Gorodeski George I
Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Endocrinology. 2007 Jan;148(1):279-92. doi: 10.1210/en.2006-1045. Epub 2006 Oct 5.
The study tested the hypothesis that estrogen controls epithelial paracellular resistance through modulation of myosin. The objective was to understand how estrogen modulates nonmuscle myosin-II-B (NMM-II-B), the main component of the cortical actomyosin in human epithelial cervical cells. Experiments used human cervical epithelial cells CaSki as a model, and end points were NMM-II-B phosphorylation, filamentation, and MgATPase activity. The results were as follows: 1) treatment with estrogen increased phosphorylation and MgATPase activity and decreased NMM-II-B filamentation; 2) estrogen effects could be blocked by antisense nucleotides for the estrogen receptor-alpha and by ICI-182,780, tamoxifen, and the casein kinase-II (CK2) inhibitor, 5,6-dichloro-1-beta-(D)-ribofuranosylbenzimidazole and attenuated by AG1478 and PD98059 (inhibitors of epithelial growth factor receptor and ERK/MAPK) but not staurosporine [blocker of protein kinase C (PKC)]; 3) treatments with the PKC activator sn-1,2-dioctanoyl diglyceride induced biphasic effect on NMM-II-B MgATPase activity: an increase at 1 nm to 1 microM and a decrease in activity at more than 1 microM; 4) sn-1,2-dioctanoyl diglyceride also decreased NMM-II-B filamentation in a monophasic and saturable dose dependence (EC(50) 1-10 microM); 5) when coincubated directly with purified NMM-II-B filaments, both CK2 and PKC decreased filamentation and increased MgATPase activity; 6) assays done on disassembled NMM-II-B filaments showed MgATPase activity in filaments obtained from estrogen-treated cells but not estrogen-depleted cells; and 7) incubations in vitro with CK2, but not PKC, facilitated MgATPase activity, even in disassembled NMM-II-B filaments. The results suggest that estrogen, in an effect mediated by estrogen receptor-alpha and CK2 and involving the epithelial growth factor receptor and ERK/MAPK cascades, increases NMM-II-B MgATPase activity independent of NMM-II-B filamentation status.
该研究检验了雌激素通过调节肌球蛋白来控制上皮细胞旁通透性的假说。目的是了解雌激素如何调节非肌肉肌球蛋白-II-B（NMM-II-B），这是人类宫颈上皮细胞中皮质肌动球蛋白的主要成分。实验以人宫颈上皮细胞CaSki为模型，终点指标为NMM-II-B的磷酸化、丝化及MgATP酶活性。结果如下：1）雌激素处理可增加磷酸化和MgATP酶活性，并减少NMM-II-B丝化；2）雌激素受体-α的反义核苷酸、ICI-182,780、他莫昔芬以及酪蛋白激酶-II（CK2）抑制剂5,6-二氯-1-β-(D)-呋喃核糖基苯并咪唑可阻断雌激素的作用，而AG1478和PD98059（上皮生长因子受体和ERK/MAPK的抑制剂）可减弱其作用，但星形孢菌素[蛋白激酶C（PKC）的阻断剂]则无此作用；3）PKC激活剂sn-1,2-二辛酰甘油对NMM-II-B MgATP酶活性产生双相效应：在1 nM至1 μM时活性增加，在超过1 μM时活性降低；4）sn-1,2-二辛酰甘油还以单相且饱和的剂量依赖性方式降低NMM-II-B丝化（半数有效浓度为1-10 μM）；5）当与纯化的NMM-II-B丝直接共孵育时，CK2和PKC均可降低丝化并增加MgATP酶活性；6）对解离的NMM-II-B丝进行的检测显示，从雌激素处理的细胞中获得的丝具有MgATP酶活性，而从雌激素缺乏的细胞中获得的丝则无此活性；7）即使在解离的NMM-II-B丝中，体外与CK2而非PKC孵育也可促进MgATP酶活性。结果表明，雌激素通过雌激素受体-α和CK2介导的效应，涉及上皮生长因子受体和ERK/MAPK级联反应，可独立于NMM-II-B丝化状态增加其MgATP酶活性。
