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.

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 is an important mediator of tumor metastatic behavior. We have previously demonstrated that endotoxin-dependent OPN gene transcription is regulated by a constitutive transcriptional repressor protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNP-A/B). However, in the context of cancer, the role of hnRNP-A/B in the transcriptional regulation of OPN and its metastasis-promoting functions has not been previously studied. We examined hnRNP-A/B in the 4T1 murine mammary epithelial tumor cell line, a thioguanine resistant subline which closely mimics stage IV breast cancer in humans. Our data indicate that hnRNP-A/B p37 binds to the OPN promoter, significantly decreases OPN promoter activity and mRNA levels, ablates OPN protein expression, and inhibits OPN dependent in vitro correlates of metastatic behavior, motility, and invasion. These results are unique and may suggest new therapies to re-establish loco-regional control of cancers.

Cancer progression depends on an accumulation of metastasis-supporting physiological changes which are regulated by cell signaling molecules. One such molecule, osteopontin (OPN), is a secreted phosphoprotein which mediates increased cellular migratory and invasive behavior, increased metastasis, protection from apoptosis, promotion of colony formation and 3D growth ability, induction of tumor-associated inflammatory cells, and induction of expression of angiogenic factors. Studies show that OPN expression is controlled by complex regulatory pathways at the transcriptional level in several cancers, but the molecular mechanisms which determine expression of OPN in HCC are largely unknown. In HepG2 and Hep3B tumor cell lines that differentially express OPN mRNA and protein, we identify elongation translation factor-1A1 (EF1A1) to be the trans-acting factor regulating differential OPN mRNA stability between HepG2 and Hep3B cell lines and characterize its interactions with G- and F-actin. EF1A1 binds to the OPN 5-UTR to regulate OPN mRNA half-life. EF1A1 binds to actin in Hep3B cells. Pharmacologic manipulation to increase the G:F actin ratio in Hep3B increases OPN mRNA half-life and proteinex pression with simultaneous decrease in EF1A1 binding to OPN 5′-UTR. The converse findings were noted in HepG2 cells. Overall, our results suggest that EF1A1 regulation of OPN mRNA stability is actin dependent. EF1A1 has not been previously identified as a regulatory factor in OPN expression in cancer.

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.

Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two CAM molecules: avb3-integrin and CD44. OPN initiates a number of signal transduction pathwaysthat control cell survival, proliferation, and migration. In this study, utilizing RAW 264.7 murine macrophages, we demonstrate that expression of the mitochondrial protein, CCOI, is significantly decreased in the setting of OPN stimulation. This effect is blocked by the CD44 competitive ligand, hylauronate; GRGDSP, a hexapeptide that blocks OPN–integrin binding, had no effect. CCOI mRNA and transcription were significantly decreased in the presence of OPN; CCOI mRNA half-life was unaltered by OPN. Additional mitochondrial run-on studies, which included genes from L-strand and H-strand, suggest that OPN terminates transcription of the distal H-strand. CCO enzyme activity was also significantly decreased by OPN. Our results indicate that OPN inhibits CCOI expression as the result of a novel CD44-dependent transcriptional regulatory mechanism of the mitochondrial H strand.

Background: We hypothesized that acetylation of the Stat1 regulates interferon-γ(IFN-γ) mediated macrophage expression of inducible nitric oxide synthase (iNOS). Methods: RAW 264.7 iNOS expression was induced with IFN-γ. Deacetylase inhibitors trichostatin A (TSA) or valproic acid (VPA) were added. Stat1 and iNOS mRNA and protein were measured. Acetylated Stat1 was determined by immunoprecipitation. Chromatin immunoprecipitation assessed in vivo binding of Stat1 to the iNOS promoter. Results: IFN-γ significantly increased nitrite, iNOS protein and iNOS mRNA, and iNOS promoter activation. (P<.01 vs control for nitrite, protein, and mRNA). TSA-mediated acetylation decreased these to levels that were not different from controls. IFN-γ increased acetylated Stat1 by 5-fold (P<.02 vs control); TSA+IFN-γ caused an additional 4-fold increase in acetylated Stat1 (P<.05 vs IFN alone). Stat1 binding to the iNOS promoter increased 8-fold with IFN-γ(P<.01 vs control). In TSA+IFN-γ, Stat1 binding was not different from controls. Although less potent than TSA, VPA also significantly decreased nitrite, iNOS protein, iNOS mRNA, Stat1 acetylation, and Stat1 binding. Conclusions: Acetylation of Stat1 protein correlates with decreased Stat1 binding to the iNOS promoter with resultant inhibition of IFN-γ-mediated iNOS expression. Acetylation of the Stat1 protein may downregulate iNOS expression in proinflammatory states.

Background. Osteopontin (OPN) represses inducible nitric oxide synthase (iNOS) expression by increasing ubiquitin (Ub)-proteasome degradation of Stat1, a critical transcription factor for iNOS expression. We investigated the in vivo relevance of our findings in a cecal ligation and puncture model. Methods and Results. A total of 129 wild-type (WT; n = 24) and OPN null (n = 24) mice were used. Bone marrow macrophages and whole liver tissue were isolated. iNOS and phosphorylated Stat-1 (P-Stat1) protein were significantly greater in OPN null than WT. Cecal ligation and puncture increased Ub-P-Stat1; Ub-P-Stat1 was significantly less in OPN null than WT. In chromatin immunoprecipitation assays, P-Stat1 binding to the iNOS promoter was increased in OPN null. Ex vivo studies with bone marrow macrophages were performed with MG132 (10 mM), an inhibitor of 26S proteasome function, and/or exogenous OPN (50 mM). Ub-P-Stat1 was decreased in OPN null bone marrow macrophages treated with LPS; iNOS was increased. Exogenous OPN or MG132 restored Ub-P-Stat1 and iNOS to levels seen in WT. Our results indicate that absence of OPN does the following: (1) increases iNOS and P-Stat1 protein, (2) decreases ubiquitination and degradation of P-Stat1, and (3) increases iNOS transcription. Conclusions. We conclude that OPN downregulates iNOS expression by accelerating ubiquitination and degradation of Stat1. (Surgery 2008;144:182-8.)

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.

Background. Osteopontin (OPN) mediates cancer metastasis. Mechanisms regulating OPN expression in human colorectal cancer are unknown. Using SW480 colon adenocarcinoma cells, we hypothesized that transcription determines OPN expression. Methods. SW480 constitutively express OPN. Transient transfection and deletion analysis of human OPN promoter (full-length 2.1 kb)-luciferase constructs identified cis-regulatory regions. Gelshift and chromatin immunoprecipitation (ChIP) assays identified the trans-regulatory nuclear protein. Using in vitro adhesion, migration, and invasion studies, siRNA was used to determine the functional effect of decreased nuclear protein expression. Results. A cis-regulatory promoter region, nt-80 to nt-108, upregulated OPN transcription. Gelshift assays demonstrated specific binding of nuclear proteins. Competition with unlabeled mutant oligonucleotides indicated that the region, nt-94 to nt-104 (TGGGCTGGGC), was essential for protein binding in gelshift assays. Confirmatory ChIP assays showed the corresponding nuclear protein to be Sp1. Sp1 expression was ablated with siRNA (si-Sp1), resulting in decreased OPN-dependent adhesion, migration, and invasion by 50%, 70%, and 65%, respectively. Exogenous addition of OPN to si-Sp1 cells restored adhesion, migration, and invasion indices. Conclusions. In SW480 human colon cancer cells, we conclude that Sp1 mediated expression of the tumor metastasis protein, OPN, regulates in vitro functional correlates of tumor metastasis.