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Ischemia and reperfusion enhance ATF-2 and c-Jun binding to cAMP response elements and to an AP-1 binding site from
H Morooka1, J V Bonventre, C M Pombo
1Medical Services, Massachusetts General Hospital, Charlestown 02129, USA.
Abstract:
The transcription factors controlling the complex genetic response to ischemia and their modes of regulation are poorly understood. We found that ATF-2 and c-Jun DNA binding activity is markedly enhanced in post-ischemic kidney or in LLC-PK1 renal tubular epithelial cells exposed to reversible ATP depletion. After 40 min of renal ischemia followed by reperfusion for as little as 5 min, binding of ATF-2 and c-Jun, but not ATF-3 or CREB (cAMP response element binding protein), to oligonucleotides containing either an ATF/cAMP response element (ATF/CRE) or the jun2TRE from the c-jun promoter, was significantly increased. Binding to jun2TRE and ATF/CRE oligonucleotides occurred with an identical time course. In contrast, nuclear protein binding to an oligonucleotide containing a canonical AP-1 element was not detected until 40 min of reperfusion, and although c-Jun was present in the complex, ATF-2 was not. Incubating nuclear extracts from reperfused kidney with protein phosphatase 2A markedly reduced binding to both the ATF/CRE and jun2TRE oligonucleotides, compatible with regulation by an ATF-2 kinase. An ATF-2 kinase, which phosphorylated both the transactivation and DNA binding domains of ATF-2, was activated by reversible ATP depletion. This kinase coeluted on Mono Q column chromatography with a c-Jun amino-terminal kinase and with the peak of stress-activated protein kinase, but not p38, immunoreactivity. In conclusion, DNA binding activity of ATF-2 directed at both ATF/CRE and jun2TRE motifs is modulated in response to the extreme cellular stress of ischemia and reperfusion or reversible ATP depletion. Phosphorylation-dependent activation of the DNA binding activity of ATF-2, which appears to be regulated by the stress-activated protein kinases, may play an important role in the earliest stages of the genetic response to ischemia/reperfusion by targeting ATF-2 and c-Jun to specific promoters, including the c-jun promoter and those containing ATF/CREs.
Insights
The transcription factors ATF-2 and c-Jun show increased DNA binding activity during kidney ischemia and reperfusion. This activation is phosphorylation-dependent and regulated by stress-activated protein kinases, influencing early genetic responses.
Area of Science:
- Molecular Biology
- Cellular Stress Response
- Renal Physiology
Background:
- The genetic regulation underlying ischemia-reperfusion injury is not fully understood.
- Transcription factors play a crucial role in cellular responses to stress.
Purpose of the Study:
- To investigate the role of transcription factors ATF-2 and c-Jun in the genetic response to renal ischemia-reperfusion.
- To elucidate the regulatory mechanisms controlling their DNA binding activity.
Main Methods:
- Assessing DNA binding activity of transcription factors using oligonucleotide probes (ATF/CRE, jun2TRE, AP-1).
- Utilizing renal tubular epithelial cells (LLC-PK1) and post-ischemic kidney models.
- Investigating the role of protein phosphatases and kinases, including ATF-2 kinase and stress-activated protein kinases (SAPKs).
Main Results:
- ATF-2 and c-Jun DNA binding activity significantly increased within minutes of reperfusion following ischemia.
- This enhanced binding targeted specific DNA motifs (ATF/CRE, jun2TRE) but not canonical AP-1 elements early on.
- An ATF-2 kinase, activated by ATP depletion and co-eluting with SAPKs, was identified as a key regulator.
Conclusions:
- Phosphorylation-dependent activation of ATF-2 DNA binding is a critical early event in the ischemia-reperfusion response.
- Stress-activated protein kinases likely regulate this activation pathway.
- ATF-2 and c-Jun target specific gene promoters, contributing to the early genetic reprogramming during cellular stress.