Fine-Tuning of Smad Protein Function by Poly(ADP-Ribose) Polymerases and Poly(ADP-Ribose) Glycohydrolase during Transforming Growth Factor β Signaling

Background
Initiation, amplitude, duration and termination of transforming growth factor β (TGFβ) signaling via Smad proteins is regulated by post-translational modifications, including phosphorylation, ubiquitination and acetylation. We previously reported that ADP-ribosylation of Smads by poly(ADP-ribose) polymerase 1 (PARP-1) negatively influences Smad-mediated transcription. PARP-1 is known to functionally interact with PARP-2 in the nucleus and the enzyme poly(ADP-ribose) glycohydrolase (PARG) can remove poly(ADP-ribose) chains from target proteins. Here we aimed at analyzing possible cooperation between PARP-1, PARP-2 and PARG in regulation of TGFβ signaling.
Methods
A robust cell model of TGFβ signaling, i.e. human HaCaT keratinocytes, was used. Endogenous Smad3 ADP-ribosylation and protein complexes between Smads and PARPs were studied using proximity ligation assays and co-immunoprecipitation assays, which were complemented by in vitro ADP-ribosylation assays using recombinant proteins. Real-time RT-PCR analysis of mRNA levels and promoter-reporter assays provided quantitative analysis of gene expression in response to TGFβ stimulation and after genetic perturbations of PARP-1/-2 and PARG based on RNA interference.
Results
TGFβ signaling rapidly induces nuclear ADP-ribosylation of Smad3 that coincides with a relative enhancement of nuclear complexes of Smads with PARP-1 and PARP-2. Inversely, PARG interacts with Smads and can de-ADP-ribosylate Smad3 in vitro. PARP-1 and PARP-2 also form complexes with each other, and Smads interact and activate auto-ADP-ribosylation of both PARP-1 and PARP-2. PARP-2, similar to PARP-1, negatively regulates specific TGFβ target genes (fibronectin, Smad7) and Smad transcriptional responses, and PARG positively regulates these genes. Accordingly, inhibition of TGFβ-mediated transcription caused by silencing endogenous PARG expression could be relieved after simultaneous depletion of PARP-1.
Conclusion
Nuclear Smad function is negatively regulated by PARP-1 that is assisted by PARP-2 and positively regulated by PARG during the course of TGFβ signaling.

研究背景
转化生长因子β（transforming growth factor β，TGFβ）通过Smad蛋白（Smad proteins）介导的信号通路，其激活强度、持续时长与终止过程受到磷酸化、泛素化及乙酰化等翻译后修饰（post-translational modifications）的调控。我们此前的研究表明，多聚ADP核糖聚合酶1（poly(ADP-ribose) polymerase 1，PARP-1）对Smad蛋白的ADP核糖基化（ADP-ribosylation）修饰会负向调控Smad介导的转录过程。已知PARP-1可在细胞核内与PARP-2发生功能互作，而多聚ADP核糖水解酶（poly(ADP-ribose) glycohydrolase，PARG）可将靶蛋白上的多聚ADP核糖链移除。本研究旨在分析PARP-1、PARP-2与PARG在调控TGFβ信号通路过程中可能存在的协同作用。
实验方法
本研究采用稳定成熟的TGFβ信号通路细胞模型——人HaCaT角质形成细胞。通过邻近连接测定法（proximity ligation assays）与免疫共沉淀实验（co-immunoprecipitation assays）分析内源性Smad3的ADP核糖基化修饰，以及Smad蛋白与PARPs之间的蛋白复合物，并辅以使用重组蛋白的体外ADP核糖基化实验进行验证。通过实时荧光定量逆转录聚合酶链反应（real-time RT-PCR）检测mRNA水平，结合启动子报告基因实验，定量分析TGFβ刺激后，以及通过RNA干扰（RNA interference）对PARP-1、PARP-2及PARG进行基因扰动后的基因表达情况。
实验结果
TGFβ信号通路可快速诱导Smad3的核内ADP核糖基化修饰，该过程伴随Smad蛋白与PARP-1、PARP-2形成的核内复合物相对增多。反之，PARG可与Smad蛋白结合，并在体外对Smad3进行去ADP核糖基化修饰。PARP-1与PARP-2之间也可形成复合物，且Smad蛋白可与二者结合并激活它们的自身ADP核糖基化过程。与PARP-1类似，PARP-2同样可负向调控特定TGFβ靶基因（纤连蛋白（fibronectin）、Smad7（Smad7））的表达以及Smad的转录响应，而PARG则对这些基因起到正向调控作用。据此，沉默内源性PARG表达所导致的TGFβ介导的转录抑制，可通过同时敲除PARP-1得以缓解。
结论
在TGFβ信号传导过程中，核内Smad的功能由PARP-1负向调控（该过程可由PARP-2辅助完成），并由PARG正向调控。