Abstract
Chondrocytes that are embedded within the growth plate or the intervertebral disc are sensitive to environmental stresses, such as inflammation and hypoxia. However, little is known about the molecular signalling pathways underlining the hypoxia-induced mitochondrial dysfunction and apoptosis in chondrocytes. In the present study, we firstly examined the hypoxia-induced apoptosis, mitochondrial dysfunction and the activation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) signalling in human chondrocyte cell line, C28/I2 and then investigated the regulatory role of RhoA, a well-recognized apoptosis suppressor, in such process, with gain-of-function strategy. Our results indicated that hypoxia induced apoptosis and inhibited CREB phosphprylation in chondrocytes, meanwhile, the dysfunctional mitochondria with up-regulated mitochondrial superoxide and reactive oxygen species (ROS) levels, whereas with a reduced mitochondrial membrane potential (MMP) and Complex IV activity were observed in the hypoxia-treated C28/I2 cells. However, the overexpressed RhoA blocked the hypoxia-mediated reduction in CREB phosphprylation and inhibited the apoptosis induction, along with an ameliorated mitochondrial function in the hypoxia-treated C28/I2 cells. In conclusion, the present study confirmed the reduced CREB phosphorylation, along with the apoptosis induction and mitochondrial dysfunction in the hypoxia-treated chondrocyte cells. And the overexpression of RhoA ameliorated the hypoxia-induced mitochondrial dysfunction and apoptosis via blocking the hypoxia-mediated reduction in CREB phosphorylation.
Highlights
Chondrocytes which are embedded within the growth plate or the intervertebral disc survive in an almost avascular and hypoxic milieu and are sensitive to environmental and multiple cellular stresses, such as inflammation, endoplasmic reticulum (ER) stress and hypoxia [1,2]
In order to investigate the apoptosis induction by hypoxia in chondrocytes, we examined the cellular viability with MTT assay, the apoptosis induction with flow cytometric analysis and the caspase 3 (CASP 3) activity with Caspase SenSolyte kit in the chondrocyte C28/I2 cells under hypoxia or normoxia
cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) has been well recognized to function as a prosurvival signal [18], and our study has investigated the activation of CREB signalling in the hypoxia-treated C28/I2 cells (Figure 1F)
Summary
Chondrocytes which are embedded within the growth plate or the intervertebral disc survive in an almost avascular and hypoxic milieu and are sensitive to environmental and multiple cellular stresses, such as inflammation, endoplasmic reticulum (ER) stress and hypoxia [1,2]. Hypertrophic chondrocytes die through the induction of programmed cell death (apoptosis) in the background of cervical spondylosis or the joint disorder containing osteoarthritis (OA) characterized by progressive breakdown of articular cartilage [3,4]. Reactive oxygen species (ROS) generated from the hypoxia/ischaemia trigger apoptotic cell death [5]. The elevated ROS production causes mitochondrial damage, such as collapse of the mitochondrial membrane potential (MMP), complex IV inactivation, which opens up the mitochondrial permeability transition pores, leading to the release of cytochrome c (Cyt c) into the cytoplasm, to the cleavage of pro-apoptotic proteins [6,7]. The released Cyt c initiates the mitochondria-dependent c 2017 The Author(s).
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