Bhattacharya Sujoy, Ray Ramesh M, Johnson Leonard R
Department of Physiology, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Biochem J. 2005 Dec 1;392(Pt 2):335-44. doi: 10.1042/BJ20050465.
Activation of STAT3 (signal transducer and activator of transcription 3) plays a crucial role in cell survival and proliferation. The aim of the present study was to clarify the role of STAT3 signalling in the protection of polyamine-depleted intestinal epithelial cells against TNF-alpha (tumour necrosis factor-alpha)-induced apoptosis. Polyamine depletion by DFMO (alpha-difluoromethylornithine) caused phosphorylation of STAT3 at Tyr-705 and Ser-727. Phospho-Tyr-705 STAT3 was immunolocalized at the cell periphery and nucleus, whereas phospho-Ser-727 STAT3 was predominantly detected in the nucleus of polyamine-depleted cells. Sustained phosphorylation of STAT3 at tyrosine residues was observed in polyamine-depleted cells after exposure to TNF-alpha. Inhibition of STAT3 activation by AG490 or cell-membrane-permeant inhibitory peptide (PpYLKTK; where pY represents phospho-Tyr) increased the sensitivity of polyamine-depleted cells to apoptosis. Expression of DN-STAT3 (dominant negative-STAT3) completely eliminated the protective effect of DFMO against TNF-alpha-induced apoptosis. Polyamine depletion increased mRNA and protein levels for Bcl-2, Mcl-1 (myeloid cell leukaemia-1) and c-IAP2 (inhibitor of apoptosis protein-2). Significantly higher levels of Bcl-2 and c-IAP2 proteins were observed in polyamine-depleted cells before and after 9 h of TNF-alpha treatment. Inhibition of STAT3 by AG490 and DN-STAT3 decreased Bcl-2 promoter activity. DN-STAT3 decreased mRNA and protein levels for Bcl-2, Mcl-1 and c-IAP2 in polyamine-depleted cells. siRNA (small interfering RNA)-mediated inhibition of Bcl-2, Mcl-1 and c-IAP2 protein levels increased TNF-alpha-induced apoptosis. DN-STAT3 induced the activation of caspase-3 and PARP [poly(ADP-ribose) polymerase] cleavage in polyamine-depleted cells. These results suggest that activation of STAT3 in response to polyamine depletion increases the transcription and subsequent expression of anti-apoptotic Bcl-2 and IAP family proteins and thereby promotes survival of cells against TNF-alpha-induced apoptosis.
信号转导与转录激活因子3（STAT3）的激活在细胞存活和增殖中起着至关重要的作用。本研究的目的是阐明STAT3信号通路在保护多胺缺乏的肠上皮细胞免受肿瘤坏死因子-α（TNF-α）诱导的凋亡中的作用。α-二氟甲基鸟氨酸（DFMO）导致多胺缺乏，引起STAT3在酪氨酸705位点和丝氨酸727位点的磷酸化。磷酸化的酪氨酸705 STAT3免疫定位在细胞周边和细胞核，而磷酸化的丝氨酸727 STAT3主要在多胺缺乏细胞的细胞核中检测到。在多胺缺乏的细胞中，暴露于TNF-α后观察到STAT3在酪氨酸残基处持续磷酸化。AG490或细胞膜穿透性抑制肽（PpYLKTK；其中pY代表磷酸化酪氨酸）抑制STAT3激活，增加了多胺缺乏细胞对凋亡的敏感性。显性负性STAT3（DN-STAT3）的表达完全消除了DFMO对TNF-α诱导凋亡的保护作用。多胺缺乏增加了Bcl-2、髓样细胞白血病-1（Mcl-1）和凋亡抑制蛋白-2（c-IAP2）的mRNA和蛋白质水平。在TNF-α处理9小时之前和之后，多胺缺乏细胞中观察到Bcl-2和c-IAP2蛋白水平显著更高。AG490和DN-STAT3抑制STAT3降低了Bcl-2启动子活性。DN-STAT3降低了多胺缺乏细胞中Bcl-2、Mcl-1和c-IAP2的mRNA和蛋白质水平。小干扰RNA（siRNA）介导的Bcl-2、Mcl-1和c-IAP2蛋白水平抑制增加了TNF-α诱导的凋亡。DN-STAT3诱导多胺缺乏细胞中半胱天冬酶-3的激活和聚（ADP-核糖）聚合酶（PARP）的裂解。这些结果表明，多胺缺乏时STAT3的激活增加了抗凋亡Bcl-2和IAP家族蛋白的转录及随后的表达，从而促进细胞在TNF-α诱导的凋亡中存活。
