Inhibition of lysosomal function enhances inflammatory gene expression, induce oxidative stress and apoptosis in human chondrocytes

Abstract

Purpose: Lysosomes are the major catabolic organelle of the cells and are essential for the clearance of autophagosomes. Loss of lysosomal function with age has been associated with mitochondrial dysfunction, oxidative stress and apoptosis of the cells. However, the role of lysosomes in chondrocytes homeostasis under pathological conditions has not been explored. Here, we used Chloroquine (CQ) and Bafilomycin A1 (Baf) to inhibit lysosomal function and investigated its consequences on chondrocyte homeostasis and viability in vitro. Methods: Human articular chondrocytes and cartilage explants were prepared from OA knee cartilage obtained from donors undergoing knee arthroplasty at the SUMMA Health Barberton Hospital. Human normal cartilage was obtained from NDRI. The expression of lysosomal associated membrane protein 1 (LAMP1) in normal and OA cartilage was determined by immunohistochemistry. Primary human OA chondrocytes or cartilage explants were maintained in complete DMEM/F12 and treated with CQ or Baf for 24 h or indicated time in DMEM/F12 medium without serum. Induction of apoptosis in chondrocytes treated with CQ or Baf was determined by measuring the activity of LDH released in the culture supernatant, caspase 3/7-luciferase reporter assay and TUNEL staining. The level of IL-1β and IL-6 was determined by TaqMan assays. The level of reactive oxygen species (ROS) was determined by DHR123 staining followed by flow cytometry and MitoSOX Red staining followed by confocal microscopy. The loss of mitochondrial membrane potential was determined by JC-1 dye staining and ATP levels were determined using a kit. The accumulation of dysfunctional mitochondria was determined by staining with MitoTracker Deep Red followed by flow cytometry. The signaling pathway activation was investigated by immunoblot analysis of NFκB and MAPK pathways and its involvement was confirmed by using small molecule inhibitors of the induced pathway. The role of ROS in CQ and Baf induced expression of IL-1β and IL-6 was determined by using anti-oxidants, MitoTempo and Trolox. Results: The expression of LAMP1 was highly downregulated in OA cartilage compared to normal cartilage. CQ and Baf significantly induced apoptosis of primary human OA chondrocytes as determined by LDH activity assay, caspase3/7 luciferase reporter assay and TUNEL staining. Similar results were obtained with mouse chondrocytes. Human OA cartilage explants as well as mouse femoral head cartilage explant culture treated with CQ or Baf showed significant increase in LDH release and TUNEL staining. The CQ and Baf induced inhibition of lysosomal function resulted in the loss of mitochondrial membrane potential, excessive levels of ROS, lower levels of ATP and the accumulation of dysfunctional mitochondria in the treated chondrocytes. Treatment of primary human OA chondrocytes with CQ or Baf significantly increased the gene and protein expression of IL-1β and IL-6. Investigation of the signaling pathways showed no role of NFκB signaling in the CQ and Baf induced upregulation of IL-1β and IL-6 expression. However, the JNK-MAPK, but not ERK- or p38-MAPK pathway, was activated in response to Baf treatment. Inhibition of JNK activation by using a specific inhibitor abrogated the upregulation of IL-1β and IL-6 expression in Baf treated human chondrocytes. The use of anti-oxidants, MitoTempo and Trolox also suppressed the CQ and Baf induced expression of IL-1β and IL-6. JNK inhibition and treatment with MitoTempo or Trolox failed to suppress apoptosis suggesting that this was independent of ROS levels and JNK pathway in chondrocytes with inhibited lysosomal function. Conclusions: We demonstrate here that inhibition of lysosomal function promotes inflammation, oxidative stress and chondrocyte apoptosis through JNK activation. Our data suggest that protecting lysosomal function may be a therapeutic target for OA management.