Department of Pharmaceutical Sciences, Jerry H. Hodge School of Pharmacy, Texas Tech University Health Sciences Center, ARB Suite 2116, 1406 South Coulter Street, Amarillo, TX, 79106, USA.
Stem Cell Rev Rep. 2023 Nov;19(8):2886-2900. doi: 10.1007/s12015-023-10607-0. Epub 2023 Aug 29.
Transplantation of stem cells for treating neurodegenerative disorders is a promising future therapeutic approach. However, the molecular mechanism underlying the neuronal differentiation of dental pulp-derived stem cells (DPSC) remains inadequately explored. The current study aims to define the regulatory role of KLF2 (Kruppel-like factor 2) during the neural differentiation (ND) of DPSC.
We first investigated the transcriptional and translational expression of KLF2, autophagy, and mitophagy-associated markers during the ND of DPSC by using quantitative RT-PCR and western blot methods. After that, we applied the chemical-mediated loss- and gain-of-function approaches using KLF2 inhibitor, GGPP (geranylgeranyl pyrophosphate), and KLF2 activator, GGTI-298 (geranylgeranyl transferase inhibitor-298) to delineate the role of KLF2 during ND of DPSC. The western blot, qRT-PCR, and immunocytochemistry were performed to determine the molecular changes during ND after KLF2 deficiency and KLF2 sufficiency. We also analyzed the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) using the Seahorse XFe24 analyzer.
Our study demonstrated that the expression level of KLF2, autophagy, and mitophagy-associated markers were significantly elevated during the ND of DPSC. Next, we found that the KLF2 inhibitor, GGPP significantly reduced the ND of DPSC. Inversely, KLF2 overexpression accelerated the molecular phenomenon of DPSC's commitment towards ND, indicating the crucial role of KLF2 in neurogenesis. Moreover, we found that the KLF2 positively regulated autophagy, mitophagy, and the Wnt5a signaling pathway during neurogenesis. Seahorse XFe24 analysis revealed that the ECAR and OCR parameters were significantly increased during ND, and inhibition of KLF2 marginally reversed them towards DPSC's cellular bioenergetics. However, KLF2 overexpression shifted the cellular energy metabolism toward the quiescent stage.
Collectively, our findings provide the first evidence that the KLF2 critically regulates the neurogenesis of DPSC by inducing autophagy and mitophagy.
干细胞移植治疗神经退行性疾病是一种很有前途的未来治疗方法。然而，牙髓干细胞（DPSC）向神经元分化的分子机制仍未得到充分探索。本研究旨在确定 KLF2（Kruppel 样因子 2）在 DPSC 神经分化（ND）过程中的调控作用。
我们首先通过定量 RT-PCR 和 Western blot 方法研究了 KLF2、自噬和线粒体自噬相关标志物在 DPSC 向神经元分化过程中的转录和翻译表达。然后，我们应用 KLF2 抑制剂 GGPP（香叶基香叶基焦磷酸）和 KLF2 激活剂 GGTI-298（法尼基转移酶抑制剂-298）的化学介导的基因缺失和过表达方法，来描绘 KLF2 在 DPSC 向神经元分化过程中的作用。在 KLF2 缺乏和 KLF2 充足的情况下进行 Western blot、qRT-PCR 和免疫细胞化学分析，以确定 ND 过程中的分子变化。我们还使用 Seahorse XFe24 分析仪分析了耗氧率（OCR）和细胞外酸化率（ECAR）。
我们的研究表明，KLF2、自噬和线粒体自噬相关标志物的表达水平在 DPSC 的 ND 过程中显著升高。接下来，我们发现 KLF2 抑制剂 GGPP 显著降低了 DPSC 的 ND。相反，KLF2 的过表达加速了 DPSC 向 ND 分化的分子现象，表明 KLF2 在神经发生中的关键作用。此外，我们发现 KLF2 正向调节神经发生过程中的自噬、线粒体自噬和 Wnt5a 信号通路。 Seahorse XFe24 分析显示，ND 过程中 ECAR 和 OCR 参数显著增加，而 KLF2 抑制则使它们向 DPSC 的细胞生物能量学方向略有逆转。然而，KLF2 的过表达将细胞能量代谢向静止期转移。
总之，我们的研究结果首次提供了证据，表明 KLF2 通过诱导自噬和线粒体自噬，对 DPSC 的神经发生起关键调节作用。
