Alpha ketoglutarate exerts a pro-osteogenic effect in osteoblast cell lines through activation of JNK and mTOR/S6K1/S6 signaling pathways

• Published in: Toxicology and applied pharmacology 2019-07, Vol.374, p.53-64
• Main Authors: Żurek, Aleksandra, Mizerska-Kowalska, Magdalena, Sławińska-Brych, Adrianna, Kaławaj, Katarzyna, Bojarska-Junak, Agnieszka, Kandefer-Szerszeń, Martyna, Zdzisińska, Barbara
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Alpha ketoglutarate; Animals; C-Jun N-terminal protein kinase; Cell Line; Cell Proliferation - drug effects; Cell Survival - drug effects; Core Binding Factor Alpha 1 Subunit - genetics; Core Binding Factor Alpha 1 Subunit - metabolism; Differentiation; G-protein-coupled receptor 99; Gene Expression Regulation - drug effects; Humans; Ketoglutaric Acids - pharmacology; Mammalian target of rapamycin; Mice; Osteoblasts; Osteoblasts - drug effects; Osteogenesis - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sp7 Transcription Factor - genetics; Sp7 Transcription Factor - metabolism
• ISSN: 0041-008X; 1096-0333
• DOI: 10.1016/j.taap.2019.04.024

Although numerous in vivo studies have suggested that alpha-ketoglutarate (AKG), i.e. the key intermediate in the Krebs cycle, may have an anabolic effect on bone tissue, the direct influence of AKG on osteoblasts and the underlying mechanism of its action have not been investigated so far. The aim of this study was to assess the impact of AKG (disodium salt dihydrate) on osteogenesis in vitro and identification of some signaling mechanisms involved in this activity. The human and mouse normal osteoblast cell lines hFOB 1.19 and MC3T3-E1 were used in this study. The results showed that AKG did not increase the proliferation of osteoblasts; however, it upregulated the expression of transcription factors RUNX2 and Osterix, the mRNA and protein levels of osteoblast differentiation markers (alkaline phosphatase, type I collagen, bone sialoprotein II, osteopontin, osteocalcin), and the mineralization levels in the hFOB 1.19 and MC3T3-E1 cell cultures. Moreover, AKG increased JNK, mTOR, S6K1, and S6 phosphorylation and decreased ERK1/2 phosphorylation in both osteoblast cell lines. The JNK inhibitor and rapamycin, but not the ERK inhibitor, abolished the AKG-promoted osteoblast differentiation. Using immunofluorescence staining, qRT-PCR, and Western blot analysis, we detected the presence of an AKG receptor GPR99 activated by alpha ketoglutaric acid in the tested osteoblast cell lines. However, AKG salt did not activate GPR99. Our findings suggest that AKG salt activates the JNK and mTOR/S6K1/S6 signaling pathways to promote differentiation of osteoblasts, independently of GPR99 activation. We can conclude that AKG salts might be promising candidates for bone anabolic drugs used for prevention or/and treatment of osteoporosis. •AKG promotes differentiation and enhances mineralization in osteoblasts.•AKG increases JNK, mTOR, S6K1, and S6 phosphorylation in osteoblasts.•Functional AKG receptor GPR99 is present on osteoblasts.•AKG promotes osteogenesis independently of GPR99 activation.