METTL14-mediated HOXA5 m6A modification alleviates osteoporosis via promoting WNK1 transcription to suppress NLRP3-dependent macrophage pyroptosis

Background

Osteoporosis is a commonly diagnosed metabolic bone disease. NLRP3 inflammasome activation and pyroptosis are observed during osteoporosis. However, the mechanism by which NLRP3-mediated pyroptosis contributes to osteoporosis remains largely undefined.

Methods

Ovariectomized (OVX) mice were employed as an in vivo model of osteoclastogenesis. H&E staining and micro-CT detected the histological changes and bone parameters in the femur tissues. RANKL-treated macrophages were used as the in vitro model of osteoclastogenesis, and LPS/ATP treatment was used as the macrophage pyroptosis model. The cytotoxicity, cytokine secretion and caspase-1 activity were assessed by LDH release assay, ELISA and flow cytometry, respectively. The osteoclast formation ability was detected by TRAP staining. qRT-PCR, IHC and Western blotting detected the expression and localization of METTL14, pyroptosis-related or osteoclast-specific molecules in femur tissues or macrophages. Mechanistically, MeRIP assessed the m⁶A modification of HOXA5. Luciferase and ChIP assays were employed to detect the direct association between HOXA5 and WNK1 promoter in macrophages.

Results

METTL14, HOXA5 and WNK1 were decreased in OVX mice, which was associated with pyroptosis. METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis, along with the upregulation of WNK1. METTL14-mediated m⁶A modification stabilized HOXA5 mRNA and increased its expression, and HOXA5 regulated WNK1 expression via direct binding to its promoter. Functional studies showed that WNK1 knockdown counteracted METTL14- or HOXA5-suppressed pyroptosis and macrophage-osteoclast differentiation. In OVX mice, overexpression of METTL14 or HOXA5 alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling.

Conclusion

METTL14-mediated HOXA5 m⁶A modification increased its expression, thereby inducing WNK1 expression and suppressing NLRP3-dependent pyroptosis to alleviate osteoporosis. The combination of METTL14 or HOXA5 agonist with pyroptosis targeted therapy may be a promising therapeutic approach for osteoporosis.

The Translational Potential of this Article·

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  METTL14 or HOXA5 overexpression suppressed macrophage-osteoclast differentiation and pyroptosis in macrophages.·

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  METTL14-mediated m6A modification stabilized HOXA5 mRNA and increased its expression.

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  HOXA5 regulated WNK1 expression via direct binding to its promoter.

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  Silencing of WNK1 reversed METTL14- or HOXA5-suppressed pyroptosis and macrophageosteoclast differentiation.·

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  METTL14 or HOXA5 overexpression alleviated osteoporosis via suppressing WNK1-dependent NLRP3 signaling in OVX mice.