Nitric oxide post-transcriptionally up-regulates LPS-induced IL-8 expression through p38 MAPK activation

Abstract

Nitric oxide (NO(.-)) contributes to vascular collapse in septic shock and regulates inflammation. Here, we demonstrate in lipopolysaccharide (LPS)-stimulated human THP-1 cells and monocytes that NO(.-) regulates interleukin (IL)-8 and tumor necrosis factor alpha (TNF-alpha) by distinct mechanisms. Dibutyryl-cyclic guanosine 5'-monophosphate (cGMP) failed to simulate NO(.-)-induced increases in TNF-alpha or IL-8 production. In contrast, dibutyryl-cyclic adenosine monophosphate blocked NO(.-)-induced production of TNF-alpha (P=0.009) but not IL-8. NO(.-) increased IL-8 (5.7-fold at 4 h; P=0.04) and TNF-alpha mRNA levels (2.2-fold at 4 h; P=0.037). However, nuclear run-on assays demonstrated that IL-8 transcription was slightly decreased by NO(.-) (P=0.08), and TNF-alpha was increased (P=0.012). Likewise, NO(.-) had no effect on IL-8 promoter activity (P=0.84) as measured by reporter gene assay. In THP-1 cells and human primary monocytes treated with actinomycin D, NO(.-) had no effect on TNF-alpha mRNA stability (P>0.3 for both cell types) but significantly stabilized IL-8 mRNA (P=0.001 for both cell types). Because of its role in mRNA stabilization, the p38 mitogen-activated protein kinase (MAPK) pathway was examined and found to be activated by NO(.-) in LPS-treated THP-1 cells and human monocytes. Further, SB202190, a p38 MAPK inhibitor, was shown to block NO(.-)-induced stabilization of IL-8 mRNA (P<0.02 for both cell types). Thus, NO(.-) regulates IL-8 but not TNF-alpha post-transcriptionally. IL-8 mRNA stabilization by NO(.-) is independent of cGMP and at least partially dependent on p38 MAPK activation.

Nitric Oxide Down-regulates Polo-like Kinase 1 through a Proximal Promoter Cell Cycle Gene Homology Region

“…Target gene mRNA levels were normalized to the housekeeping gene GAPDH. To measure protein expression levels for p21/ Waf1 and PLK1, 30 g of whole cell lysate was extracted from cells under various conditions as indicated and used for Western blotting following procedures described previously (14). The housekeeping gene ␣-tubulin was measured at the same time to control for protein loading.…”

“…Nitric oxide (NO ⅐ ) regulates a wide range of cellular activities including gene expression (12)(13)(14)(15)(16), cell proliferation (17,18), and apoptosis (19) that are closely linked to its anti-tumor and anti-atherosclerotic properties (17,20). Using a microarraybased approach in soluble guanylate cyclase-deficient cells, we recently demonstrated that NO ⅐ predominately regulated a large set of cell cycle genes independent cGMP (15).…”

Increased NOS2 predicts poor survival in estrogen receptor–negative breast cancer patients

“…human monocytes, and lung epithelial cells (53)(54)(55). IL-8 is of particular importance in breast tumor biology and is associated with increased breast cancer cell invasion, neutrophil infiltration, and microvessel density (35).…”

Effect of Rifampin on Production of Inflammatory Mediators in HepG2 Liver Epithelial Cells

“…It acts as an intercellular and intracellular messenger, regulating expression of many genes and the activity of a wide range of proteins and signal transduction pathways. NO modulates the production of several cytokines and chemokines, among them gamma interferon (IFN-␥)-induced protein-10 (IP-10) (CXCL-1), regulated on activation, normal Tcell expressed and secreted protein (RANTES), monocyte chemotactic protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor alpha (TNF-␣), interleukin-1 beta (IL-1␤), macrophage inflammatory protein-1 (MIP-1), and IL-8, in different cell types by different mechanisms (8,17,20,22). The production of NO is controlled by constitutive and inducible isoforms of nitric oxide synthase (NOS), each associated with a different mode of regulation and a different amount of NO produced.…”