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Translation-linked mRNA destabilization accompanying serum-induced Nox4 expression in human endothelial cells
Hitesh Peshavariya1, Fan Jiang, Caroline J Taylor
1Bernard O'Brien Institute of Microsurgery, University of Melbourne, Victoria 3065, Australia.
Abstract:
NADPH oxidase is involved in cell signaling, regulating proliferation of vascular cells, especially in endothelium. The Nox4 catalytic subunit has a major role in endothelial cells, but growth arrest of cultured endothelial cells following serum deprivation paradoxically increases mRNA for Nox4. We investigated the relationships between Nox4 mRNA stability and protein expression in human microvascular endothelial cells. Serum starvation increased the steady-state level of Nox4 mRNA but paradoxically diminished Nox4 protein expression. mRNA transcription in the absence of serum is maintained by the p38MAP kinase pathway, for inhibition of p38MAP kinase reduced both Nox4 mRNA and Nox4 promoter activity. In serum-starved cells, reintroduction of serum increased Nox4 protein levels within 30 min and up to 24 h. In contrast, the mRNA decreased equally rapidly after serum stimulation. Inhibition of Nox4 translation by cycloheximide blocked serum-induced mRNA degradation and Nox4 protein synthesis, and actinomycin-D also delayed Nox4 mRNA decay. Therefore, Nox4 mRNA level falls after serum stimulation because of a translation-initiated mRNA destabilization program. Clearly Nox4 mRNA is regulated at both transcriptional and post-transcriptional levels, and the steady state level of Nox4 mRNA does not accurately reflect Nox4 protein abundance and functions, with implications for regulation of cell proliferation and survival.
Insights
NADPH oxidase 4 (Nox4) mRNA levels do not reflect protein abundance in endothelial cells. Serum starvation increases Nox4 mRNA but decreases protein, while serum reintroduction rapidly decreases mRNA and increases protein via translation-initiated decay.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- NADPH oxidase (NOX) enzymes are critical in cell signaling and vascular endothelial cell proliferation.
- The Nox4 subunit plays a significant role in endothelial cells, but its regulation is complex.
- Serum deprivation paradoxically increases Nox4 mRNA while decreasing its protein levels in cultured endothelial cells.
Purpose of the Study:
- To investigate the relationship between Nox4 mRNA stability and protein expression in human microvascular endothelial cells.
- To elucidate the regulatory mechanisms controlling Nox4 expression under varying serum conditions.
Main Methods:
- Utilized human microvascular endothelial cells.
- Manipulated serum concentration (starvation and reintroduction).
- Assessed mRNA and protein levels of Nox4.
- Employed p38MAP kinase pathway inhibitors, cycloheximide (translation inhibitor), and actinomycin-D (transcription inhibitor).
Main Results:
- Serum starvation elevated steady-state Nox4 mRNA levels but reduced Nox4 protein expression.
- p38MAP kinase pathway inhibition decreased both Nox4 mRNA and promoter activity, indicating transcriptional regulation.
- Serum reintroduction rapidly increased Nox4 protein while decreasing Nox4 mRNA.
- Inhibition of translation and mRNA decay studies revealed a translation-initiated mRNA destabilization program following serum stimulation.
Conclusions:
- Nox4 expression is regulated at both transcriptional and post-transcriptional levels.
- Steady-state Nox4 mRNA levels do not accurately predict Nox4 protein abundance or function.
- These findings have significant implications for understanding the regulation of endothelial cell proliferation and survival.
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