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.

Circulating tumor cells (CTCs) are important markers of metastatic cancer. The isolation and detection of CTCs from peripheral blood provides valuable information for cancer diagnosis and precision medicine. However, cost-efficient targeted separation of CTCs of different origins with clinically significant specificity and efficiency remains a major challenge. In this study, a facile approach was developed to fabricate a thin sheet of hyaluronic acid (HA)-functionalized PLGA nanofibrous membrane and integrate it into a microfluidic chamber. The HA was covalently conjugated onto polyethyleneimine (PEI)-modified electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers. Different techniques were employed to characterize the resulted nanofibers. The results show that the CD44+ carcinoma of various origins (HeLa, KB, A549, and MCF-7 cells) could be selectively captured by the PLGA-PEI-HA nanofibers in the microfluidic platform. Importantly, the PLGA-PEI-HA nanofibrous membrane was more efficient to capture HeLa cancer cells under flowing conditions than in static dishes, and at a really low density (20 cells per mL). Furthermore, with constant media perfusion, the captured HeLa cells could grow on the nanofibrous membrane in the microchip for days without compromised cell viability. This is the first trial of using HA-functionalized electrospun nanofibers in a lab-chip device for cancer cell capture and culture. Compared to conventional CTC capture methods, the integration of inexpensive functional electrospun nanofibers and microfluidic technologies may expand the frontiers of using advanced nanomaterials in portable diagnostic applications.

The ubiquitination and deubiquitination enzymes ensure the stability and proper function of most cellular proteins. Disturbance of either enzyme compromises tissue homeostasis. We recently have identified that the ubiquitin-specific protease 34 (USP34) contributes to bone formation by promoting osteogenic differentiation of mesenchymal stem cells. However, its role in bone resorption, which couples bone formation, remains unknown. Here we show that knockdown of Usp34 promotes osteoclast differentiation of RAW264.7 cells. Conditional knockout of Usp34 in bone marrow-derived macrophages (BMMs) or in osteoclasts leads to elevated osteoclast function and low bone mass. Mechanically, we identify that USP34 restrains NF-κB signaling by deubiquitinating and stabilizing the NF-κB inhibitor alpha (IκBα). Overexpression of IκBα represses osteoclastic hyperfunction of Usp34-deficient RAW264.7 cells. Collectively, our results show that USP34 inhibits osteoclastogenesis by regulating NF-κB signaling.