Tranque P A, Calle R, Naftolin F, Robbins R
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conneticut 06510.
Endocrinology. 1992 Oct;131(4):1948-54. doi: 10.1210/endo.131.4.1396338.
Insulin-like growth factor-I (IGF-I) stimulates the proliferation of many cell types, including astrocytes. Astrocytes are a population of brain cells highly enriched in IGF-I receptors, which unlike neurons, retain the ability to proliferate in the adult brain. Although astrocyte proliferation in response to IGF-I is well documented, the intracellular mechanisms that mediate this phenomenon are poorly defined. Interestingly, activation of protein kinase-C (PKC) by IGF-I has been observed in several cell types. In this report we first characterized the mitogenic effects of IGF-I on highly purified type I rat astrocyte cultures. Next, we determined whether IGF-I activates PKC in our cultures. Finally, since astrocyte proliferation is stimulated by both IGF-I and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA), we decided to test the possible involvement of PKC in the mitogenic activity of IGF-I on astrocytes. IGF-I stimulated the DNA synthesis rate in rat astrocytes. Analysis of the time course revealed that IGF-I (10 nM) induces maximal stimulation of [3H]thymidine incorporation (a 4-fold increase) 16-18 h after exposure. TPA also stimulated mitogenesis in our cultures. The dose-response of [3H]thymidine incorporation induced by IGF-I and TPA indicated that 10 nM was the lowest concentration producing a maximal effect for both agents. Analysis of proteins by Western blot revealed that 10 nM IGF-I translocates PKC(alpha), the predominant PKC isoform in astrocyte cultures, from the cytosol to the membrane fraction within 20 min. A similar activation of PKC was achieved with 100 nM TPA. When astrocytes were exposed to IGF-I (10 nM) and TPA (10 nM) in combination, [3H]thymidine uptake was significantly higher than the uptake induced by either IGF-I (10 nM) or TPA (10 nM) alone. However, the effect of IGF-I plus TPA was not fully additive. In a second experiment, the mitogenic effect of IGF-I was partially abolished in cells depleted of PKC by preincubation with high concentrations of TPA (300 nM). Finally, incubation of astrocytes with the PKC inhibitor H-7 at 20 microM, a concentration that completely blocked the mitogenic action of TPA, only reduced the ability of IGF-I to stimulate DNA synthesis by 50%. In summary, our results demonstrate that IGF-I can rapidly activate PKC in astrocytes, and that PKC activation is involved in the mitogenic effect of IGF-I on these cells. However, we conclude that IGF-I also stimulates astrocyte proliferation through PKC-independent pathways.
胰岛素样生长因子-I（IGF-I）可刺激包括星形胶质细胞在内的多种细胞类型的增殖。星形胶质细胞是一类在大脑细胞中高度富集IGF-I受体的细胞群体，与神经元不同，它们在成年大脑中仍保留增殖能力。尽管IGF-I诱导星形胶质细胞增殖已有充分记录，但介导这一现象的细胞内机制仍不清楚。有趣的是，在几种细胞类型中均观察到IGF-I可激活蛋白激酶-C（PKC）。在本报告中，我们首先对IGF-I对高度纯化的I型大鼠星形胶质细胞培养物的促有丝分裂作用进行了表征。接下来，我们确定IGF-I是否能在我们的培养物中激活PKC。最后，由于IGF-I和佛波酯12-O-十四酰佛波醇-13-乙酸酯（TPA）均可刺激星形胶质细胞增殖，我们决定测试PKC是否可能参与IGF-I对星形胶质细胞的促有丝分裂活性。IGF-I刺激大鼠星形胶质细胞的DNA合成速率。对时间进程的分析表明，IGF-I（10 nM）在暴露后16 - 18小时诱导[3H]胸苷掺入的最大刺激（增加4倍）。TPA也刺激了我们培养物中的有丝分裂。IGF-I和TPA诱导[3H]胸苷掺入的剂量反应表明，10 nM是两种试剂产生最大效应的最低浓度。通过蛋白质印迹分析蛋白质发现，10 nM IGF-I在20分钟内可使星形胶质细胞培养物中主要的PKC同工型PKC（α）从细胞质转移至膜部分。100 nM TPA也能实现类似的PKC激活。当星形胶质细胞同时暴露于IGF-I（10 nM）和TPA（10 nM）时，[3H]胸苷摄取显著高于单独由IGF-I（10 nM）或TPA（10 nM）诱导的摄取。然而，IGF-I加TPA的效应并非完全相加。在第二个实验中，通过用高浓度TPA（300 nM）预孵育使PKC耗竭的细胞中，IGF-I的促有丝分裂作用部分被消除。最后，用20 μM的PKC抑制剂H-7孵育星形胶质细胞，该浓度完全阻断了TPA的促有丝分裂作用，仅使IGF-I刺激DNA合成的能力降低了50%。总之，我们的结果表明IGF-I可在星形胶质细胞中快速激活PKC，且PKC激活参与了IGF-I对这些细胞的促有丝分裂作用。然而，我们得出结论，IGF-I也通过不依赖PKC的途径刺激星形胶质细胞增殖。
