Immediate Early Genes

C-fos, C-jun, and Activator Protein-1

Members of the IEG families, such as c-fos, c-jun, and zinc finger (zif), were among the first genes observed after ischemia onset and are known to be highly upregulated within minutes (72). Mitochondrial membrane depolarization, calcium uptake, and release of factors (e.g., cytochrome c) can ultimately activate activator protein-1 (AP-1) to induce gene expression (73). The induction of IEGs includes several consecutive steps. Ischemia-associated increases in extracellular glutamate and intracellular calcium can lead to the activation of protein kinases (e.g., protein kinase A and protein kinase C), which can phosphorylate DNA binding proteins. DNA binding proteins recognize specific binding domains, which then initiate transcription of the downstream target genes (74). The c-fos gene, an IEG that encodes the Fos protein, was found to be upregulated within 30 min after stroke onset (72). Fos protein contains several important structural features, such as a DNA binding region and a leucine zipper. The latter is a region containing leucine residues every 7 amino acids and forms an a-helix with the leucines aligned on one side. Leucine zippers can align with other proteins containing this structure (such as Jun protein families, another IEG) to form dimers. These dimers bind to a specific DNA region known as the AP-1 domain, which regulates the expression of a number of target genes, including the so-called late response genes. Combinations of c-fos and c-jun family proteins form various dimers that consist of different subunits under certain ischemic circumstances. The composition of the dimer might determine whether the late response gene is turned on or off (75). AP-1, a transcription factor, is formed through dimerization of fos and jun and is thought to be an important component of brain responses to ischemia (76). Whether fos/jun/AP-1 is beneficial or damaging following ischemia is still unclear. Consistent with a beneficial effect, hypothermia, an established neuroprotectant in the laboratory, was associated with early increases in c-fos expression and AP-1 DNA binding activity in peri-infarct cortex (77). AP-1 activity is also related to neuronal cell tolerance in ischemia models (78). Furthermore, compounds known to protect the brain were also associated with c-fos upregulation (79), and suppression of c-fos by an antisense oligonucleotide led to increased tissue damage following cerebral ischemia (80). On the other hand, other studies have shown that transgenic mice that overexpress SOD1 and are protected from experimental stroke have attenuated AP-1 activation, which would be more consistent with a damaging effect (81). The time and regional differences in AP1-binding protein complexes appear to influence final postischemic outcome (82) and might explain why c-jun expression has been linked to neuronal apoptosis (83) and to neuronal survival (84 ).

Activating Transcription Factor

The activating transcription factor (ATF) family includes ATF-1, ATF-2, ATF-3, among which ATF-2 and ATF-3 are reported to be induced by focal cerebral ischemia (85,86). ATF encodes a member of the cAMP response element-binding (CREB) protein family of transcription factors. Members of this family share the same DNA-binding domain (leucine zipper domain) and bind to consensus DNA sequence (TGACGTCA) to form a variety of selective heterodimers with each other via the leucine zipper region (87). ATF-3 is not expressed in the brain under normal conditions but is markedly induced after permanent focal cerebral ischemia. Recent data revealed concurrent expression of ATF-3 and phospho-c-jun in neurons (85). ATF-3 represses transcription as a homodimer and activates transcription as a heterodimer with ATF-2 and Jun families (88). Furthermore, ATF-3 has many characteristics of IEGs (89). After MCAO, 98% of ATF-3 immu-noreactive neurons simultaneously expressed damage-induced neuronal endopeptidase (DINE) mRNA. In that DINE expression promotes antioxidant activity, these observations suggest that ATF-3 activation upregulates DINE expression and is neuroprotective under ischemic conditions (54 ).

Additional Immediate Early Genes

Several other IEGs, such as nerve growth factor-induced gene A (NGFI-A, also known as krox24, egr1, and zif268) (74,90) and the orphan nuclear receptor NGFI-B (also known as nur/77) (90,91), have been identified with different structural elements and functions following focal cerebral ischemia. NGFI-B is a member of the steroid receptor superfamily and might have a role coordinating the regulation of the hypothalamic-pituitary-adrenal axis (74). The NGFI-A protein contains a zif that binds to a guanylate-rich DNA consensus sequence. It has been suggested that prolonged NGFI-A expression after an ischemic insult is associated with delayed neuronal degeneration (92 ).

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