Induced Signal Transduction Research Articles

Angiogenesis is induced by airway smooth muscle strain

Angiogenesis is an important feature of airway remodeling in both chronic asthma and chronic obstructive pulmonary disease (COPD). Airways in those conditions are exposed to excessive mechanical strain during periods of acute exacerbations. We recently reported that mechanical strain of human airway smooth muscle (HASM) led to an increase in their proliferation and migration. Sustained growth in airway smooth muscle in vivo requires an increase in the nutritional supply to these muscles, hence angiogenesis. In this study, we examined the hypothesis that cyclic mechanical strain of HASM produces factors promoting angiogenic events in the surrounding vascular endothelial cells. Our results show: 1) a significant increase in human lung microvascular endothelial cell (HMVEC-L) proliferation, migration, and tube formation following incubation in conditioned media (CM) from HASM cells exposed to mechanical strain; 2) mechanical strain of HASM cells induced VEGF expression and release; 3) VEGF neutralizing antibodies inhibited the proliferation, migration, and tube formations of HMVEC-L induced by the strained airway smooth muscle CM; 4) mechanical strain of HASM induced a significant increase in hypoxia-inducible factor-1alpha (HIF-1alpha) mRNA and protein, a transcription factor required for VEGF gene transcription; and 5) mechanical strain of HASM induced HIF-1alpha/VEGF through dual phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) and ERK pathways. In conclusion, exposing HASM cells to mechanical strain induces signal transduction pathway through PI3K/Akt/mTOR and ERK pathways that lead to an increase in HIF-1alpha, a transcription factor required for VEGF expression. VEGF release by mechanical strain of HASM may contribute to the angiogenesis seen with repeated exacerbation of asthma and COPD.

Unique Signaling Properties of CTAR1 in LMP1-Mediated Transformation

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-kappaB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G(1)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.

8-Prenylnaringenin, inhibits estrogen receptor-α mediated cell growth and induces apoptosis in MCF-7 breast cancer cells

The discovery that the hop constituent 8-prenylnaringenin (8PN) shows potent estrogenic activity, higher than that of the known phytoestrogens coumestrol, genistein and daidzein, has spurred an intense activity aimed at elucidating its biological profile and its dietary relevance connected with the consumption of beer. We have investigated if 8PN can induce signal transduction pathways via rapid estrogen receptor (ER) activation. Under conditions of estrogen-dependent growth, treatment of MCF-7 human breast cancer cells with 8PN induced a rapid and transient activation of the MAP kinase Erk-1 and Erk-2, with kinetics similar to those induced by 17β-estradiol (E2). 8PN could trigger the MAP kinase pathway via dual c-Src kinase activation and association with ERα. Co-treatment with the ER antagonist ICI 182,780 blocked each step of this transduction pathway, confirming its ER dependence. However, and in striking contrast with E2, 8PN could not induce the PI3K/Akt pathway, resulting in altered kinetics and levels of cyclin D1 expression. In accordance with these observations, flow cytometric and biochemical analysis showed that 8PN inhibited cell cycle progression and induced apoptosis in MCF-7 cells. Interference with an ER associated PI3K pathway is proposed as a possible mechanism underlying the inhibition of survival and proliferation of estrogen responsive cells by 8PN. Taken together, our finding show that 8PN is an interesting new chemotype to explore the biology of ERs.

Interferon-alpha-2b induced signal transduction and gene regulation in patient immune cells is not enhanced by a dose increase from 5 MU/m2 to 10 MU/m2

3026 Background: High dose interferon-alpha-2b (IFN-a) is employed as an adjuvant in melanoma patients who have had surgery for high-risk lesions. It mediates its anti-tumor effects via activation of the transcription factor STAT1 (signal transducer and activator of transcription) within host immune cells. We hypothesized that intermediate doses of IFN-a would be just as effective as higher doses in stimulating the activation of STAT1 and gene transcription in immune cells. Methods: Samples for analysis were obtained from patients with metastatic melanoma who were enrolled in a clinical trial of bevacizumab in combination with escalating doses of IFN-a (5 MU/m^2 and then 10 MU/m^2). Peripheral blood mononuclear cells (PBMCs) were procured before and 1 hour after the administration of IFN-a and analyzed for the presence of phosphorylated STAT1 (P-STAT1) and P-STAT2 by intracellular flow cytometric analysis and the induction of interferon stimulated gene (ISG) transcripts by Real Time PCR. Results: P-STAT1 in response to 5 MU/m^2 IFN-a was higher than that for the 10 MU/m^2 dose (p = 0.0617). The 5 MU/m^2 dose also led to a greater activation of STAT2 (p = 0.0388). The induction of interferon stimulated genes (ISGs; IFIT1, IFIT2, OAS3) within PBMCs was not enhanced following the increase in IFN-a dose to 10 MU/m^2 at 2 weeks although inhibitors of IFN-a-signaling (SOCS1 and SOCS3) were activated to a greater degree. In addition, microarray analysis was performed on 4 patients and revealed that only one of 36 interferon regulated genes was expressed to a greater extent following treatment with 10 MU/m^2 IFN-a as compared to 5 MU/m^2. Conclusions: These results suggest that 5 MU/m^2 of IFN-a are as effective as higher doses with respect to the induction of STAT signal transduction and ISGs within immune effector cells. No significant financial relationships to disclose.

Platelet-Derived Growth Factor Receptor β–Mediated Phosphorylation of MUC1 Enhances Invasiveness in Pancreatic Adenocarcinoma Cells

MUC1 is a heterodimeric transmembrane glycoprotein that is overexpressed and aberrantly glycosylated in ductal adenocarcinomas. Differential phosphorylation of the MUC1 cytoplasmic tail (MUC1CT) has been associated with signaling events that influence the proliferation and metastasis of cancer cells. We identified a novel tyrosine phosphorylation site (HGRYVPP) in the MUC1CT by mass spectrometric analysis of MUC1 from human pancreatic adenocarcinoma cell lines. Analyses in vitro and in vivo showed that platelet-derived growth factor receptor beta (PDGFRbeta) catalyzed phosphorylation of this site and of tyrosine in the RDTYHPM site. Stimulation of S2-013.MUC1F cells with PDGF-BB increased nuclear colocalization of MUC1CT and beta-catenin. PDGF-BB stimulation had no significant effect on cell proliferation rate; however, it enhanced invasion in vitro through Matrigel and in vivo tumor growth and metastases. Invasive properties of the cells were significantly altered on expression of phosphorylation-abrogating or phosphorylation-mimicking mutations at these sites. We propose that interactions of MUC1 and PDGFRbeta induce signal transduction events that influence the metastatic properties of pancreatic adenocarcinoma.

Response to “Binding of the C‐terminal amino acids of VEGF121 directly with neuropilin‐1 should be considered”

We have recently found that neuropilin-1 and neuropilin-2 enhance VEGF121 induced signal transduction mediated by the VEGFR-2 tyrosine kinase receptor (1). This observation was surprising because several previous studies have shown that VEGF121 does not bind to native neuropilins of endothelial cells or to recombinant neuropilins (2, 3). Our observation suggests that neuropilins may enhance VEGFR2-mediated VEGF signaling even when the VEGF binding site of neuropilin-1 is not occupied by VEGF. In their letter, Drs. Von-Wronski, Tweedle, and Nunn point out that several small peptides containing sequences homologous to the sequence found at the C-terminal of exon-8 of the VEGF gene are able to inhibit the binding of VEGF165 to neuropilin-1 and inhibit VEGF165 induced signaling. These results suggest that all VEGF forms containing this exon, including VEGF121, should be able to bind to neuropilin-1. Knowing that VEGF121 does not normally bind to neuropilins the authors of the letter suggest that neuropilin-1 may display increased affinity towards VEGF121 when in complex with VEGFR-2, thereby enabling binding of VEGF121 to neuropilin-1. This alternative hypothesis predicts that these peptides should be able to inhibit neuropilin-1mediated enhancement of VEGFR-2 signaling induced by VEGF121, provided that the mechanism by which these peptides inhibit the biological effect of neuropilin-1 on VEGF signaling is indeed dependent on their ability to inhibit VEGF binding to neuropilin-1. When we noticed that neuropilins enhance VEGFR2-mediated VEGF121 signaling, we did think of the possibility suggested by the authors of the letter. We published a manuscript several years ago in which we showed that VEGF121 is able to bind to neuropilin-1 and neuropilin-2 in the presence of VEGFR-1 (4). Therefore, following our initial observation, we conducted cross-linking experiments in which we cross-linked IVEGF121 to PAE cells coexpressing VEGFR-2 and either neuropilin-1 or neuropilin-2. We used I-VEGF121 at concentrations of up to 100 ng/ml. However, we could not detect I-VEGF121/np1 or I-VEGF121/np2 complexes in these experiments. We did not show a figure containing these data in the manuscript due to their negative nature, but we described these experiments and their outcome in the results section. The results of these experiments represent a major reason for favoring the model we proposed in the manuscript. Secondly, neuropilin-1 enhanced I-VEGF165 binding to VEGFR-2 at low I-VEGF165 concentrations, but at these concentrations we could not observe binding to neuropilin-1, arguing independently that the VEGF binding site does not need to be occupied for neuropilin-1 mediated enhancement. Nevertheless, it is still possible, although we think less likely, that when in complex with VEGFR-2, neuropilins assume a conformation that allows VEGF121 to bind to neuropilins, but does not allow the homobifunctional cross-linkers we used to cross-link the bound VEGF121 to neuropilins, resulting in incorrect conclusions. If that is what happens, than this neuropilin conformation must differ considerably from the neuropilin conformation induced by VEGFR-1 since in that case we were able to cross-link VEGF121 to neuropilins (4). Examining this possibility may be worthwhile but it may not be easy to design such an experiment. It should be noted that it is not sufficient to show that a peptide such as Tuftsin inhibits neuropilin-enhanced VEGF121 activity, because the mechanism by which these peptides inhibit the biological effect of neuropilin-1 may be unrelated to their effect on VEGF165 binding to neuropilin-1.

ErbB-2 Induces the Cyclin D1 Gene in Prostate Epithelial Cells In vitro and In vivo

The receptor tyrosine kinase ErbB-2 plays an important role in the regulation of growth factor-induced signal transduction cascades in the epithelium, and ErbB-2 is frequently overexpressed in epithelial tumors. Our previous studies on clinical prostate cancer specimens indicated that ErbB-2 expression was increased in patients undergoing hormone ablation therapy. We had also shown that the critical cell cycle regulatory gene cyclin D1 and its promoter were targets of proliferative signaling in prostate cancer cell lines, and that cyclin D1 was required for ErbB-2-induced mammary tumorigenesis. In the current studies, we found that increased ErbB-2 membrane expression correlated with increased nuclear cyclin D1 staining in clinical prostate cancer specimens, and that expression of ErbB-2 was capable of inducing cell cycle progression in human prostate cancer cell lines. We further showed that ErbB-2 induced the cyclin D1 promoter in DU145 cells, and that small interfering RNA knockdown of cyclin D1 protein levels blocked a significant proportion of the heregulin-induced cell cycle progression in LNCaP cells. Probasin promoter-targeted expression of an activated ErbB-2 isoform induced cyclin D1 expression in the mouse prostate, commensurate with prostate intraepithelial neoplasia. Together, these in vitro and in vivo studies identify cyclin D1 as a critical downstream target of ErbB-2 in the prostate epithelium, both of which are possible therapeutic targets for cancer intervention. Furthermore, our novel mouse model provides a useful platform for ongoing in vivo investigations of ErbB-2 signaling in the prostate epithelium.

109: Suppressors of cytokine signaling (SOCS) negatively regulate IFN-alpha-induced signal transduction, gene expression, and anti-tumor activity of immune effector cells

Introduction: Suppressors of cytokine signaling (SOCS) regulate cytokine signaling in many cells, but their role in modulating the response of immune cells to exogenous interferon-alpha (IFNa) has not been defined in the context of cancer immunotherapy. We hypothesized that SOCS proteins are negative regulators of IFNa induced signal transduction, gene expression, and anti-tumor activity. Methods: SOCS1-knockout mice (that were also IFNγ−/−) and SOCS3+/−mice were bred to produce SOCS-competent and SOCS-deficient counterparts.

Two axes in platelet-derived growth factor signaling: tyrosine phosphorylation and reactive oxygen species.

The tyrosine phosphorylation cascade is a hallmark of platelet-derived growth factor (PDGF)- induced signal transduction. The amplitude and propagation of the tyrosine phosphorylation signal relies on the balance between tyrosine kinase and tyrosine phosphatase. The tyrosine kinase is latent in the absence of stimulation, whereas the tyrosine phosphatase is highly and constitutively active. Therefore, the kinase activation should be accompanied by temporal and spatial inactivation of tyrosine phosphatase to achieve the robust amplification of tyrosine phosphorylation. For the past decade, reactive oxygen species have been receiving a great deal of attention with regard to their ability to shut down tyrosine phosphatase activities in a reversible manner. In this article, the crosstalk between tyrosine phosphorylation and reactive oxygen species in PDGF signaling is discussed.

Float on: lipid rafts in the lifecycle of HIV

All steps in the HIV lifecycle--entry, assembly, budding, induction of signal transduction and subsequent cell activation--are complex multifactorial mechanisms where cholesterol and sphingolipids (glycosphingolipids--GSLs and sphingomyelins in mammalian cells) are closely involved. Here we will review the contribution of these heterogeneous membrane lipid microdomains, referred to as lipid rafts, DIGs (detergent-insoluble glycolipid-enriched complexes), DRMs (detergent-resistant membranes), GEMs (glycolipid-enriched membranes), caveolae, TIMs (Triton-insoluble membranes) for interactions of HIV with the host cell. The accurate terminology was discussed elsewhere, and to simplify matters we will use rafts or lipid rafts throughout the review.

A Functional Screen of Genes Expressed in AML Patients Identifies Novel Transforming Properties of IL27R.

A thorough understanding of the signaling pathways required for AML formation is necessary in order to develop much needed therapeutic treatments for AML patients. While there are many known mutations that contribute to AML, additional unknown genes likely exist, since the leukemic cells of a significant number of AML patients are cytogenetically normal and AML likely forms as a result of two mutations in different classes of oncogenes. Therefore, in order to identify novel mutations and signaling pathways that contribute to AML, we undertook functional genetic screening for genes, expressed in the leukemic cells of AML patients, that could transform myeloid cells in culture. To do this we generated cDNA libraries from cytogenetically normal AML patients and screened 32D myeloid cells for cytokine independent growth. We did not identify any genes capable of transforming 32D cells to cytokine independence by this approach, likely because the apoptotic response upon cytokine removal is very rapid. We then performed screens in 32D cells that exogenously express the anti-apoptotic Bcl2 protein in order to attempt to sensitize these cells to transformation by unknown oncogenes. These cells undergo cell death much slower than parental 32D cells in the absence of cytokine. From these screens, we identified IL27Ra (also known as TCCR and WSX1) as a gene that can induce the cytokine-independent growth of 32D cells. Interestingly, IL27R is capable of transforming 32D cells that lack exogenous Bcl2 expression, suggesting that our use of Bcl2 sensitized 32D cells to transformation in the context of expressing a library of genes in these cells. Therefore, this approach may allow for the identification of AML oncogenes in a relevant cell system. IL27Ra (IL27R) is a type I cytokine receptor that functions as the ligand-binding component for the receptor for IL-27 and functions with the gp130 co-receptor to induce signal transduction in response to IL-27. Cytokine independent 32D/IL27R cells contain elevated levels of activated forms of various signaling proteins, including JAK1, JAK2, STAT1, STAT5, ERK1/2, and SHP-2. Activation of these signaling proteins is dependent on the kinase activity of JAK family proteins as the pan JAK inhibitor, JAK inhibitor I, blocked activation of these signaling proteins. JAK inhibitor I also induced apoptotic cell death in 32D cells transformed to cytokine independence by IL27R, suggesting the transforming properties of IL27R are dependent on the activity of JAK family members. In addition, IL27R can transform BaF3 pro-B cells to cytokine independence. Since BaF3 cells lack expression of the gp130 co-receptor for IL-27, this suggests that IL27R-mediated transformation of hematopoietic cells is gp130-independent. This is a novel finding for IL27R-dependent signal transduction. IL27R has primarily been studied in the context of T-cell regulation. Our results are very exciting because our work is the first to suggest that IL27R may function as a hematopoietic cell oncogene. This is also interesting because type I cytokine receptors are required for activation of a JAK2 mutant (V617F) found in various myeloproliferative disorders (MPDs). We also show that IL27R is capable of activating JAK2-V617F in cells. Our data demonstrate that IL27R possesses hematopoietic cell transforming properties and suggest that type I cytokine receptors, such as IL27R, may play unappreciated roles in MPDs, including AML.

Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis

Transforming growth factor-beta (TGF-beta) has been implicated in the pathogenesis of liver disease. TGF-beta is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded-only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-beta- and c-Jun-induced signaling cascades. The effects of either SHDAg or LHDAg on TGF-beta- and c-Jun-induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. The LHDAg, but not the SHDAg, potentiated TGF-beta- and c-Jun-induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein-mediated TGF-beta and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-beta-induced plasminogen activator inhibitor-1. LHDAg may induce liver fibrosis through the regulation of TGF-beta-induced signal transductions. This regulation of TGF-beta-mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis.

Stereochemical and regiochemical trends in the selective detection of saccharides.

Several discreet sugar-boronate complexes exist in solution. This is due to the complex equilibria between isomeric species of even the simplest monosaccharides. In the current investigation, we determine the regio- and stereochemical features of the various equilibrating sugar isomers that induce signal transduction in boronic acid chemosensors such as 1 as well as 2 and 3. We present a unique example of a chemosensor (1) that is selective for ribose, adenosine, nucleotides, nucleosides, and congeners. As a result of this study, we are able to predict and achieve selective fluorescence and colorimetric responses to specific disaccharides as a consequence of their terminal sugar residue linkage patterns and configurations. We also find that the combined use of chemosensors exhibiting complementary reactivities may be used cooperatively to obtain enhanced selectivity for ribose and rare saccharides.

Prion protein modifies TGF-β induced signal transduction

Members of the transforming growth factor-beta (TGF-β) superfamily regulate a multitude of cellular processes as well as the expression of various proteins such as, e.g., matrix metalloproteinases (MMPs). These endopeptidases selectively degrade components of the extracellular matrix as well as non-matrix substrates like growth factors and cell surface receptors. MMPs are activated during embryonic development, morphogenesis, and tissue resorption/remodeling as well as in pathological conditions such as deranged wound healing and cancer metastasis. In this report we demonstrate that over-expression of cellular prion protein in mouse mammary gland epithelial cells is able to modulate TGF-β induced signal transduction leading to a synergistic increase of secreted MMP-2 activity. This correlates with elevated substrate detachment of cells grown as an epithelial monolayer as well as interfering with morphogenesis of cells cultured in a three-dimensional collagen type I matrix.

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.

Adenovirus expressing interleukin-1 receptor antagonist alleviates allergic airway inflammation in a murine model of asthma

Interleukin-1 (IL-1) is a proinflammatory cytokine and IL-1 receptor antagonist (IL-1ra) is a natural inhibitor that binds to IL-1 receptor type I without inducing signal transduction. It is suggested that IL-1 is required for allergen-specific T helper type 2 cell activation and the development of airway hyper-responsiveness (AHR), but the immunologic effect of exogenous IL-1ra in allergic asthma remains unclear. To examine the effect of IL-1ra on airway inflammation and immunoeffector cells in allergic asthma, recombinant adenovirus expressing human IL-1ra (Ad-hIL-1ra) was delivered intranasally into ovalbumin (OVA)-immunized mice. Single intranasal administration of Ad-hIL-1ra before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and reduced pulmonary infiltration of eosinophils and neutrophils. Suppression of IL-5 and eotaxin with concomitant enhancement of interferon gamma in bronchoalveolar lavage fluid was also noted in OVA-immunized mice by administration of Ad-hIL-1ra. In addition, histological studies showed that Ad-hIL-1ra was able to decrease OVA-induced peribronchial inflammation. Taken together, our results indicated that administration of Ad-hIL-1ra may have therapeutic potential for the immunomodulatory treatment of allergic asthma.

Mitogen-activated protein kinase (MAPK) signalling pathways in HepG2 cells infected with a virulent strain of Klebsiella pneumoniae

Klebsiella pneumoniae (KP), an enterobacterium, usually causes urinary tract infection or pneumonia; however, it has caused severe liver abscess in diabetic patients in recent years. How this emerging virulent KP strain causes liver abscess is not known. This study investigates signalling pathways in HepG2 cells infected by virulent KP. Cells were infected with bacteria for various durations and harvested to screen for signalling molecules by Western blotting. Our results showed that phosphorylated mitogen-activated protein kinase (MAPK) kinase (MEK) 1/2, p44/p42 MAPK and p90 ribosomal S6 kinase (p90RSK) were observed and this pathway was inhibited by MEK1/2 inhibitors U0126 and PD98059. Phosphorylation of MEK3/6, p38 kinase and ATF-2 was also observed and this pathway was inhibited by p38 kinase inhibitors SB203850 and SB202190. Toll-like receptor (TLR) 2 and 4 expressions were increased and maximized 2-4 h post infection. The JNK pathway, Elk, MAPKAPK-2 and HSP27 were not activated. These results suggest that KP infections induce signal transduction through TLR2 and TLR4 and activate two downstream MAP kinase pathways, MEK1/2-p44/p42 MAPK-p90RSK and MEK3/6-p38 kinase-ATF-2, but not the JNK pathway in HepG2 cells. The infected HepG2 eventually showed apoptosis and died.

Yaba-like disease virus chemokine receptor 7L, a CCR8 orthologue

Yaba-like disease virus (YLDV) gene 7L encodes a seven-transmembrane G protein-coupled receptor with 53 % amino acid identity to human CC chemokine receptor 8 (CCR8). Initial characterization of 7L showed that this 56 kDa cell-surface glycoprotein binds human CCL1 with high affinity (Kd=0.6 nM) and induces signal transduction by activation of heterotrimeric G proteins and downstream protein kinases. Further characterization of YLDV 7L is presented here and shows that murine CC chemokines can induce G-protein activation via the 7L receptor, despite having a low binding affinity for this receptor. In addition, when expressed by recombinant vaccinia virus (VACV), YLDV 7L was found on the outer envelope of VACV extracellular enveloped virus. The contribution of 7L to poxvirus pathogenesis was investigated by infection of mice with a recombinant VACV expressing 7L (vDeltaB8R-7L) and was compared with the outcome of infection by parental and revertant control viruses. In both intranasal and intradermal models, expression of 7L caused attenuation of VACV. The role of this protein in viral virulence is discussed.

Differential Rho‐kinase dependency of full and partial muscarinic receptor agonists in airway smooth muscle contraction

In airway smooth muscle (ASM), full and partial muscarinic receptor agonists have been described to have large differences in their ability to induce signal transduction, including Ca2+-mobilization. Despite these differences, partial agonists are capable of inducing a submaximal to maximal ASM contraction. To further elucidate transductional differences between full and partial muscarinic receptor agonists, we investigated the contribution of Rho-kinase (an important regulator of Ca2+-sensitization) to methacholine-, pilocarpine- and McN-A-343-induced bovine tracheal smooth muscle (BTSM) contraction, using the selective Rho-kinase inhibitor Y-27632. In addition, we measured Ca2+-mobilization and -influx in BTSM cells in response to these agonists in the absence and presence of Y-27632. Whereas treatment with Y-27632 (1 microM) significantly decreased potency (pEC50) for all agonists, maximal contraction (Emax) was reduced by 23.4+/-2.8 and 50.4+/-7.9% for the partial agonists pilocarpine and McN-A-343, respectively, but was unaffected for the full agonist methacholine. However, Emax of methacholine became Rho-kinase dependent after taking away its receptor reserve using the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard. Pilocarpine and McN-A-343 induced a very small Ca2+-mobilization and -influx as compared to methacholine. In addition, an inverse relationship of these two parameters with the Rho-kinase dependency was observed. Interestingly, no inhibitory effects of Y-27632 were observed on Ca2+-mobilization and-influx for all three agonists, indicating that the effects of Y-27632 on contraction are most likely on the level of Ca2+-sensitization. In conclusion, in contrast to the full agonist methacholine, the partial muscarinic receptor agonists pilocarpine and McN-A-343 are dependent on Rho-kinase for their maximal contractile effects, presumably as a consequence of differences in transductional reserve, indicating an agonist-dependent role for Rho-kinase in ASM contraction. Moreover, an inverse relationship exists between Rho-kinase dependency and both Ca2+-mobilization and Ca2+-influx for these agonists.

Photoprotective effect of isoflavone genistein on ultraviolet B-induced pyrimidine dimer formation and PCNA expression in human reconstituted skin and its implications in dermatology and prevention of cutaneous carcinogenesis

Genistein, the most abundant isoflavone of the soy derived phytoestrogen compounds, is a potent antioxidant and inhibitor of tyrosine kinase. We previously reported the antiphotocarcinogenic effects of genistein in SKH-1 murine skin, including its capacity for scavenging reactive oxygen species, inhibiting photodynamic DNA damage and downregulating UVB(ultra violet B)-induced signal transduction cascades in carcinogenesis. In this study we elucidate genistein's photoprotective efficacy within the context of full thickness human reconstituted skin relative to acute challenges with ultraviolet-B irradiation. Skin samples were pre-treated with three concentrations of genistein (10, 20 and 50 microM) 1 h prior to UVB radiation at 20 and 60 mJ/cm2. Proliferating cell nuclear antigen (PCNA) and pyrimidine dimer (PD) expression profiles were localized using immunohistochemical analysis on paraffin embedded samples 6 and 12 h post UVB exposure. Genistein dose dependently preserved cutaneous proliferation and repair mechanics at 20 and 60 mJ/cm2, as evidenced by the preservation of proliferating cell populations with increasing genistein concentrations and noticeable paucity in PCNA immunoreactivity in the absence of genistein. Genistein inhibited UV-induced DNA damage, evaluated with PD immunohistochemical expression profiles, demonstrated an inverse relationship with increasing topical genistein concentrations. Irradiation at 20 and 60 mJ/cm2 substantially induced PD formation in the absence of genistein, and a dose dependent inhibition of UVB-induced PD formation was observed relative to increasing genistein concentrations. Collectively all genistein pre-treated samples demonstrated appreciable histologic architectural preservation when compared with untreated specimens. These findings represent a critical link between our animal and cell culture studies with those of human skin and represent the first characterization of the dynamic alterations of UV-induced DNA damage and proliferating cell populations relative to pretreatment with genistein in human reconstituted skin. The implications of our findings serve as compelling validation to our conclusions that genistein may serve as a potent chemopreventive agent against photocarcinogenesis.