The occlusion of blood vessels in the setting of cardiovascular disease leads to ischemia, initiating processes that promote neovascularization to restore blood flow and preserve tissue function. Our in vivo studies show that Osteopontin (OPN) is a critical mediator of post-ischemic neovascularization and that ischemia-induced increases in OPN expression are H
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O
2
-dependent. However, the mechanisms by which H
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O
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increases OPN expression are poorly defined. To determine if H
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O
2
mediates transcriptional, post-transcriptional, and/or translational regulation of OPN expression in vitro, we used rat aortic smooth muscle cells as an in vitro system and stimulated with H
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O
2
. Dose response studies showed OPN expression increased with 50 μM H
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O
2
(51.9%±2.2, p<0.05). Using 50 μM H
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O
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, we performed time courses and measured OPN mRNA by qRT-PCR and protein by Western blot. OPN mRNA levels significantly increased in response to H
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O
2
at 8 (70.4%±5.7, p<0.05) and 18 hours (120.2%±5.2, p<0.005). Interestingly, the increases in OPN protein expression in response to H
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O
2
occurred in an unusual bi-phasic pattern, with significant increases at 6 (96.9%±1.5, p<0.001) and 18 hours (234.0%±3.6, p<0.001), with a return to baseline in between. An increase in OPN mRNA preceded the increase in OPN protein at 18 hours, suggesting transcriptional regulation; however, the acute increase in OPN at 6 hours was not preceded by increased mRNA, suggesting multiple mechanisms of OPN regulation by H
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O
2
. To determine if the increase in OPN at 6 hours is due to increased mRNA stability or translation, we performed an RNA stability assay. H
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stimulation did not alter OPN stability or the rate of OPN RNA degradation, leading us to conclude the increase in OPN expression at 6 hours is due to increased translation. Further studies reveal H
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2
-mediated increases in phosphorylation of 4E-BP1 at the redox-sensitive Ser65 site (89.4%±6.1, p<0.05), allowing for the subsequent release of eukaryotic initiation factor eIF4E and increased phosphorylation at Ser209 (139.2%±3.9, p<0.05), resulting in increased OPN translation. In conclusion, H
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O
2
enhances OPN expression through acute increases in translation, while long-term increases in OPN occur through increased transcriptional regulation.
The poly(A) tail inhibits the assembly of a 3'-to-5' exonuclease in an in vitro RNA stability system.