Hyperosmotic stress-induced apoptotic signaling pathways in chondrocytes
Introduction
Articular chondrocytes are constantly exposed to changes in osmotic environment and the ability of cells to adapt to these changes influences survival under both hyposmotic and hyperosmotic conditions [1], [2]. Due to the high concentration of macromolecules, the interstitial osmolarity of cartilage ranges between 350 and 450 mOsm [3], [4]. Load bearing cartilages regularly experience changes in mechanical compression which leads to changes in the osmotic environment and thus, influences metabolic activity, physical properties and complex biochemical responses [3], [5]. Such alterations of osmotic pressure under cyclic and static loading require a well-developed osmoregulatory system within the cartilage [2], [6]. However, when osmotic loading exceeds that occurring under physiological conditions, such as in case of intraarticular fluid accumulation, hemorrhages, prolonged mechanical loading and traumatic injuries, chondrocytes are not able to compensate and undergo degenerative changes [1], [2], [3], [7], [8], [9].
Members of the mitogen-activated protein kinase (MAPK) family regulate cell growth, differentiation and apoptosis. Three subfamilies have been identified in mammalian cells: extracellular signal-regulated kinase-p44/42 MAPK (ERK1/2), p38 MAP kinase (p38) and c-Jun N-terminal kinase (JNK). Although many MAPK-activating stimuli are proapoptotic or antiapoptotic, the biological outcome of MAPK activation is highly divergent and appears to be largely dependent on the cell type [10]. ERK1/2 pathways are usually linked to growth factor action and are associated with cellular differentiation, proliferation and survival [10], but inhibition of chondrocyte proteoglycan synthesis and protein synthesis has also been described. p38 MAPK and JNK1/2 are serine and threonine protein kinases that are activated by various stress stimuli, including UV irradiation, osmotic shock, toxic compounds and proinflammatory cytokines, which may lead to chondrocyte apoptosis and cartilage degradation [11], [12]. Caspase-3 plays an essential role during apoptotic cell death and in doing so it exacerbates disease pathogenesis. Some recent studies have shown that caspase inhibitors can block chondrocyte apoptosis in cultured cells [13], [14].
The intracellular signaling pathways involved in the hyperosmotic stress-induced chondrocyte damage are not yet clear. Therefore, the aim of the present work was to investigate the hyperosmotic stress-induced cell death by means of cell viability and flow cytometry assays and to study the changes in the apoptotic signaling molecules by means of Western blotting.
Section snippets
Cell culture
Chondrocytes were isolated from knees of healthy, 6-month-old female Yorkshire pigs, weighing 20–25 kg and kept under standard diurnal housing conditions, fed ad libitum (Szarsomlyo Kft., Villany, Hungary). The animals were sedated with ketamine (15 mg/kg) and euthanized with intra-cardiac injections of sodium pentobarbital (120 mg/kg) [15]. All procedures were performed in accordance with the ethical guidelines approved by the University of Pécs (No: BA02/2000-31/2001). Whole thickness
Results
Phenotypic characterization of the primary chondrocytes cultured for 72 h in vitro was based on their morphology as well as on RT-PCR analysis of their chondrogenic and dedifferentiation markers (Fig. 1, Fig. 2). As it is shown on Fig. 2a, the cells retained their chondrocyte-like shape and did not show morphologic characteristics suggesting dedifferentiation to fibroblast phenotype. The gene-expression pattern of the primary chondrocyte cultures also suggested the retainment of the chondrocyte
Discussion
In the present study, we showed that hyperosmotic stress leads mainly to apoptotic cell death that involves changes in the apoptotic signaling molecules in a primary chondrocyte cell culture. Hyperosmotic environment induced the activation of proapoptotic signaling factors such as JNK, p38 and caspase-3 and also led to an increased level of ERK1/2. Several pieces of evidence suggest that in vitro culturing and passaging of primary chondrocytes induce dedifferentiation of the cells to a more
Acknowledgments
The present work was supported by Grants OTKA T046589, T048851, F046504, F048908, ETT 596/2003, 439/2006 and the Hungarian Academy of Sciences.
References (46)
- et al.
Adaptive cellular response to osmotic stress in pig articular chondrocytes
Tissue Cell
(1995) - et al.
Differential effects of hyperosmotic challenge on interleukin-1-activated pathways in bovine articular cartilage
Arch. Biochem. Biophys.
(2006) - et al.
The effects of osmotic stress on the viscoelastic and physical properties of articular chondrocytes
Biophys. J.
(2002) - et al.
Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton
Biophys. J.
(2002) - et al.
Short-term exposure of cartilage to blood results in chondrocyte apoptosis
Am. J. Pathol.
(2003) - et al.
Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes
J. Biol. Chem.
(2004) - et al.
Signal transduction pathways regulated by mitogen-activated/extracellular response kinase kinase kinase induce cell death
J. Biol. Chem.
(1996) - et al.
Chondrocyte apoptosis induced by collagen degradation: inhibition by caspase inhibitors and IGF-1
J. Orthop. Res.
(2004) - et al.
Curcumin protects human chondrocytes from IL-11beta-induced inhibition of collagen type II and beta1-integrin expression and activation of caspase-3: an immunomorphological study
Ann. Anat.
(2005) - et al.
Pituitary adenylate cyclase activating polypeptide protects cardiomyocytes against oxidative stress-induced apoptosis
Peptides
(2006)