Oliver H. Voss

Apigenin Blocks Lipopolysaccharide-Induced Lethality In Vivo and Proinflammatory Cytokines Expression by Inactivating NF-κB through the Suppression of p65 Phosphorylation

LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1β, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-κB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-κB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-κB inhibitor IκBα was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-κB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-κB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKβ. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.

Binding of Caspase-3 Prodomain to Heat Shock Protein 27 Regulates Monocyte Apoptosis by Inhibiting Caspase-3 Proteolytic Activation

Caspase-3 is an essential executioner of apoptosis responsible for regulating many important cellular processes, among them the number of circulating monocytes, central players in the innate immune response. The activation of caspase-3 requires its processing from an inactive precursor. Here we show that the small heat shock protein 27 (Hsp27) associates with caspase-3 and protein-protein interaction experiments in vivo and with purified proteins demonstrate a direct interaction between Hsp27 and the amino-terminal prodomain of caspase-3. Using an in vitro caspase-3 activation assay, our results further establish that the interaction of Hsp27 with the caspase-3 prodomain inhibits the second proteolytic cleavage necessary for caspase-3 activation, revealing a novel mechanism for the regulation of this effector caspase. Hsp27 expression in monocytes is constitutive. Consistent with a central role of Hsp27 in blocking caspase-3 activation, Hsp27 down-regulation by double-stranded RNA interference induces apoptosis of macrophages, whereas Hsp27 overexpression increases the life span of monocytes by inhibiting apoptosis. Highlighting the importance of cell partitioning in the regulation of apoptosis, immunofluorescence, and subcellular fractionation studies revealed that whereas both caspase-3 and Hsp27 are cytoplasmic in fresh monocytes (i.e. not undergoing apoptosis), Hsp27 moves to the nucleus during apoptosis, a relocalization that can be blocked by promoting the differentiation of monocytes to macrophages or by inhibiting cell death. These results reveal a novel mechanism of caspase-3 regulation and underscore a novel and fundamental role of Hsp27 in the regulation of monocyte life span.

Regulation of Monocyte Apoptosis by the Protein Kinase Cδ-dependent Phosphorylation of Caspase-3

Monocytes are central components of the innate immune response and normally circulate for a short period of time before undergoing spontaneous apoptosis. During inflammation, differentiation, or oncogenic transformation, the life span of monocytes is prolonged by preventing the activation of the apoptotic program. Here we showed that caspase-3, a cysteine protease required for monocyte apoptosis, is a phosphoprotein. We identified protein kinase Cδ (PKCδ) as a member of the protein kinase C family that associates with and phosphorylates caspase-3. The PKCδ-dependent phosphorylation of caspase-3 resulted in an increase in the activity of caspase-3. This effect of PKCδ is specific to caspase-3, as evidenced by the absence of similar effects on caspase-9. The activity of PKCδ precedes the activation of caspase-3 during spontaneous monocyte apoptosis and in monocyte-induced apoptosis. We found that the overexpression of PKCδ resulted in an increase of apoptosis, whereas its inhibition blocked caspase-3 activity and decreased apoptosis. Our results provided evidence that the PKCδ-dependent phosphorylation of caspase-3 provided a novel pro-apoptotic mechanism involved in the regulation of monocyte life span.

Chaperone Peptides of α-Crystallin Inhibit Epithelial Cell Apoptosis, Protein Insolubilization, and Opacification in Experimental Cataracts

Background: Peptides derived from the core domain of human α-crystallin act as molecular chaperones. Results: Chaperone peptides of α-crystallin inhibit stress-induced apoptosis in cultured cells and prevent experimental cataracts in rats. Conclusion: Chaperone peptides of α-crystallin are anti-apoptotic and retain biological activity when injected into animals. Significance: α-Crystallin peptides could be used as therapeutic agents to inhibit protein aggregation and apoptosis in diseases. α-Crystallin is a member of the small heat-shock protein (sHSP) family and consists of two subunits, αA and αB. Both αA- and αB-crystallin act as chaperones and anti-apoptotic proteins. Previous studies have identified the peptide 70KFVIFLDVKHFSPEDLTVK88 in αA-crystallin and the peptide 73DRFSVNLDVKHFSPEELKVK92 in αB-crystallin as mini-chaperones. In the human lens, lysine 70 (Lys70) of αA and Lys92 of αB (in the mini-chaperone sequences) are acetylated. In this study, we investigated the cellular effects of the unmodified and acetyl mini-chaperones. The αA- and αB-crystallin peptides inhibited stress-induced aggregation of four client proteins, and the αA-acetyl peptide was more effective than the native peptide against three of the client proteins. Both the acetyl and native crystallin peptides inhibited stress-induced apoptosis in two mammalian cell types, and this property was directly related to the inhibition of cytochrome c release from mitochondria and the activity of caspase-3 and -9. In organ-cultured rat lenses, the peptides inhibited calcimycin-induced epithelial cell apoptosis. Intraperitoneal injection of the peptides inhibited cataract development in selenite-treated rats, which was accompanied by inhibition of oxidative stress, protein insolubilization, and caspase activity in the lens. These inhibitory effects were more pronounced for acetyl peptides than native peptides. A scrambled αA-crystallin peptide produced no such effects. The results suggest that the α-crystallin chaperone peptides could be used as therapeutic agents to treat cataracts and diseases in which protein aggregation and apoptosis are contributing factors.

Apigenin-induced apoptosis of leukemia cells is mediated by a bimodal and differentially regulated residue-specific phosphorylation of heat-shock protein-27

Apigenin, a natural plant flavonoid with antiproliferative activity, is emerging as a promising compound for cancer prevention and therapy, but its mechanism of action remains unclear. High expression of the small heat-shock protein-27 (Hsp27) in leukemia contributes to the resistance of these cells to cancer treatments. Changes in Hsp27 phosphorylation have been associated with heat and metabolic stress, but its role in flavonoid anticancer activity has not been investigated. In this study, we examined the effect of apigenin in the regulation of Hsp27 on leukemia. We showed that apigenin does not affect Hsp27 expression but induces a bimodal phosphorylation on Ser78 and Ser82. The phosphorylation at early times was regulated by p38. At later times, Hsp27 phosphorylation was dependent on p38 activity and for some residues on PKCδ. Silencing of p38 expression reduced apigenin-induced phosphorylation on Ser15, Ser78, and Ser82, whereas silencing of PKCδ expression reduced the phosphorylation on Ser15 and Ser82 without affecting Ser78. In addition, we found that apigenin-induced PKCδ activity is mediated by p38. We also showed that the phosphorylation of Hsp27 significantly increased the susceptibility of leukemia cells to apigenin-induced apoptosis. Together, these results identify a complex signaling network regulating the cytotoxic effect of apigenin through Hsp27 phosphorylation.

Inhibition of ROS-induced apoptosis in endothelial cells by nitrone spin traps via induction of phase II enzymes and suppression of mitochondria-dependent pro-apoptotic signaling

Oxidative stress is the main etiological factor behind the pathogenesis of various diseases including inflammation, cancer, cardiovascular and neurodegenerative disorders. Due to the spin trapping abilities and various pharmacological properties of nitrones, their application as therapeutic agent has been gaining attention. Though the antioxidant properties of the nitrones are well known, the mechanism by which they modulate the cellular defense machinery against oxidative stress is not well investigated and requires further elucidation. Here, we have investigated the mechanisms of cytoprotection of the nitrone spin traps against oxidative stress in bovine aortic endothelial cells (BAEC). Cytoprotective properties of both the cyclic nitrone 5,5-dimethyl-pyrroline N-oxide (DMPO) and linear nitrone α-phenyl N-tert-butyl nitrone (PBN) against H₂O₂-induced cytotoxicity were investigated. Preincubation of BAEC with PBN or DMPO resulted in the inhibition of H₂O₂-mediated cytotoxicity and apoptosis. Nitrone-treatment resulted in the induction and restoration of phase II antioxidant enzymes via nuclear translocation of NF-E2-related factor 2 (Nrf-2) in oxidatively-challenged cells. Furthermore, the nitrones were found to inhibit the mitochondrial depolarization and subsequent activation of caspase-3 induced by H₂O₂. Significant down-regulation of the pro-apoptotic proteins p53 and Bax, and up-regulation of the anti-apoptotic proteins Bcl-2 and p-Bad were observed when the cells were preincubated with the nitrones prior to H₂O₂-treatment. It was also observed that Nrf-2 silencing completely abolished the protective effects of nitrones. Hence, these findings suggest that nitrones confer protection to the endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade.

Dietary Apigenin Exerts Immune-Regulatory Activity in Vivo by Reducing NF-κB Activity, Halting Leukocyte Infiltration and Restoring Normal Metabolic Function

The increasing prevalence of inflammatory diseases and the adverse effects associated with the long-term use of current anti-inflammatory therapies prompt the identification of alternative approaches to reestablish immune balance. Apigenin, an abundant dietary flavonoid, is emerging as a potential regulator of inflammation. Here, we show that apigenin has immune-regulatory activity in vivo. Apigenin conferred survival to mice treated with a lethal dose of Lipopolysaccharide (LPS) restoring normal cardiac function and heart mitochondrial Complex I activity. Despite the adverse effects associated with high levels of splenocyte apoptosis in septic models, apigenin had no effect on reducing cell death. However, we found that apigenin decreased LPS-induced apoptosis in lungs, infiltration of inflammatory cells and chemotactic factors’ accumulation, re-establishing normal lung architecture. Using NF-κB luciferase transgenic mice, we found that apigenin effectively modulated NF-κB activity in the lungs, suggesting the ability of dietary compounds to exert immune-regulatory activity in an organ-specific manner. Collectively, these findings provide novel insights into the underlying immune-regulatory mechanisms of dietary nutraceuticals in vivo.

The Small Heat Shock Protein 27 Is a Key Regulator of CD8+CD57+ Lymphocyte Survival

Differences in CD8+CD57− and CD8+CD57+ lymphocyte lifespan have been documented. Lower numbers and shorter lifespan are characteristic of CD8+CD57+ in normal individuals. However, CD8+CD57+ are expanded in certain disease states including T cell large granular leukemia and other hematologic malignancies. The mechanisms responsible for the differences in CD8+CD57− and CD8+CD57+ lifespan remain elusive. In this study, we demonstrate that the small heat shock protein (Hsp) 27 is a key regulator of CD8+CD57+ lymphocyte lifespan. We found that Hsp27 expression is significantly lower in CD8+CD57+ than in CD8+CD57− lymphocytes. In contrast, Hsp60 and Hsp70 are expressed at comparable levels. Unlike other antiapoptotic Bcl-2–like molecules, the expression of Hsp27 tightly correlates with CD8+CD57+ and CD8+CD57− lifespan. We demonstrate that Hsp27 overexpression in CD8+CD57+ lymphocytes to levels found normally in CD8+CD57− lymphocytes decreased apoptosis. Accordingly, silencing of Hsp27 in CD8+CD57− lymphocytes increased apoptosis. Collectively these results demonstrate that Hsp27 is a critical regulator of normal CD8+CD57+ lifespan supporting its use as a marker of lifespan in this lineage, and suggest a mechanism responsible for the decreased apoptosis and clonal expansion characteristic of certain disease states.

Distinct contribution of protein kinase Cδ and protein kinase Cε in the lifespan and immune response of human blood monocyte subpopulations

Monocytes, key components of the immune system, are a heterogeneous population comprised of classical monocytes (CD16−) and non‐classical monocytes (CD16+). Monocytes are short lived and undergo spontaneous apoptosis, unless stimulated. Dysregulation of monocyte numbers contribute to the pathophysiology of inflammatory diseases, yet the contribution of each subset remains poorly characterized. Protein kinase C (PKC) family members are central to monocyte biology; however, their role in regulating lifespan and immune function of CD16− and CD16+ monocytes has not been studied. Here, we evaluated the contribution of PKCδ and PKCε in the lifespan and immune response of both monocyte subsets. We showed that CD16+ monocytes are more susceptible to spontaneous apoptosis because of the increased caspase‐3, ‐8 and ‐9 activities accompanied by higher kinase activity of PKCδ. Silencing of PKCδ reduced apoptosis in both CD16+ and CD16− monocytes. CD16+ monocytes express significantly higher levels of PKCε and produce more tumour necrosis factor‐α in CD16+ compared with CD16− monocytes. Silencing of PKCε affected the survival and tumour necrosis factor‐α production. These findings demonstrate a complex network with similar topography, yet unique regulatory characteristics controlling lifespan and immune response in each monocyte subset, helping define subset‐specific coordination programmes controlling monocyte function.