Role of putative membrane receptors in the effect of androgens on human vascular cell growth.
Role of putative membrane receptors in the effect of androgens on human vascular cell growth.
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DOI：
10.1677/joe.0.1800097
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发表时间：
2004
期刊：
影响因子：
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通讯作者：
D. Somjen;F. Kohen;B. Gayer;T. Kulik;E. Knoll;N. Stern
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文献类型：
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作者：
D. Somjen;F. Kohen;B. Gayer;T. Kulik;E. Knoll;N. Stern
我们已经报道了双氢睾酮(DHT)对人血管平滑肌细胞(VSMC)DNA合成的双向作用，即低浓度的刺激和高浓度的抑制。相反，DHT剂量依赖性地刺激人内皮细胞系(ECV304)的[(3)H]胸腺嘧啶核苷掺入。此外，DHT还增加了两种血管细胞中肌酸激酶(CK)的比活性。在目前的研究中，我们已经确定了其中一些效应是否通过膜结合位点发挥作用。我们测定了DHT和去膜的牛血清白蛋白(T-BSA)处理后DNA合成和CK的变化。高浓度的DHT和T-BSA均可抑制VSMC增殖(分别为52±22%和51±25%)。DHT和T-BSA可剂量依赖性地促进ECV304细胞DNA合成。相反，T-BSA对两种细胞类型的CK均无影响。在这两种类型的细胞中，DHT和T-BSA增加了丝裂原活化蛋白激酶(MAPK)的活性，这是通过总的MAPK来衡量的。此外，游离或蛋白质结合的雄激素对DNA合成的抑制作用可被MAPK激酶活性的抑制剂UO126阻断。Eu标记的T-BSA结合物与整个VSMC结合，可被过量的T-BSA取代，但不能被雌二醇-BSA或游离激素所取代。最后，使用T-BSA连接到荧光染料Cy3.5，我们直接证明了VSMC中雄激素的膜结合位点的存在。因此，睾酮对VSMC DNA合成的抑制作用显然是通过雄激素的膜结合部位发挥的，而不需要激素进入细胞内并与经典的核受体结合，并与MAPK激活有关。
We have reported previously that dihydrotestosterone (DHT) induces a biphasic effect on DNA synthesis in human vascular smooth muscle cells (VSMC), i.e. stimulation at low concentrations and inhibition at high concentrations. In contrast, DHT dose-dependently stimulated [(3)H]thymidine incorporation in a human endothelial cell line (ECV304). Additionally, DHT increased the specific activity of creatine kinase (CK) in both vascular cell types. In the present study, we have determined whether some of these effects are exerted via membrane-binding sites. We measured changes in DNA synthesis and CK after treatment with DHT and the membrane-impermeant testosterone-3-carboxymethyl oxime conjugated to bovine serum albumin (BSA) (T-BSA). High concentrations of either DHT or T-BSA inhibited VSMC proliferation (by 52+22% and 51+25% respectively). DHT as well as T-BSA increased DNA synthesis in ECV304 cells dose-dependently. In contrast, T-BSA did not affect CK in either cell type. In both cell types, DHT as well as T-BSA increased mitogen-activated protein kinase (MAPK) kinase activity as measured by total phosphorylated MAPK. Further, the inhibitory effect of either the free or protein-bound androgens on DNA synthesis was blocked by UO126, an inhibitor of MAPK kinase activity. T-BSA conjugate labeled with Europium showed binding to whole VSMC, which could be displaced by excess T-BSA, but not by estradiol-BSA or the free hormones. Finally, using T-BSA linked to the fluorescent dye Cy3.5, we directly demonstrated the presence of membrane-binding sites for androgen in VSMC. Hence, the inhibitory effects of testosterone on DNA synthesis in VSMC are apparently exerted by membrane-binding sites for androgen, do not require intracellular entry of the hormone and its binding to the classical nuclear receptors and are linked to MAPK activation.