Proliferation of the human urothelium is induced by atypical β1-adrenoceptors

M. Winder, C. Wasén, P. Aronsson, D. Giglio
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引用次数: 4

Abstract

  1. We wanted to assess whether β-adrenoceptors mediate proliferation in the normal and malignant urothelial cell lines UROtsa and T24, respectively.
  2. Urothelial cells were cultured for 24 h in the presence of the β-adrenoceptor agonists isoprenaline (β1/2/3), dobutamine (β1), salbutamol (β2), BRL 37344 (β3), CGP 12177 (a partial β-agonist) or β-adrenoceptor antagonists (metoprolol; β1, propranolol; β1/2). Phosphorylation of kinases was screened with a Human Phospho-Kinase Array Kit (R&D systems). Intracellular pathways activated by proliferation of urothelial cells were characterized by incubating cells with the MEK1/2 inhibitor PD 98,059, the p38 kinase inhibitor losmapimod or with the Akt 1/2 kinase inhibitor. Proliferation was assessed with the MTT proliferation assay (ATCC). Western blot and immunocytochemistry were used for detection of the β1-adrenoceptor.
  3. Isoprenaline and dobutamine induced proliferation, while salbutamol and BRL 37344 did not. Dobutamine-induced proliferation was not affected by metoprolol or propranolol but was instead antagonized by CGP 12177 in T24 but not in UROtsa. In response to stimulation with dobutamine, Akt1/2/3 was phosphorylated in UROtsa, while ERK1/2 and p38 were phosphorylated in T24. MEK1/2 inhibition blocked basal and dobutamine-induced proliferation in T24 but only basal proliferation in UROtsa. Losmapimod slightly inhibited basal proliferation in T24 but not dobutamine-induced proliferation. Akt 1/2 inhibitor blocked basal and dobutamine-induced proliferation in UROtsa. Immunocytochemistry and Western blot revealed expression of β1-adrenoceptors in both urothelial cell lines.
  4. The present data show that the urothelium expresses atypical β1-adrenoceptors that activate intracellular kinases inducing urothelial proliferation.
非典型β1-肾上腺素受体可诱导人尿路上皮的增殖
我们想要评估β-肾上腺素受体是否分别介导正常和恶性尿路上皮细胞系UROtsa和T24的增殖。尿路上皮细胞在β-肾上腺素受体激动剂异丙肾上腺素(β1/2/3)、多巴酚丁胺(β1)、沙丁胺醇(β2)、BRL 37344 (β3)、CGP 12177(部分β激动剂)或β-肾上腺素受体拮抗剂(美托洛尔;β1、普萘洛尔;β1/2)。使用Human Phospho-Kinase Array Kit (R&D systems)筛选激酶的磷酸化。通过MEK1/2抑制剂PD 98059、p38激酶抑制剂losmapimod或Akt 1/2激酶抑制剂孵育细胞,研究了尿路上皮细胞增殖激活的细胞内通路。用MTT增殖试验(ATCC)评估增殖情况。Western blot和免疫细胞化学检测β1-肾上腺素能受体。异丙肾上腺素和多巴酚丁胺可诱导细胞增殖,而沙丁胺醇和BRL 37344则无此作用。美托洛尔和心得安对多巴酚丁胺诱导的增殖没有影响,而CGP 12177对T24有拮抗作用,而对UROtsa无拮抗作用。多巴酚丁胺刺激后,UROtsa中Akt1/2/3被磷酸化,而T24中ERK1/2和p38被磷酸化。MEK1/2抑制可阻断T24细胞基底细胞和多巴酚丁胺诱导的增殖,但仅阻断UROtsa细胞基底细胞的增殖。Losmapimod对T24的基底细胞增殖有轻微抑制作用,但对多巴酚丁胺诱导的增殖没有抑制作用。Akt 1/2抑制剂阻断基底和多巴酚丁胺诱导的UROtsa细胞增殖。免疫细胞化学和Western blot显示两种尿路上皮细胞系均表达β1-肾上腺素受体。目前的数据表明,尿路上皮表达非典型β1-肾上腺素受体,激活细胞内激酶,诱导尿路上皮增殖。
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