Interferon γ induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells
Interferon γ induces upregulation and activation of caspases 1, 3, and 8 to produce apoptosis in human erythroid progenitor cells
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DOI：
10.1182/blood.v93.10.3309.410k04_3309_3316
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发表时间：
1999-05-15
期刊：
影响因子：
20.3
通讯作者：
Krantz, SB
中科院分区：
文献类型：
--
作者：
Dai, CH;Krantz, SB
干扰素γ（IFN γ）诱导纯化的人红系集落形成细胞（ECFC）凋亡并抑制细胞生长。Fas（APO-1; CD 95）和Fas配体（FasL）介导IFN γ诱导的细胞凋亡，因为IFN γ显著上调Pas，而Pas配体组成性地存在于ECFC中，并且FasL的中和大大减少了细胞凋亡。由于半胱天冬酶从其休眠酶原形式转化为活性酶在转导导致细胞凋亡的级联中具有关键作用，因此我们对正常人和IFN γ处理的第6天ECFC中半胱天冬酶的表达和活化进行了进一步研究，以更好地理解IFN γ在产生这种细胞死亡中的作用机制。RNA酶保护试验表明，caspase-1，-2，-6，-8，和-9的mRNA被IFN γ上调，而caspase-5和-7的mRNA没有增加。Western印迹显示，FLICE/caspase-8被上调，并通过与IFN γ孵育24小时激活。FADD与IFN γ孵育后没有类似的改变。ICE/半胱天冬酶-1的蛋白质印迹，这可能是扩增初始FLICE激活信号所必需的，显示用IFN γ处理24小时后pro-ICE表达显著增加，pro-ICE的切割也增加。还研究了负责聚（ADP）核糖聚合酶（PARP）蛋白水解切割的CPP 32/apopain/caspase-3，用IFN γ处理ECFC导致caspase-3浓度在24小时内增加，并在48小时内明显诱导酶活化，这通过其p17-kD肽片段的出现来鉴定。PARP的裂解通过89-kD PARP裂解产物的明显增加来证明，这与IFN γ处理的细胞中的半胱天冬酶-3活化几乎同时观察到，而未处理的ECFC几乎没有变化。将胱天蛋白酶蛋白的肽抑制剂DEVD-factase、DEVD-cho、YVAD-cho和IETD-factase与ECFC一起孵育，以获得胱天蛋白酶参与IFN γ诱导的细胞凋亡的进一步证据。FLICE/caspase-8和CPP 32/caspase-3的活化和PARP的裂解明显受到抑制，但IFN γ诱导的细胞凋亡引起的细胞生长减少仅部分被抑制剂的存在所阻断。这些结果表明，IFN γ作用于ECFC不仅上调Pas，而且选择性上调caspase-1、-3和-8，其被激活并产生凋亡，而Fast和FADD的浓度没有明显变化。(C)1999年，美国血液学会。
Interferon gamma (IFN gamma) induces apoptosis in purified human erythroid colony-forming cells (ECFC) and inhibits cell growth. Fas (APO-1; CD95) and Fas ligand (FasL) mediate apoptosis induced by IFN gamma, because Pas is significantly upregulated by IFN gamma, whereas Pas ligand is constitutively present in the ECFC and neutralization of FasL greatly reduces the apoptosis. Because conversion of caspases from their dormant proenzyme forms to active enzymes has a critical role in transducing a cascade leading to apoptosis, we performed further studies of the expression and activation of caspases in normal human and IFN gamma-treated day-6 ECFC to better understand the mechanism of IFN gamma action in producing this cell death. RNase protection assays showed that the caspase-1, -2, -6, -8, and -9 mRNAs were upregulated by IFN gamma, whereas the caspase-5 and -7 mRNAs were not increased. Western blots showed that FLICE/caspase-8 was upregulated and activated by 24 hours of incubation with IFN gamma. FADD was not similarly altered by incubation with IFN gamma. Western blots of ICE/caspase-1, which might be required for amplification of the initial FLICE activation signal, showed that pro-ICE expression significantly increased after treatment with IFN gamma for 24 hours and cleavage of pro-ICE also increased. CPP32/apopain/caspase-3, responsible for the proteolytic cleavage of poly (ADP) ribose polymerase (PARP), was also studied and treatment of ECFC with IFN gamma resulted in an increased concentration of caspase-3 by 24 hours and a clear induction of enzyme activation by 48 hours, which was identified by the appearance of its p17-kD peptide fragment. The cleavage of PARP was demonstrated by an obvious increase of the 89-kD PARP cleavage product, which was observed at almost the same time as caspase-3 activation in the IFN gamma-treated cells, whereas untreated ECFC showed little change. Peptide inhibitors of the caspase proteins, DEVD-fmk, DEVD-cho, YVAD-cho, and IETD-fmk, were incubated with the ECFC to obtain further evidence for the involvement of caspases in IFN gamma-induced apoptosis. The activation of FLICE/caspase-8 and CPP32/caspase-3 and cleavage of PARP clearly were inhibited, but the reduction of cell growth due to apoptosis, induced by IFN gamma, was only partially blocked by the presence of the inhibitors. These results indicate that IFN gamma acts on ECFC not only to upregulate Pas, but also to selectively upregulate caspases-1, -3, and -8, which are activated and produce apoptosis, whereas the concentrations of Fast and FADD are not demonstrably changed. (C) 1999 by The American Society of Hematology.