Mammalian tissues produce nitric oxide (NO) to modify proteins at heme and sulfhydryl sites, thereby regulating vital cell functions. The majority of NO produced is widely assumed to be neutralized into supposedly inert oxidation products including nitrite (NO2(-)). Here we show that nitrite, also ubiquitous in dietary sources, is remarkably efficient at modifying the same protein sites, and that physiological nitrite concentrations account for the basal levels of these modifications in vivo. We further find that nitrite readily affects cyclic GMP production, cytochrome P450 activities, and heat shock protein 70 and heme oxygenase-1 expression in a variety of tissues. These cellular activities of nitrite, combined with its stability and abundance in vivo, suggest that this anion has a distinct and important signaling role in mammalian biology, perhaps by serving as an endocrine messenger and synchronizing agent. Thus, nitrite homeostasis may be of great importance to NO biology.

The DF3/MUC1 mucin-like glycoprotein is aberrantly overexpressed in human breast carcinomas. The functional role of DF3 is unknown. The present studies demonstrate that DF3 associates with ␤-catenin. Similar findings have been obtained for ␥-catenin but not ␣-catenin. DF3, like E-cadherin and the adenomatous polyposis coli gene product, contains an SXXXXXSSL site that is responsible for direct binding to ␤-catenin. The results further demonstrate that interaction of DF3 and ␤-catenin is dependent on cell adhesion. These findings and the role of ␤-catenin in cell signaling support a role for DF3 in the adhesion of epithelial cells.The human DF3 (MUC1, episialin, PEM) gene encodes a high molecular mass membrane-associated glycoprotein with a mucin-like external domain. The DF3 glycoprotein is expressed on the apical borders of secretory mammary epithelial cells and aberrantly expressed over the entire surface of carcinoma cells (1). The ectodomain consists of varying numbers of 20-amino acid tandem repeats that are subject to O-glycosylation and that contribute to the expression of a polymorphic protein (2-4). The N-terminal region contains hydrophobic signal sequences that vary as a consequence of alternate splicing (5-7). The C-terminal region includes a transmembrane domain and a 72-amino acid cytoplasmic tail that contains tyrosine phosphorylation sites (8, 9). The function of DF3 is unclear. However, high levels found on carcinoma cells reduce cell-cell and cell-extracellular matrix adhesion in a nonspecific manner (10 -12). These studies have suggested that DF3 interferes with cellular adhesion by steric hindrance from the rigid ectodomain (11).Cadherin cell adhesion molecules form complexes with the cytoplasmic ␣-, ␤-, and ␥-catenin proteins (13). ␣-Catenin is required for cadherin-mediated cell adhesion and links cadherins to the actin cytoskeleton (14, 15). ␤-Catenin links ␣-catenin to the cadherins and is highly related to plakoglobin (␥-catenin) (16 -18). ␤-Catenin is homologous to the Drosophila segment polarity gene product Armadillo (19) that acts downstream of Wingless (20). Armadillo forms complexes with Drosophila E-cadherin and ␣-catenin (21, 22). These findings have supported a role for ␤-catenin in morphogenetic signals. Other studies have demonstrated that ␤-catenin binds directly to the adenomatous polyposis coli (APC) 1 gene product (23-25). The APC protein and E-cadherin form independent complexes with ␤-catenin (25). ␥-Catenin mediates similar interactions among APC, ␣-catenin, and the cytoskeleton (16).The present results demonstrate that DF3 interacts directly with ␤-catenin. An SXXXXXSSL motif in the DF3 cytoplasmic domain is responsible for binding to ␤-catenin. We also demonstrate that cell adhesion induces the interaction between DF3 and ␤-catenin. MATERIALS AND METHODSCell Culture-Human ZR-75-1 breast carcinoma cells were grown in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum, 100 g/ml streptomycin, 100 units/ml penicillin, and 2 mM Lglutamine. Cells were grown...

Cytochrome C is a mitochondrial protein that induces apoptosis when released into the cytosol or when added to cell-free extracts. Here we show that cells that overexpress the Bcl-2-related protein Bcl-xL fail to accumulate cytosolic cytochrome C or undergo apoptosis in response to genotoxic stress. Coimmunoprecipitation studies demonstrate that Bcl-xL associates with cytochrome C. Cytochrome C binds directly and specifically to Bcl-xL and not to the proapoptotic Bcl-xs protein. The results also demonstrate that Bcl-xs blocks binding of cytochrome C to Bcl-xL. Our findings support a role for Bcl-xL in protecting cells from apoptosis by inhibiting the availability of cytochrome C in the cytosol.