Peptide IDR-1002 Inhibits NF-κB Nuclear Translocation by Inhibition of IκBα Degradation and Activates p38/ERK1/2-MSK1-Dependent CREB Phosphorylation in Macrophages Stimulated with Lipopolysaccharide.

The inflammatory response is a critical molecular defense mechanism of the innate immune system that mediates the elimination of disease-causing bacteria. Repair of the damaged tissue, and the reestablishment of homeostasis, must be accomplished after elimination of the pathogen. The innate defense regulators (IDRs) are short cationic peptides that mimic natural host defense peptides and are effective in eliminating pathogens by enhancing the activity of the immune system while controlling the inflammatory response. Although the role of different IDRs as modulators of inflammation has been reported, there have been only limited studies of the signaling molecules regulated by this type of peptide. The present study investigated the effect of IDR-1002 on nuclear factor κB (NF-κB) and cAMP-response element-binding protein (CREB) transcription factors that are responsible for triggering and controlling inflammation, respectively, in macrophages. We found that TNF-α and COX-2 expression, IκBα phosphorylation, and NF-κB nuclear translocation were strongly inhibited in macrophages pre-incubated with IDR-1002 and then stimulated with lipopolysaccharide (LPS). IDR-1002 also increased CREB phosphorylation at Ser133 via activation of the p38/ERK1/2-MSK1 signaling pathways without detectable expression of the cytokines IL-4, IL-10, and IL-13 involved is suppressing inflammation or alternative activation. Transcriptional activation of NF-κB and CREB is known to require interaction with the transcriptional coactivator CREB-binding protein (CBP). To test for CBP-NF-κB and CBP-CREB complex formation, we performed co-immunoprecipitation assays. These assays showed that IDR-1002 inhibited the interaction between CBP and NF-κB in macrophages stimulated with LPS, which might explain the inhibition of TNF-α and COX-2 expression. Furthermore, the complex between CBP and CREB in macrophages stimulated with IDR-1002 was also inhibited, which might explain why IDR-1002 did not lead to expression of IL-4, IL-10, and IL-13, even though it induced an increase in phospho-CREB relative abundance. In conclusion, our results indicated that IDR-1002 has a dual effect. On one hand, it inhibited NF-κB nuclear translocation through a mechanism that involved inhibition of IκBα phosphorylation, and on the other, it activated a protein kinase signaling cascade that phosphorylated CREB to selectively influence cytokine gene expression. Based on these results, we think IDR-1002 could be a potential good biopharmaceutical candidate to control inflammation.

The Glycogen Synthase Kinase 3α and ß Isoforms Differentially Regulates Interleukin-12p40 Expression in Endothelial Cells Stimulated with Peptidoglycan from Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutively active regulatory enzyme that is important in cancer, diabetes, and cardiovascular, neurodegenerative, and psychiatric diseases. While GSK3α is usually important in neurodegenerative and psychiatric diseases GSK3ß is fundamental in the inflammatory response caused by bacterial components. Peptidoglycan (PGN), one of the most abundant cell-wall structures of Gram-positive bacteria, is an important inducer of inflammation. To evaluate whether inhibition of GSK3α and GSK3ß activity in bovine endothelial cells (BEC) regulates the expression of the pro-inflammatory cytokine IL-12p40, we treated BEC with SDS-purified PGN from Staphylococcus aureus. We found that PGN triggered a TLR2/PI3K/Akt-dependent phosphorylation of GSK3α at Ser21, GSK3ß at Ser9, and NF-κB p65 subunit (p65) at Ser536, and the phosphorylation of GSK3α was consistently higher than that of GSK3ß. The expression of IL-12p40 was inhibited in BEC stimulated with PGN and pre-treated with a specific neutralizing anti-TLR2 antibody that targets the extracellular domain of TLR2 or by the addition of Akt-i IV (an Akt inhibitor). Inhibition of GSK3α and GSK3ß with LiCl or SB216763 induced an increase in IL-12p40 mRNA and protein. The effect of each isoform on IL-12p40 expression was evaluated by siRNA-gene expression silencing of GSK3α and GSK3ß. GSK3α gene silencing resulted in a marked increase in IL-12p40 mRNA and protein while GSK3ß gene silencing had the opposite effect on IL-12p40 expression. These results indicate that the TLR2/PI3K/Akt-dependent inhibition of GSK3α activity also plays an important role in the inflammatory response caused by stimulation of BEC with PGN from S. aureus.

The phosphoinositide-3-kinase-Akt signaling pathway is important for Staphylococcus aureus internalization by endothelial cells.

Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.