In systemic inflammation induced by endotoxin (LPS), the macrophage produces the majority of the circulating NO metabolites. However, while the molecular pathways which up-regulate iNOS expression have been extensively studied in the macrophage, little is known of the parallel counterregulatory pathways which repress or inhibit macrophage iNOS expression. Using both in vivo and in vitro murine models of endotoxin (LPS) stimulation, we have previously demonstrated that NO feedback inhibits its own synthesis by increasing transcription of osteopontin (OPN), a potent transrepressor of inducible NO synthase expression. In this current study, using a system of LPS-treated RAW264.7 macrophages, we go on to demonstrate that OPN increases STAT1 ubiquitination and subsequent 26s proteasome-mediated degradation to inhibit STAT1 dependent iNOS promoter activity, transcription, and protein expression. In addition, we identify STAT-interacting LIM protein as the critical STAT ubiquitin E3 ligase critical for STAT1 degradation in this setting. OPN has not been linked previously to STAT1 degradation. This regulation of STAT1 degradation underlies OPNs effect as an inhibitor of iNOS gene transcription. These are novel findings and define OPN as a unique and as yet, poorly characterized, transactivator of STAT1 degradation by the ubiquitin-proteasome system.

Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of 298 amino acids which contains a Gly-Arg-Gly-Asp-Ser sequence. It is a secreted protein with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. Despite the many recognized functions of OPN, very little is known of the transcriptional regulation of OPN. In this regard, we have previously demonstrated that OPN transcription and promoter activity are significantly upregulated in response to nitric oxide (NO) in a system of endotoxin-stimulated murine macrophages. However, the specific cis- and trans-regulatory elements that determine the extent of endotoxin- and NO-mediated induction of OPN synthesis are unknown. In this followup study, we demonstrate that: 1) OPN gene transcription is regulated by a constitutive transcriptional repressor protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNP-A/B), 2) inhibition of in vivo hnRNP DNA binding activity is accompanied by increased S-nitrosylation of hnRNP A/B in the setting of LPS-mediated NO synthesis, 3) inhibition of LPS mediated NO synthesis restores hnRNP DNA binding and decreases the extent of S-nitrosylation, and 4) Snitrosylation of hnRNP at cysteine-104 inhibits in vitro DNA binding activity, which is reversed by DTT. Our findings suggest that LPS induced S-nitrosylation of hnRNP inhibits its activity as a constitutive repressor of the OPN promoter and results in enhanced OPN expression.

Background: Osteopontin (OPN) is a secreted glycoprotein that mediates cell–matrix interactions and cellular signaling by binding with integrin (primarily AvB3) and CD44 receptors. OPN regulates cell adhesion, chemotaxis, macrophage-directed IL-10 suppression, stressdependent angiogenesis, apoptosis prevention, and anchorage-independent growth of tumor cells. However, the molecular mechanisms that define the role of OPN in tumor progression and metastasis are incompletely understood. Methods: In this study, we use a system of 4T1 and 4T07 murine mammary epithelial tumor cell lines that are divergent in both metastatic phenotype and OPN expression. 4T1 expresses OPN and hematogeneously metastasizes, whereas 4T07 does not express OPN and is highly tumorigenic but fails to metastasize. Results: Our results demonstrate that OPN regulates Stat1 protein degradation through the ubiquitin–proteasome pathway to alter interferon-;–dependent growth inhibition and p21 expression. We identify Stat-interacting LIM protein as the critical Stat ubiquitin E3 ligase in this setting. Conclusions: OPN regulates Stat1-dependent functions, such as growth inhibition and p21 expression, in the murine mammary epithelial cells lines 4T1 and 4T07. This relationship between OPN and Stat1 in the context of tumor biology has not been previously examined.

The interaction of osteopontin (OPN) with CD44 and avb3-integrin has been implicated in numerous signal transduction pathways that may promote cancer metastasis. CD44v6 is a splice variant of CD44 which has been identified as a marker of cancer progression. In this study, immortalized liver carcinoma cells (HepG2) were used to examine the effect of OPN on two isoforms of CD44: CD44 standard (CD44s) and CD44v6. Western blots demonstrated that OPN up-regulated plasma membrane CD44v6 protein expression in a concentrationand time-dependent fashion. CD44v6 levels returned to control levels when OPN±avb3-integrin binding was blocked by an RGD peptide or tyrosine kinase activity was inhibited. OPN significantly increased CD44v6 protein synthesis, while simultaneously decreasing protein degradation. Steady-state mRNA levels of both CD44s and CD44v6 were unaltered in the presence of OPN stimulation. OPN increased HepG2 in vitro adhesion to hyaluronate (HA); excess soluble HA extinguished OPN-mediated HepG2 adhesion, indicating CD44 dependence. In conclusion, OPN binds to the avb3-integrin to increase plasma membrane CD44v6 expression and augment in vitro adhesion to HA. This may contribute to the mechanism by which OPN enhances metastatic behavior in hepatocellular cancer cells.

Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identiWcation of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identiWcation of the"universe"of S-nitrsoylated proteins. In this study using endotoxinstimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identiWed as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins speciWc to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not suYcient.