Abstract
Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.
Highlights
Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase and Nitric oxide (NO) production in endothelial cells
NO generation was markedly increased in only the L-arginine treatment group; CRIF1 knockdown significantly inhibited L-arginine-induced NO production. These results suggest that CRIF1 deficiency limited the common substrate L-arginine to NO synthesis and resulted in endothelial nitric oxide synthase (eNOS) uncoupling
These results were determined by the inhibition of both de novo and recycling pathways, which resulted in eNOS uncoupling and reactive oxygen species (ROS) generation (Fig. 7)
Summary
Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Exactly how CRIF1-deficiency-induced mitochondrial dysfunction mediates the uncoupling of eNOS vascular endothelial cells remains unknown. We used siRNA-mediated knockdown of CRIF1 to explore the relative roles of CRIF1 deficiency and mitochondrial dysfunction in BH4 biosynthesis and recycling, as well as eNOS activity in vascular endothelial cells.
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