Insulin signaling in vascular endothelial cells: a key role for heterotrimeric G proteins revealed by siRNA-mediated Gbeta1 knockdown

Activation of insulin receptors stimulates the phosphoinositide 3-kinase (PI3-K)/Akt signaling pathway in vascular endothelial cells. Heterotrimeric G proteins appear to modulate some of the cellular responses that are initiated by receptor tyrosine kinases, but the roles of specific G protein subunits in signaling are less clearly defined. We found that insulin treatment of cultured bovine aortic endothelial cells (BAEC) activates the alpha isoform of PI3-K (PI3-Kalpha) and discovered that purified G protein Gbeta1gamma2 inhibits PI3-Kalpha enzyme activity. Transfection of BAEC with a duplex siRNA targeting bovine Gbeta1 leads to a 90% knockdown in Gbeta1 protein levels, with no effect on expression of other G protein subunits. siRNA-mediated Gbeta1 knockdown markedly and specifically potentiates insulin-dependent activation of kinase Akt, likely reflecting the removal of the inhibitory effect of Gbetagamma on PI3-Kalpha activity. Insulin-induced tyrosine phosphorylation of insulin receptors is unaffected by Gbeta1 siRNA. By contrast, Gbeta1 knockdown leads to a significant decrease in the level of serine phosphorylation of the insulin receptor substrate IRS-1. We explored the effects of siRNA on several serine/threonine protein kinases that have been implicated in insulin signaling. Gbeta1 siRNA significantly attenuates phosphorylation of the 70 kDa ribosomal protein S6 kinase (p70S6K) in the basal state and following insulin treatment. We also found that IGF-1-initiated activation of Akt is significantly enhanced after siRNA-mediated Gbeta1 knockdown, while IGF-1-induced p70S6K activation is markedly suppressed following transfection of Gbeta1 siRNA. We propose that Gbeta1 participates in the activation of p70S6K, which in turn promotes the serine phosphorylation and inhibition of IRS-1. Taken together, these studies suggest that Gbeta1 plays an important role in insulin and IGF-1 signaling in endothelial cells, both by inhibiting the activity of PI3-Kalpha and by stimulating pathways that lead to activation of protein kinase p70S6K and to the serine phosphorylation of IRS-1.     

Biochemical characterization of UDP-GlcNAc/Glc 4-epimerase from Escherichia coli O86:B7

The O-antigen of lipopolysaccharide in Gram-negative bacteria plays an important role in bacterium-host interactions. Escherichia coli O86:B7 O-unit contains five sugar residues: one fucose (Fuc) and two each of N-acetylgalactosamine (GalNAc) and galactose (Gal). The entire O-antigen gene cluster was previously sequenced: orf1 was assigned the gne gene for the biosynthesis of UDP-GalNAc. To confirm this annotation, overexpression, purification, and biochemical characterization of Gne were performed. By using capillary electrophoresis, we showed that Gne can catalyze the interconversion of both UDP-GlcNAc/GalNAc and UDP-Glc/Gal almost equally well. The Km values of Gne for UDP-Glc, UDP-Gal, UDP-GlcNAc, and UDP-GalNAc are 370, 295, 323, and 373 microM, respectively. The comparison of kinetic parameters of Gne from Escherichia coli O86:B7 to those of other characterized UDP-GlcNAc/Glc 4-epimerases indicated that it has relaxed specificity toward the four substrates, the first characterized enzyme to have this activity in the O-antigen biosynthesis. Moreover, the calculated kcat/Km values for UDP-GalNAc and UDP-Gal are approximately 2-4 times higher than those for UDP-GlcNAc and UDP-Glc, suggesting that Gne is slightly more efficient for the epimerization of UDP-GalNAc and UDP-Gal. One mutation (S306Y) resulted in a loss of epimerase activity for non-acetylated substrates by about 5-fold but totally abolished the activity for N-acetylated substrates, indicating that residue S306 plays an important role in the determination of substrate specificity.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

The role of tuberous sclerosis complex 1 in regulating innate immunity

The mechanisms that control TLR-induced responses, including endotoxin tolerance, have been not well understood. The tuberous sclerosis complex 1 (TSC1) is a tumor suppressor that inhibits the mammalian target of rapamycin (mTOR). We show in this study that deficiency of TSC1 results in enhanced activation of not only mTOR complex 1 (mTORC1), but also JNK1/2, following LPS stimulation in macrophages. TSC1-deficient macrophages produce elevated proinflammatory cytokines and NO in response to multiple TLR ligands. Such enhanced TLR-induced responses can be inhibited by reducing mTORC1 and JNK1/2 activities with chemical inhibitors or small hairpin RNA, suggesting that TSC1 negatively controls TLR responses through both mTORC1 and JNK1/2. The impact of TSC1 deficiency appeared not limited to TLRs, as NOD- and RIG-I/MDA-5-induced innate responses were also altered in TSC1-deficient macrophages. Furthermore, TSC1 deficiency appears to cause impaired induction of endotoxin tolerance in vitro and in vivo, which is correlated with increased JNK1/2 activation and can be reversed by JNK1/2 inhibition. Our results reveal a critical role of TSC1 in regulating innate immunity by negative control of mTORC1 and JNK1/2 activation.     

Interaction between M-like protein and macrophage thioredoxin facilitates antiphagocytosis for Streptococcus equi ssp. zooepidemicus

Streptococcus equi ssp. zooepidemicus (S. zooepidemicus, S.z) is one of the common pathogens that can cause septicemia, meningitis, and mammitis in domesticated species. M-like protein (SzP) is an important virulence factor of S. zooepidemicus and contributes to bacterial infection and antiphagocytosis. The interaction between SzP of S. zooepidemicus and porcine thioredoxin (TRX) was identified by the yeast two-hybrid and further confirmed by co-immunoprecipitation. SzP interacted with both reduced and the oxidized forms of TRX without inhibiting TRX activity. Membrane anchored SzP was able to recruit TRX to the surface, which would facilitate the antiphagocytosis of the bacteria. Further experiments revealed that TRX regulated the alternative complement pathway by inhibiting C3 convertase activity and associating with factor H (FH). TRX alone inhibited C3 cleavage and C3a production, and the inhibitory effect was additive when FH was also present. TRX inhibited C3 deposition on the bacterial surface when it was recruited by SzP. These new findings indicated that S. zooepidemicus used SzP to recruit TRX and regulated the alternative complement pathways to evade the host immune phagocytosis.     

Comparative proteomic analysis of Streptococcus suis biofilms and planktonic cells that identified biofilm infection-related immunogenic proteins

Streptococcus suis (SS) is a zoonotic pathogen that causes severe disease symptoms in pigs and humans. Biofilms of SS bind to extracellular matrix proteins in both endothelial and epithelial cells and cause persistent infections. In this study, the differences in the protein expression profiles of SS grown either as planktonic cells or biofilms were identified using comparative proteomic analysis. The results revealed the existence of 13 proteins of varying amounts, among which six were upregulated and seven were downregulated in the Streptococcus biofilm compared with the planktonic controls. The convalescent serum from mini-pig, challenged with SS, was applied in a Western blot assay to visualize all proteins from the biofilm that were grown in vitro and separated by two-dimensional gel electrophoresis. A total of 10 immunoreactive protein spots corresponding to nine unique proteins were identified by MALDI-TOF/TOF-MS. Of these nine proteins, five (Manganese-dependent superoxide dismutase, UDP-N-acetylglucosamine 1-carboxyvinyltransferase, ornithine carbamoyltransferase, phosphoglycerate kinase, Hypothetical protein SSU05_0403) had no previously reported immunogenic properties in SS to our knowledge. The remaining four immunogenic proteins (glyceraldehyde-3-phosphate dehydrogenase, hemolysin, pyruvate dehydrogenase and DnaK) were identified under both planktonic and biofilm growth conditions. In conclusion, the protein expression pattern of SS, grown as biofilm, was different from the SS grown as planktonic cells. These five immunogenic proteins that were specific to SS biofilm cells may potentially be targeted as vaccine candidates to protect against SS biofilm infections. The four proteins common to both biofilm and planktonic cells can be targeted as vaccine candidates to protect against both biofilm and acute infections.     

Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.     

Plasmin potentiates synaptic N-methyl-D-aspartate receptor function in hippocampal neurons through activation of protease-activated receptor-1

Protease-activated receptor-1 (PAR1) is activated by a number of serine proteases, including plasmin. Both PAR1 and plasminogen, the precursor of plasmin, are expressed in the central nervous system. In this study we examined the effects of plasmin in astrocyte and neuronal cultures as well as in hippocampal slices. We find that plasmin evokes an increase in both phosphoinositide hydrolysis (EC(50) 64 nm) and Fura-2/AM fluorescence (195 +/- 6.7% above base line, EC(50) 65 nm) in cortical cultured murine astrocytes. Plasmin also activates extracellular signal-regulated kinase (ERK1/2) within cultured astrocytes. The plasmin-induced rise in intracellular Ca(2+) concentration ([Ca(2+)](i)) and the increase in phospho-ERK1/2 levels were diminished in PAR1(-/-) astrocytes and were blocked by 1 microm BMS-200261, a selective PAR1 antagonist. However, plasmin had no detectable effect on ERK1/2 or [Ca(2+)](i) signaling in primary cultured hippocampal neurons or in CA1 pyramidal cells in hippocampal slices. Plasmin (100-200 nm) application potentiated the N-methyl-D-aspartate (NMDA) receptor-dependent component of miniature excitatory postsynaptic currents recorded from CA1 pyramidal neurons but had no effect on alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate- or gamma-aminobutyric acid receptor-mediated synaptic currents. Plasmin also increased NMDA-induced whole cell receptor currents recorded from CA1 pyramidal cells (2.5 +/- 0.3-fold potentiation over control). This effect was blocked by BMS-200261 (1 microm; 1.02 +/- 0.09-fold potentiation over control). These data suggest that plasmin may serve as an endogenous PAR1 activator that can increase [Ca(2+)](i) in astrocytes and potentiate NMDA receptor synaptic currents in CA1 pyramidal neurons.     

Differential expression of microRNAs in mouse liver under aberrant energy metabolic status

Despite years of effort, exact pathogenesis of nonalcoholic fatty liver disease (NAFLD) remains obscure. To gain an insight into the regulatory roles of microRNAs (miRNAs) in aberrant energy metabolic status and pathogenesis of NAFLD, we analyzed the expression of miRNAs in livers of ob/ob mice, streptozotocin (STZ)-induced type 1 diabetic mice, and normal C57BL/6 mice by miRNA microarray. Compared with normal C57BL/6 mice, ob/ob mice showed upregulation of eight miRNAs and downregulation of four miRNAs in fatty livers. Upregulation of miR-34a and downregulation of miR-122 was found in livers of STZ-induced diabetic mice. These results demonstrate that distinct miRNAs are strongly dysregulated in NAFLD and hyperglycemia. Comparison between miRNA expressions in livers of ob/ob mice and STZ-administered mice further revealed upregulation of four miRNAs and downregulation of two miRNAs in livers of ob/ob mice, indicating that these miRNAs may represent a molecular signature of NAFLD. A distinctive miRNA expression pattern was identified in ob/ob mouse liver, and hierarchical clustering of this pattern could clearly discriminate ob/ob mice from either normal C57BL/6 mice or STZ-administered mice. These findings suggest an important role of miRNAs in hepatic energy metabolism and implicate the participation of miRNAs in the pathophysiological processes of NAFLD.