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Neuropilin-1 (Nrp1) encodes the transmembrane cellular receptor neuropilin-1, which is associated with cardiovascular and neuronal development and was within the peak SNP interval on chromosome 8 in our prior GWAS study on age-related hearing loss (ARHL) in mice. In this study, we generated and characterized an inner ear-specific Nrp1 conditional knockout (CKO) mouse line because Nrp1 constitutive knockouts are embryonic lethal. In situ hybridization demonstrated weak Nrp1 mRNA expression late in embryonic cochlear development, but increased expression in early postnatal stages when cochlear hair cell innervation patterns have been shown to mature. At postnatal day 5, Nrp1 CKO mice showed disorganized outer spiral bundles and enlarged microvessels of the stria vascularis (SV) but normal spiral ganglion cell (SGN) density and presynaptic ribbon body counts; however, we observed enlarged SV microvessels, reduced SGN density, and a reduction of presynaptic ribbons in the outer hair cell region of 4-month-old Nrp1 CKO mice. In addition, we demonstrated elevated hearing thresholds of the 2-month-old and 4-month-old Nrp1 CKO mice at frequencies ranging from 4 to 32kHz when compared to 2-month-old mice. These data suggest that conditional loss of Nrp1 in the inner ear leads to progressive hearing loss in mice. We also demonstrated that mice with a truncated variant of Nrp1 show cochlear axon guidance defects and that exogenous semaphorin-3A, a known neuropilin-1 receptor agonist, repels SGN axons in vitro. These data suggest that Neuropilin-1/Semaphorin-3A signaling may also serve a role in neuronal pathfinding in the developing cochlea. In summary, our results here support a model whereby Neuropilin-1/Semaphorin-3A signaling is critical for the functional and morphological integrity of the cochlea and that Nrp1 may play a role in ARHL.

Besides scavenging free radicals, antioxidants inhibit signaling enzymes such as protein kinase C (PKC) that play a crucial role in tumor promotion. By having different oxidation susceptible regions, PKC can respond to both oxidant tumor promoters and cancer-preventive antioxidants to elicit opposite cellular responses. Oxidant tumor promoters activate PKC by reacting with zinc-thiolates present within the regulatory domain. In contrast, the oxidized forms of some cancer-preventive agents, such as polyphenolics (ellagic acid, 4-hydroxytamoxifen and curcumin) and selenocompounds, can inactivate PKC by oxidizing the vicinal thiols present within the catalytic domain. This brings an efficient counteractive mechanism to block the signal transduction induced by tumor promoters at the first step itself. Because prostate cancer prevention clinical trials in large human population are under way, we have focused more on understanding the cancer-preventive mechanism of selenium. Methylselenol, the postulated cancer-preventive metabolite, has no direct effect on PKC activity. However, methylseleninic acid, locally generated by the reaction of membrane methylselenol with PKC-bound tumor-promoting fatty acid hydroperoxides, selectively inactivates PKC. This mechanism clarifies how the volatile methylselenol that is present in a low concentration induces the inactivation of PKC selectively in the promoting precancer cells. Selenoprotein thioredoxin reductase reverses selenium-induced inactivation of PKC, suggesting that selenoproteins may serve as a safeguard against the toxicity induced by selenometabolites. Moreover, this also explains how a resistance to selenium develops in advanced malignant cells. The redox-mediated inactivation of PKC may, at least in part, be responsible for the antioxidant-induced inhibition of tumor promotion and cell growth, as well as for the induction of cell death. J. Nutr. 132: 3819S-3823S, 2002. KEY WORDS: * antioxidants * selenium * cancer prevention * protein kinase C * thioredoxin reductase

Besides scavenging free radicals, antioxidants such as selenium inhibit cell signaling and inhibit tumor promotion. However, it is not clear how selenium interferes with cell signaling selectively in precancer cells but not in normal cells. Methylselenol was previously postulated to be the active metabolite that mediates cancer-preventive actions of selenium. Nevertheless, it is a volatile compound and its mechanism of action, especially that occurring at bioavailable low concentrations, is not known. We previously showed that redox-active selenocompounds directly inactivate protein kinase C (PKC) by inducing sulfhydryl oxidation. Because selenium is required at low concentrations to inhibit tumor promotion, the oxidants present in promoting cells may enhance cellular retention and the preventive actions of volatile methylselenol. Methylselenol, benzeneselenol, and selenol metabolites of ebselen at catalytically low concentrations induced PKC inactivation only in the presence of peroxides such as arachidonic acid hydroperoxide and t-butyl hydroperoxide. By reducing peroxides, selenol was oxidized to seleninic acid, which in turn oxidized protein sulfhydryls and thus was reduced back to selenol. Although this peroxidatic redox-cycling process nonspecifically oxidizes many thiols including glutathione, the lipophilic selenols and organic peroxides present in the membrane can selectively oxidize sulfhydryls of key membrane-bound enzymes such as PKC. Application of sublethal doses of these peroxides and organic selenols to JB6 epidermal cells resulted in inactivation of PKC α isoenzyme, induction of apoptosis, and inhibition of cell transformation. Conceivably, the reaction of selenols with tumor-promoting peroxides provides one of the specific mechanisms by which cancer-preventive selenocompounds selectively interfere with signaling in the promoting cells but not in the normal cells. [ABSTRACT FROM AUTHOR]