Calcium signaling of primary chondrocytes and ATDC5 chondrogenic cells under osmotic stress and mechanical stimulation

Chondrocytes, the sole cell population in articular cartilage, play essential roles in cartilage homeostasis by mediating the biosynthesis, assembly, and organization of the extracellular matrix (ECM) (Griffin and Guilak, 2005). When cartilage is mechanically loaded, chondrocytes are subject to a cascade of physical stimuli, e.g., stress, strain, fluid pressure, osmotic stress, and electric potential. Due to the negatively charged nature of proteoglycans, deformation of the ECM results in an osmotic stress on chondrocytes (Guilak et al., 2014, Mow and Huiskers, 2005). One of the earliest biochemical responses occurring within seconds in chondrocytes upon mechanical or osmotic stimuli is a transient oscillation of intracellular calcium concentration ([Ca2+]i), known as [Ca2+]i responses as revealed by Guilak and colleagues (Chao et al., 2006, Erickson et al., 2003, Guilak et al., 1999, Hung et al., 1997, O'Conor et al., 2014, Pritchard et al., 2008, Zhou et al., 2015). Hydrostatic pressure (Mizuno, 2005), membrane deformation (Donahue et al., 1995), fluid flow-induced shear stress (Yellowley et al., 1997), and electric field (Chao et al., 2000) can all impact [Ca2+]i oscillations in chondrocytes. Although both mechanical loading and osmotic stress are major regulators of chondrocyte activities, few studies have systematically compared their effects on the calcium responses of chondrocytes and chondrogenic cell line.

Increases of cytosol [Ca2+]i usually rely on two sources, extracellular Ca2+ and intracellular calcium stores, such as the endoplasmic reticulum (ER) (Berridge, 2005). Upon physical stimulation, extracellular Ca2+ can flux into cytoplasm through various cation channels or pumps on the plasma membrane, such as ligand-gated ion channels, and mechano- and voltage-sensitive ion channels. For chondrocytes, Guilak and colleagues revealed that TRPV4 and Piezo channels play essential roles in chondrocyte calcium signaling induced by load, stretch, and osmotic stress (Lee et al., 2017, Phan et al., 2009). Ca2+ can also be released from the ER calcium store. Extracellular ATP can trigger the purinergic receptors of the G protein-coupled P2Y receptors that further activate phospholipase C, resulting in the generation of IP3 and intracellular calcium release from IP3-sensitive ER calcium stores (Berridge, 2005). After the elevation of [Ca2+]i levels, this dangerous divalent ion is rapidly retaken into intracellular stores though Ca2+-ATPase, and extruded to the extracellular space through Ca2+ pumps and Na+/Ca2+ exchangers (Clapham, 2007). After the [Ca2+]i recovers to the resting level, a [Ca2+]i peak is completed.

Besides primary chondrocytes from animal or human joints, a chondrogenic cell line, ATDC5, is often used in regenerative and mechanobiology research (Atsumi et al., 1990, McDonough and Price, 2022, Yao and Wang, 2013). ATDC5 cells retain the properties of chondroprogenitor cells that are at an early phase of differentiation. After cultured in differentiation media, ATDC5 cells can reproduce multi-steps of chondrocyte differentiation and become chondrocyte-like cells (Weiss et al., 2012). Limited knowledge is available about the calcium signaling of ATDC5 cells and its comparison with primary chondrocytes. The objectives of this study are 1) to compare the [Ca2+]i responses in chondrocytes triggered by mechanical loading and osmotic stress, 2) to compare the [Ca2+]i responses of ATDC5 cells upon mechanical loading and osmotic stress with those in primary chondrocytes, and 3) to investigate the roles of three types of essential pathways in the [Ca2+]i responses of chondrocytes.

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