A role for cytoglobin in regulating intracellular hydrogen peroxide and redox signals in the vasculature

Abstract

The oxidant hydrogen peroxide serves as a signaling molecule that alters many aspects of cardiovascular functions. Recent studies suggest that cytoglobin, a hemoglobin expressed in the vasculature, may promote electron transfer reactions with proposed functions in hydrogen peroxide decomposition. Here, we determined the extent to which cytoglobin regulates intracellular hydrogen peroxide and established mechanisms. We found that cytoglobin decreased the hyperoxidation of peroxiredoxins and maintained the activity of peroxiredoxin 2 following challenge with exogenous hydrogen peroxide. Cytoglobin promoted a reduced intracellular environment and facilitated the reduction of the thiol-based hydrogen peroxide sensor Hyper7 after bolus addition of hydrogen peroxide. Cytoglobin also limited the inhibitory effect of hydrogen peroxide on glycolysis and reversed the oxidative inactivation of the glycolytic enzyme GAPDH. Our results indicate that cytoglobin in cells exists primarily as oxyferrous cytoglobin (CygbFe2+O2) with its cysteine residues in the reduced form. We found that the specific substitution of one of two cysteine residues on cytoglobin (C83A) inhibited the reductive activity of cytoglobin on Hyper7 and GAPDH. Carotid arteries from cytoglobin knockout mice were more sensitive to glycolytic inhibition by hydrogen peroxide than arteries from wildtype mice. Together, these results support a role for cytoglobin in regulating intracellular redox signals associated with hydrogen peroxide through oxidation of its cysteine residues, independent of hydrogen peroxide reaction at its heme center.

Nuclear cytoglobin associates with HMGB2 and regulates DNA damage and genome-wide transcriptional output in the vasculature

“…Overall, these results suggest that cytoglobin is required in the nucleus to affect gene programs related to DNA repair and mitigate both oxidant and replicativeinduced DNA damage. Moreover, we show that these effects occur through a mechanism separate from the previously established oxidant scavenging activity of cytoglobin 10 .…”

“…As such, cytoglobin has been proposed to regulate NO dependent-vasorelaxation and NO-dependent cytotoxicity in vessels during conditions associated with increased inducible NO synthase (NOS2) expression 4,8,9 . More recently, we found that isolated vessels from cytoglobin knockout mice also displayed decreased rates of hydrogen peroxide consumption, suggesting a role for cytoglobin as a peroxidase in the vasculature 10 . In models of vascular injury, we proposed that loss of cytoglobin limited the survival capacity of a cell population that expresses cytoglobin 2-3 days after initiation of the injury 9 .…”

“…Although we obtained clear evidence of regulated nuclear accumulation of cytoglobin, this did not establish that nuclear cytoglobin was required to inhibit hydrogen peroxide induced DNA damage and regulate gene expression. This is not trivial since we recently found that cytoglobin scavenges intracellular hydrogen peroxide through redox cycling of its two cysteine residues 10 . Thus, in this case even cytosolic cytoglobin might be sufficient to limit the diffusion of hydrogen peroxide into the nucleus and inhibit the reaction of this oxidant with DNA.…”

“…In a recent study, we found that cytoglobin can function as an antioxidant through scavenging of cytosolic hydrogen peroxide 10 . To test whether cytoglobin inhibits DNA damage through hydrogen peroxide scavenging in the nucleus, we expressed HyPer7 in HEK293 cells, a genetically encoded fluorescent probe specific for hydrogen peroxide detection 23 (Fig.…”

“…We used the Ion AmpliSeq Transcriptome Mouse Gene Expression Kit according to manufactures directions Generation of HEK 293 clones, cell culture, and treatments. Human cytoglobin was stably expressed in HEK293 cells as previously described using either pCDNA 3.1 plasmids or pCMV6 DYKDDDDK tagged plasmids 9,10 . Briefly, stably expressing human cytoglobin cell lines were established through limiting dilution cloning.…”