N6-methyladenosine (m6A) is the most abundant epigenetic modification in eukaryotic mRNAs and is essential for multiple RNA processing events during mammalian development and disease control. Here we show that conditional knockout of the m6A methyltransferase Mettl3 in bone marrow mesenchymal stem cells (MSCs) induces pathological features of osteoporosis in mice. Mettl3 loss-of-function results in impaired bone formation, incompetent osteogenic differentiation potential and increased marrow adiposity. Moreover, Mettl3 overexpression in MSCs protects the mice from estrogen deficiency-induced osteoporosis. Mechanistically, we identify PTH (parathyroid hormone)/Pth1r (parathyroid hormone receptor-1) signaling axis as an important downstream pathway for m6A regulation in MSCs. Knockout of Mettl3 reduces the translation efficiency of MSCs lineage allocator Pth1r, and disrupts the PTH-induced osteogenic and adipogenic responses in vivo. Our results demonstrate the pathological outcomes of m6A mis-regulation in MSCs and unveil novel epitranscriptomic mechanism in skeletal health and diseases.

The osteogenic differentiation of mesenchymal stem cells (MSCs) is governed by multiple mechanisms. Growing evidence indicates that ubiquitin-dependent protein degradation is critical for the differentiation of MSCs and bone formation; however, the function of ubiquitin-specific proteases, the largest subfamily of deubiquitylases, remains unclear. Here, we identify USP34 as a previously unknown regulator of osteogenesis. The expression of USP34 in human MSCs increases after osteogenic induction while depletion of USP34 inhibits osteogenic differentiation. Conditional knockout of Usp34 from MSCs or pre-osteoblasts leads to low bone mass in mice. Deletion of Usp34 also blunts BMP2-induced responses and impairs bone regeneration. Mechanically, we demonstrate that USP34 stabilizes both Smad1 and RUNX2 and that depletion of Smurf1 restores the osteogenic potential of Usp34-deficient MSCs Taken together, our data indicate that USP34 is required for osteogenic differentiation and bone formation.

Objectives Growth differentiation factor 11 (GDF11), an emerging secreted member of the TGF‐beta superfamily, plays essential roles in development, physiology and multiple diseases; however, its role during adipogenic differentiation and the underlying mechanisms remains poorly understood. Materials and methods Bone marrow‐derived human mesenchymal stem cells (hMSCs) and 3T3‐L1 pre‐adipocytes were induced with adipogenic culture medium supplementing with different concentrations of recombinant GDF11 (rGDF11 0, 10, 50, 100 ng mL −1 ). Oil Red O staining, qRT‐PCR analysis, Western blot analysis and immunofluorescence staining were performed to assay adipogenesis. Results For both hMSCs and 3T3‐L1 pre‐adipocytes, the presence of rGDF11 leads to a dose‐dependent reduction of intracellular lipid droplet accumulation and suppressed adipogenic‐related gene expression. Mechanically, GDF11 inhibits adipogenesis by activating Smad2/3‐dependent TGF‐beta signalling pathway, and these inhibitory effects could be restored by SB‐431542, a pharmacological TGF‐beta type I receptor inhibitor. Conclusions Taken together, our data indicates that GDF11 inhibits adipogenic differentiation in both hMSCs and 3T3‐L1 pre‐adipocytes by activating Smad2/3‐dependent TGF‐beta signalling pathway.

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Growth differentiation factor 11 (GDF11), a secreted member of the transforming growth factor-␤ (TGF-␤) superfamily, has been reported to have the capacity to reverse age-related pathologic changes and regulate organ regeneration after injury; however, the role of GDF11 in fracture healing and bone repair is still unclear. Here, we established a fracture model in 12-week-old male mice to observe two healing states: the cartilaginous callus and bony callus formation phases. Our results showed that recombinant GDF11 (rGDF11) injection inhibits cartilaginous callus maturation and hard callus formation, thereby impairing fracture healing in vivo. In vitro, rGDF11 administration inhibited chondrocyte differentiation and maturation by phosphorylating SMAD2/3 protein and inhibiting RUNX2 expression. Notably, inhibition of TGF-␤ activity by a SMAD-specific inhibitor attenuated GDF11 effects. Thus, our study demonstrates that, rather than acting as a rejuvenating agent, rGDF11 impairs fracture healing by inhibiting chondrocyte differentiation and maturation.