瘢痕疙瘩中PTEN/PPM1A复合体调控PPM1A磷酸酶活性介导TGF-β1/Smad信号通路的研究

Transforming growth factor β1(TGF-β1)/Smad signaling plays a key role in keloid formation. Our preliminary work found that the decrease of PPM1A expression and phosphatase activity mediate the activation of TGF-β1/Smad signaling. However, the regulatory mechanism of PPM1A in the keloid pathogenesis remains uncertain. Our further work found that the expression of PTEN in keloid is lower than in normal skin using proteomic technology and western blotting. PTEN overexpression upregulated PPM1A expression and increased PPM1A phosphatase activity, as well as decreased the levels of phosphorylated Smad2/3 (P-Smad2/3). PTEN and PPM1A were interacting protein. Therefore, we speculate that PTEN stabilizes PPM1A expression and phosphatase activity by linking PPM1A to form a complex, negatively regulating TGF-β1/Smad signaling by promoting the dephosphorylation of P-Smad2/3. This research will adopt methods of specimen collection, deletion mutantion , FRET and animal experiment to obtain the reliable evidence of PTEN suppressing PPM1A degradation by protein-protein interactions, and demonstrate the mechanism that PTEN/PPM1A complex regulates the expression and phosphatase activity of PPM1A-mediated activation of TGF-β1/Smad signaling in keloid pathogenesis , thus provide clues for development of PTEN supplement strategies for therapy or prophylaxis of keloids.

TGF-β1/Smad与瘢痕疙瘩形成密切相关。我们前期工作发现PPM1A蛋白表达下调，磷酸酶活性降低，使得P-Smad2/3去磷酸化受到抑制，介导了TGF-β1/Smad通路的激活。然而，PPM1A表达下调的机制不清楚。进一步研究发现：运用蛋白质组学技术筛选到PTEN蛋白；PTEN在瘢痕疙瘩中低表达；过表达PTEN，上调了PPM1A蛋白水平及磷酸酶活性，下调了P-Smad2/3水平；PTEN与PPM1A为相互作用蛋白。因此，我们推测：瘢痕疙瘩中，PTEN与PPM1A形成复合体，抑制PPM1A下调，稳定PPM1A磷酸酶活性，通过对P-Smad2/3去磷酸化，实现TGF-β1/Smad信号通路的负性调节。本课题将采用临床标本检测、缺失突变、FRET、动物实验等方法，获得PTEN通过蛋白-蛋白相互作用抑制PPM1A降解的可靠证据，阐明PTEN与PPM1A形成复合体，稳定PPM1A表达及其磷酸酶活性，进而抑制TGF-β1/Smad信号通路持续激活的新机制，为防治瘢痕疙瘩提供新的靶点和理论依据。

瘢痕疙瘩（Keloid）系临床常见的病理性瘢痕，是继发于皮肤损伤的一种胶原过度沉积性疾病，是烧伤、整形外科和康复医学领域的难治性疾病。TGF-β1/Smad信号通路与瘢痕疙瘩形成密切相关。负反馈调节信号分子PPM1A，通过促进磷酸化Smad2/3 (P-Smad2/3)去磷酸化，进而Smad2/3与Smad4分离，Smad2/3出核，实现TGF-β1/Smad信号通路的负性调节。我们前期工作发现PPM1A蛋白表达下调，磷酸酶活性降低，使得P-Smad2/3去磷酸化受到抑制，介导了TGF-β1/Smad通路的激活。然而，PPM1A表达下调的机制不清楚。我们的研究证实：1. 蛋白酪氨酸磷酸酶PTEN在瘢痕疙瘩中表达下调。过表达PTEN，可以上调PPM1A蛋白水平及其磷酸酶活性，下调了P-Smad2/3水平。2. 采用免疫共沉淀、荧光素酶实验等方法，明确PTEN 与 PPM1A 的结合位点在86-134aa序列。3. 明确了PTEN、PPM1A 和 Smad 三者的相互作用关系 。4. 明确PTEN通过稳定PPM1A的表达及其磷酸酶活性，介导PI3K/Akt/mTOR信号通路，从而促进了瘢痕疙瘩的发生发展。我们的研究证实了瘢痕疙瘩中PTEN通过蛋白-蛋白相互作用抑制PPM1A降解的可靠证据，阐明PTEN与PPM1A形成复合体，稳定PPM1A表达及其磷酸酶活性，进而调控TGFβ1/Smad信号通路的新机制。