Tamazato Longhi Mariana, Magalhães Magna, Reina Jeffrey, Morais Freitas Vanessa, Cella Nathalie
Departamento de Biologia Celular e do Desenvolvimento, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brazil.
PLoS One. 2016 Jul 22;11(7):e0159856. doi: 10.1371/journal.pone.0159856. eCollection 2016.
Maspin (SerpinB5) is a non-inhibitory serpin (serine protease inhibitor) with very diverse biological activities including regulation of cell adhesion, migration, death, control of gene expression and oxidative stress response. Initially described as a tumor and metastasis suppressor, clinical data brought controversies to the field, as some studies reported no correlation between SerpinB5 expression and prognosis value. These data underscore the importance of understanding SerpinB5 function in a normal physiological context and the molecular mechanism involved. Several SerpinB5 phosphoforms have been detected in different cell lines, but the signaling pathways involved and the biological significance of this post-translational modification in vivo remains to be explored. In this study we investigated SerpinB5 expression, subcellular localization and phosphorylation in different stages of the mouse mammary gland development and the signaling pathway involved. Here we show that SerpinB5 is first detected in late pregnancy, reaches its highest levels in lactation and remains at constant levels during post-lactational regression (involution). Using high resolution isoelectric focusing followed but immunoblot, we found at least 8 different phosphoforms of SerpinB5 during lactation, which decreases steadily at the onset of involution. In order to investigate the signaling pathway involved in SerpinB5 phosphorylation, we took advantage of the non-transformed MCF-10A model system, as we have previously observed SerpinB5 phosphorylation in these cells. We detected basal levels of SerpinB5 phosphorylation in serum- and growth factor-starved cells, which is due to amphiregulin autocrine activity on MCF-10A cells. EGF and TGF alpha, two other EGFR ligands, promote important SerpinB5 phosphorylation. Interestingly, EGF treatment is followed by SerpinB5 nuclear accumulation. Altogether, these data indicate that SerpinB5 expression and phosphorylation are developmentally regulated. In vitro analyses indicate that SerpinB5 phosphorylation is regulated by EGFR ligands, but EGF appears to be the only able to induce SerpinB5 nuclear localization.
乳腺丝氨酸蛋白酶抑制剂（Maspin，即丝氨酸蛋白酶抑制剂B5）是一种非抑制性丝氨酸蛋白酶抑制剂（serpin），具有多种生物学活性，包括调节细胞黏附、迁移、死亡、控制基因表达以及氧化应激反应。最初它被描述为肿瘤和转移抑制因子，但临床数据给该领域带来了争议，因为一些研究报告称丝氨酸蛋白酶抑制剂B5的表达与预后价值之间没有相关性。这些数据凸显了在正常生理背景下理解丝氨酸蛋白酶抑制剂B5功能及其相关分子机制的重要性。在不同细胞系中已检测到几种丝氨酸蛋白酶抑制剂B5的磷酸化形式，但体内涉及的信号通路以及这种翻译后修饰的生物学意义仍有待探索。在本研究中，我们调查了小鼠乳腺发育不同阶段丝氨酸蛋白酶抑制剂B5的表达、亚细胞定位和磷酸化以及相关的信号通路。我们发现丝氨酸蛋白酶抑制剂B5在妊娠后期首次被检测到，在哺乳期达到最高水平，并在哺乳后退化（ involution）期间保持恒定水平。使用高分辨率等电聚焦随后进行免疫印迹分析，我们发现在哺乳期丝氨酸蛋白酶抑制剂B5至少有8种不同的磷酸化形式，在退化开始时其数量稳步减少。为了研究参与丝氨酸蛋白酶抑制剂B5磷酸化的信号通路。我们利用了未转化的MCF - 10A模型系统，因为我们之前在这些细胞中观察到了丝氨酸蛋白酶抑制剂B5的磷酸化。我们在血清和生长因子饥饿的细胞中检测到了丝氨酸蛋白酶抑制剂B5磷酸化的基础水平，这是由于双调蛋白对MCF - 10A细胞的自分泌活性所致。另外两种表皮生长因子受体（EGFR）配体，表皮生长因子（EGF）和转化生长因子α（TGFα），可促进丝氨酸蛋白酶抑制剂B5的重要磷酸化。有趣的是，EGF处理后丝氨酸蛋白酶抑制剂B5会在细胞核中积累。总之，这些数据表明丝氨酸蛋白酶抑制剂B5的表达和磷酸化受到发育调控。体外分析表明丝氨酸蛋白酶抑制剂B5的磷酸化受EGFR配体调控，但EGF似乎是唯一能够诱导丝氨酸蛋白酶抑制剂B5细胞核定位的配体。
