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Wnt5a mediates the effects of Bushen Huoxue decoction on the migration of bone marrow mesenchymal stem cells in vitro.

ABSTRACT: Bushen Huoxue decoction (BHD) has a significant effect on fracture rehabilitation, yet its underlying mechanism is still unknown. The purpose of this study was to explore whether BHD promotes bone marrow mesenchymal stem cell (BMSC) migration through the Wnt5a signalling pathway.BHD was extracted by petroleum, and its composition was analysed. Cell viability in the presence of various concentrations of BHD for 24, 48 and 72 h was measured using a Cell Counting Kit-8 assay. Transwell assays and wound healing assays were used to observe the migration ability of BMSCs. Lentiviral vectors were used to knock down Wnt5a. Polymerase chain reaction and Western blot analyses were used to further compare Wnt5a signalling components at the mRNA and protein levels between groups.BHD treatment groups showed increased migration ability and Wnt5a expression. Knocking down Wnt5a using a lentivirus significantly inhibited the effects of BHD, which implies that BHD promotes BMSC migration ability through activation of Wnt5a.BHD can enhance BMSC migration, possibly by activating Wnt5a signalling.

SUBMITTER: Shen W

PROVIDER: S-EPMC6114862 | biostudies-other | 2018

REPOSITORIES: biostudies-other

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Wnt5a mediates the effects of Bushen Huoxue decoction on the migration of bone marrow mesenchymal stem cells in vitro.

Shen Wei W Luo Hui H Xu Liangliang L Wu Zhifang Z Chen Hongtai H Liu Yamei Y Yu Lijuan L Hu Liuchao L Wang Bin B Luo Yiwen Y

Chinese medicine 20180829

<h4>Background</h4>Bushen Huoxue decoction (BHD) has a significant effect on fracture rehabilitation, yet its underlying mechanism is still unknown. The purpose of this study was to explore whether BHD promotes bone marrow mesenchymal stem cell (BMSC) migration through the Wnt5a signalling pathway.<h4>Methods</h4>BHD was extracted by petroleum, and its composition was analysed. Cell viability in the presence of various concentrations of BHD for 24, 48 and 72 h was measured using a Cell Counting ...[more]

PMID: 30181770

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Project description:Rationale: Bone marrow-derived mesenchymal stem cells (BM-MSCs) recruited into breast tumors regulate the behavior of tumor cells via various mechanisms and affect clinical outcomes. Although signaling molecules, such as transforming growth factor β (TGF-β), are known to transmit signals between BM-MSCs and breast tumor cells for recruiting BM-MSCs, it is unclear which specific intrinsic molecules involved in cell motility mediate the migration of BM-MSCs into breast tumor. It is also unclear as to how specific intrinsic molecules contribute to the migration. Methods: Conditioned medium (CM) from breast tumor cells (MCF-7 and MDA-MB-231) that simulates breast tumor secreting TGF-β was used to examine the migration of BM-MSCs into breast tumors. A three-dimensional migration assay was performed to investigate the collective migration of BM-MSCs, maintaining cell-cell adhesion, toward breast tumor cells. Results: N-cadherin formed adherens junction-like structures on the intercellular borders of BM-MSCs, and TGF-β increased the expression of N-cadherin on these borders. Knockdown of Smad4 impaired the TGF-β-mediated increase in N-cadherin expression in BM-MSCs, but inhibitors of non-canonical TGF-β pathways, such as extracellular signal-regulated kinases, Akt, and p38, did not affect it. siRNA-mediated knockdown of N-cadherin and Smad4 impaired the migration of BM-MSCs in response to TGF-β. Conditioned medium from breast tumor cells also enhanced the expression of N-cadherin in BM-MSCs, but inactivation of TGF-β type 1 receptor (TGFBR1) with SB505124 and TGFBR1 knockdown abolished the increase in N-cadherin expression. BM-MSCs collectively migrated toward CM from MDA-MB-231 in vitro while maintaining cell-cell adhesion through N-cadherin. Knockdown of N-cadherin abolished the migration of BM-MSCs toward the CM from breast tumor cells. Conclusion: In the present study, we identified N-cadherin, an intrinsic transmembrane molecule in adherens junction-like structures, on BM-MSCs as a mediator for the migration of these cells toward breast tumor. The expression of N-cadherin increases on the intercellular borders of BM-MSCs through the TGF-β canonical signaling and they collectively migrate in response to breast tumor cells expressing TGF-β via N-cadherin-dependent cell-cell adhesion. We, herein, introduce a novel promising strategy for controlling and re-engineering the breast tumor microenvironment.

Project description:Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3‑phosphoinositide‑dependent protein kinase‑1 (PDK‑1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK‑1 in the process of mouse osteoblast differentiation in vitro. In the BX‑912 group, BX‑912, a specific inhibitor of PDK‑1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd‑cre‑EGFP) was used to disrupt the PDK‑1 gene in BMSCsPDK1flox/flox; this served as the pHBAd‑cre‑EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)‑PDK1 and p‑AKT were gradually increased during the osteoblast differentiation process. Notably, BX‑912 treatment and disruption of the PDK‑1 gene with pHBAd‑cre‑EGFP effectively reduced the number of alkaline phosphatase (ALP)‑positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK‑1 in the pHBAd‑cre‑EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3‑7. The mRNA expression levels of the osteoblast‑related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX‑912 and pHBAd‑cre‑EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX‑912 and disruption of the PDK‑1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK‑1 in osteoblasts.

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Wnt5a mediates the effects of Bushen Huoxue decoction on the migration of bone marrow mesenchymal stem cells in vitro.

Publications

Wnt5a mediates the effects of Bushen Huoxue decoction on the migration of bone marrow mesenchymal stem cells in vitro.

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