STAT1-induced Apoptosis Is Mediated by Caspases 2, 3, and 7

Abstract

STAT1(signal transducer and activator of transcription 1) has been implicated as a mediator of a variety of biological responses in response to stimulation by specific growth factors and cytokines. To understand better the role of STAT1 in the interferon-␥ (IFN-␥)-induced phenotype, we generated an active form of STAT1 (STAT1C) by substituting Cys residues for both Arg-656 and Asn-658 within the C-terminal loop of the STAT1 SH2 domain. The IFN-␥ activation site element was stimulated and bound efficiently by STAT1C without IFN-␥ treatment. STAT1C was found to be tyrosine-phosphorylated in the nucleus for more than 30 h after IFN-␥ stimulation. STAT1-negative U3A cells reexpressing STAT1C showed retarded cell growth and underwent apoptosis when treated with IFN-␥. Further analysis demonstrated that apoptosis was preceded by proteolytic cleavage of caspases 2, 3, and 7, and wild type STAT1 also induced cleavage of caspase 7 when expressed in STAT1-negative U3A cells, indicating that STAT1C augments potential activity of wild type STAT1. Studies with cycloheximide treatment showed that protein synthesis induced in the first 24 h after IFN-␥ treatment was required for apoptosis under these conditions. Finally, we found that STAT1C-induced apoptosis was, in part, mediated by caspase 2, 3, and 7 because benzyloxycarbonyl-valyl-aspartyl-valyl-alanyl-aspartic acid fluoromethyl ketone (Z-VDVAD-FMK) treatment partially blocked apoptosis. These results suggest that prolonged nuclear localization of activated STAT1 results in apoptosis involving specific regulation of caspase pathway.Signal transducer and activator of transcription (STAT) 1 1 plays an important role in such IFN-␥-induced biological responses as tumor surveillance (1-4), the immune response (5, 6), and cell growth control (7,8). It has been suggested that it serves as a tumor suppressor by activating proapoptotic pathways (3, 9 -14).In response to IFN-␥ stimulation, STAT1 becomes phosphorylated on Tyr-701 and forms a homodimer through reciprocal SH2-phosphotyrosine interactions. Phosphorylation on Tyr-701 is required for subsequent nuclear translocation, DNA binding, and gene activation (15)(16)(17). Tyrosine phosphorylation of STAT1 is regulated both by Jak1/Jak2 protein-tyrosine kinases and by protein-tyrosine phosphatase. Significantly, treatment with sodium vanadate, a protein-tyrosine phosphatase inhibitor, can sustain the phosphorylation of Tyr-701, preventing down-regulation of STAT1 transcriptional activity (18,19). Biochemical analysis has demonstrated that dephosphorylated STAT1 is rapidly exported back into the cytoplasm and takes part in subsequent activation-inactivation cycles and that loss of dephosphorylation leads to prolonged STAT1 activation in the nucleus (20 -27).STAT1 has been implicated in apoptosis in response to IFN-␥ stimulation. Mutant forms of STAT1 lacking the N-terminal 62 amino acid residues or carrying a single amino acid substitution of Ala-31 for Arg-31 inhibited STAT1 tyrosine dephosphorylation and greatly enhanced the anti...

Innate Immunity in Pluripotent Human Cells

“…The specific action of type I IFN-mediated PI3K signaling on human pluripotent cells remains unclear and thus requires further examination. More importantly, several lines of evidence suggest that STAT1 signaling plays a pro-apoptotic role by upregulating the expression of caspases (43)(44)(45) or by repressing pro-survival NF-B signaling (46). Interestingly, hESCs are primed to undergo rapid apoptosis (47).…”

Crif1 is a novel transcriptional coactivator of STAT3

“…Whereas STAT3-mediated transcriptional activity by OSM stimulation was readily observed in the Crif1 þ /D MEFs in a dose-dependent manner, its activity was severely decreased in the Crif1 À/D MEFs ( Figure 6B). Since OSM activates other factors besides STAT3 (Kuropatwinski et al, 1997), and the m67-luc reporter is also responsive to STAT1 (Sironi and Ouchi, 2004), the residual response by OSM in Crif1 À/D MEFs might be due to STAT1 activation. To exclude the influence of STAT1 after OSM stimulation, we used a constitutively active form of STAT3, STAT3-C (bridged by S-S linkages between cysteines, instead of phosphotyrosines), which can induce cellular transformation of fibroblasts (Bromberg et al, 1999).…”

Oncostatin M inhibits myoblast differentiation and regulates muscle regeneration

“…In contrast, both the full-length and an N-terminus-truncated STAT1 (i.e., aa 317-750) interacted with His-MEF2 ( Figure 5D), suggesting that the C-terminal portion of STAT1 is involved in interacting with MEF2. To reveal the functional significance of MEF2 interaction with STAT1, we first measured the effect of a constitutively active STAT1 (i.e., STAT1c) on two MEF2-dependent luciferase reporter genes: one is 3xMEF2-Luc used in Figure 3 and the other is a luciferase gene driven by 133-bp proximal mouse myogenin promoter (i.e., G133-luc) [57,62]. As shown in Figure 5E, STAT1c inhibited the activity of both MEF2-dependent reporters.…”