Protein kinase C-ERK1/2 signal pathway switches glucose depletion-induced necrosis to apoptosis by regulating superoxide dismutases and suppressing reactive oxygen species production in A549 lung cancer cells

Cells typically die by either apoptosis or necrosis. However, the consequences of apoptosis and necrosis are quite different for a whole organism. In the case of apoptosis, the cell content remains packed in the apoptotic bodies that are removed by macrophages, and thereby inflammation does not occur; during necrosis, the cell membrane is ruptured, and the cytosolic constituents are released into the extracellular space provoking inflammation. Recently, inflammation and necrosis have been suggested to promote tumor growth. We investigated the molecular mechanism underlying cell death in response to glucose depletion (GD), a common characteristic of the tumor microenvironment. GD induced necrosis through production of reactive oxygen species (ROS) in A549 lung carcinoma cells. Inhibition of ROS production by N-acetyl-L-cysteine and catalase prevented necrosis and switched the cell death mode to apoptosis that depends on mitochondrial death pathway involving caspase-9 and caspase-3 activation, indicating a critical role of ROS in determination of GD-induced cell death mode. We demonstrate that protein kinase C-dependent extracellular regulated kinase 1/2 (ERK1/2) activation also switched GD-induced necrosis to apoptosis through inhibition of ROS production possibly by inducing manganese superoxide dismutase (SOD) expression and by preventing GD-induced degradation of copper zinc SOD. Thus, these results suggest that GD-induced cell death mode is determined by the protein kinase C/ERK1/2 signal pathway that regulates MnSOD and CuZnSOD and that these antioxidants may exert their known tumor suppressive activities by inducing necrosis-to-apoptosis switch.     

A distinct role of neutrophil lactoferrin in RelA/p65 phosphorylation on Ser536 by recruiting TNF receptor-associated factors to IkappaB kinase signaling complex

The activation of NF-kappaB by neutrophil lactoferrin (Lf) is regulated via the IkappaB kinase (IKK) signaling cascade, resulting in the sequential phosphorylation and degradation of IkappaB. In this study, we observed that Lf protein augmented p65 phosphorylation at the Ser(536), but not the Ser(276) residue, and stimulated the translocation of p65 into the nucleus. Lf was also shown to enhance the association between p65 and CREB-binding protein/p300 in vivo. To elucidate the mechanism by which Lf triggers these signaling pathways, we attempted to delineate the roles of the upstream components of the IKK complex, using their dominant-negative mutants and IKKalpha(-/-) and IKKbeta(-/-) mouse embryonic cells. We demonstrated that both IKKalpha and IKKbeta as well as NF-kappaB-inducing kinase are indispensable for Lf-induced p65 phosphorylation. However, MAPK kinase kinase 1 is not essentially required for this activation. We also observed that Lf-induced p65 phosphorylation was either partially or completely abrogated as the result of treatment with the mutant forms of TNFR-associated factor (TRAF) 2, TRAF5, or TRAF6. Moreover, we demonstrated that Lf directly interacted with TRAF5. Expression of the dominant-negative mutant of TRAF5 or its small interfering RNA almost completely abrogated the Lf-induced p65 phosphorylation. These results suggest that signaling pathways, including TRAFs/NF-kappaB-inducing kinase/IKKs, may be involved in the regulation of Lf-induced p65 activation, thereby resulting in the activation of members of the NF-kappaB family.     

Triamcinolone acetonide protects the rat retina from STZ-induced acute inflammation and early vascular leakage

Streptozotocin (STZ) has been commonly used to induce in vivo and in vitro hyperglycemic diabetes and its toxicity leads to inflammation and vascular injury. Triamcinolone acetonide (TA), as an anti-angiogenic/anti-inflammatory drug, is clinically used to improve the visual acuity in neovascular and edematous ocular diseases. The aim of this study was to investigate the effect of TA on early inflammation and vascular leakage in the retina of STZ-induced hyperglycemic rats. Hyperglycemia was induced in 8-week-old male Sprague-Dawley (SD) rats by a single intraperitoneal injection of STZ (65 mg/kg); only rats with blood glucose levels >13.9 mmol/l 1 day after STZ injection were included in STZ-hyperglycemic group. Sex- and age-matched SD rats injected with buffer were used as the control group. One day before STZ and buffer injection, 2 microl TA (4 mg/ml in saline) and 2 microl saline were intravitreal-injected into the right and the left eyes of rats, respectively. Retinal vascular leakage was measured using the Evans-blue method. Changes in pro-inflammatory target genes, such as tumor necrotic factor (TNF)-alpha, intracellular adhesion molecule (ICAM)-1, and vascular endothelial growth factor (VEGF) were assessed by immunoblottings, immunostaining, and ELISA analyses. Vascular hyperleakage and up-regulation of most pro-inflammatory genes peaked within a few days after STZ injection and had recovered. However, these changes were blocked by TA pretreatment. Our data suggest that TA controls STZ-induced early vascular leakage and temporary pro-inflammatory signals in the rat retina.     

Inhibition by epigallocatechin gallate of CoCl2-induced apoptosis in rat PC12 cells

Epigallocatechin-3-gallate (EGCG) is a major constituent of green tea polyphenols. This study was aimed to investigate the possible mechanisms of EGCG-mediated inhibition against apoptosis in rat pheochromocytoma PC12 cells by exposure to CoCl(2). Exposure to CoCl(2) caused the generation of ROS and induced cell death with appearance of apoptotic morphology and DNA fragmentation. However, EGCG rescued the loss of viability in the cells exposed to CoCl(2) and led the reduction of DNA fragmentation and sub-G(1) fraction of cell cycle. Also, EGCG attenuated the CoCl(2)-induced disruption of mitochondrial membrane potential (DeltaPsim), release of cytochrome c from the mitochondria to cytosol and abolished the CoCl(2)-stimulated activities of the caspase cascades, caspase-9 and caspase-3. In addition, EGCG ameliorated the increase in the Bax to Bcl-2 ratio, a marker of apoptosis proceeding, induced by CoCl(2) treatment. Taken together, the present results suggest that EGCG inhibit the CoCl(2)-induced apoptosis of PC12 cells through the mitochondria-mediated apoptosis pathway involved in modulating the Bcl-2 family.     

Annexin II binds to SHP2 and this interaction is regulated by HSP70 levels

The protein tyrosine phosphatase SHP2 is a positive effector of EGFR signaling. To improve our understanding of SHP2's function, we searched for additional binding proteins of SHP2. We found that Annexin II is an SHP2-binding protein. Physical interactions of SHP2 with Annexin II were confirmed in vivo. Furthermore, binding of SHP2 with Annexin II was regulated somewhat by EGF treatment and the extracellular Ca2+ chelator, EGTA. Previously, we reported that HSP70 levels can influence the binding of SHP2 with EGFR. Interestingly, increased HSP70 levels also inhibited the binding of SHP2 with Annexin II after EGF treatment in vivo. In addition, immunostaining experiments indicated that a fraction of SHP2 and Annexin II co-localized in the cell membrane region after EGF treatment. Our findings indicate that Annexin II is binding partner of SHP2 and the binding of SHP2 with Annexin II is affected by EGF stimulation, extracellular calcium levels, and the levels of HSP70.     

Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran

TLR4 and MD-2 form a heterodimer that recognizes LPS (lipopolysaccharide) from Gram-negative bacteria. Eritoran is an analog of LPS that antagonizes its activity by binding to the TLR4-MD-2 complex. We determined the structure of the full-length ectodomain of the mouse TLR4 and MD-2 complex. We also produced a series of hybrids of human TLR4 and hagfish VLR and determined their structures with and without bound MD-2 and Eritoran. TLR4 is an atypical member of the LRR family and is composed of N-terminal, central, and C-terminal domains. The beta sheet of the central domain shows unusually small radii and large twist angles. MD-2 binds to the concave surface of the N-terminal and central domains. The interaction with Eritoran is mediated by a hydrophobic internal pocket in MD-2. Based on structural analysis and mutagenesis experiments on MD-2 and TLR4, we propose a model of TLR4-MD-2 dimerization induced by LPS.