Identification of potent small-molecule inhibitors of STAT3 with anti-inflammatory properties in RAW 264.7 macrophages.

Signal transducer and activator of transcription 3 (STAT3) is a key mediator of the inflammatory response of macrophages and other immune cell types. The naturally occurring polyphenol resveratrol is associated with anti-proliferative and anti-inflammatory properties via mechanisms implicating inhibition of STAT3 signaling. Here, we report that the small-molecule analogs of resveratrol, RSVA314 and RSVA405, are potent inhibitors of STAT3. RSVA314 and RSVA405 inhibited both constitutive and stimulated STAT3 activity in HEK293 cells and lipopolysaccharide-stimulated RAW 264.7 macrophages, respectively. The small-molecule analogs inhibited STAT3 nearly 50 times more potently than did resveratrol (apparent IC(50) ~0.5 µM). We further show that RSVA405 interfered with the inflammatory response by RAW 264.7 cells upon lipopolysaccharide stimulation by inhibiting IκB kinase and IκBα phosphorylation and by decreasing the expression of several cytokines, including the NF-κB target genes tumor necrosis factor α and interleukin-6. Downstream activation of STAT1 upon lipopolysaccharide stimulation was also inhibited by RSVA405. Consequently, RSVA405 significantly interfered with the phagocytotic activity and proliferation of lipopolysaccharide-activated RAW 264.7 macrophages. Finally, we found that the effect of the two small-molecule analogs on STAT3 phosphorylation could be prevented by inhibitors of protein tyrosine phosphatases, indicating that the small molecules acted by promoting dephosphorylation of STAT3 by protein tyrosine phosphatases.

Gas1 interferes with AßPP trafficking by facilitating the accumulation of immature AßPP in endoplasmic reticulum-associated raft subdomains.

The amyloid-ß protein precursor (AßPP) is a type I transmembrane protein that undergoes maturation during trafficking in the secretory pathway. Proper maturation and trafficking of AßPP are necessary prerequisites for AßPP processing to generate amyloid-ß (Aß), the core component of Alzheimer's disease senile plaques. Recently, we reported that the glycosylphosphatidylinositol (GPI)-anchored protein growth arrest-specific 1 (Gas1) binds to and interferes with the maturation and processing of AßPP. Gas1 expression led to a trafficking blockade of AßPP between the endoplasmic reticulum (ER) and the Golgi. GPI-anchored proteins can exit the ER by transiting through raft subdomains acting as specialized sorting platforms. Here, we show that Gas1 co-partitioned and formed a complex with AßPP in raft fractions, wherein Gas1 overexpression triggered immature AßPP accumulation. Pharmacological interference of ER to Golgi transport increased immature AßPP accumulation upon Gas1 expression in these raft fractions, which were found to be positive for the COPII protein complex component Sec31A, a specific marker for ER exit sites. Furthermore, a Gas1 mutant lacking the GPI anchor that could not transit through rafts was still able to form a complex with AßPP but did not lead to immature AßPP accumulation in rafts. Together these data show that Gas1 interfered with AßPP trafficking by interacting with AßPP to facilitate its translocation into specialized ER-associated rafts where immature AßPP accumulated.

Resveratrol mitigates lipopolysaccharide- and Aß-mediated microglial inflammation by inhibiting the TLR4/NF-κB/STAT signaling cascade.

Activation of microglia, the resident macrophages of the brain, around the amyloid plaques is a key hallmark of Alzheimer's disease (AD). Recent evidence in mouse models indicates that microglia are required for the neurodegenerative process of AD. Amyloid-ß (Aß) peptides, the core components of the amyloid plaques, can trigger microglial activation by interacting with several Toll-like receptors (TLRs), including TLR4. In this study, we show that resveratrol, a natural polyphenol associated with anti-inflammatory effects and currently in clinical trials for AD, prevented the activation of murine RAW 264.7 macrophages and microglial BV-2 cells treated with the TLR4 ligand, lipopolysaccharide (LPS). Resveratrol preferentially inhibited nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) activation upon LPS stimulation by interfering with IKK and IκB phosphorylation, an effect that potently reduced the transcriptional stimulation of several NF-κB target genes, including tumor necrosis factor-α and interleukin-6. Consequently, downstream phosphorylation of signal transducer and activator of transcription (STAT)1 and STAT3 upon LPS stimulation was also inhibited by resveratrol. We found that resveratrol acted upstream in the activation cascade by interfering with TLR4 oligomerization upon receptor stimulation. Resveratrol treatment also prevented the pro-inflammatory effect of fibrillar Aß on macrophages by potently inhibiting the effect of Aß on IκB phosphorylation, activation of STAT1 and STAT3, and on tumor necrosis factor-α and interleukin-6 secretion. Importantly, orally administered resveratrol in a mouse model of cerebral amyloid deposition lowered microglial activation associated with cortical amyloid plaque formation. Together this work provides strong evidence that resveratrol has in vitro and in vivo anti-inflammatory effects against Aß-triggered microglial activation. Further studies in cell culture systems showed that resveratrol acted via a mechanism involving the TLR4/NF-κB/STAT signaling cascade.