Local and remote tissue injury upon intestinal ischemia and reperfusion depends on the TLR/MyD88 signaling pathway

Innate immune responses against microorganisms may be mediated by Toll-like receptors (TLRs). Intestinal ischemia-reperfusion (i-I/R) leads to the translocation of bacteria and/or bacterial products such as endotoxin, which activate TLRs leading to acute intestinal and lung injury and inflammation observed upon gut trauma. Here, we investigated the role of TLR activation by using mice deficient for the common TLR adaptor protein myeloid differentiation factor 88 (MyD88) on local and remote inflammation following intestinal ischemia. Balb/c and MyD88(-/-) mice were subjected to occlusion of the superior mesenteric artery (45 min) followed by intestinal reperfusion (4 h). Acute neutrophil recruitment into the intestinal wall and the lung was significantly diminished in MyD88(-/-) after i-I/R, which was confirmed microscopically. Diminished neutrophil recruitment was accompanied with reduced concentration of TNF-alpha and IL-1 beta level. Furthermore, diminished microvascular leak and bacteremia were associated with enhanced survival of MyD88(-/-) mice. However, neither TNF-alpha nor IL-1 beta neutralization prevented neutrophil recruitment into the lung but attenuated intestinal inflammation upon i-I/R. In conclusion, our data demonstrate that disruption of the TLR/MyD88 pathway in mice attenuates acute intestinal and lung injury, inflammation, and endothelial damage allowing enhanced survival.

Extracellular Signal-Regulated Kinase 1/2 Activation, via Downregulation of Mitogen-Activated Protein Kinase Phosphatase 1, Mediates Sex Differences in Desoxycorticosterone Acetate-Salt Hypertension Vascular Reactivity

Extracellular signal-regulated kinase (ERK) 1/2 has been reported to play a role in vascular dysfunction associated with mineralocorticoid hypertension. We hypothesized that, compared with female rats, an upregulation of ERK1/2 signaling in the vasculature of male rats contributes to augmented contractile responses in mineralocorticoid hypertension. Uninephrectomized male and female Sprague-Dawley rats received desoxycorticosterone acetate (DOCA) pellets (200 mg per animal) and saline to drink for 3 weeks. Control uninephrectomized rats received tap water to drink. Blood pressure, measured by telemetry, was significantly higher in male DOCA rats (191 +/- 3 mm Hg) compared with female DOCA rats (172 +/- 7 mm Hg; n=5). DOCA treatment resulted in augmented contractile responses to phenylephrine in aorta (22 +/- 3 mN; n=6) and small mesenteric arteries (13 +/- 2 mN; n=6) from male DOCA rats versus uninephrectomized male rats (16 +/- 3 and 10 +/- 2 mN, respectively; P<0.05) and female DOCA rats (15 +/- 1 and 11 +/- 1 mN, respectively). ERK1/2 inhibition with PD-98059 (10 mu mol/L) abrogated increased contraction to phenylephrine in aorta (14 +/- 2 mN) and small mesenteric arteries (10 +/- 2 mN) from male DOCA rats, without any effects in arteries from male uninephrectomized or female animals. Compared with the other groups, phosphorylated ERK1/2 levels were increased in the aorta from male DOCA rats, whereas mitogen-activated protein kinase phosphatase 1 expression was decreased. Interleukin-10 plasma levels, which positively regulate mitogen-activated protein kinase phosphatase 1 activity, were reduced in male DOCA-salt rats. We speculate that augmented vascular reactivity in male hypertensive rats is mediated via activation of the ERK1/2 pathway. In addition, mitogen-activated protein kinase phosphatase 1 and interleukin 10 play regulatory roles in this process. (Hypertension. 2010; 55: 172-179.)

Melatonin Protects CD4(+) T Cells from Activation-Induced Cell Death by Blocking NFAT-Mediated CD95 Ligand Upregulation

Over the past 20 y, the hormone melatonin was found to be produced in extrapineal sites, including cells of the immune system. Despite the increasing data regarding the biological effects of melatonin on the regulation of the immune system, the effect of this molecule on T cell survival remains largely unknown. Activation-induced cell death plays a critical role in the maintenance of the homeostasis of the immune system by eliminating self-reactive or chronically stimulated T cells. Because activated T cells not only synthesize melatonin but also respond to it, we investigated whether melatonin could modulate activation-induced cell death. We found that melatonin protects human and murine CD4(+) T cells from apoptosis by inhibiting CD95 ligand mRNA and protein upregulation in response to TCR/CD3 stimulation. This inhibition is a result of the interference with calmodulin/calcineurin activation of NFAT that prevents the translocation of NFAT to the nucleus. Accordingly, melatonin has no effect on T cells transfected with a constitutively active form of NFAT capable of migrating to the nucleus and transactivating target genes in the absence of calcineurin activity. Our results revealed a novel biochemical pathway that regulates the expression of CD95 ligand and potentially other downstream targets of NFAT activation. The Journal of Immunology, 2010, 184: 3487-3494.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway

Giachini FR, Zemse SM, Carneiro FS, Lima VV, Carneiro ZN, Callera GE, Ergul A, Webb RC, Tostes RC. Interleukin-10 attenuates vascular responses to endothelin-1 via effects on ERK1/2-dependent pathway. Am J Physiol Heart Circ Physiol 296: H489-H496, 2009. First published December 12, 2008; doi:10.1152/ajpheart.00251.2008.-Interleukin-10 (IL-10) is an anti-inflammatory cytokine with protective actions on the vasculature. On the other hand, endothelin ( ET)-1 has potent vasoconstrictor, mitogenic, and proinflammatory activities, which have been implicated in the pathophysiology of a number of cardiovascular diseases. We hypothesized that, in a condition where ET-1 expression is upregulated, i.e., on infusion of TNF-alpha, IL-10 confers vascular protection from ET-1-induced injury. Aortic rings and first-order mesenteric arteries from male C57BL/6 (WT) and IL-10-knockout (IL-10(-/-)) mice were treated with human recombinant TNF-alpha (220 ng.kg(-1).day(-1)) or vehicle (saline) for 14 days. TNF-alpha infusion significantly increased blood pressure in IL-10(-/-), but not WT, mice. TNF-alpha augmented vascular ET-1 mRNA expression in arteries from WT and IL-10(-/-) mice. ET type A (ETA) receptor expression was increased in arteries from IL-10(-/-) mice, and TNF-alpha infusion did not change vascular ETA receptor expression in control or IL-10(-/-) mice. Aorta and mesenteric arteries from TNF-alpha-infused IL-10(-/-) mice displayed increased contractile responses to ET-1, but not the ET type B receptor agonist IRL-1620. The ETA receptor antagonist atrasentan completely abolished responses to ET-1 in aorta and mesenteric vessels, whereas the ERK1/2 inhibitor PD-98059 abrogated increased contractions to ET-1 in arteries from TNF-alpha-infused IL-10(-/-) mice. Infusion of TNF-alpha, as well as knockdown of IL-10 (IL-10(-/-)), induced an increase in total and phosphorylated ERK1/2. These data demonstrate that IL-10 counteracts ET(A)-mediated vascular responses to ET-1, as well as activation of the ERK1/2 pathway.

Islet neogenesis-associated protein signaling in neonatal pancreatic rat islets: involvement of the cholinergic pathway

Islet neogenesis associated protein (INGAP) increases islet mass and insulin secretion in neonatal and adult rat islets. lit the Present Study, we measured the short- and long-term effects of INGAP-PP (a pentadecapeptide having the 104-118 amino acid sequence of INGAP) upon islet protein expression and phosphorylation of components of the PI3K, MAPK and cholinergic pathways, and on insulin secretion. Short-term exposure of neonatal islets to INGAP-PP (90 s, 5, 15, and 30 min) significantly increased Akt1(-Ser473) and MAPK3/1(-Thr202/Tyr204) phosphorylation and INGAP-PP also acutely increased insulin secretion from islets perifused with 2 and 20 mM glucose. Islets cultured for 4 days in the presence of INGAP-PP showed an increased expression of Akt1, Frap1, and Mapk1 mRNAs as well as of the muscarinic M3 receptor subtype, and phospholipase C (PLC)-beta 2 proteins. These islets also showed increased Akt1 and MAPK3/1 protein phosphorylation. Brief exposure of INGAP-P-treated islets to carbachol (Cch) significantly increased P70S6K(-Thr389) and MAPK3/1 phosphorylation and these islets released more insulin when challenged with Cch that was prevented by the M3 receptor antagonist 4-DAMP in a concentration-dependent manner. In conclusion, these data indicate that short- and long-term exposure to INGAP-PP significantly affects the expression and the phosphorylation of proteins involved in islet PI3K and MAPK signaling pathways. The observations of INGAPP-PP-stimulated up-regulation of cholinergic M3 receptors and PLC-beta 2 proteins, enhanced P70S6K and MAIIK3/1 phosphorylation and Cch-induced insulin secretion suggest a participation of the cholinergic pathway in INGAP-PP-mediated effects.

Exercise Training Reduces Insulin Resistance and Upregulates the mTOR/p70S6k Pathway in Cardiac Muscle of Diet-Induced Obesity Rats

Obesity and insulin resistance are rapidly expanding public health problems. These disturbances are related to many diseases, including heart pathology. Acting through the Akt/mTOR pathway, insulin has numerous and important physiological functions, such as the induction of growth and survival of many cell types and cardiac hypertrophy. However, obesity and insulin resistance can alter mTOR/p70S6k. Exercise training is known to induce this pathway, but never in the heart of diet-induced obesity subjects. To evaluate the effect of exercise training on mTOR/p70S6k in the heart of obese Wistar rats, we analyzed the effects of 12 weeks of swimming on obese rats, induced by a high-fat diet. Exercise training reduced epididymal fat, fasting serum insulin and plasma glucose disappearance. Western blot analyses showed that exercise training increased the ability of insulin to phosphorylate intracellular molecules such as Akt (2.3-fold) and Foxo1 (1.7-fold). Moreover, reduced activities and expressions of proteins, induced by the high-fat diet in rats, such as phospho-JNK (1.9-fold), NF-kB (1.6-fold) and PTP-1B (1.5-fold), were observed. Finally, exercise training increased the activities of the transduction pathways of insulin-dependent protein synthesis, as shown by increases in Raptor phosphorylation (1.7-fold), p70S6k phosphorylation (1.9-fold), and 4E-BP1 phosphorylation (1.4-fold) and a reduction in atrogin-1 expression (2.1-fold). Results demonstrate a pivotal regulatory role of exercise training on the Akt/ mTOR pathway, in turn, promoting protein synthesis and antagonizing protein degradation. J. Cell. Physiol. 226: 666-674, 2011. (C) 2010 Wiley-Liss, Inc.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Angiotensin type 1 receptor mediates thyroid hormone-induced cardiomyocyte hypertrophy through the Akt/GSK-3 beta/mTOR signaling pathway

Several studies have implicated the renin angiotensin system in the cardiac hypertrophy induced by thyroid hormone. However, whether Angiotensin type 1 receptor (AT(1)R) is critically required to the development of T(3)-induced cardiomyocyte hypertrophy as well as whether the intracellular mechanisms that are triggered by AT(1)R are able to contribute to this hypertrophy model is unknown. To address these questions, we employed a selective small interfering RNA (siRNA, 50 nM) or an AT(1)R blocker (Losartan, 1 mu M) to evaluate the specific role of this receptor in primary cultures of neonatal cardiomyocytes submitted to T(3) (10 nM) treatment. The cardiomyocytes transfected with the AT(1)R siRNA presented reduced mRNA (90%, P < 0.001) and protein (70%, P < 0.001) expression of AT(1)R. The AT(1)R silencing and the AT(1)R blockade totally prevented the T(3)-induced cardiomyocyte hypertrophy, as evidenced by lower mRNA expression of atrial natriuretic factor (66%, P < 0.01) and skeletal alpha-actin (170%, P < 0.01) as well as by reduction in protein synthesis (85%, P < 0.001). The cardiomyocytes treated with T(3) demonstrated a rapid activation of Akt/GSK-3 beta/mTOR signaling pathway, which was completely inhibited by the use of PI3K inhibitors (LY294002, 10 mu M and Wortmannin, 200 nM). In addition, we demonstrated that the AT(1)R mediated the T(3)-induced activation of Akt/GSK-3 beta/mTOR signaling pathway, since the AT(1)R silencing and the AT(1)R blockade attenuated or totally prevented the activation of this signaling pathway. We also reported that local Angiotensin I/II (Ang I/II) levels (120%, P < 0.05) and the AT(1)R expression (180%, P < 0.05) were rapidly increased by T(3) treatment. These data demonstrate for the first time that the AT(1)R is a critical mediator to the T(3)-induced cardiomyocyte hypertrophy as well as to the activation of Akt/GSK-3 beta/mTOR signaling pathway. These results represent a new insight into the mechanism of T(3)-induced cardiomyocyte hypertrophy, indicating that the Ang I/II-AT(1)R-Akt/GSK-3 beta/mTOR pathway corresponds to a potential mediator of the trophic effect exerted by T(3) in cardiomyocytes.

Evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii

The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor N omega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(center dot)NO-cGMP signaling pathway playing a role in zoospore biogenesis. (C) 2009 Elsevier Inc. All rights reserved.

Arginine vasopressin controls p27(KiP1) protein expression by PKC activation and irreversibly inhibits the proliferation of K-Ras-dependent mouse Y1 adrenocortical malignant cells

The neurohypophyseal hormone arginine vasopressin (AVP) is a classic mitogen in many cells. In K-Ras-dependent mouse Y1 adrenocortical malignant cells, AVP elicits antagonistic responses such as the activation of the PKC and the ERK1/2 mitogenic pathways to down-regulate cyclin D1 gene expression, which induces senescence-associated beta-galactosidase (SA-beta Gal) and leads to cell cycle arrest. Here, we report that in the metabolic background of Y1 cells, PKC activation either by AVP or by PMA inhibits the PI3K/Akt pathway and stabilises the p27(Kip1) protein even in the presence of the mitogen fibroblast growth factor 2 (FGF2). These results suggest that p27(Kip1) is a critical signalling node in the mechanisms underlying the survival of the Y1 cells. In Y1 cells that transiently express wild-type p27(Kip1), AVP caused a severe reduction in cell survival, as shown by clonogenic assays. However, AVP promoted the survival of Y1 cells transiently expressing mutant p27-S10A or mutant p27-T187A, which cannot be phosphorylated at Ser10 and Thr187, respectively. In addition, PKC activation by PMA mimics the toxic effect caused by AVP in Y1 cells, and inhibition of PKC completely abolishes the effects caused by both PMA and AVP in clonogenic assays. The vulnerability of Y1 cells during PKC activation is a phenotype conditioned upon K-ras oncogene amplification because K-Ras down-regulation with an inducible form of the dominant-negative mutant H-RasN17 has resulted in Y1 cells that are resistant to AVP`s deleterious effects. These data show that the survival destabilisation of K-Ras-dependent Y1 malignant cells by AVP requires large quantities of the p27(Kip1) protein as well as phosphorylation of the p27(Kip1) protein at both Ser10 and Thr187. (C) 2011 Elsevier B.V. All rights reserved.

Susceptibility to Yersinia pseudotuberculosis Infection is Linked to the Pattern of Macrophage Activation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modulatory effect of prostaglandins on human monocyte activation for killing of high- and low-virulence strains of Paracoccidioides brasiliensis

The effect of indomethacin (Indo), a cyclo-oxygenase inhibitor, on the monocyte-mediated killing of a low-(Pb265) and a high-(Pb18) virulence strain of Paracoccidioides brasiliensis was examined. The Pb18 strain was not killed by either non-activated or interferon-gamma (IFN-gamma)-activated human monocytes but these cells did show fungicidal activity if pretreated with Indo. In contrast with IFN-gamma tumour necrosis factor-alpha (TNF-alpha) was very effective at stimulating the fungicidal activity of monocytes. While the low-virulence strain, Pb265, could not be killed by monocytes, cells preincubated with IFN-gamma demonstrated fungicidal activity. The killing of this strain was also induced by pretreatment of monocytes with Indo. The results suggest a negative role for prostaglandins, which are synthesized via the cyclo-oxygenase pathway, in the regulation of monocyte-mediated killing of virulent and avirulent strains of P. brasiliensis and that TNF-alpha generation during the fungus-monocyte interaction is more important in the killing of Pb265 than Pb18.

Silibinin modulates the NF-kappa b pathway and pro-inflammatory cytokine production by mononuclear cells from preeclamptic women

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of the Interleukin-10 Signaling Pathway Genes in Individuals With Down Syndrome and Periodontitis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)