Olfactomedin 4 Is Essential for Superoxide Production and Sensitizes Oxidative Stress-Induced Apoptosis in Neutrophils.

Abstract 1356 Poster Board I-378 Introduction Olfactomedin 4 (OLFM4), also called hGC-1, GW112 and pDP4, was first identified and specifically expressed in hematopoietic myeloid cells. OLFM4 expression in myeloid cells is regulated by transcription factors, PU1 and NF-κB. It has significant homology in its C-terminal domain with other olfactomedin-related proteins. OLFM4 encodes a 510 amino acid N-linked glycoprotein. The exact biological function of OLFM4, especially in neutrophils, is currently undefined. To characterize the in vivo function of OLFM4, we generated OLFM4 deficient mice (OLFM4-/-) and investigated its potential role in neutrophil functioins. Results 1) In this study, we showed that OLFM4 is a secreted glycoprotein and is also localized in the mitochondria, cytoplasm and cell membrane fractions of neutrophils. We demonstrated that OLFM4 interacts with GRIM-19 (Genes associated with Retinoid-IFN-induced Mortality-19), an apoptosis related protein, in the neutrophil mitochondria using co-immuoprecipitation assay. GRIM-19 is a subunit of complex I of mitochondrial respiratory chain and is essential for maintenance of mitochondrial membrane potential. Our result suggests that OLFM4 appears to be a novel component of complex I of mitochondrial respiratory chain and may be involved in regulation of mitochondrial membrane potential. 2) Mice heterozygous (OLFM4+/-) and homozygous (OLFM4-/-) for the null mutation in OLFM4 appeared to have normal development, fertility, and viability relative to wild-type (WT) mice. Whole blood analysis, differential leukocyte counts, blood chemistry and bone marrow smears were normal in OLFM4-/- mice, suggesting that OLFM4 is not essential for normal development and hematopoiesis in mice. 3) In response to LPS, fMLP and E.coli bacteria challenge, neutrophils from OLFM4-/- mice showed significantly reduced superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) production compared with WT mice. These results suggest that OLFM4 is an essential component to mediate O 2 − and H 2 O 2 production in the neutrophil mitochondria under inflammation stimuli. 4) Exogenous H 2 O 2 induced neutrophil apoptosis in a time and dose dependent manner in WT mice, but this induction of apoptosis was significantly reduced in OLFM4-/- mice. This result suggests that OLFM4 sensitizes and mediates H 2 O 2 -induced apoptosis in neutrophils. 5) Furthermore, we demonstrated that H 2 O 2 -stimulated mitochondrial membrane permeability reduction and caspase-3 and caspase-9 activation were inhibited in the neutrophils of OLFM4-/- mice. This result confirmed our hypothesis that OLFM4 may be involved in maintenance of mitochondrial membrane potential and suggests that OLFM4 may have opposite role as GRIM-19. 6) Moreover, Bax association with mitochondria and the cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO in response to H 2 O 2 were inhibited in the neutrophils of OLFM4-/- mice. Conclusion Our results suggest: 1) OLFM4 has multiple subcellular localizations including mitochondria, cytoplasm, and cell membrane in neutrophils. The interaction of OLFM4 with GRIM-19 in the mitochondria suggests that OLFM4 is novel component of complex I of mitochondrial respiratory chain in the mitochondria of neutrophils, 2) OLFM4 is a novel mitochondrial molecule that is essential for O 2 − and H 2 O 2 production in the neutrophils in the presence of inflammation stimuli, 3) Loss of OLFM4 in neutrophils does not trigger spontaneous apoptosis. However, OLFM4 sensitizes oxidative stress-induced apoptosis in mouse neutrophils. OLFM4 is involved in the regulation of mitochondria membrane potential and sensitizes cytoplasmic translocation of Omi/HtrA2 and Smac/DIABLO and caspases-3 and caspase-9 mediated apoptosis in the presence of oxidative stress. Disclosures No relevant conflicts of interest to declare.