CCN6 regulates mitochondrial function

Abstract

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.

CCN6 regulates mitochondrial respiratory complex assembly and activity

“…Previously, we demonstrated that CCN6 regulates the production of reactive oxygen species (ROS) and localizes to mitochondria. 20,21 In the current study, we demonstrated using chondrocyte lines that CCN6 controls the assembly and activity of mitochondrial respiratory Complex I, an entity of 45 subunits present also in the form of Super Complexes, and crucial for mitochondrial electron transport. [22][23][24] The level of cell associated CCN6 protein and its mitochondrial distribution are important for the regulation of mitochondrial respiratory complex assembly and activity.…”

“…Such a detailed concept of mitochondrial respiration by CCN6 remained unexplained in our previous study although we observed closer association of mitochondria with the ER in CCN6 depleted cells. 21 Generation of a CCN6 knock out cell line for evaluating how essential CCN6 is for mitochondrial function was difficult. However, we generated cell lines expressing CCN6 C-terminal truncation mutants similar to those linked with PPRD so as to decipher the effect of these mutants on mitochondrial function and integrity.…”

“…C-28/I2 and C20A4 chondrocyte cells were transfected separately with CCN6 siRNA (50 nM) and Control siRNA (50 nM) (Dharmacon, Horizon Discovery) using RNAimax transfection reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) following previously published protocol. 21 In brief, the required amount of each of RNAimax and siRNA was mixed separately with 150 μL of Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) following which the siRNA mix was slowly added to the RNAimax solution and mixed well. Incubation was continued for 20 minutes.…”

“…Immunoblotting was performed following previously published procedures. 9,21 In brief, for each experiment, the protein was transferred to PVDF membrane after separation through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and thereafter blocked for 2 hours with 5% BSA in Tris-buffered saline with 0.1% Tween 20 (TBST). Blots generated were incubated at 4°C overnight with the primary antibodies appropriate for the different experiments executed: anti-CCN6 (custom synthesis targeting residues 201 aa to 372 aa of CCN6, from BioBharati Lifescience, Kolkata, West Bengal, India), anti-β-actin (Santa Cruz, Dallas, TX, USA), anti-OXPHOS cocktail comprising mitochondrial respiratory complex subunits (Abcam, Cambridge, MA, USA), anti-NDUFB8 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), anti-NDUFS1 (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), anti-VDAC1 antibody (Abcam, Cambridge, MA, USA) and anti-COX 4 antibody (Abcam, Cambridge, MA, USA).…”

Regulation and bioactivity of the CCN family of genes and proteins in obesity and diabetes

“…Potential roles of CCNs acutely in regulation of macronutrients of protein, lipids and carbohydrate, and cellular downstream pathways, for example of glucose metabolism, have been minimally examined. Of much intrigue, a recent abstract identified CCN6 as protein that localizes to mitochondria and may act as a regulator mitochondrial electron transport in eukaryotic cells (Patra et al 2017). At the same recent international CCN Workshop, it was reported that in chondrocytes CCN2 and CCN3 may differentially regulate glycolysis in a sub-acute time course (Kubota et al 2017).…”