Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling
Smoothened-dependent and -independent pathways in mammalian noncanonical Hedgehog signaling
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DOI：
10.1074/jbc.ra119.007956
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发表时间：
2019-06-21
影响因子：
4.8
通讯作者：
Peppelenbosch, Maikel P.
中科院分区：
文献类型：
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作者：
Faria, Alessandra V. de S.;Akyala, Adamu Ishaku;Peppelenbosch, Maikel P.
Hedgehog蛋白是通过典型途径起作用的关键形态发生剂，该途径涉及首先激活配体与Patched的结合，然后缓解Smoothened受体抑制，导致Gli转录因子的激活。非规范Hedgehog信号传导仍然缺乏特征，但被认为主要依赖于Smoothened。然而，Smoothened抑制剂在对抗Hedgehog信号转导依赖性癌症方面仅取得了部分成功，这表明非经典Smoothened独立途径也具有临床相关性。此外，一些Smoothened依赖性效应（例如神经突投射）不需要转录激活，进一步表明非经典Smoothened依赖性途径的生物学重要性。我们全面表征了刺猬攻击的小鼠WT和Smoothened（-/-）成纤维细胞以及Smoothened激动剂刺激的细胞中的细胞激酶组。基于肽分析的激酶组分析（其中细胞裂解物用于磷酸化特定激酶底物），沿着内吞作用、基于荧光黄的和免疫印迹测定，鉴定了Smoothened依赖性和非依赖性途径的复杂信号网络，其通过Src介导肌动蛋白重组样激酶，激活各种促炎信号级联，并伴随刺激Wnt和Notch信号传导，同时抑制骨形态发生蛋白（BMP）信号传导。非典型的平滑独立信号转导对Hedgehog对细胞生理学的整体影响的贡献似乎比以前设想的要大得多，并且可以解释Hedgehog信号转导对细胞骨架的转录独立影响。Patched依赖性、Smoothened非依赖性、非经典Hedgehog信号传导增加Wnt/Notch信号传导的观察结果为Smoothened拮抗剂在对抗Hedgehog依赖性但Smoothened拮抗剂耐药的癌症中失败提供了可能的解释。我们的研究结果表明，抑制刺猬补丁相互作用可能会导致更有效的治疗相比，传统的平滑导向疗法。
Hedgehog proteins are pivotal morphogens acting through a canonical pathway involving first activation of ligand binding to Patched followed by alleviation of Smoothened receptor inhibition, leading to activation of Gli transcription factors. Noncanonical Hedgehog signaling remains poorly characterized but is thought to be mainly dependent on Smoothened. However, Smoothened inhibitors have yielded only partial success in combating Hedgehog signal transduction-dependent cancer, suggesting that noncanonical Smoothened-independent pathways also are clinically relevant. Moreover, several Smoothened-dependent effects (e.g. neurite projection) do not require transcriptional activation, further suggesting biological importance of noncanonical Smoothened-dependent pathways. We comprehensively characterized the cellular kinome in Hedgehog-challenged murine WT and Smoothened(-/-) fibroblasts as well as Smoothened agonist-stimulated cells. A peptide assay-based kinome analysis (in which cell lysates are used to phosphorylate specific kinase substrates), along with endocytosis, Lucifer Yellow-based, and immunoblotting assays, identified an elaborate signaling network of both Smoothened-dependent and -independent pathways that mediates actin reorganization through Src-like kinases, activates various proinflammatory signaling cascades, and concomitantly stimulates Wnt and Notch signaling while suppressing bone morphogenetic protein (BMP) signaling. The contribution of noncanonical Smoothened-independent signaling to the overall effects of Hedgehog on cellular physiology appears to be much larger than previously envisioned and may explain the transcriptionally independent effects of Hedgehog signaling on cytoskeleton. The observation that Patched-dependent, Smoothened-independent, noncanonical Hedgehog signaling increases Wnt/Notch signaling provides a possible explanation for the failure of Smoothened antagonists in combating Hedgehog-dependent but Smoothened inhibitor-resistant cancer. Our findings suggest that inhibiting Hedgehog-Patched interaction could result in more effective therapies as compared with conventional Smoothened-directed therapies.