Canesi Laura, Ciacci Caterina, Betti Michele, Lorusso Lucia Cecilia, Marchi Barbara, Burattini Sabrina, Falcieri Elisabetta, Gallo Gabriella
Istituto di Scienze Fisiologiche, Università di Urbino Carlo Bo, Loc. Crocicchia, 61029 Urbino (PU), Italy.
Gen Comp Endocrinol. 2004 Mar;136(1):58-71. doi: 10.1016/j.ygcen.2003.12.003.
Estrogens affect the functioning of several non-reproductive tissues, the immune system in particular. In mammalian immunocytes, 17beta-estradiol (E2) has both dose- and cell-type specific effects and the responses to E2 seem to be mediated by rapid, non-genomic mechanisms; these may be initiated at either membrane or cytosolic locations, and can result in both direct local effects, such as modification of ion fluxes, and regulation of gene transcription secondary to activation of different kinase cascades, including mitogen activated protein kinases (MAPKs). In this work, the short-term effects of E(2) and the possible mechanisms of estrogen-mediated cell signaling were investigated in the hemocytes, the immune cells of the bivalve mollusc, the mussel Mytilus galloprovincialis Lam. The results show that E2 (25nM) caused a rapid and significant increase in hemocyte cytosolic [Ca2+]; lower concentrations (5 nM) showed a smaller, not significant effect. Both E2 concentrations affected the phosphorylation state of the components of tyrosine kinase-mediated signal transduction MAPK- and STAT- (signal transducers and activators of transcription) like proteins within 5-15 min from E2 addition. A greater effect and clearer time course were observed with 25 nM E2: in particular, E2 induced a transient increase in p-ERK2 MAPK and a persistent increase in p-p38 MAPK. Moreover, both STAT3 and STAT5 were tyrosine phosphorylated in response to E2. E2 (5 nM) induced both morphological (as evaluated by SEM) and functional changes (such as extracellular release of hydrolytic enzymes, lysosomal membrane destabilisation, and stimulation of the bactericidal activity) within 10-30 min from addition. Lysosomal membrane destabilisation induced by both E2 concentrations was abolished by hemocyte preincubation with the p38 MAPK inhibitor SB203580, and significantly reduced by PD98059 and Wortmannin (inhibitors of ERK MAPK and PI3-K, respectively), this suggesting that rapid activation of kinase cascades is involved in mediating the effects of E2 in mussel hemocytes. The antiestrogen Tamoxifen prevented or strongly reduced most, but not all, the effects of E2. Western blotting with heterologous anti-ERalpha-anti-ERbeta-antibodies revealed the presence of immunoreactive ERalpha- and ERbeta-like proteins in hemocyte protein extracts. Overall, our data support the hypothesis that the rapid effects and mechanisms of action of 17beta-estradiol are extremely conserved and that they may play a crucial role in endocrine-immune interactions in invertebrates.
雌激素会影响多个非生殖组织的功能，尤其是免疫系统。在哺乳动物免疫细胞中，17β-雌二醇（E2）具有剂量和细胞类型特异性效应，对E2的反应似乎是由快速的非基因组机制介导的；这些机制可能在细胞膜或胞质中启动，并可导致直接的局部效应，如离子通量的改变，以及继发于不同激酶级联反应（包括丝裂原活化蛋白激酶（MAPK））激活的基因转录调控。在本研究中，我们研究了E2的短期效应以及雌激素介导的细胞信号传导的可能机制，所用材料为双壳贝类贻贝（Mytilus galloprovincialis Lam.）的血细胞，即其免疫细胞。结果表明，E2（25nM）可使血细胞胞质[Ca2+]迅速显著升高；较低浓度（5 nM）的作用较小且不显著。两种E2浓度均在添加E2后的5 - 15分钟内影响酪氨酸激酶介导的信号转导MAPK和STAT（信号转导子和转录激活子）样蛋白组分的磷酸化状态。25 nM E2的作用更明显且时间进程更清晰：特别是，E2诱导p-ERK2 MAPK短暂升高，p-p38 MAPK持续升高。此外，STAT3和STAT5均因E2而发生酪氨酸磷酸化。E2（5 nM）在添加后10 - 三十分钟内诱导了形态学变化（通过扫描电子显微镜评估）和功能变化（如水解酶的细胞外释放、溶酶体膜不稳定以及杀菌活性的刺激）。两种E2浓度诱导的溶酶体膜不稳定可通过血细胞预先与p38 MAPK抑制剂SB203580孵育而消除，并被PD98059和渥曼青霉素（分别为ERK MAPK和PI3-K的抑制剂）显著降低，这表明激酶级联反应的快速激活参与介导E2对贻贝血细胞的作用。抗雌激素他莫昔芬可预防或强烈降低E2的大部分但并非全部作用。用异源抗ERα-抗ERβ抗体进行的蛋白质免疫印迹显示，血细胞蛋白提取物中存在免疫反应性ERα和ERβ样蛋白。总体而言，我们的数据支持以下假设：17β-雌二醇的快速效应和作用机制具有高度保守性，且它们可能在无脊椎动物的内分泌-免疫相互作用中起关键作用。
