WNT5A 通过 rho 相关激酶 ROCK 诱导人脂肪干细胞的成骨分化。

WNT5A induces osteogenic differentiation of human adipose stem cells via rho-associated kinase ROCK.

机构信息

Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, Research Institute MOVE, Amsterdam, the Netherlands.

出版信息

Cytotherapy. 2010 Nov;12(7):924-32. doi: 10.3109/14653241003774011.

DOI:10.3109/14653241003774011

PMID:20429785

Abstract

BACKGROUND AIMS

Human (h) adipose tissue-derived mesenchymal stromal cells (ASC) constitute an interesting cellular source for bone tissue engineering applications. Wnts, for example Wnt5a, are probably important regulators of osteogenic differentiation of stem cells, but the role of Wnt5a in hASC lineage commitment and the mechanisms activated upon Wnt5a binding are unknown. We examined whether Wnt5a induces osteogenic and/or adipogenic differentiation of hASC.

METHODS

hASC were incubated for 7 days with or without Wnt5a, rho-associated kinase (ROCK)-activity inhibitor Y27632 or Wnt3a. Cells were lysed for total RNA isolation, DNA content and alkaline phosphatase (ALP) activity. Mineralized nodule formation and gene expression of osteogenic markers osteocalcin and runt-related protein-2 (RUNX2), and adipogenic markers peroxisome proliferator activator receptor-γ (PPARγ) and transcription factor apetala-2 (aP2), were analyzed. hASC were incubated with Wnt5a or Wnt3a to determine activation of canonical and/or non-canonical Wnt signaling pathways, and protein kinase C activity (PKC), total ß-catenin content and gene expression of connexin 43 and cyclin D1 were quantified.

RESULTS

Wnt5a increased ALP activity and RUNX2 and osteocalcin gene expression, and down-regulated adipogenic markers through ROCK activity. Wnt5a also induced mineralized nodule formation. Wnt3a only enhanced RUNX2 and osteocalcin gene expression, and did not induce osteogenic differentiation. Wnt5a activated the non-canonical Wnt signaling pathway by increasing PKC activity, while Wnt3a mildly activated the Wnt canonical pathway by increasing total ß-catenin content and connexin 43 and cyclin D1 gene expression.

CONCLUSIONS

Our data illustrate the importance of Wnt5a as a stimulator of hASC osteogenic differentiation, and show that changes in actin cytoskeleton controlled by ROCK are determinants for Wnt5a-induced osteogenic differentiation of hASC.

摘要

背景目的

人（h）脂肪组织来源的间充质基质细胞（ASC）构成了骨组织工程应用中一种有趣的细胞来源。例如，Wnt5a 可能是干细胞成骨分化的重要调节因子，但 Wnt5a 在 hASC 谱系分化中的作用以及 Wnt5a 结合后激活的机制尚不清楚。我们研究了 Wnt5a 是否诱导 hASC 的成骨和/或成脂分化。

方法

用或不用 Wnt5a、rho 相关激酶（ROCK）活性抑制剂 Y27632 或 Wnt3a 孵育 hASC 7 天。细胞裂解液用于总 RNA 分离、DNA 含量和碱性磷酸酶（ALP）活性分析。矿化结节形成和骨形成标志物骨钙素和 runt 相关蛋白-2（RUNX2）以及脂肪形成标志物过氧化物酶体增殖物激活受体-γ（PPARγ）和转录因子 apetala-2（aP2）的基因表达进行分析。用 Wnt5a 或 Wnt3a 孵育 hASC 以确定经典和/或非经典 Wnt 信号通路的激活以及蛋白激酶 C 活性（PKC）、总 β-连环蛋白含量和连接蛋白 43 和细胞周期蛋白 D1 的基因表达。

结果

Wnt5a 通过 ROCK 活性增加 ALP 活性和 RUNX2 和骨钙素基因表达，并下调脂肪形成标志物。Wnt5a 还诱导矿化结节形成。Wnt3a 仅增强 RUNX2 和骨钙素基因表达，不诱导成骨分化。Wnt5a 通过增加 PKC 活性激活非经典 Wnt 信号通路，而 Wnt3a 通过增加总 β-连环蛋白含量和连接蛋白 43 和细胞周期蛋白 D1 的基因表达轻度激活经典 Wnt 通路。