Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.
Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells.
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DOI：
10.1371/journal.pone.0146124
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
van der Kraan PM
中科院分区：
文献类型：
--
作者：
de Kroon LM;Narcisi R;Blaney Davidson EN;Cleary MA;van Beuningen HM;Koevoet WJ;van Osch GJ;van der Kraan PM
骨髓间充质干细胞（BMSC）有望用于软骨再生，因为 BMSC 可以分化为产生软骨组织的软骨细胞。转化生长因子 β (TGFβ) 对于诱导 BMSC 的软骨分化至关重要，并且已知通过激活素受体样激酶 (ALK) 受体 ALK5 和 ALK1 发出信号。由于这两种TGFβ受体在软骨形成中的具体作用尚不清楚，我们研究了ALK5和ALK1是否在BMSC中表达以及这两种受体是否是BMSC的软骨分化所必需的。使用 RT-qPCR 测定人 BMSC 中的 ALK5 和 ALK1 基因表达。为了诱导软骨形成，将人 BMSC 在含有 TGFβ1 的无血清软骨形成培养基中进行颗粒培养。通过聚集蛋白聚糖和 II 型胶原蛋白 RT-qPCR 分析以及蛋白多糖和 II 型胶原蛋白的组织学染色来评估软骨形成。为了过度表达组成型活性 (ca) 受体，用 caALK5 或 caALK1 转导 BMSC。通过用针对 ALK5 或 ALK1 的 shRNA 转导 BMSC，下调 ALK5 和 ALK1 的表达。 ALK5 和 ALK1 在来自 5 个供体的体外扩增以及颗粒培养的 BMSC 中表达，但两种 TGFβ 受体的 mRNA 水平与软骨形成诱导没有明显关联。在成软骨分化的 BMSC 颗粒中，TGFβ 增加了 ALK5 并降低了 ALK1 基因表达。 caALK5 和 caALK1 过表达诱导软骨基质形成的效率均不及 TGFβ 诱导的效率。此外，短发夹介导的 ALK5 或 ALK1 下调导致对 TGFβ 诱导的软骨形成的强烈抑制。 TGFβ 诱导 BMSC 软骨形成分化需要 ALK5 和 ALK1，TGFβ 不仅直接诱导软骨形成，还调节 BMSC 中 ALK5 和 ALK1 受体信号传导。这些结果意味着间充质干细胞优化软骨形成将取决于两种受体的激活。
Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.