Icariin upregulates methyltransferase-like 14-mediated prolyl 4-hydroxylase beta subunit m6A modification to promote osteogenic differentiation of bone marrow stem cells.

Prolyl 4-hydroxylase beta subunit (P4HB) plays a vital role in bone formation. This study intends to clarify the role of P4HB in the therapeutic effect of Icariin (ICA) on osteoporosis. Herein, in vivo and in vitro models were constructed by performing ovariectomy (OVX) in rats and inducing osteogenic differentiation in bone marrow stem cells (BMSCs), respectively. Hematoxylin and eosin staining and micro-computed tomography analysis were performed to evaluate osteoporosis in OVX rats. Alizarin Red staining, alkaline phosphatase staining, and the ALP activity test were employed to assess osteogenesis. m6A dot blotting and methylated RNA immunoprecipitation were used to determine m6A modification. We found that P4HB was downregulated in bone tissues of patients with osteoporosis and OVX rats. P4HB facilitated osteogenic differentiation of BMSCs. What's more, ICA upregulated P4HB expression, promoted osteogenic differentiation of BMSCs, and alleviated osteoporosis in OVX rats, which were reversed by knocking down P4HB. ICA enhanced the stability and m6A modification of P4HB. METTL14 mediated m6A modification of P4HB mRNA. In addition, METTL14 knockdown overturned the promotive effects of ICA on P4HB m6A level and BMSC osteogenic differentiation. To sum up, ICA elevated the METTL14-mediated m6A modification of P4HB to facilitate BMSC osteogenic differentiation.

Jin Y ，Wu A ，Bian S ，Teng J ... -  《-》

METTL14 Regulates Osteogenesis of Bone Marrow Mesenchymal Stem Cells via Inducing Autophagy Through m6A/IGF2BPs/Beclin-1 Signal Axis.

The development of osteoporosis is often accompanied by autophagy disturbance, which also causes new osteoblast defects from bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are still not fully understood. Methyltransferase-like 14 (METTL14) is the main enzyme for N6-methyladenosine (m6A), the most prevalent internal modification in mammalian mRNAs, and it has been implicated in many bioprocesses. Herein, we demonstrate that METTL14 plays a critical role in autophagy induction and hinders osteoporosis process whose expression is decreased both in human osteoporosis bone tissue and ovariectomy (OVX) mice. In vivo, METTL14+/- knockdown mice exhibit elevated bone loss and impaired autophagy similar to the OVX mice, while overexpression of METTL14 significantly promotes bone formation and inhibits the progression of osteoporosis caused by OVX surgery. In vitro, METTL14 overexpression significantly enhances the osteogenic differentiation ability of BMSCs through regulating the expression of beclin-1 depending on m6A modification and inducing autophagy; the opposite is true with METTL14 silencing. Subsequently, m6A-binding proteins IGF2BP1/2/3 recognize m6A-methylated beclin-1 mRNA and promote its translation via mediating RNA stabilization. Furthermore, METTL14 negatively regulates osteoclast differentiation. Collectively, our study reveals the METTL14/IGF2BPs/beclin-1 signal axis in BMSCs osteogenic differentiation and highlights the critical roles of METTL14-mediated m6A modification in osteoporosis.

He M ，Lei H ，He X ，Liu Y ，Wang A ，Ren Z ，Liu X ，Yan G ，Wang W ，Wang Y ，Li G ，Wang T ，Pu J ，Shen Z ，Wang Y ，Xie J ，Du W ，Yuan Y ，Yang L ... -  《-》

Icariin promotes osteogenic differentiation of bone marrow stromal cells and prevents bone loss in OVX mice via activating autophagy.

Osteoporosis (OP) results from the impaired function of endogenous bone marrow mesenchymal stem cells (BMSCs). Icariin (ICA) has shown potential osteoprotective effects. However, the molecular mechanism for the anabolic action of ICA remains largely unknown. The objective of the present study is to investigate whether ICA prevents bone loss by acting on BMSCs via affecting the level of autophagy after ovariectomy (OVX). The BMSCs were extracted from BALB/c mice treated with ICA, chloroquine (CQ, an autophagy inhibitor) or ICA + CQ. The OVX mice were injected with ICA, CQ, or ICA + CQ for 1 month. We performed Alizarin Red staining and alkaline phosphatase staining to detect osteogenic differentiation of BMSCs. Micro-CT, hematoxylin and eosin staining, Oil Red O staining, and tartrate-resistant acid phosphatase staining were used to assess the bone mass, lipid droplets and osteoclasts in femurs. Autophagy activity in BMSCs from different groups was evaluated by Western blot analysis. The osteogenic differentiation of BMSCs from OVX-induced OP mice was decreased. Treatment with ICA reduced bone loss and formation of osteoclasts and increased osteogenic differentiation of BMSCs in vitro and vivo. In addition, autophagy was enhanced in BMSCs of OVX mice treated with ICA. Our results indicate that ICA prevents OVX-induced bone loss possibly by strengthening the osteogenic differentiation of BMSCs via increasing autophagic activity.

Liang X ，Hou Z ，Xie Y ，Yan F ，Li S ，Zhu X ，Cai L ... -  《-》

Icariin recovers the osteogenic differentiation and bone formation of bone marrow stromal cells from a rat model of estrogen deficiency-induced osteoporosis.

Luo Z ，Liu M ，Sun L ，Rui F ... -  《-》

ALKBH5 regulates etoposide-induced cellular senescence and osteogenic differentiation in osteoporosis through mediating the m(6)A modification of VDAC3.

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis.

Huang Y ，Wang S ，Hu D ，Zhang L ，Shi S ... -  《Scientific Reports》