Balancing Inflammatory, Lipid, and Xenobiotic Signaling Pathways by VSL#3, a Biotherapeutic Agent, in the Treatment of Inflammatory Bowel Disease

<p>Background: The interleukin 10 knockout mouse (IL10-KO) is a model of human inflammatory bowel disease (IBD) used to Study host microbial interactions and the action of potential therapeutics. Using Affymetrix data analysis, important signaling pathways and transcription factors relevant to gut inflammation and antiinflammatory probiotics were identified.</p><p>Methods: Affymetrix microarray analysis on both wildtype (WT) and IL10-KO mice orally administered with and without the probiotic VSL#3 was performed and the results validated by real-time polymerase chain reaction (PCR), immunocytochemistry, proteomics, and histopathology. Changes in metabolically active bacteria were assessed with denaturing gradient gel electrophoresis (DGGE).</p><p>Results: Inflammation in IL10-KO mice was characterized by differential regulation of inflammatory, nuclear receptor, lipid, and xenobiotic signaling pathways. Probiotic intervention resulted in downregulation of CXCL9 (fold change [FC] = -3.98, false discovery rate [FDR] = 0.019), CXCL10 (FC = -4.83, FDR = 0.0008), CCL5 (FC -3.47 FDR = 0.017), T-cell activation (Itgal [FC = -4.72, FDR = 0.00009], Itgae [FC = -2.54 FDR = 0.0044]) and the autophagy gene IRGM (FC = -1.94, FDR = 0.01), a recently identified susceptibility gene in human IBD. Consistent with a marked reduction in integrins, probiotic treatment decreased the number of CCL5+ CD3+ double-positive T Cells and upregulated galectin2, which triggers apoptosis of activated T cells. Importantly, genes associated with lipid and PPAR signaling (PPAR alpha [FC = 2.36, FDR = 0.043], PPARGC1 alpha [FC 2.58, FDR = 0.016], Nrld2 [FC = 3.11, FDR = 0.0067]) were also upregulated. Altered microbial diversity was noted in probiotic-treated mice.</p><p>Conclusions: Bioinformatics analysis revealed important immune response. phagocytic and inflammatory pathways dominated by elevation of T-helper cell 1 type (TH1) transcription factors in IL10-KO mice. Probiotic intervention resulted in a site-specific reduction of these pathways but importantly upregulated PPAR, xenobiotic, and lipid signaling genes. potential antagonists of NF-kappa B inflammatory pathways.</p>

HIV-1 Nef limits communication between linker of activated T cells and SLP-76 to reduce formation of SLP-76-signaling microclusters following TCR stimulation

Signal initiation by engagement of the TCR triggers actin rearrangements, receptor clustering, and dynamic organization of signaling complexes to elicit and sustain downstream signaling. Nef, a pathogenicity factor of HIV, disrupts early TCR signaling in target T cells. To define the mechanism underlying this Nef-mediated signal disruption, we employed quantitative single-cell microscopy following surface-mediated TCR stimulation that allows for dynamic visualization of distinct signaling complexes as microclusters (MCs). Despite marked inhibition of actin remodeling and cell spreading, the induction of MCs containing TCR-CD3 or ZAP70 was not affected significantly by Nef. However, Nef potently inhibited the subsequent formation of MCs positive for the signaling adaptor Src homology-2 domain-containing leukocyte protein of 76 kDa (SLP-76) to reduce MC density in Nef-expressing and HIV-1-infected T cells. Further analyses suggested that Nef prevents formation of SLP-76 MCs at the level of the upstream adaptor protein, linker of activated T cells (LAT), that couples ZAP70 to SLP-76. Nef did not disrupt pre-existing MCs positive for LAT. However, the presence of the viral protein prevented de novo recruitment of active LAT into MCs due to retargeting of LAT to an intracellular compartment. These modulations in MC formation and composition depended on Nef's ability to simultaneously disrupt both actin remodeling and subcellular localization of TCR-proximal machinery. Nef thus employs a dual mechanism to disturb early TCR signaling by limiting the communication between LAT and SLP-76 and preventing the dynamic formation of SLP-76-signaling MCs.

Apoptosis induction by oxidized glycated LDL in human retinal capillary pericytes is independent of activation of MAPK signaling pathways

Pericyte loss is a cardinal feature of early diabetic retinopathy. We previously reported that highly oxidized-glycated low density lipoprotein (HOG-LDL) induces pericyte apoptosis in vitro. In this study, we investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathways in HOG-LDL-induced apoptosis in human pericytes.

Sirolimus Stimulates Vascular Stem/Progenitor Cell Migration and Differentiation Into Smooth Muscle Cells via Epidermal Growth Factor Receptor/Extracellular Signal-Regulated Kinase/β-Catenin Signaling Pathway

Sirolimus-eluting stent therapy has achieved considerable success in overcoming coronary artery restenosis. However, there remain a large number of patients presenting with restenosis after the treatment, and the source of its persistence remains unclarified. Although recent evidence supports the contribution of vascular stem/progenitor cells in restenosis formation, their functional and molecular responses to sirolimus are largely unknown.

Involvement of mitochondrial and B-RAF/ERK signaling pathways in berberine-induced apoptosis in human melanoma cells

The natural isoquinoline alkaloid berberine exhibits a wide spectrum of biological activities including antitumor activity, but its mechanism of action remains to be fully elucidated. Here, we report that berberine induced apoptosis in human melanoma cells, through a process that involved mitochondria and caspase activation. Berberine-induced activation of a number of caspases, including caspases 3, 4, 7, 8, and 9. Pan-caspase inhibitor, z-VAD-fmk, and caspase-8 and caspase-9 inhibitors prevented apoptosis. Berberine also led to the generation of the p20 cleavage fragment of BAP31, involved in directing proapoptotic signals between the endoplasmic reticulum and the mitochondria. Treatment of SK-MEL-2 melanoma cells with berberine induced disruption of the mitochondrial transmembrane potential, release of cytochrome c and apoptosis-inducing factor from the mitochondria to the cytosol, generation of reactive oxygen species (ROS), and a decreased ATP/ADP ratio. Overexpression of bcl-xL by gene transfer prevented berberine-induced cell death, mitochondrial transmembrane potential loss, and cytochrome c and apoptosis-inducing factor release, but not ROS generation. N-acetyl-L-cysteine inhibited the production of ROS, but did not abrogate the berberine-induced apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) phosphorylation, by using the mitogen-activated protein kinase/ERK kinase inhibitor PD98059, and reduction of B-RAF levels by silencing RNA induced cell death of SK-MEL-2 cells, and diminished the berberine concentration required to promote apoptosis. These data show that berberine-induced apoptosis in melanoma cells involves mitochondria and caspase activation, but ROS generation was not essential. Our results indicate that inhibition of B-RAF/ERK survival signaling facilitates the cell death response triggered by berberine. © 2011 Wolters Kluwer Health | Lippincott Williams &amp; Wilkins.

Regulator of G-protein signaling 4 (RGS4) gene is associated with schizophrenia in Irish high density families

The regulator of the G-protein signaling 4 (RGS4) gene was shown to have a different expression pattern in schizophrenia patients in a microarray study. A family-based study subsequently implicated the association of this gene with schizophrenia. We replicated the study with our sample from the Irish Study of High Density Schizophrenia Families (ISHDSF). Single marker transmission disequilibrium tests (TDT) for the four core SNPs showed modest association for SNP 18 (using a narrow diagnostic approach with FBAT P = 0.044; with PDT P = 0.0073) and a trend for SNP 4 (with FBAT P = 0.1098; with PDT P = 0.0249). For SNP 1 and 7, alleles overtransmitted to affected subjects were the same as previously reported. Haplotype analyses suggested that haplotype G-G-G for SNP1-4-18, which is the most abundant haplotype (42.3%) in the Irish families, was associated with the disease (narrow diagnosis, FBAT P = 0.0061, PDT P = 0.0498). This was the same haplotype implicated in the original study. While P values were not corrected for multiple testing because of the clear prior hypothesis, these results could be interpreted as supporting evidence for the association between RGS4 and schizophrenia.

Implications of Intercellular Signaling for Radiation Therapy: A Theoretical Dose-Planning Study

Purpose <br/>Recent in vitro results have shown significant contributions to cell killing from signaling effects at doses that are typically used in radiation therapy. This study investigates whether these in vitro observations can be reconciled with in vivo knowledge and how signaling may have an impact on future developments in radiation therapy. <br/>Methods and Materials <br/>Prostate cancer treatment plans were generated for a series of 10 patients using 3-dimensional conformal therapy, intensity modulated radiation therapy (IMRT), and volumetric modulated arc therapy techniques. These plans were evaluated using mathematical models of survival following modulated radiation exposures that were developed from in vitro observations and incorporate the effects of intercellular signaling. The impact on dose-volume histograms and mean doses were evaluated by converting these survival levels into "signaling-adjusted doses" for comparison. <br/>Results <br/>Inclusion of intercellular communication leads to significant differences between the signalling-adjusted and physical doses across a large volume. Organs in low-dose regions near target volumes see the largest increases, with mean signaling-adjusted bladder doses increasing from 23 to 33 Gy in IMRT plans. By contrast, in high-dose regions, there is a small decrease in signaling-adjusted dose due to reduced contributions from neighboring cells, with planning target volume mean doses falling from 74 to 71 Gy in IMRT. Overall, however, the dose distributions remain broadly similar, and comparisons between the treatment modalities are largely unchanged whether physical or signaling-adjusted dose is compared. Conclusions Although incorporating cellular signaling significantly affects cell killing in low-dose regions and suggests a different interpretation for many phenomena, their effect in high-dose regions for typical planning techniques is comparatively small. This indicates that the significant signaling effects observed in vitro are not contradicted by comparison with clinical observations. Future investigations are needed to validate these effects in vivo and to quantify their ranges and potential impact on more advanced radiation therapy techniques.

Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis

Mutations that result in loss of function of Nod2, an intracellular receptor for bacterial peptidoglycan, are associated with Crohn's disease. Here we found that the E3 ubiquitin ligase Pellino3 was an important mediator in the Nod2 signaling pathway. Pellino3-deficient mice had less induction of cytokines after engagement of Nod2 and had exacerbated disease in various experimental models of colitis. Furthermore, expression of Pellino3 was lower in the colons of patients with Crohn's disease. Pellino3 directly bound to the kinase RIP2 and catalyzed its ubiquitination. Loss of Pellino3 led to attenuation of Nod2-induced ubiquitination of RIP2 and less activation of the transcription factor NF-?B and mitogen-activated protein kinases (MAPKs). Our findings identify RIP2 as a substrate for Pellino3 and Pellino3 as an important mediator in the Nod2 pathway and regulator of intestinal inflammation. © 2013 Nature America, Inc. All rights reserved.

The poxvirus protein A52R targets Toll-like receptor signaling complexes to suppress host defense

Toll-like receptors (TLRs) are crucial in the innate immune response to pathogens, in that they recognize and respond to pathogen associated molecular patterns, which leads to activation of intracellular signaling pathways and altered gene expression. Vaccinia virus (VV), the poxvirus used to vaccinate against smallpox, encodes proteins that antagonize important components of host antiviral defense. Here we show that the VV protein A52R blocks the activation of the transcription factor nuclear factor kappa B (NF-kappa B) by multiple TLRs, including TLR3, a recently identified receptor for viral RNA. A52R associates with both interleukin 1 receptor-associated kinase 2 (IRAK2) and tumor necrosis factor receptor-associated factor 6 (TRAF6), two key proteins important in TLR signal transduction. Further, A52R could disrupt signaling complexes containing these proteins. A virus deletion mutant lacking the A52R gene was attenuated compared with wild-type and revertant controls in a murine intranasal model of infection. This study reveals a novel mechanism used by VV to suppress the host immunity. We demonstrate viral disabling of TLRs, providing further evidence for an important role for this family of receptors in the antiviral response.

Elevated Circulation Levels of an Antiangiogenic SERPIN in Patients with Diabetic Microvascular Complications Impair Wound Healing through Suppression of Wnt Signaling

Wound healing, angiogenesis and hair follicle maintenance are often impaired in the skin of diabetic patients, but the pathogenesis has not been well understood. Here, we report that circulation levels of kallistatin, a member of the serine proteinase inhibitor (SERPIN) superfamily with anti-angiogenic activities, were elevated in Type 2 diabetic patients with diabetic vascular complications. To test the hypothesis that elevated kallistatin levels could contribute to a wound healing deficiency via inhibition of Wnt/β-catenin signaling, we generated kallistatin-transgenic (KS-TG) mice. KS-TG mice had reduced cutaneous hair follicle density, microvascular density, and panniculus adiposus layer thickness as well as altered skin microvascular hemodynamics and delayed cutaneous wound healing. Using Wnt reporter mice, our results showed that Wnt/β-catenin signaling is suppressed in dermal endothelium and hair follicles in KS-TG mice. Lithium, a known activator of β-catenin via inhibition of glycogen synthase kinase-3β, reversed the inhibition of Wnt/β-catenin signaling by kallistatin and rescued the wound healing deficiency in KS-TG mice. These observations suggest that elevated circulating anti-angiogenic serpins in diabetic patients may contribute to impaired wound healing through inhibition of Wnt/β-catenin signaling. Activation of Wnt/β-catenin signaling, at a level downstream of Wnt receptors, may ameliorate the wound healing deficiency in diabetic patients.Journal of Investigative Dermatology accepted article preview online, 24 January 2014. doi:10.1038/jid.2014.40.

Role of ion channels and subcellular Ca2+ signalling in arachidonic acid induced dilation of pressurised retinal arterioles

Purpose: To investigate the mechanisms responsible for the dilatation of rat retinal arterioles in response to arachidonic acid (AA). Methods: Changes in the diameter of isolated, pressurized rat retinal arterioles were measured in the presence of AA alone and following pre-incubation with pharmacological agents inhibiting Ca2+ sparks and oscillations and K+ channels. Subcellular Ca2+ signals were recorded in arteriolar myocytes using Fluo-4-based confocal imaging. The effects of AA on membrane currents of retinal arteriolar myocytes were studied using whole-cell perforated patch clamp recording. Results: AA dilated pressurised retinal arterioles under conditions of myogenic tone. Eicosatetraynoic acid (ETYA) exerted a similar effect, but unlike AA, its effects were rapidly reversible. AA-induced dilation was associated with an inhibition of subcellular Ca2+ signals. Interventions known to block Ca2+ sparks and oscillations in retinal arterioles caused dilatation and inhibited AA-induced vasodilator responses. AA accelerated the rate of inactivation of the A-type Kv current and the voltage dependence of inactivation was shifted to more negative membrane potentials. It also enhanced voltage-activated and spontaneous BK currents, but only at positive membrane potentials. Pharmacological inhibition of A-type Kv and BK currents failed to block AA-induced vasodilator responses. AA suppressed L-type Ca2+ currents. Conclusions: These results suggest that AA induces retinal arteriolar vasodilation by inhibiting subcellular Ca2+ signalling activity in retinal arteriolar myocytes, most likely through a mechanism involving the inhibition of L-type Ca2+ channel activity. AA actions on K+ currents are inconsistent with a model in which K+ channels contribute to the vasodilator effects of AA.

V3 versican isoform alters the behavior of human melanoma cells by interfering with CD44/ErbB-dependent signaling

Versican is a hyaluronan-binding, extracellular chondroitin sulfate proteoglycan produced by several tumor types, including malignant melanoma, which exists as four different splice variants. The short V3 isoform contains the G1 and G3 terminal domains of versican that may potentially interact directly or indirectly with the hyaluronan receptor CD44 and the EGFR, respectively. We have previously described that overexpression of V3 in MeWo human melanoma cells markedly reduces tumor cell growth in vitro and in vivo. In this study we have investigated the signaling mechanism of V3 by silencing the expression of CD44 in control and V3-expressing melanoma cells. Suppression of CD44 had the same effects on cell proliferation and cell migration than those provoked by V3 expression, suggesting that V3 acts through a CD44-mediated mechanism. Furthermore, CD44-dependent hyaluronan internalization was blocked by V3 expression and CD44 silencing, leading to an accumulation of this glycosaminoglycan in the pericellular matrix and to changes in cell migration on hyaluronan. Furthermore, ERK1/2 and p38 activation after EGF treatment were decreased in V3-expressing cells suggesting that V3 may also interact with the EGFR through its G3 domain. The existence of a EGFR/ErbB2 receptor complex able to interact with CD44 was identified in MeWo melanoma cells. V3 overexpression resulted in a reduced interaction between EGFR/ErbB2 and CD44 in response to EGF treatment. Our results indicate that the V3 isoform of versican interferes with CD44 and the CD44-EGFR/ErbB2 interaction, altering the signaling pathways, such as ERK1/2 and p38 MAPK, that regulate cell proliferation and migration.