Yasuda H, Mine T, Shibata H, Eto Y, Hasegawa Y, Takeuchi T, Asano S, Kojima I
Cell Biology Research Unit, Gunma University, Maebashi, Japan.
J Clin Invest. 1993 Sep;92(3):1491-6. doi: 10.1172/JCI116727.
The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.
本研究旨在检测激活素A对大鼠肝细胞生长的影响。通过在培养的肝细胞中掺入[3H]胸苷来评估，表皮生长因子（EGF）可使DNA合成增加10倍。当激活素A与EGF一起添加时，EGF诱导的DNA合成受到显著抑制。在浓度为10(-10) M时可检测到抑制作用，5×10(-9) M的激活素A几乎完全阻断了EGF介导的DNA合成。同样，激活素A完全阻断了肝细胞生长因子/分散因子诱导的DNA合成。即使在添加EGF 36小时后再添加激活素A，它仍能抑制EGF介导的DNA合成。在相同的时间间隔内，转化生长因子-β（TGF-β）也阻断了EGF诱导的DNA合成。尽管激活素A和TGF-β都以类似的方式抑制肝细胞生长，但通过使用碘化配体的竞争性结合评估，激活素A和TGF-β在结合时并不相互竞争。当肝细胞与EGF一起孵育时，48小时或更晚后可检测到激活素A的生物活性释放到培养基中。激活素A的活性是从实质细胞释放的，而非实质细胞则不释放。在未受刺激的肝细胞中仅略微检测到激活素βA亚基的mRNA，但在添加EGF后48小时显著增加。为了确定内源性产生的激活素A是否影响DNA合成，我们检测了卵泡抑素的作用，卵泡抑素是一种能阻断激活素A作用的激活素结合蛋白。添加卵泡抑素可显著增强EGF诱导的DNA合成。最后，在部分肝切除的大鼠中，部分肝切除24小时后肝脏中βA亚基的mRNA表达显著增加。这些结果表明，激活素A通过作用于自身受体抑制肝细胞中DNA合成的起始，并且激活素A在大鼠肝细胞中作为DNA合成的自分泌抑制剂发挥作用。
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