Tongzheng Liu

Sensitivity of mice to lipopolysaccharide is increased by a high saturated fat and cholesterol diet

BackgroundIt was hypothesized that a pro-atherogenic, high saturated fat and cholesterol diet (HCD) would increase the inflammatory response to E. coli endotoxin (LPS) and increase its concentration in plasma after administration to mice.MethodsC57Bl/6 mice were fed a HCD or a control diet (CD) for 4 weeks, and then treated with saline, 0.5, 1 or 2 mg LPS/kg, ip. Liver injury (alanine:2-oxoglutarate aminotransferase and aspartate aminotransferase, collagen staining), circulating cytokines (tumor necrosis factor-α, interleukin-6 and interferon-γ), factors that can bind LPS (serum amyloid A, apolipoprotein A1, LPS binding protein, and CD14), and plasma levels of LPS were measured. The hepatic response was assessed by measuring vascular cell adhesion molecule (VCAM)-1, inducible nitric oxide synthase (iNOS) and signal transducer and activator of transcription-1 proteins, and VCAM-1 and iNOS mRNAs. Hepatic mRNA encoding the LPS receptor, Toll like receptor 4, was also determined.ResultsTwo mg LPS/kg killed 100% of mice fed HCD within 5 d, while no mice fed CD died. All mice treated with 0 to 1 mg LPS/kg survived 24 h. HCD increased plasma alanine:2-oxoglutarate aminotransferase and aspartate aminotransferase, and the enzymes were increased more by LPS in HCD than CD mice. Induction of plasma tumor necrosis factor-α, interleukin-6, and interferon-γ by LPS was greater with HCD than CD. Hepatic VCAM-1 and iNOS protein and mRNA were induced by LPS more in mice fed HCD than CD. Tyrosine phosphorylation of signal transducer and activator of transcription-1 caused by LPS was prolonged in HCD compared with CD mice. Despite the hepatic effects of HCD, diet had no effect on the LPS plasma concentration-time profile. HCD alone did not affect circulating levels of plasma apolipoprotein A1 or LPS binding protein. However, plasma concentrations of serum amyloid A and CD14, and hepatic toll-like receptor-4 mRNA were increased in mice fed HCD.ConclusionHCD increased the sensitivity of mice to LPS without affecting its plasma level. Although increased serum amyloid A and CD14 in the circulation may inhibit LPS actions, their overexpression, along with hepatic toll-like receptor-4 or other factors, may contribute to the heightened sensitivity to LPS.

Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells.

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.

Calpastatin is regulated by protein never in mitosis gene A interacting-1 (PIN1) in endothelial cells.

The peptidyl-proline isomerase, protein never in mitosis gene A interacting-1 (PIN1) binds and isomerizes proteins phosphorylated on serine/threonine before a proline. It was previously found that depletion of PIN1 greatly increased induction of cyclooxygenase-2 and inducible nitric oxide synthase by lowering calpain activity in murine aortic endothelial cells (MAEC). Here we investigated the effect of PIN1 on the endogenous inhibitor of heterodimeric μ- and m-calpains, calpastatin. MAEC were transduced with small hairpin (sh) RNA to knock down PIN1 (KD) or an inactive Control shRNA. Cells were also treated with non-targeted double stranded small inhibitory RNA (siRNA) or siRNA designed to deplete calpastatin. Despite reducing calpain activity, PIN1 KD did not significantly affect the expression of μ- and m-calpains, or calpastatin, compared to Control shRNA. Instead, depletion of PIN1 increased the inhibitory activity of calpastatin. Calpastatin co-immunoprecipitated with endogenous PIN1 and was pulled down with glutathione-S-transferase (GST)-PIN1 fusion protein. Adding GST-PIN1 to KD cell extracts lacking PIN1 reduced calpastatin inhibitory activity. Substrate binding and catalytic domain mutants of PIN1 failed to do so. These results suggest that protein interaction and the proline isomerase functions of PIN1 are required for it to inhibit calpastatin. Furthermore, depletion of calpastatin raised calpain activity and reduced calpain inhibitory activity to similar levels in KD and Control MAEC, indicating that calpastatin is required for PIN1 depletion to lower calpain activity. Thus, PIN1 apparently restrains the ability of calpastatin to inhibit calpain, maintaining calpain activity in endothelial cells. PIN1 may act directly via phosphorylated serine/threonine-proline motifs in calpastatin, or indirectly via other PIN1 substrates that control calpastatin.

Protein Never in Mitosis Gene A Interacting-1 regulates calpain activity and the degradation of cyclooxygenase-2 in endothelial cells

BackgroundThe peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), regulates turnover of inducible nitric oxide synthase (iNOS) in murine aortic endothelial cells (MAEC) stimulated with E. coli endotoxin (LPS) and interferon-γ (IFN). Degradation of iNOS was reduced by a calpain inhibitor, suggesting that PIN1 may affect induction of other calpain-sensitive inflammatory proteins, such as cyclooxygenase (COX)-2, in MAEC.MethodsMAEC, transduced with lentivirus encoding an inactive control short hairpin (sh) RNA or one targeting PIN1 that reduced PIN1 by 85%, were used. Cells were treated with LPS/IFN, calpain inhibitors (carbobenzoxy-valinyl-phenylalaninal (zVF), PD150606), cycloheximide and COX inhibitors to determine the effect of PIN1 depletion on COX-2 and calpain.ResultsLPS or IFN alone did not induce COX-2. However, treatment with 10 μg LPS plus 20 ng IFN per ml induced COX-2 protein 10-fold in Control shRNA MAEC. Induction was significantly greater (47-fold) in PIN1 shRNA cells. COX-2-dependent prostaglandin E2 production increased 3-fold in KD MAEC, but did not increase in Control cells. The additional increase in COX-2 protein due to PIN1 depletion was post-transcriptional, as induction of COX-2 mRNA by LPS/IFN was the same in cells containing or lacking PIN1. Instead, the loss of COX-2 protein, after treatment with cycloheximide to block protein synthesis, was reduced in cells lacking PIN1 in comparison with Control cells, indicating that degradation of the enzyme was reduced. zVF and PD150606 each enhanced the induction of COX-2 by LPS/IFN. zVF also slowed the loss of COX-2 after treatment with cycloheximide, and COX-2 was degraded by exogenous μ-calpain in vitro. In contrast to iNOS, physical interaction between COX-2 and PIN1 was not detected, suggesting that effects of PIN1 on calpain, rather than COX-2 itself, affect COX-2 degradation. While cathepsin activity was unaltered, depletion of PIN1 reduced calpain activity by 55% in comparison with Control shRNA cells.ConclusionPIN1 reduced calpain activity and slowed the degradation of COX-2 in MAEC, an effect recapitulated by an inhibitor of calpain. Given the sensitivity of COX-2 and iNOS to calpain, PIN1 may normally limit induction of these and other calpain substrates by maintaining calpain activity in endothelial cells.

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) regulates pulmonary effects of endotoxin and tumor necrosis factor-α in mice

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) modulates phospho-signaling by catalyzing rotation of the bond between a phosphorylated serine or threonine before proline in proteins. As depletion of PIN1 increased inflammatory protein expression in cultured endothelial cells treated with bacterial endotoxin (lipopolysaccharide, LPS) and interferon-γ, we hypothesized that PIN1 knockout would increase sensitivity to LPS-induced lung inflammation in mice. Mortality due to a high dose of LPS (30mg/kg) was greater in knockout than wildtype mice. Lung myeloperoxidase activity, reflecting neutrophils, was increased to a 35% higher level in PIN1 knockout mouse lung, as compared with wildtype, after treatment with a sublethal dose of 3mgLPS/kg, ip. Unexpectedly, plasma tumor necrosis factor-α (TNF) was approximately 50% less than in wildtype mice. Knockout mice, however, were more sensitive than wildtype to TNF-induced neutrophil accumulation. The neutrophil adhesion molecule, E-selectin, was also elevated in lungs of knockout mice treated with TNF, suggesting that PIN1 depletion increases endothelial sensitivity to TNF. Indeed, TNF induced more reactive oxygen species in cultured endothelial cells depleted of PIN1 with short hairpin RNA than in control cells. Collectively, the results indicate that while PIN1 normally facilitates TNF production in LPS-treated mice, it suppresses pulmonary and endothelial reactions to the cytokine. Tissue or cell-specific effects of PIN1 may affect the overall inflammatory response to LPS and other stimuli.