Abstract
Chordin-like 1 (CHRDL1) is a secreted glycoprotein with repeated cysteine-rich domains, which can bind to BMPs family ligands. Although it has been reported to play important roles in several systems, the exact roles of CHRDL1 on human bone mesenchymal stem cells (hBMSCs) osteogenesis remain to be explored. The present study aimed to investigate the roles of CHRDL1 on the osteogenic differentiation of hBMSCs and the underlying molecular mechanisms. We found that CHRDL1 was upregulated during hBMSCs osteogenesis, and rhBMP-4 administration could enhance CHRDL1 mRNA expression in a dose and time dependent manner. Knockdown of CHRDL1 did not affect hBMSCs proliferation, but inhibited the BMP-4-dependent osteogenic differentiation, showing decreased mRNA expression levels of osteogenic markers and reduced mineralization. On the contrary, overexpression of CHRDL1 enhanced BMP-4 induced osteogenic differentiation of hBMSCs. Moreover, in vivo experiments by transplanting CHRDL1 gene modified hBMSCs into nude mice defective femur models displayed higher new bone formation in CHRDL1 overexpression groups, but lower new bone formation in CHRDL1 knockdown groups, compared with control groups. In consistent with the bone formation rate, there were increased CHRDL1 protein expression in new bone formation regions of defective femur in CHRDL1 overexpression groups, while reduced CHRDL1 protein expression in CHRDL1 knockdown groups compared with control groups. These indicate that CHRDL1 can promote osteoblast differentiation in vivo. Furthermore, the mechanisms study showed that CHRDL1 improved BMP-4 induced phosphorylation of SMAD1/5/9 during osteogenic differentiation of hBMSCs. Besides, promotion of osteogenic differentiation and the activation of SMAD phosphorylation by CHRDL1 can be blocked by BMP receptor type I inhibitor LDN-193189. In conclusion, our results suggested that CHRDL1 can promote hBMSCs osteogenic differentiation through enhancing the activation of BMP-4-SMAD1/5/9 pathway.
Highlights
Bone size and shape are precisely modeled and remodeled throughout life to ensure the structure and integrity of the skeleton [1]
To verify the relationship of Chordin-like 1 (CHRDL1) and bone morphogenetic proteins (BMPs)-4 during the osteogenic differentiation induced by them, we investigated the effect of CHRDL1 on downstream of the BMP-4-SMAD signaling pathway events during osteoblastic differentiation 48 h after pLVX-CHRDL1 transduction
To further confirm the functional connection between CHRDL1 and BMP-4, we examined the effect of LDN-193189 on pLVX- CHRDL1 transfection and rhBMP-4 administrated human bone mesenchymal stem cells (hBMSCs) osteogenesis. 24 h after pLVX- CHRDL1 or pLVXvector transfection, LDN193189 (100 nM) in its vehicle DMSO was applied during rhBMP-4 induced osteogenesis. 72 h after transfection, osteoblastic genes, such as COL1A1, ALP, OCN, and OPN mRNA expressions, were detected
Summary
Bone size and shape are precisely modeled and remodeled throughout life to ensure the structure and integrity of the skeleton [1]. Bone remodeling is maintained by the regulation of two essential cell types, namely, the bone resorption osteoclasts and matrix-forming osteoblasts [2]. Osteoporosis develops when the rate of osteoclastic bone resorption exceeds that of osteoblastic bone formation, which leads to loss of BMD and deterioration of bone structure and strength [3]. Bone formation is mediated by osteoblasts recruited from bone mesenchymal cells [5], which can differentiate into cells of other lineages, including myoblasts, chondrocytes, and adipocytes. The fate determination of bone marrow mesenchymal cells and their differentiation toward cells of the osteoblastic lineage is tightly controlled by several early regulators including: Wnt/β-catenin signaling, bone morphogenetic proteins (BMPs), hedgehog proteins, endocrine hormones, epigenetic regulators, and various growth factors. BMPs are known to exhibit high osteogenic activity [6]
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