Abstract
BackgroundOsteoporosis is a common bone disease in elderly population caused by imbalanced bone formation and bone resorption. Mesenchymal stem cells (MSCs) are responsible for maintaining this bone homeostasis. The phenotype of transmembrane 9 superfamily 4 (TM9SF4) knockout mice suggests a relationship between TM9SF4 proteins and bone homeostasis. But the effect of TM9SF4 in osteology has never been reported. In the present study, we investigated the function of TM9SF4 in MSC differentiation commitment, as well as its role in osteoporosis.MethodsPrimary bone marrow MSCs, isolated from TM9SF4 wildtype (TM9SF4+/+) and knockout (TM9SF4−/−) mice, were induced to differentiate into osteoblasts or adipocytes, respectively. The osteogenesis was examined by qRT-PCR detection of osteogenic markers, ALP staining and Alizarin Red S staining. The adipogenesis was tested by qRT-PCR quantification of adipogenic markers and Oil Red O staining. The cytoskeletal organization of MSCs was observed under confocal microscope. The osteoporotic model was induced by ovariectomy in TM9SF4+/+ and TM9SF4−/− mice, followed by Toluidine blue and H&E staining to assess lipid accumulation in trabecular bones, as well as micro-computed tomography scanning and immunohistochemistry staining for bone mass density assessment. The experiments on signaling pathways were conducted using qRT-PCR, Western blot and Alizarin Red S staining.ResultsWe determined the role of TM9SF4 in MSC differentiation and found that TM9SF4−/− MSCs had higher potential to differentiate into osteoblasts and lower capability into adipocytes, without affecting osteoclastogenesis in vitro. In ovariectomy-induced osteoporotic model, TM9SF4−/− mice retained higher bone mass and less lipid accumulation in trabecular bones, indicating an important role of TM9SF4 in the regulation of osteoporosis. Mechanistically, TM9SF4-depleted cells showed elongated actin fibers, which may act through mTORC2/Akt/β-catenin pathway to promote their commitment into osteoblasts. Furthermore, TM9SF4-depleted cells showed higher activity of canonical Wnt pathway, suggesting the participation of Wnt/β-catenin during TM9SF4-regulated osteogenesis.ConclusionsOur study demonstrates TM9SF4 as a novel regulator for MSC lineage commitment. Depletion of TM9SF4 preferentially drives MSCs into osteoblasts instead of adipocytes. Furthermore, TM9SF4−/− mice show delayed bone loss and reduced lipid accumulation during ovariectomy-induced osteoporosis. Our results indicate TM9SF4 as a promising target for the future clinical osteoporotic treatment.
Highlights
Osteoporosis is a skeletal disease characterized by decreased bone density and increased fracture risk
The results clearly showed that genetic knockout of transmembrane 9 superfamily 4 (TM9SF4) elevated the expression levels of most osteogenic makers (Fig. 1A), stimulated the activity of alkaline phosphatase (ALP) (Fig. 1C, D), and increased the calcium deposition as detected by Alizarin Red S staining (Fig. 1E, F) in differentiated osteoblasts
Involvement of actin polymerization in TM9SF4 depletion‐induced osteogenic differentiation Based on the great importance of stress fibers on Mesenchymal stem cells (MSC) lineage commitment [12,13,14,15], as well as the role of TM9SF4 in cytoskeleton remodeling [21], we explored the possible involvement of F-actin polymerization in TM9SF4 depletion-induced osteogenic differentiation
Summary
Osteoporosis is a skeletal disease characterized by decreased bone density and increased fracture risk. As the common progenitor cells of osteoblasts and adipocytes, MSCs are delicately balanced in their differentiation commitment. Their differentiation selection between osteogenesis and adipogenesis is often competing and reciprocal [3]. MSCs exhibit a reduced capacity to differentiate into osteoblasts and an increased capacity into adipocytes, which results in a reduction in bone formation and an increase in marrow fat accumulation [4, 5]. Such a shift of MSC differentiation into adipocytes rather than osteoblasts contributes to osteoporosis [2, 4]. We investigated the function of TM9SF4 in MSC differentiation commitment, as well as its role in osteoporosis
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