermore, on the exterior of the cell membrane, fibronectin was altered by substrate stiffness. The fibronectin then induced a change in paxillin on the inner membrane of the cell via protein–protein interaction through transmembrane processing. Paxillin led to changes in connexin 43 via protein–protein binding, thereby influencing osteocyte gap junction elongation. Conclusion: This process -from mechanosensing and mechanotransduction to cell function - not only indicates that the effects of mechanical factors on osteocytes can be directly sensed from the cell body, but also indicates the involvement of paxillin transduction.

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“…The death rate for a patient that has co-morbidities and gets infected by COVID-19 are higher than a healthy patient. (7,10, 22,24,26,28,29,31,36,37,38,40,42,44,45)…”

Biomaterial Stiffness Guides Cross-talk between Chondrocytes: Implications for a Novel Cellular Response in Cartilage Tissue Engineering

“…43 Likewise, fibronectin could be regulated by biomaterial stiffnesses in the study of osteocytes, and then the mechanical signal was transmitted through a direct interaction with paxillin, a signal transduction adaptor protein, to regulate osteocyte functions. 10 Similarly, studies into the influences of biomaterial stiffnesses on dental papilla cells (DPCs) identified that the odontogenic differentiation potential of DPCs could be regulated via a fibronectin/paxillin/β-catenin pathway. 44 Based on the previous studies and the observations that biomaterial stiffnesses had a positive correlation to intercellular links among a collective of chondrocytes (Figure 3A,B), to protein levels of fibronectin and type I collagen (Figure 3C,D), we further verified decreased expressions of fibronectin in chondrocytes plated on softer substrates in immunofluorescent images (Figure 3E).…”

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