Rapid Effects of 17beta-estradiol on Cell Signaling and Function of Mytilus Hemocytes

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.