Role And Mechanism Of Bone Marrow Megakaryocyte-derived CXCL12 In Mediating Postmenopausal Osteoporosis

Objective: To determine the role and mechanism of megakaryocyte(MK)-derived chemokine(C-X-C motif)ligand 12(CXCL12)in mediating postmenopausal osteoporosis(PMOP).Methods: Ovariectomy(OVX)was performed to build a mouse model of PMOP.Single-cell RNA-sequencing was performed to analyze the variation and differentially expressed genes of bone marrow cells between OVX and sham operated(SHAM)mice.Flow cytometry was used to verify the increase of MKs and MK-derived CXCL12 in OVX mice.The effect of MK-derived CXCL12 on osteoclastogenesis was measured for RAW264.7 monocyte/macrophage cells.Then,OVX mice were treated with AMD3100,a CXCL12 receptor antagonist,or 17 β-estradiol,analyzed for femoral bone mass and trabecular osteoclasts by μCT and tartrate-resistant acid phosphatase(TRAP)staining,respectively.Hematopoietic stem/progenitor cells were induced for MK differentiation in vitro to observe the effects of estrogen on the proliferation,CXCL12 secretion and expression of activator protein-1(AP-1)transcription factors of MKs.Results:(1)Single-cell RNA-sequencing and flow cytometry revealed a significant increase of bone marrow MK progenitors and mature MKs,secreting a large amount of CXCL12,in the OVX mice.A hematopoietic stem cell mobilization phenotype was identified in the OVX mice resulting in a significant increase of hematopoietic stem/progenitor cells in bone marrow and periphery circulation,and bone marrow monocyte/macrophages.(2)Through CXCL12,MKs promoted the RANKL-induced osteoclastic differentiation of RAW 264.7monocyte/macrophage cell line.(3)AMD3100 prevented the bone loss and decreased osteoclastogenesis of trabecular bone in OVX mice.(4)17 β-estradiol inhibited the MKs differentiation of hematopoietic stem/progenitor cells and decreased the CXCL12 secretion and expression of AP-1 transcription factor Junb of MKs in vitro.Conclusion:(1)The number and CXCL12 secretion of MKs were significantly increased in OVX mice,accompanying with a hematopoietic stem cell mobilization phenotype in the bone marrow.(2)MK-derived CXCL12 enhanced the osteoclastogenesis and thereby mediated the progress of PMOP.(3)Estrogen inhibited the MKs differentiation of hematopoietic stem cells,and decreased the CXCL12 secretion and AP-1transcription factor expression of MKs.