Experimental Rheumatology, Department of Rheumatology, Radboud University Medical Center, Nijmegen, 6500 HB, The Netherlands.
Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, 3015 CN, The Netherlands.
Sci Rep. 2017 Feb 27;7:43164. doi: 10.1038/srep43164.
To improve cartilage formation by bone marrow-derived mesenchymal stem cells (BMSCs), the signaling mechanism governing chondrogenic differentiation requires better understanding. We previously showed that the transforming growth factor-β (TGFβ) receptor ALK5 is crucial for chondrogenesis induced by TGFβ. ALK5 phosphorylates SMAD2 and SMAD3 proteins, which then form complexes with SMAD4 to regulate gene transcription. By modulating the expression of SMAD2, SMAD3 and SMAD4 in human BMSCs, we investigated their role in TGFβ-induced chondrogenesis. Activation of TGFβ signaling, represented by SMAD2 phosphorylation, was decreased by SMAD2 knockdown and highly increased by SMAD2 overexpression. Moreover, TGFβ signaling via the alternative SMAD1/5/9 pathway was strongly decreased by SMAD4 knockdown. TGFβ-induced chondrogenesis of human BMSCs was strongly inhibited by SMAD4 knockdown and only mildly inhibited by SMAD2 knockdown. Remarkably, both knockdown and overexpression of SMAD3 blocked chondrogenic differentiation. Chondrogenesis appears to rely on a delicate balance in the amount of SMAD3 and SMAD4 as it was not enhanced by SMAD4 overexpression and was inhibited by SMAD3 overexpression. Furthermore, this study reveals that TGFβ-activated phosphorylation of SMAD2 and SMAD1/5/9 depends on the abundance of SMAD4. Overall, our findings suggest a more dominant role for SMAD3 and SMAD4 than SMAD2 in TGFβ-induced chondrogenesis of human BMSCs.
为了提高骨髓间充质干细胞（BMSCs）的软骨形成能力，需要更好地了解调控软骨分化的信号机制。我们之前已经证明，转化生长因子-β（TGFβ）受体 ALK5 对于 TGFβ 诱导的软骨形成至关重要。ALK5 磷酸化 SMAD2 和 SMAD3 蛋白，然后这些蛋白与 SMAD4 形成复合物，从而调节基因转录。通过调节人 BMSCs 中 SMAD2、SMAD3 和 SMAD4 的表达，我们研究了它们在 TGFβ 诱导的软骨形成中的作用。SMAD2 敲低降低了 TGFβ 信号的激活，代表为 SMAD2 磷酸化，而 SMAD2 过表达则显著增加了 SMAD2 的磷酸化。此外，SMAD4 敲低强烈降低了 TGFβ 信号的替代 SMAD1/5/9 通路。SMAD4 敲低强烈抑制了人 BMSCs 的 TGFβ 诱导的软骨形成，而 SMAD2 敲低仅轻度抑制了软骨形成。值得注意的是，SMAD3 的敲低和过表达均阻断了软骨分化。软骨形成似乎依赖于 SMAD3 和 SMAD4 的数量之间的微妙平衡，因为 SMAD4 的过表达并没有增强软骨形成，而 SMAD3 的过表达则抑制了软骨形成。此外，这项研究揭示了 TGFβ 激活的 SMAD2 和 SMAD1/5/9 的磷酸化依赖于 SMAD4 的丰度。总的来说，我们的研究结果表明，在 TGFβ 诱导的人 BMSCs 软骨形成中，SMAD3 和 SMAD4 的作用比 SMAD2 更为重要。
