Junoy Brice, Maccario Helene, Mas Jean-Louis, Enjalbert Alain, Drouva Sophia V
Centre National de la Recherche Scientifique UMR 6544, Université de la Méditerranée, Faculté de Médecine, 13916 Marseille, France.
Endocrinology. 2002 Apr;143(4):1386-403. doi: 10.1210/endo.143.4.8752.
We investigated mechanisms underlying selective down-modulation of PKC isoforms (alpha, epsilon, zeta): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10(-7) M) or GnRH (10(-7) M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (alpha T(3)-1, L beta T(2)) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in alpha T(3)-1 cells, in a more differentiated gonadotrope cell line L beta T(2) the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (alpha, beta II, delta, epsilon, zeta) were selectively and differentially down-regulated by TPA (alpha, beta II, delta, epsilon) or GnRH (delta, epsilon). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, beta-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC alpha, epsilon, and the GnRH-elicited PKC epsilon down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC alpha, epsilon depletion. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in alpha T(3)-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (alpha, epsilon) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, Gö6976), selectively prevented the TPA-evoked PKC alpha depletion in both mixed pituitary cells and alpha T(3)-1 gonadotropes; in alpha T(3)-1 subcellular fractions, PKC alpha inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses.
我们研究了蛋白激酶C（PKC）亚型（α、ε、ζ）选择性下调的潜在机制：1）在两种促性腺激素细胞系（αT3-1、LβT2）中，于12-O-十四烷酰佛波醇-13-乙酸酯（TPA）（10-7 M）或促性腺激素释放激素（GnRH）（10-7 M）脱敏条件下（处理2至6小时）；2）在雄性大鼠原代垂体细胞培养物中进行长期佛波酯给药期间。我们证明，与αT3-1细胞一样，在更分化的促性腺激素细胞系LβT2中，GnRH受体偶联（磷脂酶C、磷脂酶A2、磷脂酶D）产生了PKC激活所必需的第二信使；所鉴定的亚型（α、βII、δ、ε、ζ）被TPA（α、βII、δ、ε）或GnRH（δ、ε）选择性地、差异性地下调。在全细胞裂解物中，蛋白酶体抑制剂（蛋白酶体抑制剂I和II、乳胞素、β-内酯、钙蛋白酶抑制剂I）在两种促性腺激素细胞系中均能阻止TPA诱导的PKCα、ε耗竭以及GnRH引起的PKCε下调；它们在混合垂体细胞培养物中也能抵消TPA引起的PKCα、ε耗竭。相比之下，钙蛋白酶和溶酶体蛋白酶抑制剂（钙蛋白酶抑制肽、E64d、钙蛋白酶抑制剂II和PD150606）则无效。如在αT3-1亚细胞组分中所示，蛋白酶体的废除并不影响TPA和GnRH作用靶点亚型（α、ε）的膜转位，但通过阻止它们的降解，有利于酶在膜区室的积累。PKC的蛋白水解加工可能取决于其磷酸化状态和/或催化活性。抑制PKC催化活性（GF109203X、Gö6976）在混合垂体细胞和αT3-1促性腺激素细胞中均选择性地阻止了TPA诱导的PKCα耗竭；在αT3-1亚细胞组分中，PKCα失活通过诱导该亚型在膜上的显著积累克服了TPA诱导的同工酶降解，而不影响其膜重新定位。因此，蛋白酶体系统通过调节PKC细胞水平，可能代表了一种参与时间依赖性细胞反应适应性机制的调节性蛋白水解途径。
