Osteopontin Is a Negative Feedback Regulator of Nitric Oxide Synthesis in Murine Macrophages

Abstract

In a system of endotoxin (LPS)-mediated NO production in ANA-1 murine macrophages, suppression subtractive hybridization was used to identify genes up-regulated by NO. Osteopontin (OPN), a secreted acidic phosphoprotein that binds to a cell surface RGD integrin-binding motif, was found to be differentially expressed in the presence of NO. OPN has been demonstrated to inhibit NO production in a variety of cell types. Northern blot and nuclear run-on analyses demonstrated that OPN mRNA levels and gene transcription were significantly increased in the presence of LPS-induced NO synthesis. Transient transfection of an OPN promoter-luciferase reporter plasmid construct showed that promoter activity is increased in the presence of LPS and NO. Immunoblot analysis showed that OPN protein is secreted into the extracellular fluid. Similar results were noted with an alternative cell system, RAW 264.7 macrophages, and alternative inducers of NO synthesis, IFN-γ and IL-1β. In the presence of GRGDSP, a hexapeptide that blocks binding of RGD-containing proteins to cell surface integrins, NO production is significantly increased in the presence of LPS stimulation. These data suggest a unique trans-regulatory mechanism in which LPS-induced NO synthesis feedback regulates itself through up-regulation of OPN promoter activity and gene transcription.

Osteopontin Induces Ubiquitin-Dependent Degradation of STAT1 in RAW264.7 Murine Macrophages

“…A siRNA MM with limited homology to mouse genes served as the siRNA-negative control. As we and others (2,(12)(13)(14) have previously demonstrated, LPS induced iNOS, Tap1, and OPN protein expression in RAW264.7 cells. Ablation of OPN expression with siRNA in the presence of LPS was associated with a 6-fold increase iNOS and Tap1 protein in comparison to LPS alone ( p Ͻ 0.01 LPS vs LPSϩsiRNA OPN).…”

“…M acrophage-inducible NO synthase (iNOS) 3 expression is central to many of the systemic effects associated with endotoxin (LPS) stimulation (1). 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 iNOS expression (2)(3)(4). OPN transcription and promoter activity are significantly up-regulated in response to NO in LPS-stimulated ANA-1 and RAW264.7 murine macrophages (5).…”

S-Nitrosylation of Heterogeneous Nuclear Ribonucleoprotein A/B Regulates Osteopontin Transcription in Endotoxin-stimulated Murine Macrophages

“…However, the converse relationship, the role of NO in the induction of OPN synthesis, has not been well studied. We have previously found that LPS induced NO synthesis up-regulates OPN promoter activity and protein expression (3). In this study, we provide evidence that S-nitrosylation of hnRNP A/B inhibits DNA binding activity to the OPN promoter.…”

“…Studies indicate that the OPN promoter contains various motifs including a purine-rich sequence, an Ets-like sequence, glucocorticoid and vitamin D response elements, and interferon-inducible elements (1,2). In this regard, we have previously demonstrated that OPN transcription and promoter activity are significantly up-regulated in response to NO in a system of endotoxin-stimulated murine macrophages (3). However, the specific cis-and trans-regulatory elements that determine the extent of endotoxin-and NO-mediated induction of OPN synthesis are unknown.…”

“…In RAW 264.7 and ANA-1 murine macrophages, we have demonstrated that LPS and/or pro-inflammatory cytokine induced NO synthesis is a potent mediator of OPN promoter activation, gene transcription, and protein expression (3). In this 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) in vivo hnRNP DNA binding activity is significantly inhibited in the setting of LPS-mediated NO synthesis; and 3) S-nitrosylation of hnRNP A/B at a cysteine residue in the DNA-binding region inhibits in vitro DNA binding activity.…”