Kochendoerfer S K, Krishnan N, Buckley D J, Buckley A R
Department of Pharmacology and Toxicology, University of North Dakota, Grand Forks, North Dakota, USA.
J Endocrinol. 2003 Aug;178(2):265-73. doi: 10.1677/joe.0.1780265.
Prolactin (PRL)-dependent rat pre-T Nb2 (Nb2-11) cell lines serve as a useful model for investigation of mechanisms underlying lactogen-mediated suppression of apoptosis. Glucocorticoids, such as dexamethasone (DEX), induce apoptosis in Nb2-11 cells; the addition of PRL abrogates the cytolytic actions of DEX in this model, presumably because of increased expression of survival genes. In the present study, we investigated whether inhibition of DEX-induced apoptosis by PRL in Nb2-T cells was accompanied by altered expression of Bcl-2 family members, mcl-1, bad or bcl-x(L) determined by Northern and immunoblot analysis. The results indicated that a 0.9 kb bcl-x(L) transcript was rapidly induced by PRL. It reached maximal levels within 2 to 4 h (>20-fold) before declining toward basal values. Similar results were obtained in primary cultures of mouse thymocytes exposed to DEX in combination with PRL. In addition to increasing its mRNA expression, PRL also increased Bcl-xL protein levels by 6 h. Moreover, the effect of PRL to increase bcl-x(L) appeared to reflect direct and indirect mechanisms, since it was attenuated by the inhibition of protein synthesis. Results from other experiments suggest that PRL signaling to bcl-x(L) expression was independent of the Jak2/Stat pathway but appeared to require activation of a Src tyrosine kinase. In contrast, while a 1.1 kb mcl-1 transcript was detected in proliferating and quiescent cells, PRL did not alter its expression at either mRNA or protein levels. Moreover, neither bad mRNA nor its protein product were detectable under any of the experimental conditions evaluated. We have concluded that bad and mcl-1 are unlikely candidates for apoptosis regulatory genes modulated by PRL. However, the kinetic pattern of PRL-provoked bcl-x(L) expression is consistent with its playing a role as an apoptosis suppressor in Nb2-T cells and primary cultures of mouse thymocytes exposed to glucocorticoids.
催乳素（PRL）依赖的大鼠前T细胞系Nb2（Nb2-11）可作为研究催乳素介导的细胞凋亡抑制机制的有用模型。糖皮质激素，如地塞米松（DEX），可诱导Nb2-11细胞凋亡；在该模型中加入PRL可消除DEX的细胞溶解作用，这可能是由于存活基因表达增加所致。在本研究中，我们通过Northern印迹和免疫印迹分析，研究了PRL对Nb2-T细胞中DEX诱导的细胞凋亡的抑制作用是否伴随着Bcl-2家族成员、mcl-1、bad或bcl-x(L)表达的改变。结果表明，PRL可迅速诱导0.9 kb的bcl-x(L)转录本。在2至4小时内达到最高水平（>20倍），然后降至基础值。在与PRL联合暴露于DEX的小鼠胸腺细胞原代培养物中也获得了类似的结果。除了增加其mRNA表达外，PRL还可在6小时内增加Bcl-xL蛋白水平。此外，PRL增加bcl-x(L)的作用似乎反映了直接和间接机制，因为它被蛋白质合成抑制所减弱。其他实验结果表明，PRL向bcl-x(L)表达的信号传导独立于Jak2/Stat途径，但似乎需要Src酪氨酸激酶的激活。相反，虽然在增殖和静止细胞中检测到1.1 kb的mcl-1转录本，但PRL在mRNA或蛋白质水平上均未改变其表达。此外，在任何评估的实验条件下均未检测到bad mRNA及其蛋白质产物。我们得出结论，bad和mcl-1不太可能是受PRL调节的细胞凋亡调控基因的候选者。然而，PRL诱导的bcl-x(L)表达的动力学模式与其在Nb2-T细胞和暴露于糖皮质激素的小鼠胸腺细胞原代培养物中作为细胞凋亡抑制因子的作用一致。
