Department of Systems Pharmacology and Translational Therapeutics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
Leeds Institute of Cancer and Pathology and School of Molecular and Cellular Biology, University of Leeds, United Kingdom.
PLoS One. 2018 May 15;13(5):e0197442. doi: 10.1371/journal.pone.0197442. eCollection 2018.
The Hedgehog family of morphogens has long been known to utilize, through the 7-transmembrane protein Smoothened (Smo), the heterotrimeric G protein Gi in both canonical and noncanonical forms of signaling. Other G proteins, while not specifically utilized by Smo, may nonetheless provide access to some of the events controlled by it. We reported several years ago that the G protein G13 activates one or more forms of the Gli family of transcription factors. While the Gli transcription factors are well known targets for Smo, the uncertain mechanism of activation by G13 and the identity of the targeted Gli(s) limited predictions as to the extent to which G13 might mimic Smo's actions. We evaluate here the potential for overlap in G13 and Smo signaling using C3H10T1/2 and 3T3-L1 cells as models of osteogenesis and adipogenesis, respectively. We find in C3H10T1/2 cells that a constitutively active form of Gα13 (Gα13QL) increases Gli1 mRNA, as does a constitutively active form of Smo (SmoA1). We find as well that Gα13QL induces alkaline phosphatase activity, a marker of osteogenesis, albeit the induction is far less substantial than that achieved by SmoA1. In 3T3-L1 cells both Gα13QL and SmoA1 markedly suppress adipogenic differentiation as determined by triglyceride accumulation. RNA sequencing reveals that Gα13QL and SmoA1 regulate many of the same genes but that quantitative and qualitative differences exist. Differences also exist, we find, between SmoA1 and purmorphamine, an agonist for Smo. Therefore, while comparisons of constitutively active proteins are informative, extrapolations to the setting of agonists require care.
刺猬家族的形态发生素有很长一段时间一直被认为是通过 7 跨膜蛋白 Smoothened（Smo）利用异三聚体 G 蛋白 Gi 来进行信号转导，无论是在经典还是非经典信号转导形式中。其他 G 蛋白虽然不是 Smo 特异性利用的，但它们可能为其控制的一些事件提供途径。我们几年前曾报道过，G 蛋白 G13 可以激活一个或多个 Gli 家族转录因子。虽然 Gli 转录因子是 Smo 的众所周知的靶点，但 G13 激活的机制不确定，以及靶向 Gli(s)的身份限制了对 G13 可能在多大程度上模拟 Smo 作用的预测。我们在这里使用 C3H10T1/2 和 3T3-L1 细胞分别作为成骨和成脂的模型，评估了 G13 和 Smo 信号转导之间的重叠的可能性。我们在 C3H10T1/2 细胞中发现，组成型激活的 Gα13（Gα13QL）会增加 Gli1 mRNA，与组成型激活的 Smo（SmoA1）一样。我们还发现，Gα13QL 诱导碱性磷酸酶活性，这是成骨的标志物，尽管诱导程度远低于 SmoA1。在 3T3-L1 细胞中，Gα13QL 和 SmoA1 都显著抑制了脂肪生成分化，这是通过甘油三酯积累来确定的。RNA 测序显示，Gα13QL 和 SmoA1 调节许多相同的基因，但存在数量和质量上的差异。我们发现，SmoA1 和 purmorphamine 之间也存在差异，purmorphamine 是 Smo 的激动剂。因此，虽然比较组成型激活蛋白很有启发性，但对激动剂的推断需要谨慎。
