Variant analysis of the first Lebanese SARS-CoV-2 isolates.

Recently the first genome sequences for 11 SARS-CoV-2 isolates from Lebanon became available. Here, we report the detection of variants within the genome of these strains. Pairwise alignment analysis using blastx was performed between these sequences and the UniProtKB data for the SARS-CoV-2 coronavirus to identify amino acid variations. Variants analysis was performed using multiple Bioinformatics tools. We noticed for the first time 18 mutations that have never been reported before. Among those, a frame shift (8651A>) in NSP4, a stop codon 6887A > T in NSP3 and two missense mutations in spike S2 were found. In addition, we found 28 variants in ORF1ab alone. A previously reported variant, 23403A > G, in the spike protein S2 was mostly seen. Two other known mutations 25563G > T in ORF3a and 14408C > T in ORF1ab were detected respectively in 6 and 8 out of the 11 isolates. Our results may help to prognose forthcoming infections in this region.

VIP modulates human macrophages phenotype via FPRL1 via activation of RhoA-GTPase and PLC pathways.

OBJECTIVE AND DESIGN: This study is aimed at uncovering the signaling pathways activated by vasoactive intestinal peptide in human macrophages MATERIALS: Human peripheral blood mononuclear cell-derived macrophages were used for the in vitro investigation of the VIP-activated signaling pathways. METHODS AND TREATMENT: Time-course and dose-response experiments and siRNA were used in human macrophages co-challenged with various concentrations of VIP and different MAPK pharmacologic inhibitors to investigate signaling pathways activated by VIP. Flow analysis was performed to assess the levels of CD11b, CD35 and CD66. Luminescence spectrometry was used to measure the levels of the released hydrogen peroxide and the intracellular calcium levels in the media. RESULTS: Macrophages incubated with VIP showed increased phospho-AKT and phospho-ERK1/2 levels in a GTP-RhoA-GTPase-dependent manner. Similarly, VIP increased intracellular release of H2O2 and calcium via PLC and GTP-RhoA-GTPase, in addition to inducing the expression of CD11b, CD35, CD66 and MMP9. Furthermore, VIP activated P38 MAPK through the cAMP/PKA pathway but was independent of both PLC and RhoA signaling. The above-mentioned VIP effects were mediated via activation of the FPRL1 receptor. CONCLUSION: VIP/FPRL1/VPAC/GTP-RhoA-GTPase signaling modulated macrophages phenotype through activation of multiple signaling pathways including ERK1/2, AKT, P38, ROS, cAMP and calcium.

Human CD8+ CD25 + CD127 low regulatory T cells: microRNA signature and impact on TGF-ß and IL-10 expression.

Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8+ CD25 + CD127 low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 + CD25 + CD127 low Tregs in comparison to CD8 + CD25 - T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3'-untranslated region (3'-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Having identified potential miR target sites in the 3'-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-ß (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3'-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-ß expression. These results highlighted an important impact of the CD8 + Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.

Glucose restriction reverses the Warburg effect and modulates PKM2 and mTOR expression in breast cancer cell lines.

Aerobic glycolysis, known as the "Warburg effect", is one of several hallmarks of cancer cells. The conversion of phosphoenolpyruvate (PEP) to pyruvate can be down regulated by the re-expression of the embryonic isoform 2 of pyruvate kinase (PKM2). This mechanism allows the accumulation of glycolytic intermediates for the biosynthesis of macromolecules, such as proteins, lipids and nucleic acids. PKM2 is favored by the well-known PI3K/Akt/mTOR proliferative pathway. This pathway is induced by high glucose levels, and the mTOR kinase is the central activator of the Warburg effect. In this study, we investigated the role of glucose restriction (GR) and mTOR inhibition  in reversing the Warburg effect in MDA-MB 231 and MCF-7 breast cancer cell lines. PKM2 expression was measured by western blot. Lactate production by cells was determined by a colorimetric assay. The concentration of glucose in the supernatant of cells was measured using the Trinder method. ATP level  was evaluated by using a Colorimetric/Fluorometric ATP Assay Kit. Our results showed that MDA-MB 231 cells increased glucose consumption when the glucose concentration was 0 g/L (P <0.01). In MCF-7 cells, glucose deprivation reduced lactate secretion by 80% (P =0.0001) but tripled glucose consumption (P = 0.0041). ATP concentration increased approximately when MCF-7 cells were deprived of glucose (P = 0.02). GSK1059615 does not significantly modulate lactate secretion and glucose uptake in both cell lines. Glucose restriction contribute to the reduction of the Warburg effect through mTOR inhibition and regulation of PKM2 kinases.

Ghrelin modulates intracellular signalling pathways that are critical for podocyte survival.

Damage to podocytes is a key event in glomerulopathies. While energy dense food can contribute to kidney damage, the role of the orixegenic hormone "ghrelin" in podocyte biology is still unknown. In the present study, we investigated the effect of ghrelin on podocyte survival as well as the signalling pathways mediating ghrelin effect in immortalized cultured rat podocytes. RT-PCR analysis revealed that GHS-R1 is expressed in rat podocytes. Western blot analysis showed that ghrelin upregulated COX-2 protein expression in a time and dose-dependent manner. Additionally, ghrelin activated P38 MAPK, AKT, and ERK1/2 pathways and also induced P38 MAPK phosphorylation in high glucose conditions. Ghrelin induced ROS release and dose dependently reduced podocyte survival. Ghrelin mediated podocyte cell death was partially reversed by pharmacologically inhibiting P38 MAPK or phospholipase C (PLC). Furthermore, PLC inhibitor (U73122) inhibited ghrelin induced P38 MAPK activation. While PI3K inhibitor (LY294002) was without effect on cell survival or P38 MAPK activation, it inhibited ghrelin induced ERK1/2 phosphorylation. Finally, ghrelin induced TAU phosphorylation was reversed by pharmacologic inhibitors of either P38 MAPK or PKA. In conclusion, ghrelin activated harmful molecular pathways in podocytes that can be damaging to the glomerular filtration barrier SIGNIFICANCE OF THE STUDY: Endocrine derangements secondary to obesity are major players in the aetiology of renal injuries. Furthermore, energy dense diet is thought to be the major element in developing obesity. Appetite and increase in energy intake are regulated by complex hormonal pathways which mainly include the orexigenic hormone "ghrelin" in addition to leptin. To date no study have highlighted a significant role for ghrelin in kidney biology, and therefore, it is thought that its endocrine effect is mostly limited to adipose tissue metabolism and appetite regulation. In this study, we first showed that ghrelin receptor is expressed on glomerular podocytes. Also, ghrelin showed negative impact on podocyte survival through modulating signalling pathways such as P38 MAPK and AKT known to play a key role in podocyte health. Moreover, the negative effects of ghrelin on podocytes were further exacerbated in hyperglycemic conditions. Of note, podocytes contribute to the formation and the maintenance of the glomerular filtration barrier and thus are important for normal renal function. Therefore, ghrelin secretion in the context of obesity could be involved in the aetiology of kidney injury, a well-known hallmark found in obese patients.

fMLP-dependent activation of Akt and ERK1/2 through ROS/Rho A pathways is mediated through restricted activation of the FPRL1 (FPR2) receptor.

OBJECTIVE AND DESIGN: The objective of this study is to uncover the signal transduction pathways of N-formyl methionyl-leucyl-phenylalanine (fMLP) in monocyte. MATERIALS OR SUBJECTS: Freshly isolated human peripheral blood monocytes (PBMC) were used for in vitro assessment of signal transduction pathways activated by fMLP. TREATMENT: Time-course and dose-response experiments were used to evaluate the effect of fMLP along with the specific inhibitors/stimulators on the activation of downstream signaling kinases. METHODS: Freshly isolated human PBMC were stimulated with fMLP for the desired time. Western blot and siRNA analysis were used to evaluate the activated intracellular signaling kinases, and flow analysis was performed to assess the levels of CD11b. Furthermore, luminescence spectrometry was performed to measure the levels of released hydrogen peroxide in the media. RESULTS: fMLP strongly stimulated the activation of AKT and ERK1/2 through a RhoA-GTPase-dependent manner and also induced H2O2 release by monocytes. Furthermore, fMLP mediated its effects through restricted activation of formylpeptide receptor-like 1 (FPRL1/FPR2), but independently of either EGFR transactivation or intracellular calcium release. In addition, NAC reversed fMLP- and H2O2-induced activation of Akt and RhoA-GTPase. CONCLUSION: Collectively, these data suggested that fMLP-activated ERK1/2 and Akt pathways through specific activation of the FPRL1/ROS/RoA-GTPase pathway.

Detection of SARS-CoV-2 B.1.1.529 (Omicron) variant by SYBR Green-based RT-qPCR.

The coronavirus disease 2019 (COVID-19) pandemic is unceasingly spreading across the globe, and recently a highly transmissible Omicron SARS-CoV-2 variant (B.1.1.529) has been discovered in South Africa and Botswana. Rapid identification of this variant is essential for pandemic assessment and containment. However, variant identification is mainly being performed using expensive and time-consuming genomic sequencing. In this study, we propose an alternative RT-qPCR approach for the detection of the Omicron BA.1 variant using a low-cost and rapid SYBR Green method. We have designed specific primers to confirm the deletion mutations in the spike (S Δ143-145) and the nucleocapsid (N Δ31-33) which are characteristics of this variant. For the evaluation, we used 120 clinical samples from patients with PCR-confirmed SARS-CoV-2 infections, and displaying an S-gene target failure (SGTF) when using TaqPath COVID-19 kit (Thermo Fisher Scientific, Waltham, USA) that included the ORF1ab, S, and N gene targets. Our results showed that all the 120 samples harbored S Δ143-145 and N Δ31-33, which was further confirmed by whole-genome sequencing of 10 samples, thereby validating our SYBR Green-based protocol. This protocol can be easily implemented to rapidly confirm the diagnosis of the Omicron BA.1 variant in COVID-19 patients and prevent its spread among populations, especially in countries with high prevalence of SGTF profile.

MicroRNA profile of circulating CD4-positive regulatory T cells in human adults and impact of differentially expressed microRNAs on expression of two genes essential to their function.

Regulatory T cells (Tregs) are characterized by a high expression of IL-2 receptor α chain (CD25) and of forkhead box P3 (FOXP3), the latter being essential for their development and function. Another major player in the regulatory function is the cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) that inhibits cytotoxic responses. However, the regulation of CTLA-4 expression remains less well explored. We therefore studied the microRNA signature of circulating CD4(+) Tregs isolated from adult healthy donors and identified a signature composed of 15 differentially expressed microRNAs. Among those, miR-24, miR-145, and miR-210 were down-regulated in Tregs compared with controls and were found to have potential target sites in the 3'-UTR of FOXP3 and CTLA-4; miR-24 and miR-210 negatively regulated FOXP3 expression by directly binding to their two target sites in its 3'-UTR. On the other hand, miR-95, which is highly expressed in adult peripheral blood Tregs, positively regulated FOXP3 expression via an indirect mechanism yet to be identified. Finally, we showed that miR-145 negatively regulated CTLA-4 expression in human CD4(+) adult peripheral blood Tregs by binding to its target site in CTLA-4 transcript 3'-UTR. To our knowledge, this is the first identification of a human adult peripheral blood CD4(+) Treg microRNA signature. Moreover, unveiling one mechanism regulating CTLA-4 expression is novel and may lead to a better understanding of the regulation of this crucial gene.

Involvement of caveolae in hyperglycemia-induced changes in adiponectin and leptin expressions in vascular smooth muscle cells.

Hyperglycemia exerts various harmful effects on the vasculature. Studies have shown an association between the levels of the adipokines leptin and adiponectin (APN) and vascular complications in diabetes mellitus. The aim of our study was to investigate the molecular mechanisms mediated by APN and leptin that are involved in hyperglycemia-induced vascular remodeling, especially at the level of oxidative stress and actin cytoskeleton dynamics. Rat aorta organ culture was used to investigate the effect of hyperglycemia on APN and leptin protein expression in vascular smooth muscle cells (VSMCs) using Western blot analysis and immunohistochemistry. Hyperglycemia lead to a significant increase in APN synthesis in VSMCs, mainly through caveolae, but this increase failed to provide vascular protection because of the decreased expression of APN receptors, especially AdipoR2, which was assessed by qPCR. In addition, hyperglycemia significantly upregulated leptin expression in VSMCs through caveolae and the RhoA/ROCK pathway. These variations lead to a marked increase in reactive oxygen species (ROS) production, detected by dihydroethidium (DHE) staining, and in NADPH oxidase type 4 (Nox4) expression. Moreover, Nox4 mediated the synthesis of APN in hyperglycemia in VSMCs. Finally, hyperglycemia activated the RhoA/ROCK pathway and subsequently induced the polymerization of globular actin (G-actin) into filamentous actin (F-actin), decreasing the G/F-actin ratio. Taken together, these data show that hyperglycemia increases oxidative stress and changes actin cytoskeleton dynamics in the aorta via caveolae, favoring vascular remodeling.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Human natural Treg microRNA signature: role of microRNA-31 and microRNA-21 in FOXP3 expression.

Treg are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate currently. However, a human microRNA (miR) Treg signature has not been described yet. We investigated human natural Treg and identified a signature composed of five miR (21, 31, 125a, 181c and 374). Among those, two were considerably under-expressed (miR-31 and miR-125a). We identified a functional target sequence for miR-31 in the 3' untranslated region (3' UTR) of FOXP3 mRNA. Using lentiviral transduction of fresh cord blood T cells, we demonstrated that miR-31 and miR-21 had an effect on FOXP3 expression levels. We showed that miR-31 negatively regulates FOXP3 expression by binding directly to its potential target site in the 3' UTR of FOXP3 mRNA. We next demonstrated that miR-21 acted as a positive, though indirect, regulator of FOXP3 expression. Transduction of the remaining three miR had no direct effect on FOXP3 expression or on the phenotype and will remain the subject of future investigations. In conclusion, not only have we identified and validated a miR signature for human natural Treg, but also unveiled some of the mechanisms by which this signature was related to the control of FOXP3 expression in these cells.

VIP differentially activates beta2 integrins, CR1, and matrix metalloproteinase-9 in human monocytes through cAMP/PKA, EPAC, and PI-3K signaling pathways via VIP receptor type 1 and FPRL1.

The neuropeptide vasoactive intestinal peptide (VIP) regulates the exocytosis of secretory granules in a wide variety of cells of neuronal and non-neuronal origin. In human monocytes, we show that the proinflammatory effects of VIP are associated with stimulation of exocytosis of secretory vesicles as well as tertiary (gelatinase) granules with, respectively, up-regulation of the membrane expression of the beta2 integrin CD11b, the complement receptor 1 (CD35), and the matrix metalloproteinase-9 (MMP-9). Using the low-affinity formyl peptide receptor-like 1 (FPRL1) antagonist Trp-Arg-Trp-Trp-Trp-Trp (WRW4) and the exchange protein directly activated by cAMP (EPAC)-specific compound 8CPT-2Me-cAMP and measuring the expression of Rap1 GTPase-activating protein as an indicator of EPAC activation, we found that the proinflammatory effect of VIP is mediated via the specific G protein-coupled receptor VIP/pituitary adenylate cyclase-activating protein (VPAC1) receptor as well as via FPRL1: VIP/VPAC1 interaction is associated with a cAMP increase and activation of a cAMP/p38 MAPK pathway, which regulates MMP-9, CD35, and CD11b exocytosis, and a cAMP/EPAC/PI-3K/ERK pathway, which regulates CD11b expression; VIP/FPRL1 interaction results in cAMP-independent PI-3K/ERK activation with downstream integrin up-regulation. In FPRL1-transfected Chinese hamster ovary-K1 cells lacking VPAC1, VIP exposure also resulted in PI-3K/ERK activation. Thus, the proinflammatory effects of VIP lie behind different receptor interactions and multiple signaling pathways, including cAMP/protein kinase A, cAMP/EPAC-dependent pathways, as well as a cAMP-independent pathway, which differentially regulates p38 and ERK MAPK and exocytosis of secretory vesicles and granules.

The neuropeptide pituitary adenylate cyclase activating polypeptide modulates Ca2+ and pro-inflammatory functions in human monocytes through the G protein-coupled receptors VPAC-1 and formyl peptide receptor-like 1.

In human neutrophils, the neuropeptide pituitary adenylate cyclase activating polypeptide (PACAP) acting via the G protein-coupled receptors vasoactive intestinal peptide/PACAP receptor 1 (VPAC-1) and formyl peptide receptor-like 1 (FPRL1) modulates Ca2+ and pro-inflammatory activities. We evaluated in human monocytes the importance of the Ca2+ signal and the participation of FPRL1 in PACAP-associated signaling pathways and pro-inflammatory activities. PACAP-evoked Ca2+ transient involved both Ca2+ influx and intracytoplasmic Ca2+ mobilisation. This was pertussis toxin, protein kinase A and adenylate cyclase dependent indicating the participation of Galphai and Galphas with mobilisation of both InsP3 sensitive and insensitive stores. Intra- or extracellular Ca2+ depletion resulted in the inhibition of PACAP-induced, Akt, ERK, p38 and NF-kappaB activations as well as a decrease in PACAP-associated reactive oxygen species (ROS) production and integrin CD11b membrane upregulation. The FPRL1 antagonist, Trp-Arg-Trp-Trp-Trp (WRW4), decreased PACAP-evoked Ca2+ signal, Akt, ERK phosphorylation, ROS and CD11b upregulation without affecting p38 phosphorylation. NF-kappaB inhibitors prevented PACAP-induced Ca2+ mobilisation. Monocytes pre-treatment with fMLP but not with LPS desensitised cells to the pro-inflammatory effects of PACAP. Thus, both intra- and extracellular Ca2+ play a role in controlling pro-inflammatory functions stimulated by PACAP which acts through a VPAC-1, FPRL1/Galphai/PI3K/ERK pathway and a VPAC-1/Galphas/PKA/p38 pathway to fully activate monocytes.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

Signaling pathways activated by PACAP in MCF-7 breast cancer cells.

PACAP has opposing roles ranging from activation to inhibition of tumor growth and PACAP agonists/antagonists could be used in tumor therapy. In this study, the effect of PACAP stimulation on signaling pathways was investigated in MCF-7 human adenocarcinoma breast cancer cells. Results showed that MCF-7 cells express VPAC1 and VPAC2, but not PAC1, receptors. In addition, PACAP increased the phosphorylation levels of STAT1, Src and Raf within seconds, confirming their involvement in early stages of PACAP signaling whereas maximal phosphorylation of AKT, ERK and p38 was reached 10 to 20 min later. Moreover, selective inhibition of Src or PI3K resulted in a significant decrease in the phosphorylation of ERK and AKT, but not p38, demonstrating that PACAP signaling follows Src/Raf/ERK and PI3K/AKT pathways. On the other hand, selective inhibition of PLC or PKA resulted in a significant decrease in the phosphorylation of p38, but not AKT or ERK, indicating that PACAP signaling also follows the PLC and PKA/cAMP pathways. Furthermore, PACAP induced ROS through H2O2 production whereas pretreatment with NAC inhibitor decreased AKT and ERK phosphorylation, but not p38. Selective NOX2 inhibition affected Src/Raf/Erk and PI3K/Akt pathways, without affecting the p38/PLC/PKA pathway whereas other inhibitors (ML171, VAS2870) had no effect on PACAP induced ROS generation. On the other hand, PACAP induced calcium release, which was decreased by pretreatment with PLC inhibitor. Finally, PACAP stimulation promoted apoptosis by increasing Bax and decreasing Bcl2 expression. In conclusion, we demonstrated that PACAP signaling in MCF-7 cells follows the Src/Raf/ERK and PI3K/AKT pathways and is VPAC1 dependent in a ROS dependent manner, whereas it follows PLC and PKA/cAMP pathways and is VPAC2 dependent through p38 MAP kinase activation involving calcium.

The neuropeptide pituitary adenylate cyclase activating protein is a physiological activator of human monocytes.

Pituitary adenylate cyclase activating protein (PACAP) and its structurally related vasointestinal peptide (VIP) bind to three G-protein-coupled receptors named VPAC1 and VPAC2 for VIP/PACAP receptors and PAC1 for PACAP preferred receptors. We report that in freshly isolated human monocytes PACAP acts as a pro-inflammatory molecule. By RT-PCR, VPAC1 mRNA was the only receptor found to be expressed; VPAC1 protein was detected by Western blotting and visualized by immunohistochemistry. Signaling pathways activated by PACAP include the extracellular regulated kinase (ERK), the stress-activated MAPK p38, the focal adhesion kinase, Pyk2 and its associated cytoskeleton protein paxillin and the phosphatidylinositol 3-kinase (PI-3K). PACAP induces a transient peak in cytoplasmic calcium associated with an increase in reactive oxygen species production and upregulation in membrane expression of the integrin CD11b as well as the complement receptor 1. Control of the different pathways and functions stimulated by PACAP were evaluated using Phospholipase C (PLC), PI-3K, ERK and p38 MAPK inhibitors and led to the conclusion that PLC and to a lesser degree PI-3K activation are upstream events occurring in VPAC1 mediated PACAP stimulation of monocytes and are in contrast to ERK and p38 mandatory for the initiation of other cellular events associated with monocytes activation.

ROS mediates interferon gamma induced phosphorylation of Src, through the Raf/ERK pathway, in MCF-7 human breast cancer cell line.

Interferon gamma (IFN-É£) is a pleiotropic cytokine which plays dual contrasting roles in cancer. Although IFN-É£ has been clinically used to treat various malignancies, it was recently shown to have protumorigenic activities. Reactive oxygen species (ROS) are overproduced in cancer cells, mainly due to NADPH oxidase activity, which results into several changes in signaling pathways. In this study, we examined IFN-É£ effect on the phosphorylation levels of key signaling proteins, through ROS production, in the human breast cancer cell line MCF-7. After treatment by IFN-É£, results showed a significant increase in the phosphorylation of STAT1, Src, raf, AKT, ERK1/2 and p38 signaling molecules, in a time specific manner. Src and Raf were found to be involved in early stages of IFN-É£ signaling since their phosphorylation increased very rapidly. Selective inhibition of Src-family kinases resulted in an immediate significant decrease in the phosphorylation status of Raf and ERK1/2, but not p38 and AKT. On the other hand, IFN-É£ resulted in ROS generation, through H2O2 production, whereas pre-treatment with the ROS inhibitor NAC caused ROS inhibition and a significant decrease in the phosphorylation levels of AKT, ERK1/2, p38 and STAT1. Moreover, pretreatment with a selective NOX1 inhibitor resulted in a significant decrease of AKT phosphorylation. Finally, no direct relationship was found between ROS production and calcium mobilization. In summary, IFN-É£ signaling in MCF-7 cell line is ROS-dependent and follows the Src/Raf/ERK pathway whereas its signaling through the AKT pathway is highly dependent on NOX1.

EGF-Induced VEGF Exerts a PI3K-Dependent Positive Feedback on ERK and AKT through VEGFR2 in Hematological In Vitro Models.

EGFR and VEGFR pathways play major roles in solid tumor growth and progression, however, little is known about these pathways in haematological tumors. This study investigated the crosstalk between EGFR and VEGFR2 signaling in two hematological in vitro models: THP1, a human monocytic leukemia, and Raji, a Burkitt's lymphoma, cell lines. Results showed that both cell lines express EGFR and VEGFR2 and responded to EGF stimulation by activating EGFR, triggering VEGF production and phosphorylating ERK, AKT, and p38 very early, with a peak of expression at 10-20min. Blocking EGFR using Tyrphostin resulted in inhibiting EGFR induced activation of ERK, AKT, and p38. In addition, EGF stimulation caused a significant and immediate increase, within 1min, in pVEGFR2 in both cell lines, which peaked at ~5-10 min after treatment. Selective inhibition of VEGFR2 by DMH4, anti-VEGFR2 antibody or siRNA diminished EGF-induced pAKT and pERK, indicating a positive feedback exerted by EGFR-induced VEGF. Similarly, the specific PI3K inhibitor LY294002, suppressed AKT and ERK phosphorylation showing that VEGF feedback is PI3K-dependent. On the other hand, phosphorylation of p38, initiated by EGFR and independent of VEGF feedback, was diminished using PLC inhibitor U73122. Moreover, measurement of intracellular [Ca2+] and ROS following VEGFR2 inhibition and EGF treatment proved that VEGFR2 is not implicated in EGF-induced Ca2+ release whereas it boosts EGF-induced ROS production. Furthermore, a significant decrease in pAKT, pERK and p-p38 was shown following the addition of the ROS inhibitor NAC. These results contribute to the understanding of the crosstalk between EGFR and VEGFR in haematological malignancies and their possible combined blockade in therapy.

Adiponectin Attenuates Angiotensin II-Induced Vascular Smooth Muscle Cell Remodeling through Nitric Oxide and the RhoA/ROCK Pathway.

INTRODUCTION: Adiponectin (APN), an adipocytokine, exerts protective effects on cardiac remodeling, while angiotensin II (Ang II) induces hypertension and vascular remodeling. The potential protective role of APN on the vasculature during hypertension has not been fully elucidated yet. Here, we evaluate the molecular mechanisms of the protective role of APN in the physiological response of the vascular wall to Ang II. METHODS AND RESULTS: Rat aortic tissues were used to investigate the effect of APN on Ang II-induced vascular remodeling and hypertrophy. We investigated whether nitric oxide (NO), the RhoA/ROCK pathway, actin cytoskeleton remodeling, and reactive oxygen species (ROS) mediate the anti-hypertrophic effect of APN. Ang II-induced protein synthesis was attenuated by pre-treatment with APN, NO donor S-nitroso-N-acetylpenicillamine (SNAP), or cGMP. The hypertrophic response to Ang II was associated with a significant increase in RhoA activation and vascular force production, which were prevented by APN and SNAP. NO was also associated with inhibition of Ang II-induced phosphorylation of cofilin. In addition, immunohistochemistry revealed that 24 h Ang II treatment increased the F- to G-actin ratio, an effect that was inhibited by SNAP. Ang II-induced ROS formation and upregulation of p22(phox) mRNA expression were inhibited by APN and NO. Both compounds failed to inhibit Nox1 and p47(phox) expression. CONCLUSION: Our results suggest that the anti-hypertrophic effects of APN are due, in part, to NO-dependent inhibition of the RhoA/ROCK pathway and ROS formation.