The downregulation of NADPH oxidase Nox4 during hypoxia in hemangioendothelioma cells: a possible role of p22^phox on Nox4 protein stability

Abstract

NADPH oxidase (Nox) 4 produces H₂O₂ by forming a heterodimer with p22^phox and is involved in hemangioendothelioma development through monocyte chemoattractant protein-1 (MCP-1) upregulation. Here, we show that Nox4 protein levels were maintained by p22^phox in hemangioendothelioma cells and Nox4 protein stability was dependent on p22^phox coexpression. Conversely, the degradation of Nox4 monomer was enhanced by p22^phox knockdown. Under hypoxic conditions in hemangioendothelioma cells, p22^phox was downregulated at the mRNA and protein levels. Downregulation of p22^phox protein resulted in the enhanced degradation of Nox4 protein in hypoxia-treated hemangioendothelioma cells. In contrast, Nox2, a Nox isoform, was not altered at the protein level under hypoxic conditions. Nox2 exhibited a higher affinity for p22^phox compared with Nox4, suggesting that when coexpressed with Nox4 in the same cells, Nox2 acts as a competitor. Nox2 knockdown restored Nox4 protein levels partially reduced by hypoxic treatment. Thus, Nox4 protein levels were attenuated in hypoxia-treated cells resulting from p22^phox depletion. MCP-1 secretion was decreased concurrently with hypoxia-induced Nox4 downregulation compared with that under normoxia.

Keywords:

• NADPH oxidase 4 (Nox4)
• reactive oxygen species (ROS)
• monocyte chemoattractant protein-1 (MCP-1)
• hemangioendothelioma
• p22^phox
• hypoxia

Acknowledgments

We are grateful to Aya Morihara (Kawasaki Medical School, Japan) for technical assistance and to the Central Research Institute of Kawasaki Medical School for technical support.

Author contributions

Conceptualization: KM. Investigation: KM, SO, AY, and CK. Project administration: KM and SO. Visualization: KM. Writing – original draft: KM. Writing – review & editing: KM, SO, AY, MK, TK, MI, MT, and FK.

Disclosure statement

The authors declare that they have no conflicts of interest.

Data availability statement

All the data are contained within the manuscript.

Additional information

Funding

This study was supported in part by JSPS KAKENHI grant numbers JP17K08637 (KM) and JP19K07676 (AY) in part by the Wesco Scientific Promotion Foundation (KM), in part by the Takeda Science Foundation (KM), in part by the Okayama Medical Foundation (KM), and in part by Research Project Grants [nos. R01S-003 (KM) H30Y-002 (SO), R01B-081 (FK), and R01B-093 (AY)] from the Kawasaki Medical School.