(M1130-04-22) Mitochondrial Activity and Function are Modulated by KLF2 in Rheumatoid Arthritis

Purpose: In previous studies, Kruppel-like factor 2 (KLF2) has been shown to promote autophagy during osteoclastogenesis. However, it is unclear how KLF2 regulates autophagy during the process that results in bone loss in a variety of bone disorders, such as osteoporosis, osteoarthritis, and rheumatoid arthritis. Our current study found that osteoclastic differentiation was accompanied by an increase in mitochondrial number and mass as well as mitochondrial membrane potential, mediated by the addition of soluble receptor activator of nuclear factor-kappaB ligand (sRANKL) to RAW 264.7 cells. These measures were significantly increased by the addition of GGTI298 to the chemical induction of KLF2.

Methods: Mitosox staining was used to determine mitochondrial ROS and DCFDA staining was used to determine cellular ROS. A sea horse analysis was used to determine the mitochondrial respiration and glycolytic functions. An analysis of western blots, immunocytochemistry, and qRT-PCR was used to detect autophagy and mitophagy markers.

Results: The increased number of mitochondria resulted in an increase in oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR), both of which were almost restored by the overexpression of KLF2. Additionally, scanning electron microscopy and western blot results indicated that osteoclast differentiation stimulated mitochondrial fission by stimulating mitophagy; overexpression of KLF2 reduced this process. In myeloid cells, we found a high level of autophagy molecule expression by immunological co-localization and western blot analysis.

Conclusion: We have demonstrated for the first time that mitochondrial activity and functions are significantly influenced by osteoclastic differentiation and that KLF2 plays a critical role in orchestrating these processes. The identification of the regulatory mechanisms of KLF2 could lead to the development of novel therapeutic strategies aimed at targeting osteoclast cells to treat a wide range of bone-related diseases.

References: Fearon U, Canavan M, Biniecka M, Veale DJ. Hypoxia, mitochondrial dysfunction and synovial invasiveness in rheumatoid arthritis. Nature Reviews Rheumatology. 2016 Jul;12(7):385-97.

Acknowledgements: There is no conflict of interest.

A figure shows that KLF2 as a transcription factor is significantly reduced during diseases such as osteoporosis, Paget's disease, osteosarclastoma, and rheumatoid arthritis. Symptoms of these diseases include fractures or deformities of the bones. As a result, we hypothesize that we may be able to regulate or control these diseases if we can increase the expression of the gene KLF2.
Figure 02 shows that ROS is significantly increased during the prognosis of diseases associated with mitochondrial dysfunction and cellular damage. I would like to highlight the experimental design for testing ROS generation in the following figure. DCFDA can be used to measure cellular ROS and mitosox staining can be used to measure mitochondrial ROS.

ROS and superoxide are significantly increased during osteoclast differentiation at Day 3 and Day 6 conditions which is a representation of in-vitro rheumatoid arthritis. GGTI-298 can be used as a chemical inducer of KLF2 that can be used in the regulation of OC differentiation.

In C57 BL/6 mice induced with K/BxN-serum, cellular, mitochondrial, and reactive oxygen species (ROS) levels increased. GGTI-298 was found to regulate both ROS nicely.