Lazzereschi D, Coppa A, Minicione G, Lavitrano M, Fragomele F, Colletta G
Dipartimento di Medicina Sperimentale e Patologia, Facoltà di Medicinae Chirurgia, Università La Sapienza, Rome, Italy.
Exp Cell Res. 1997 Aug 1;234(2):425-33. doi: 10.1006/excr.1997.3627.
Protein phosphorylation plays an essential role in regulating many cellular processes in eukaryotes. Signal transduction mechanisms that are reversibly controlled by protein phosphorylation require also protein phosphatases (PPs). Okadaic acid (OA), which is a potent inhibitor of protein phosphatase 2A (PP2A) and protein phosphatase 1, elicits phosphorylation of many proteins in unstimulated cells and induces different cellular responses, including transcriptional activation, shape changes, and pseudomitotic state. In this study, the effects of OA on rat thyroid cells (FRTL-5 strain) were analyzed to evaluate the role of serine/threonine phosphatases in hormone-induced thyroid cell proliferation. OA at a concentration range between 0.1 and 1 nM stimulated thyroid cell growth. Furthermore, 0.25 nM OA increased about 3.5-fold the thyrotropin (TSH)-induced DNA synthesis in quiescent cells. OA treatment also stimulated cell proliferation induced by drugs that mimic TSH effect, such as 8Br-cAMP and cholera toxin, suggesting that PP2A activity was relevant in the cAMP pathway activated by the hormone. Flow cytometry experiments showed that OA significantly increased the fraction of TSH-stimulated quiescent cells entering the S phase. In order to define the mechanisms underlying the observed stimulatory effect of OA on thyroid cell growth, expression of genes relevant in the G1-S phase transition was evaluated. A 2-fold increase in the level of cyclin D1 mRNA expression was found by Northern blot analysis in OA-treated cells. Although cdk2 gene expression was not modulated by the same OA treatment, an increase in Cdk2 protein was revealed by immunoprecipitation experiments. Moreover, OA modifies the phosphorylation pattern of the tumor suppressor retinoblastoma protein, a key event in the G1-S phase transition. Therefore, these experiments reveal that PP2A phosphatases play an important role in thyroid cell growth and can act at multiple sites in the TSH pathways driving cells to S phase.
蛋白质磷酸化在调节真核生物的许多细胞过程中起着至关重要的作用。由蛋白质磷酸化可逆控制的信号转导机制同样也需要蛋白质磷酸酶（PPs）。冈田酸（OA）是一种有效的蛋白质磷酸酶2A（PP2A）和蛋白质磷酸酶1抑制剂，它能使未受刺激的细胞中许多蛋白质发生磷酸化，并诱导不同的细胞反应，包括转录激活、形态变化和假有丝分裂状态。在本研究中，分析了OA对大鼠甲状腺细胞（FRTL-5株）的影响，以评估丝氨酸/苏氨酸磷酸酶在激素诱导的甲状腺细胞增殖中的作用。浓度在0.1至1 nM之间的OA刺激甲状腺细胞生长。此外，0.25 nM的OA使静止细胞中促甲状腺激素（TSH）诱导的DNA合成增加了约3.5倍。OA处理还刺激了由模拟TSH效应的药物（如8Br-cAMP和霍乱毒素）诱导的细胞增殖，这表明PP2A活性与激素激活的cAMP途径相关。流式细胞术实验表明，OA显著增加了受TSH刺激进入S期的静止细胞比例。为了确定OA对甲状腺细胞生长的刺激作用背后的机制，评估了与G1-S期转变相关基因的表达。通过Northern印迹分析发现，OA处理的细胞中细胞周期蛋白D1 mRNA表达水平增加了2倍。尽管相同的OA处理未调节cdk2基因表达，但免疫沉淀实验显示Cdk2蛋白增加。此外，OA改变了肿瘤抑制蛋白视网膜母细胞瘤蛋白的磷酸化模式，这是G1-S期转变中的一个关键事件。因此，这些实验表明PP2A磷酸酶在甲状腺细胞生长中起重要作用，并且可以在驱动细胞进入S期的TSH途径的多个位点发挥作用。
