Investigation of Gpx1 in chondrogenesis and its role on redox regulation in chondrocytes

Orthodontics deals with the correction of skeletal anomalies of the face occurring in the form of jaw discrepancies. There is an abundance of findings in the literature that the development and growth of the cranial base influence facial shape and jaw discrepancies. Cranial base develops by the mechanism of endochondral ossification taking place at its midline axis, where all the synchondroses are located. Chondrogenesis is the initial and indispensable part of endochondral bone formation. In the light of evidence underlying the need of reactive oxygen species in the regulation of chondrogenesis, this study aimed to investigate the ubiquitously present antioxidant enzyme Gpx1 and its contribution to redox regulation in chondrogenesis. Provided that the levels of oxidative stress were previously found to fluctuate according to the differentiation stage of chondrocytes, the gene expression of Gpx1 was measured with quantitative RT-PCR during chondrogenic differentiation. For this purpose, the chondrogenic cell line ATDC5 was utilized and cultured for 21 days. The time points of measurements were on days 0, 2, 6, 10, 14, and 21. The chondrogenic differentiation of the utilized cell line was determined with the stains Alcian blue and Alizarin red, and with the gene expression of chondrogenic biological markers Col2a1 and Col10a1. The present results suggest that the expression of Gpx1 is not of constitutive nature during chondrogenic differentiation. Taking this as a starting point, the next step was to quantitatively assess the distribution pattern of Gpx1 at the different differentiation stages of chondrogenesis. To examine this, the spheno-occipital synchondrosis from eight newborn male Wistar rats was isolated and samples were processed for formaldehyde-fixed paraffin-embedded immunohistochemistry. Photographs of the immunostained sections were analysed by two independent observers and a five-grade semiquantitative scale was used to assess Gpx1 immunoreactivity at the synchondrosis. The present findings show that Gpx1 is expressed the most at the proliferative differentiation stage and the lowest at the hypertrophic differentiation stage. Existing literature reports that an increase in oxidative levels is needed for inhibition of proliferation and initiation of hypertrophy. Further, chondrocytes at the hypertrophic stage have the highest levels of ROS compared to other differentiation stages. In this context, the present results implied that Gpx1 is involved in redox regulation in chondrogenesis. To pursue this further, the expression of Gpx1 was manipulated in ATDC5 chondrogenic cells and cells were then exposed to exogenous H2O2. The manipulation of Gpx1 expression included overexpression and silencing. A control group was also included. The apoptotic percentage of cells was measured flow cytometrically with FITC-labelled Annexin V in conjunction with PI dye. The highest apoptotic percentage was observed in Gpx1-depleted chondrocytes, followed by the control group. The lowest apoptotic percentage was presented in Gpx1-overexpressing cells. These results indicate that Gpx1 possesses an active role on the cellular enzymatic antioxidant system of chondrocytes and can regulate the cellular redox state by H2O2 scavenging. Furthermore, its presence in chondrocytes can prevent H2O2-induced apoptosis. The contribution of cranial base growth to craniofacial morphology continues until adulthood, since spheno-occipital synchondrosis is the last of the synchondroses to ossify and is active until then. This study localizes the expression of Gpx1 at the spheno-occipital synchondrosis and documents the role of Gpx1 as a redox regulator in chondrocytes.