TNFα signals via p66Shcto induce E-selectin, promote leukocyte transmigration and enhance permeability in human endothelial cells

Endothelial cells participate in inflammatory events leading to atherogenesis by regulating endothelial cell permeability via the expression of VE-Cadherin and β-catenin and leukocyte recruitment via the expression of E-Selectins and other adhesion molecules. The protein p66Shc acts as a sensor/inducer of oxidative stress and may promote vascular dysfunction. The objective of this study was to investigate the role of p66Shc in tumor necrosis factor TNFα-induced E-Selectin expression and function in human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNFα resulted in increased leukocyte transmigration through the endothelial monolayer and E-Selectin expression, in association with augmented phosphorylation of both p66Shc on Ser36 and the stress kinase c-Jun NH2-terminal protein kinase (JNK)-1/2, and higher intracellular reactive oxygen species (ROS) levels. Overexpression of p66 Shc in HUVEC resulted in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selectin levels, and amplified endothelial cell permeability and leukocyte transmigration through the HUVEC monolayer. Conversely, overexpression of a phosphorylation-defective p66 Shc protein, in which Ser36 was replaced by Ala, did not augment ROS and E-Selectin levels, nor modify cell permeability or leukocyte transmigration beyond those found in wild-type cells. Moreover, siRNA-mediated silencing of p66Shc resulted in marked reduction of E-Selectin expression and leukocyte transmigration. In conclusion, p66Shc acts as a novel intermediate in the TNFα pathway mediating endothelial dysfunction, and its action requires JNK-dependent phosphorylation of p66 Shc on Ser36. © 2013 Laviola et al

Tomato Prosystemin Is Much More than a Simple Systemin Precursor

Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity

Effects of TNFα on endothelial activation in HUVEC.

<p>Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. <i>A</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/wt, stimulated with 50 ng/ml TNFα for 4 h, and left to migrate for 18 h at 37°C. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to control. *<i>P</i><0.05 vs. no TNFα. <i>B</i>. TNFα regulation of E-Selectin mRNA levels. E-Selectin mRNA levels were evaluated by qRT-PCR, and normalized using β-actin as internal control. <i>C</i>. TNFα regulation of E-Selectin protein levels. E-Selectin protein levels were evaluated by immunoblotting, using GAPDH as internal control. <i>D</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (treated with negative siRNA) and HUVEC treated with E-Selectin siRNA#1 (50 nM) or siRNA#2 (100 nM) for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates of one experiment and are normalized to control. *<i>P</i><0.05 vs. negative siRNA. <i>E</i>. Effects of SP600125 (left) and PD98059 (right) on TNFα-induced E-Selectin mRNA expression. Cells were pre-treated with 30 mM JNK or MEK inhibitor, respectively, for 2 h and then exposed 50 ng/ml TNFα for the indicated times (untreated cells, black; inhibitor-treated cells, grey; DMSO-treated cells, light grey). E-Selectin mRNA levels were evaluated by qRT-PCR, using β-actin as internal control. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated cells.</p

Role of Ser³⁶ phosphorylation of p66^Shc in endothelial activation.

<p><i>A</i>. Basal and TNFα-stimulated phosphorylation of p66^Shc on Ser³⁶ in HUVEC overexpressing the p66^Shc Ala³⁶ mutant (HUVEC/p66^ShcAla³⁶). Cells were stimulated with 50 ng/ml TNFα for 0.5 h or left untreated. When indicated, cells were pre-incubated with 30 mM SP600125 or DMSO for 2 h prior to TNFα stimulation. Each pair of representative immunoblots shows p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left) in HUVEC/wt, HUVEC/p66^Shc, HUVEC/p66^ShcAla³⁶, and HUVEC/mock, respectively. The ratio of phosphorylated to total p66^Shc protein in the four cell lines is also shown (<i>right</i>; HUVEC/wt, filled bars; HUVEC/p66^Shc, open bars; HUVEC/p66^ShcAla³⁶, grey bars; HUVEC/mock, light grey bars). *<i>P</i><0.05, TNFα-stimulated HUVEC/p66^Shc vs. HUVEC/p66^ShcAla³⁶; ^#<i>P</i><0.05, SP600125-treated HUVEC/p66^Shc vs. untreated HUVEC/p66^Shc. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to HUVEC/w.t. and HUVEC treated with Ad/p66^Shc, Ad/p66^ShcAla³⁶, or Ad/mock for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *<i>P</i><0.05 vs. no TNFα; ^§P<0.05 vs. HUVEC/wt; ^#<i>P</i><0.05 vs. HUVEC/p66^ShcAla³⁶. <i>C</i>. Effects of Ser/Ala³⁶ mutation on E-Selectin mRNA levels in HUVEC. HUVEC/wt (filled bars), HUVEC/p66^Shc (open bars), HUVEC/<i>Ala</i>³⁶ (grey bars), and HUVEC/mock (light grey bars) were treated with 50 ng/ml TNFα for 1 h or left untreated. E-Selectin gene expression was evaluated using qRT-PCR. *<i>P</i><0.05, TNFα-stimulated cells vs. unstimulated cells; ^#<i>P</i><0.05, HUVEC/p66^Shc vs. controls. <i>D</i>. Cartoon illustrating the signaling pathway mediating the stimulatory effect of TNFα on E-Selectin gene expression in HUVEC. MKKs, MKK-7 and MKK-4; IKK, IκBα kinase; ROS, reactive oxygen species.</p

Effects of p66^Shc silencing on endothelial activation in HUVEC.

<p><i>A</i>. (top left) Representative immunoblot of p66^Shc Ser³⁶ phosphorylation in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; black bars, cells treated with siRNA#1 and siRNA#2. (bottom left) Representative immunoblot of p66^Shc protein content in HUVEC transfected with 50 nM or 100 nM of siRNA#1 or siRNA#2, respectively, and (right) quantification of multiple experiments. <i>Hatched </i><i>bar</i>, cells transfected with a negative control siRNA; grey bars, cells treated with siRNA#1; light grey bars, cells treated with siRNA#2. *<i>P</i><0.05 vs. cells transfected with negative siRNA. Neg., negative siRNA. <i>B</i>. Leukocyte transmigration test. HL-60 cells were added to control HUVEC (w.t., Lipofectamine™, and negative siRNA) and HUVEC treated with p66^Shc siRNA#1 (100 nM) or p66^Shc siRNA#2 (50 nM), and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *<i>P</i><0.05 vs. no TNFα; ^§P<0.05 vs. HUVEC/wt. <i>C</i>. Effects of siRNA-mediated knockdown of p66^Shc on E-Selectin mRNA levels under basal conditions (left) and following TNFα stimulation (right). HUVEC were transfected with 100 nM siRNA#1 (grey bars) or 50 nM siRNA#2 (light grey bars), and then left untreated or incubated with 50 ng/ml TNFα for 1 h. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated controls (wild-type, Lipofectamine™, and negative siRNA). Lysates were analyzed by qPCR 48 h following transfection, using β-actin as internal control. Lipo, Lipofectamine™.</p

Role of oxidative stress in p66^Shc signaling.

<p><i>A</i>. Levels of reactive oxygen species (ROS) in HUVEC/p66^Shc and control cells (HUVEC/wt and HUVEC/mock). Cells were pre-incubated with or without 30 mM SP600125 for 2 h and then treated with 50 ng/ml TNFα for 0.5 h or left untreated; ROS levels were evaluated by fluorimetry (HUVEC/wt, filled bars; HUVEC/mock, grey bars; HUVEC/p66^Shc, open bars). *<i>P</i><0.05 vs. untreated cells; ^#<i>P</i><0.05 vs. cells stimulated with TNFα alone; all values of HUVEC/p66^Shc were significantly different (<i>P</i><0.05) vs. HUVEC/wt. Data from multiple independent experiments (n=6) are expressed as mean±SE. <i>B</i>. Effects of N-acetyl-cysteine (NAC) on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. <i>C</i>. Effects of NAC on TNFα-induced E-Selectin expression. In <i>B</i> and <i>C</i>, HUVEC/wt (black bars), HUVEC/mock (grey bars), and HUVEC/p66^Shc (open bars) were preincubated with 10 mM NAC for 0.5 h before stimulation with 50 ng/ml TNFα. *<i>P</i><0.05 vs. untreated cells; ^#<i>P</i><0.05 vs. TNFα-stimulated cells not treated with NAC. <i>D</i>. Cells were coincubated with TNFα (50 ng/ml) and NADPH oxidase (Nox)-inhibitor 3-benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo[4,5-d]pyrimidine (VAS2810, 5 µM) or rotenone (Rot, 10 µM) + thenoyltrifluoroacetone (TTFA, 10 µM) for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ and GAPDH (left), and ratio of phosphorylated p66^Shc protein to GAPDH (<i>right</i>; untreated cells, black bars; TNFα-treated cells, grey bars). *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. TNFα-stimulated cells.</p

Effects of p66^Shc overexpression on endothelial activation in HUVEC.

<p>A. Representative images of HUVEC/p66^Shc and HUVEC/mock. Cells were infected with the adenoviral constructs and analyzed by fluorescent microscopy. The green staining identifies cells expressing the green fluorescent protein (GFP) encoded by the recombinant adenovirus. Cellular morphology was evaluated by optical microscopy. B. Leukocyte transmigration test. HL-60 cells were added to HUVEC/wt and HUVEC transduced with Ad/p66^Shc or Ad/mock for 48 h, and left to migrate for 18 h at 37°C. Studies were carried out under basal conditions or following stimulation with 50 ng/ml TNFα for 4 h. Migrated cells were stained and measured by OD at 450 nm. Data represents the mean of triplicates from two independent experiments, and are normalized to basal HUVEC/wt. *P<0.05 vs. no TNFα; ^§P<0.05 vs. controls. C. Effects of p66^Shc overexpression on E-Selectin mRNA levels in HUVEC. HUVEC/wt (filled bars), HUVEC/p66^Shc (open bars), and HUVEC/mock (grey bars) were treated with 50 ng/ml TNFα for 1 h or left untreated. When indicated, cells were pre-treated with 30 mM SP600125 or DMSO for 2 h. E-Selectin gene expression was evaluated using qRT-PCR. The inset (right) shows E-Selectin mRNA levels under basal conditions. *P<0.05, TNFα-stimulated cells vs. unstimulated cells; ^#P<0.05, HUVEC/p66^Shc vs. controls; ^§P<0.05, SP600125-treated cells vs. controls.</p

TNFα-induced phosphorylation of p66^Shc on Ser³⁶ in HUVEC.

<p><i>A</i>. Dose-response studies. Cells were incubated with TNFα for 0.5 h at the indicated doses or left untreated. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66^Shc protein (right). Cell lysates were analyzed by immunoblotting with specific antibodies. <i>B</i>. Time-course studies. Cells were incubated with 50 ng/ml TNFα for the indicated times or left untreated. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (bottom left), and ratio of phosphorylated to total p66^Shc protein (right). <i>C</i>. Effects of the JNK inhibitor SP600125 on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. Cells were pre-treated with 30 mM SP600125 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66^Shc protein (<i>right</i>; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). <i>D</i>. Effects of the MEK inhibitor PD98059 on TNFα-induced phosphorylation of p66^Shc on Ser³⁶. Cells were pre-treated with 30 mM PD98059 for 2 h and then exposed to 50 ng/ml TNFα for 0.5 h. Representative immunoblots of p66^Shc phosphorylation on Ser³⁶ (top left) and Shc protein content (middle left), and ratio of phosphorylated to total p66^Shc protein (<i>right</i>; untreated cells, black bars; inhibitor-treated cells, grey bars; DMSO-treated cells, white bars). GAPDH protein content was used as loading control. *<i>P</i><0.05 vs. basal; ^#<i>P</i><0.05 vs. controls.</p

Environmental effectiveness of GAEC cross-compliance Standard 3.1 ‘Ploughing in good soil moisture conditions’ and economic evaluation of the competitiveness gap for farmers

Within the MO.NA.CO. Project the environmental effectiveness of GAEC cross-compliance Standard 3.1 ‘Ploughing in good soil moisture conditions’ was evaluated, as well as the economic evaluation of the competitiveness gap for farmers which conform or do not conform to cross-compliance. The monitoring has been carried out at nine experimental farms with different pedoclimatic characteristics, where some indicators of soil structure degradation have been evaluated, such as bulk density, packing density and surface roughness of the seedbed, and the crop productive and qualitative parameters. In each monitoring farm two experimental plots have been set up: factual with soil tillage at proper water content (tilth), counterfactual with soil tillage at inadequate water content (no tilth). The monitoring did not exhibit univocal results for the different parameters, thus the effectiveness of the Standard 3.1 is ‘contrasting’ (class of merit B), and there was an evident practical problem to till the soil at optimum water content, even in controlled experimental condition. Bulk density was significantly lower in the factual treatment although in soils with very different textures (sandy-loam and clayey). Packing density (PD) showed a high susceptibility to compaction in soils with low PD and medium texture. The tortuosity index, indicating the roughness of the seedbed, was lower and generally significantly different in the factual treatment. Results showed that the ploughing done in excessive soil moisture conditions is more expensive due to the increased force of traction of the tractor, which causes an increase in slip of the tractor wheels, with a speed reduction and increase in the working times and fuel consumption. Moreover, the crop yield is also reduced considerably according to the cultivated species