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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 5, 2002 as doi:10.1096/fj.02-0150fje. |
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Medical Molecular Biology Unit, Institute of Child Health, University College London, London WC1N 1EH, UK;
* Hatter Institute, University College London, UK; and
Department of Cystic Fibrosis, National Heart and Lung Institute, Imperial College London, UK
2Correspondence: Medical Molecular Biology Unit, Institute of Child Health, University College London, 30 Guilford St., London WC1N 1EH. E-mail: a.stephanou{at}ich.ucl.ac.uk
SPECIFIC AIMS
Death by apoptosis of cardiac myocytes contributes significantly to the total cell loss that follows ischemia/reperfusion injury to the heart. We have previously shown that activation of the transcription factor STAT-1 by phosphorylation on serine 727 is important in the activation of genes responsible for apoptosis in cardiac myocytes exposed to ischemia/reperfusion. The present study was performed to identify the minimal region of the STAT-1 molecule necessary for this proapoptotic activity.
PRINCIPAL FINDINGS
1. The carboxyl terminus of STAT-1 is more active than full-length STAT-1 in inducing apoptosis in cardiac myocytes
We compared apoptosis (measured as TUNEL-positive cells) in primary cultures of neonatal cardiac myocytes transfected with empty vector, full-length STAT-1 (aa 1–750) and the amino- (aa 1–694) and carboxyl-terminal (aa 350–750) fragments of STAT-1. Only transfected cells (assessed by cotransfection with ß-galactosidase) were assessed. All STAT-1 variants were expressed at similar levels by Western blot. No increase in TUNEL-positive cells was seen in any control transfectants not exposed to simulated ischemia/reperfusion. However, overexpression with the carboxyl-terminal fragment resulted in significantly (P<0.01) more apoptosis than with full-length STAT-1 (Fig. 1
a) in myocytes exposed to simulated ischemia/reperfusion. In contrast, ischemia/reperfused myocytes transfected with the amino-terminal fragment (lacking the carboxyl-terminal portion) showed significantly (P<0.01) less apoptosis than cells expressing the full-length and carboxyl-terminal constructs and were not significantly different from vector-alone.
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2. Chimeric STAT constructs confirm the death-inducing properties of the carboxyl-terminal portion of STAT-1
To confirm the apoptotic activity of the carboxyl-terminal portion of STAT-1, we constructed chimeric STAT molecules in which aa 1–296 of STAT-3 were fused to aa 297–750 of STAT-1 and aa 1–297 of STAT-1 were fused to aa 298–750 of STAT-3. Consistent with the results described above, transfection of the chimera containing the carboxyl-terminal portion of STAT-1 resulted in increased apoptosis after simulated ischemia/reperfusion (Fig. 1b
). Transfection of the construct in which the amino-terminal portion of STAT-1 was fused to the carboxyl terminus of STAT-3 did not produce increased apoptosis vs. that seen in vector-only transfectants (Fig. 1b
).
3. Isolated TAD and phosphorylation of Ser 727 is essential for the apoptosis-promoting activity of the carboxyl-terminal portion of STAT-1
To determine whether the enhanced apoptosis we observed could be produced by the isolated transcriptional activation domain (TAD) of STAT-1 in the absence of the DNA binding domain, we used a construct expressing only amino acids 691 to 750 of STAT-1. This construct enhanced the level of apoptosis induced by ischemia/reperfusion even more effectively than the full-length STAT-1 (Fig. 1c
), indicating that the effect we observed requires only the TAD. Our previous experiments have shown that phosphorylation of Ser 727, and not of Tyr 701, is important for the apoptosis inducing effects of full-length STAT-1. To confirm whether this was true for the carboxyl-terminal TAD fragment, we used a mutant construct of the TAD in which serine was changed to alanine at position 727 of the carboxyl terminus (S727A). This construct induced significantly (P<0.01) less apoptosis than the isolated wild-type TAD carboxyl-terminal STAT-1 construct and was not significantly different from that in vector-only controls (Fig. 1c
).
4. The carboxyl-terminal STAT-1 fragment is a more active trans-activator of proapoptotic genes
We previously demonstrated that STAT-1 activated the promoter of the proapoptotic Fas receptor gene. Therefore, we assessed whether the isolated carboxyl-terminal TAD domain of STAT-1 is also able to activate the Fas promoter. We observed that the isolated carboxyl-terminal STAT-1 construct enhanced the Fas promoter reporter to a much greater extent than the full-length STAT-1 construct. In contrast, the isolated carboxyl-terminal mutant S727A was less efficient in enhancing the Fas promoter (see full text online). Hence, both gene activation and enhanced apoptosis can be induced by the STAT-1 TAD in the absence of the DNA binding domain.
5. STAT-1 knockout mice express a 70 kDa carboxyl-terminal STAT-1 fragment
The STAT-1 gene, which contains 24 exons, has been inactivated by gene targeting. These mice are more susceptible to infections and have apparently lost the responsiveness to interferon 
. However, analysis of these mice indicated the expression of a residual carboxyl-terminal 70 kDa fragment in cardiac myocytes from these STAT-1 -/- animals. After exposure to simulated ischemia/reperfusion, the wild-type STAT-1 from +/+ murine myocytes and the carboxyl-terminal fragment expressed by the -/- mice were phosphorylated on Tyr 701 and Ser 727 as assessed using specific antibody-phosphorylated STAT-1 (see full text online).
6. The carboxyl-terminal STAT-1 fragment expressed in STAT-1 -/- mice confers increased susceptibility to apoptosis after ischemia/reperfusion
Neonatal cardiac myocytes isolated from the STAT-1 -/- mice, which continue to express the STAT-1 carboxyl-terminal fragment, show significantly increased apoptosis after simulated ischemia/reperfusion in vitro. Isolated perfused hearts from the -/- animals showed a significantly (P<0.005) increased area of infarction (Fig. 2
a) and a significantly increased number of apoptotic cells in the risk area (Fig. 2b
) after ischemia/reperfusion ex vivo.
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CONCLUSIONS AND SIGNIFICANCE
We have previously demonstrated a role for ischemia/reperfusion-mediated activation of full-length STAT-1 in apoptosis of cardiac cells. Thus, phosphorylation of STAT-1 on Ser 727 leads to activation of proapoptotic genes such as Fas and FasL together with down-regulation of expression of antiapoptotic genes such as Bcl2 and Bclx and induces enhanced apoptosis on exposure to ischemia/reperfusion injury. In the present study, we show that the carboxyl-terminal fragment of STAT-1, which contains only the TAD and lacks the DNA binding domain, induces more pronounced apoptosis than that produced by full-length STAT-1.
Activation of caspases, the executioners of apoptosis, can be detected during ischemia and, more markedly, during subsequent reperfusion. Caspases cleave full-length STAT-1 at Asp 694 and therefore produce an amino-terminal fragment containing the DNA binding domain and the carboxyl-terminal TAD. Although the released amino-terminal fragment may act as a dominant-negative inhibitor of residual full-length STAT-1, the data reported here suggest that by further activation of proapoptotic genes, the carboxyl-terminal fragment may reinforce the apoptotic process (Fig. 3
). Previous reports show that caspase-cleaved products accentuate apoptosis. For example, caspase cleavage of the p65 subunit of NF-
B produces a fragment that acts as a dominant-negative inhibitor of the antiapoptotic action of NF-B. Similarly, caspase cleavage of the receptor interacting protein (RIP) generates a fragment that inhibits the antiapoptotic action of RIP in mediating TNF-induced NF-B activation.
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Removal of the amino-terminal DNA binding domain means that the carboxyl-terminal TAD fragment of STAT-1 must induce apoptosis and gene expression by functioning as a coactivator by interacting with other DNA-bound factors. This is supported by the increased activity of the carboxyl-terminal fragment on the Fas promoter compared with the wild-type STAT-1. Full-length STAT-1 is known to interact with transcriptional coactivators CBP, BRCA1, and MCM5, and this requires Ser 727 phosphorylation. However, since none of these coactivators of the full-length protein contains a DNA binding domain, it is likely that STAT-1 must also interact with an unknown DNA binding domain-containing factor. Experiments are in progress to identify those factors expressed in cardiac cells and that interact with the carboxyl-terminal TAD of STAT-1.
The apoptotic program is characterized by interactions between pro- and antiapoptotic factors. Caspase-mediated generation of the proapoptotic carboxyl-terminal fragment of STAT-1 suggests that this forms an amplifying loop to perpetuate apoptosis in hearts exposed to ischemia/reperfusion injury (Fig. 3)
. The requirement of both full-length STAT-1 and the carboxyl-terminal TAD for Ser 727 phosphorylation does, however, suggests that strategies based on inhibiting the responsible p38 kinase may have therapeutic potential.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0150fje; to cite this article, use FASEB J. (September 5, 2002) 10.1096/fj.02-0150fje 
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