Estrogen-driven PDK4-MYH9 phosphorylation promotes mitochondrial fission and osteogenic commitment of mesenchymal stem cells

Postmenopausal osteoporosis (PMOP), driven primarily by estrogen deficiency, represents a major public health challenge. Although estrogen's anti-resorptive effects are well established, the direct osteogenic actions by which it regulates the osteogenic differentiation of mesenchymal stem cells (MSCs) remain poorly understood.

This study aimed to elucidate a novel mechanism by which estrogen regulates osteogenic differentiation in MSCs and to identify key downstream molecular events.

We employed single-cell RNA sequencing to profile the transcriptional landscape of osteoporotic MSCs. The pro-osteogenic effects of estradiol (E2) were validated in ovariectomized mice and primary mouse bone marrow-derived MSCs through in vitro differentiation assays and mitochondrial functional analyses. Downstream targets of estrogen receptor were identified by integrating CUT&Tag-seq with transcriptomic data and validated using conditional knockout mice. Protein interactions and phosphorylation sites were mapped via co-immunoprecipitation, mass spectrometry, site-directed mutagenesis, and mitochondrial morphology assays.

Osteoporotic MSCs displayed concurrent impairments in osteogenic differentiation and mitochondrial function. E2 stimulated mitochondrial fission, enhanced maximal respiration, and induced DRP1 translocation to mitochondria. The combined evidence from our CUT&Tag-seq, RNA-seq, and the additional CUT&Tag-qPCR and ERα knockdown data supports the conclusion that Pdk4 is a direct transcriptional target of ERα. This finding is further corroborated by the compromised osteogenesis observed in conditional Pdk4 knockout mice. Mechanistically, PDK4 catalyzed MYH9 phosphorylation at serine 1943, thereby facilitating DRP1 recruitment to mitochondria and subsequent fission. Both pharmacological inhibition of PDK4 and introduction of the phosphorylation-deficient MYH9 S1943A mutant abolished the E2-induced effects. Notably, Pdk4 overexpression in ovariectomized mice reversed bone loss in the absence of exogenous estrogen.

This study identifies an unrecognized ERα-PDK4-MYH9 signaling axis through which estrogen drives mitochondrial fission to promote osteogenic differentiation of MSCs. These findings expand the current understanding of estrogen's skeletal actions by revealing a pro-osteogenic effect on MSCs, and suggest PDK4 as a potential candidate for therapeutic exploration in PMOP.