Despite established links of CCN6, or Wnt induced signaling protein-3 (WISP3), with progressive pseudo rheumatoid dysplasia, functional characterization of CCN6 remains incomplete. In light of the documented negative correlation between accumulation of reactive oxygen species (ROS) and CCN6 expression, we investigated whether CCN6 regulates ROS accumulation through its influence on mitochondrial function. We found that CCN6 localizes to mitochondria, and depletion of CCN6 in the chondrocyte cell line C-28/I2 by using siRNA results in altered mitochondrial electron transport and respiration. Enhanced electron transport chain (ETC) activity of CCN6-depleted cells was reflected by increased mitochondrial ROS levels in association with augmented mitochondrial ATP synthesis, mitochondrial membrane potential and Ca 2+. Additionally, CCN6-depleted cells display ROS-dependent PGC1α (also known as PPARGC1A) induction, which correlates with increased mitochondrial mass and volume density, together with altered mitochondrial morphology. Interestingly, transcription factor Nrf2 (also known as NFE2L2) repressed CCN6 expression. Taken together, our results suggest that CCN6 acts as a molecular brake, which is appropriately balanced by Nrf2, in regulating mitochondrial function.

Summary WISP3 (Wnt induced secreted protein 3) is a multi-domain protein of mesenchymal origin. Mutations in several domains of WISP3 cause PPRD (progressive pseudo rheumatoid dysplasia), which is associated with cartilage loss and restricted skeletal development. Despite several studies focusing on the functional characterization of WISP3, the molecular details underlying the course of PPRD remain unresolved. We are interested in analyzing the function of WISP3 in the context of cartilage integrity. The current study demonstrates that WISP3 binds to insulin-like growth factor 1 (IGF1) and inhibits IGF1 secretion. Additionally, WISP3 curbs IGF1-mediated collagen X expression, accumulation of reactive oxygen species (ROS) and alkaline phosphatase activity, all of which are associated with the induction of chondrocyte hypertrophy. Interestingly, both IGF1 and ROS in turn trigger an increase in WISP3 expression. Together, our results are indicative of an operational WISP3-IGF1 regulatory loop whereby WISP3 preserves cartilage integrity by restricting IGF1-mediated hypertrophic changes in chondrocytes, at least partly, upon interaction with IGF1.

Cellular communication network factor 6 (CCN6) mutations are linked with Progressive Pseudo Rheumatoid Dysplasia (PPRD) a debilitating musculoskeletal disorder. The function of CCN6 and the mechanism of PPRD pathogenesis remain unclear. Accordingly, we focused on the functional characterization of CCN6 and CCN6 mutants. Using size exclusion chromatography and native polyacrylamide gel electrophoresis we demonstrated that CCN6 is present as a component of the mitochondrial respiratory complex in human chondrocyte lines. By means of siRNA‐mediated transfection and electron microscopy we showed that moderate reduction in CCN6 expression decreases the RER– mitochondria inter‐membrane distance. Parallel native PAGE, immunoblotting and Complex I activity assays furthermore revealed increase in both mitochondrial distribution of CCN6 and mitochondrial respiratory complex assembly/activity in CCN6 depleted cells. CCN6 mutants resembling those linked with PPRD, which were generated by CRISPR‐Cas9 technology displayed low level of expression of mutant CCN6 protein and inhibited respiratory complex assembly/activity. Electron microscopy and MTT assay of the mutants revealed abnormal mitochondria and poor cell viability. Taken together, our results indicate that CCN6 regulates mitochondrial respiratory complex assembly/activity as part of the mitochondrial respiratory complex by controlling the proximity of RER with the mitochondria, and CCN6 mutations disrupt mitochondrial respiratory complex assembly/activity resulting in mitochondrial defects and poor cell viability.

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