Miro Caterina, Ambrosio Raffaele, De Stefano Maria Angela, Di Girolamo Daniela, Di Cicco Emery, Cicatiello Annunziata Gaetana, Mancino Giuseppina, Porcelli Tommaso, Raia Maddalena, Del Vecchio Luigi, Salvatore Domenico, Dentice Monica
1 Department of Clinical Medicine and Surgery, University of Naples "Federico II" , Napoli, Italy .
2 Istituto di Ricovero e Cura a Carattere Scientifico-SDN , Naples, Italy .
Thyroid. 2017 Apr;27(4):567-576. doi: 10.1089/thy.2016.0532. Epub 2017 Feb 22.
Thyroid hormones (THs) mediate pleiotropic cellular processes involved in metabolism, cellular proliferation, and differentiation. The intracellular hormonal environment can be tailored by the type 1 and 2 deiodinase enzymes D2 and D3, which catalyze TH activation and inactivation respectively. In many cellular systems, THs exert well-documented stimulatory or inhibitory effects on cell proliferation; however, the molecular mechanisms by which they control rates of cell cycle progression have not yet been entirely clarified. We previously showed that D3 depletion or TH treatment influences the proliferation and survival of basal cell carcinoma (BCC) cells. Surprisingly, we also found that BCC cells express not only sustained levels of D3 but also robust levels of D2. The aim of the present study was to dissect the contribution of D2 to TH metabolism in the BCC context, and to identify the molecular changes associated with cell proliferation and survival induced by TH and mediated by D2 and D3.
We used the CRISPR/Cas9 technology to genetically deplete D2 and D3 in BCC cells and studied the consequences of depletion on cell cycle progression and on cell death. Cell cycle progression was analyzed by fluorescence activated cell sorting analysis of synchronized cells, and the apoptosis rate by annexin V incorporation.
Mechanistic investigations revealed that D2 inactivation accelerates cell cycle progression thereby enhancing the proportion of S-phase cells and cyclin D1 expression. Conversely, D3 mutagenesis drastically suppressed cell proliferation and enhanced apoptosis of BCC cells. Furthermore, the basal apoptotic rate was oppositely regulated in D2- and D3-depleted cells.
Our results indicate that BCC cells constitute an example in which the TH signal is finely tuned by the concerted expression of opposite-acting deiodinases. The dual regulation of D2 and D3 expression plays a critical role in cell cycle progression and cell death by influencing cyclin D1-mediated entry into the G1-S phase. These findings reinforce the concept that TH is a potential therapeutic target in human BCC.
甲状腺激素（THs）介导参与代谢、细胞增殖和分化的多效性细胞过程。1型和2型脱碘酶D2和D3可调节细胞内激素环境，它们分别催化TH的激活和失活。在许多细胞系统中，THs对细胞增殖具有明确记载的刺激或抑制作用；然而，它们控制细胞周期进程速率的分子机制尚未完全阐明。我们之前发现，D3缺失或TH处理会影响基底细胞癌（BCC）细胞的增殖和存活。令人惊讶的是，我们还发现BCC细胞不仅持续表达高水平的D3，还表达高水平的D2。本研究的目的是剖析D2在BCC环境中对TH代谢的作用，并确定与TH诱导并由D2和D3介导的细胞增殖和存活相关的分子变化。
我们使用CRISPR/Cas9技术在BCC细胞中对D2和D3进行基因敲除，并研究敲除对细胞周期进程和细胞死亡的影响。通过对同步化细胞进行荧光激活细胞分选分析来分析细胞周期进程，通过膜联蛋白V掺入分析来分析凋亡率。
机制研究表明，D2失活会加速细胞周期进程，从而增加S期细胞比例并提高细胞周期蛋白D1的表达。相反，D3诱变会显著抑制BCC细胞的增殖并增强其凋亡。此外，D2和D3敲除细胞中的基础凋亡率受到相反的调节。
我们的结果表明，BCC细胞是一个例子，其中TH信号通过作用相反的脱碘酶的协同表达得到精细调节。D2和D3表达的双重调节通过影响细胞周期蛋白D1介导的G1-S期进入，在细胞周期进程和细胞死亡中起关键作用。这些发现强化了TH是人类BCC潜在治疗靶点的概念。
