Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells

Area of Science:

• Biochemistry and Molecular Biology
• Stem Cell Biology
• Bone Biology

Background:

• Oxysterols, cholesterol oxidation products, exhibit biological activity.
• Specific oxysterols possess osteogenic properties, influencing osteoprogenitor cells.
• Molecular mechanisms of oxysterol-induced osteoinduction in embryonic stem cells (ESCs) remain unclear.

Purpose of the Study:

• To investigate the effect of a specific oxysterol combination (22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol, denoted as SS) on ESC osteogenic differentiation.
• To elucidate the alterations in mitochondrial activity during oxysterol-induced osteogenesis in ESCs.
• To explore the involvement of Hedgehog (Hh) and Wnt/β-catenin signaling pathways in this process.

Main Methods:

• Assessed osteogenic differentiation via alkaline phosphatase (ALP) activity, matrix mineralization, and gene/protein expression of osteogenic markers (Runx2, Osterix, Osteocalcin, COLIA, OPN).
• Quantified mitochondrial activity by measuring reactive oxygen species, ATP content, mitochondrial membrane potential, mass, and DNA copy number.
• Analyzed mitochondrial biogenesis factors (PGC-1α, PGC-1β) and Hh/Gli and Wnt/β-catenin signaling pathway components.

Main Results:

• SS treatment significantly enhanced osteoinductive activity in ESCs.
• Mitochondrial activity, including biogenesis and respiratory complex levels, was upregulated during SS-induced osteogenesis.
• SS treatment activated Hh/Gli signaling, which subsequently influenced Wnt/β-catenin pathways, crucial for osteogenesis.

Conclusions:

• Lipid-based oxysterols, specifically the SS combination, promote ESC osteogenic differentiation.
• This process involves the coordinated regulation of mitochondrial activity, Hedgehog/Gli, and Wnt/β-catenin signaling pathways.
• The findings reveal novel molecular mechanisms underlying oxysterol-mediated bone formation.