Abstract
Angiogenesis is a well-characterised response to the metabolic stresses that occur during ischaemia/reperfusion, but the signalling pathways that regulate it are poorly understood. We tested whether activation of mitogen-activated protein kinases (MAPKs) was involved in regulating the expression of pro-angiogenic growth factors by the metabolic stresses associated with ischaemia/reperfusion in H9c2 rat cardiomyoblasts. Metabolic stress had no effect on vascular endothelial growth factor (VEGF) mRNA levels, but recovery after metabolic inhibition led to a strong induction of VEGF-A mRNA (3.8 ± 0.5-fold at 4 h), a modest rise in VEGF-C mRNA levels (1.7 ± 0.3-fold at 4 h), with no effect on VEGF-B or -D. A VEGF-A promoter reporter construct was unresponsive to metabolic inhibition/recovery and increases in VEGF-A mRNA were not blocked by the transcription inhibitor actinomycin D suggesting that increases in VEGF mRNA were due to enhanced VEGF-A mRNA stability. In addition, studies using reporter constructs demonstrated that regions within the 5′ untranslated region (UTR) contributed to enhanced mRNA stability following recovery from metabolic stress. Increases in VEGF-A mRNA were abolished by inhibition of extracellular signal-regulated kinase or c-jun N-terminal kinase MAPKs, suggesting that these kinases may promote angiogenesis in response to metabolic stress during ischaemia/reperfusion by increasing VEGF-A message stability.
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Abbreviations
- ATF2:
-
Activating transcription factor 2
- ATP:
-
Adenosine 5′ triphosphate
- CAT:
-
Chloramphenicol acetyl transferase
- dCTP:
-
Deoxy cytosine 5′ triphosphate
- ERK:
-
Extracellular signal-regulated kinase
- JNK:
-
c-jun N-terminal kinase
- MAPK:
-
Mitogen-activated protein kinase
- MKK6:
-
MAPK kinase 6
- PMA:
-
Phorbol myristoyl acetate
- RT-PCR:
-
Reverse transcription polymerase chain reaction
- SEM:
-
Standard error of the mean
- VEGF:
-
Vascular endothelial growth factor
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Acknowledgments
This work was supported, in part, by a grant from the British Heart Foundation (PG#98111) and studentships from the Biotechnology and Biological Sciences Research Council (#08170) and Medical Research Council (#G78/6321). We would like to thank Dr. Ben Zion-Levy for providing us with the pV3.4-CAT construct.
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Miller, B.W., Hay, J.M., Prigent, S.A. et al. Post-transcriptional regulation of VEGF-A mRNA levels by mitogen-activated protein kinases (MAPKs) during metabolic stress associated with ischaemia/reperfusion. Mol Cell Biochem 367, 31–42 (2012). https://doi.org/10.1007/s11010-012-1316-9
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DOI: https://doi.org/10.1007/s11010-012-1316-9