Ethanol dysregulates chondrocyte differentiation via different sources of reactive oxygen species in chondrocyte ATDC5 cells
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Chondrocytes are cells that produce and maintain the cartilage matrix and form the growth plate of long bones. Understanding chondrocyte differentiation under unique circumstances is the key to unlocking the mechanism by which alcohol causes osteoporosis. During differentiation, chondrocytes undergo phases of proliferation, hypertrophy and apoptosis. NADPH oxidases (NOX) enzymes, in particular NOX2 and NOX4-generated reactive oxygen species (ROS) have been found to be vital in chondrocyte differentiation. In contrast, ethanol (EtOH) has been shown to dysregulate bone turnover via NOX2 and NOX4 in osteoblasts and suppress osteoblast differentiation in vitro via mitochondrial ROS. Additionally, it has been found that alcohol dysregulates the growth plate leading to an observed shortness of long bones in vivo. This effect is reversed through co-administration of antioxidant n-acetyl cysteine (NAC). The hypothesis of these studies is that alcohol will dysregulate chondrocyte differentiation but the correct dose and type of antioxidant will mitigate that effect. To test this hypothesis, we administered ethanol (0, 25mM, or 50mM) to chondrogenic ATDC5 cells in the presence of either mitochondrial inhibitors (mitoquinone (MitoQ) and mitoTempo), a NOX-specific inhibitor (GKT), or NAC for 16 days. Starting at day 5, RNA was extracted every two days to determine expression of genes related to chondrocyte differentiation (Col2a1, Col10a1, Acan, Runx2) via rt-qPCR. At 14 days, cells were fixed and stained with alcian blue to determine cartilage formation; at day 16, cells were stained with alizarin red to determine mineral deposition. Preliminary analysis of time-dependent gene expression patterns suggest a suppression of Col2a1 and Acan by EtOH and an overall suppression of Col2a1, Col10a1, and Acan by MitoQ and NAC, where MitoQ and NAC abolished the effect of ethanol on Acan expression. Consistent with these initial results, EtOH suppressed alcian blue staining in control cultures. NAC and MitoQ independently suppressed alcian blue staining and protected against ethanol’s effect. In contrast, EtOH appears to increase chondrocyte mineralization. These initial studies will help identify important sources of ROS in chondrocytes and determine the mechanism of EtOH’s effect to dysregulate growth plate dynamics in bone.
Recommended Citation
Lewis, Jonathan, "Ethanol dysregulates chondrocyte differentiation via different sources of reactive
oxygen species in chondrocyte ATDC5 cells" (2019). Summer Research Internship Program. 20.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/20
Ethanol dysregulates chondrocyte differentiation via different sources of reactive oxygen species in chondrocyte ATDC5 cells
LSU Health Medical Education Building
Chondrocytes are cells that produce and maintain the cartilage matrix and form the growth plate of long bones. Understanding chondrocyte differentiation under unique circumstances is the key to unlocking the mechanism by which alcohol causes osteoporosis. During differentiation, chondrocytes undergo phases of proliferation, hypertrophy and apoptosis. NADPH oxidases (NOX) enzymes, in particular NOX2 and NOX4-generated reactive oxygen species (ROS) have been found to be vital in chondrocyte differentiation. In contrast, ethanol (EtOH) has been shown to dysregulate bone turnover via NOX2 and NOX4 in osteoblasts and suppress osteoblast differentiation in vitro via mitochondrial ROS. Additionally, it has been found that alcohol dysregulates the growth plate leading to an observed shortness of long bones in vivo. This effect is reversed through co-administration of antioxidant n-acetyl cysteine (NAC). The hypothesis of these studies is that alcohol will dysregulate chondrocyte differentiation but the correct dose and type of antioxidant will mitigate that effect. To test this hypothesis, we administered ethanol (0, 25mM, or 50mM) to chondrogenic ATDC5 cells in the presence of either mitochondrial inhibitors (mitoquinone (MitoQ) and mitoTempo), a NOX-specific inhibitor (GKT), or NAC for 16 days. Starting at day 5, RNA was extracted every two days to determine expression of genes related to chondrocyte differentiation (Col2a1, Col10a1, Acan, Runx2) via rt-qPCR. At 14 days, cells were fixed and stained with alcian blue to determine cartilage formation; at day 16, cells were stained with alizarin red to determine mineral deposition. Preliminary analysis of time-dependent gene expression patterns suggest a suppression of Col2a1 and Acan by EtOH and an overall suppression of Col2a1, Col10a1, and Acan by MitoQ and NAC, where MitoQ and NAC abolished the effect of ethanol on Acan expression. Consistent with these initial results, EtOH suppressed alcian blue staining in control cultures. NAC and MitoQ independently suppressed alcian blue staining and protected against ethanol’s effect. In contrast, EtOH appears to increase chondrocyte mineralization. These initial studies will help identify important sources of ROS in chondrocytes and determine the mechanism of EtOH’s effect to dysregulate growth plate dynamics in bone.
Comments
Mentors: Martin Ronis and James Watt