Distinct Regulations of HO-1 Gene Expression for Stress Response and Substrate Induction

ABSTRACT

Heme oxygenase 1 (HO-1) is the key enzyme for heme catabolism and cytoprotection. Whereas HO-1 gene expression in response to various stresses has been investigated extensively, the precise mechanisms by which HO-1 gene expression is regulated by the HO-1 substrate heme remain elusive. To systematically examine whether stress-mediated induction and substrate-mediated induction of HO-1 utilize similar or distinct regulatory pathways, we developed an HO-1–DsRed-knock-in reporter mouse in which the HO-1 gene is floxed by loxP sites and the DsRed gene has been inserted. Myeloid lineage-specific recombination of the floxed locus led to fluorescence derived from expression of the HO-1–DsRed fusion protein in peritoneal macrophages. We also challenged general recombination of the locus and generated mice harboring heterozygous recombinant alleles, which enabled us to monitor HO-1–DsRed expression in the whole body in vivo and ex vivo. HO-1 inducers upregulated HO-1–DsRed expression in myeloid lineage cells isolated from the mice. Notably, analyses of peritoneal macrophages from HO-1–DsRed mice lacking NRF2, a major regulator of the oxidative/electrophilic stress response, led us to identify NRF2-dependent stress response-mediated HO-1 induction and NRF2-independent substrate-mediated HO-1 induction. Thus, the HO-1 gene is subjected to at least two distinct levels of regulation, and the available lines of evidence suggest that substrate induction in peritoneal macrophages is independent of CNC family-based regulation.

KEYWORDS:

• HO-1
• NRF2
• stress response
• hemin

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ACKNOWLEDGMENTS

We thank Fumiki Katsuoka for discussion and advice. We also thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support.

This work was supported in part by MEXT/JSPS KAKENHI (grant no. 19H05649 to M.Y. and grant no. 19K07340 and 17KK0183 to T.S.), the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under grant no. JP20am0101095 (M.Y.), P-CREATE from AMED under grant no. JP20cm0106101 (M.Y.), and the Takeda Science Foundation (M.Y. and T.S.).

A.Z., T.S., and M.Y. designed the research and analyzed the data. A.Z., S.A., and E.N. conducted the experiments. N.S. synthesized GSK360A compound. M.B.S. synthesized compounds of CDDO-2P-Im and CDDO-3P-Im. T.H., K.I., and M.Y. generated the HO-1 flox mice. A.Z., T.S., and M.Y. wrote the paper.

M.B.S. is an employee and shareholder of Triterpenoid Therapeutics. The other authors declare no competing financial interests.