(W0930-05-25) Osteoclast Differentiation Is Controlled by KLF2 through the Regulation of Mitochondrial Dysfunction

Purpose: There is a great deal of unknown about the role of mitochondrial dysfunction in osteoclast-induced bone disorders such as osteoporosis, osteolysis, osteoarthritis (OA), and rheumatoid arthritis (RA). The function of KLF2 (kruppel-like factor 2 [lung]) in the regulation of mitochondrial repair in the current study has been investigated for the first time by using KLF2 inducer geranylgeranyl transferase inhibitor (GGTI298), following our previous study involving KLF2 and autophagy

Methods: The number, mass, and membrane potentiality of mitochondria were determined by mitotracker staining, mitochondrial ROS and superoxide formation were evaluated by DCFDA and mitosox staining respectively. Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were analyzed by sea horse analysis. Genomic and protein levels were quantified by q-RTPCR and western blot analysis.

Results: The number, mass, and membrane potentiality of mitochondria were found to be augmented in RANKL-induced mouse myeloid (RAW264.7) cells undergoing osteoclastic (OC) differentiation, which is well regulated by KLF2. An increased number of mitochondria in OC results in a higher oxygen consumption rate (OCR) and a lower extracellular acidification rate (ECAR) which are restored by overexpression of KLF2. In addition, OC states exhibit mitochondrial fission and mitophagy, as opposed to KLF2-treated states, which have healthy mitochondria. Finally, we found that the enzymes responsible for autophagy are co-localized with early endosome in macrophages, however, the position of co-localization for autophagic molecules has been switched to the late endosome.

Conclusion: Our study demonstrates for the first time that mitochondrial activity and function, as well as mitophagy, play important roles in OC differentiation, and KLF2 acts as a regulator of these processes. It is expected that identifying the regulatory mechanisms that determine KLF2's regulation of osteoclastic energy metabolism will likely lead to the development of novel therapeutic strategies targeting osteoclastic cells in a wide range of pathological bone diseases.

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Acknowledgements: National Institutes of Health grants, R01AR068279 (NIAMS), STTR R42EY031196 (NEI), and STTR 1R41AG057242 (NIA).


(A) Oxygen Consumption Rate (OCR) measurements were obtained using an extracellular flux analyzer (Seahorse Bioscience). The mitochondrial stress test was used to obtain maximum OCR, ATP-linked OCR, and spare respiratory capacity. (B) Extracellular acidification rate (ECAR) in the non-glycolytic acidification, and the glycolytic capacity conditions during OC differentiation. (C) mtDNA/nDNA was measured by using qPCR to quantitatively by relative genomic expression of Cytochrome C oxidase subunit 2 (Cox1), NADH dehydrogenase subunit 1 (Nox1), and NADH oxidase subunit 1 (Nd1).


OC differentiation reduces KLF2 expression and induces mitochondrial potential. RAW264.7 cells were differentiated into OCs (for 3 and 6 days) using soluble RANKL in presence of GGTI298. OCs (for 3 and 6 days) were produced in RAW264.7 cells with soluble RANKL and GGTI298. Undifferentiated cells were served as control. A) cells were stained with Mitotracker red and mitochondrial mass was determined. B) Cells cultured with RAW263.7 or differentiated cells were treated with GGTI298 and stained with DCF-DA to determine ROS levels using confocal microscopy. C) Using confocal microscopy, mitochondrial superoxide was measured in cells stained with MitoSox Red. D) Cellular calcium level was measured in differentiated OC cells compared with GGTI-298 treatment. (E) OC cells were subjected to glutathione peroxidase assays to assess their antioxidant capacity. All the experiments were done in triplicate. A statistical significance was considered as p < 0.01. F) A greater number of JC1 aggregates (red) were found on confocal imaging in OC during day 6, which decreased following treatment with KLF2.


(A) cells were harvested at various time points and mRNA was extracted to perform qRT-PCR for measuring the expression of fission genes, drp1, and fis1. (B) protein expression of drp1 and fis1 were determined by western blot. (C) Relative gene expression of pink1 and parkin was determined by qRT-PCR. (D) Protein was extracted from harvested cells and a western blot was performed to determine of pink1 and parkin expression.