Neuropilin-2 acts as a modulator of Sema3A-dependent glioma cell migration

Semaphorin 3A (Sema3A) is a secreted guidance molecule initially described in the nervous system. This protein is able to control axon growth but also effects on endothelial cells migration. Here, we report that Sema3A acts as a chemorepellent factor for the rat C6 glioma cells and three different human glioma cell lines. Interestingly, Sema3A triggered a chemoattractive response in a fourth human glioma cell line. The nature of the receptor complex ensuring the appropriate signaling was dissected in C6 cells by using function blocking antibodies and gain- or loss-of function experiments using recombinant receptors. Our results demonstrate that neuropilin-1, neuropilin-2 and PlexinA1 are necessary to trigger cell repulsion. The selective blockade of neuropilin-1 or Plexin-A1 switched the chemorepulsive effect of Sema3A into a chemoattractive one. Strikingly, blocking Neuropilin-2 suppressed Sema3A-induced cell migration while overexpression of neuropilin-2 was able to convert the chemorepulsive effect of Sema3A into a chemoattractive one. Our results not only provide additional evidence for a biological function of Sema3A in glioma migration but also reveal part of the receptor complex involved. Hence, our study describes a receptor-based plasticity in cancer cells leading to opposite migration behavior in response to the same extracellular signal.Key words: semaphorin, neuropilin, glioma, cell migration, signalling, cancer     

Cell surface galactosyltransferase acts as a general modulator of rat acinar cell proliferation

Summary Several physiological parameters were examined for inducing acinar cell proliferation and corresponding increased expression of 1–4 galactosyltransferase. In this study, dietary changes causing acinar cell proliferation included the following: the introduction of animals to a liquid diet (causing gland atrophy) followed by reintroduction of solid chow, gustatory stimulation provided by the introduction of 0.5% citric acid to animal drinking water, and removal of the submandibular gland with subsequent reliance on the parotid gland for salivary protein. Alterations in growth factor levels were produced by injecting animals with a chronic (3 day) regimen of either nerve growth factor or epidermal growth factor. Parotid gland proliferation could be blocked in all cases except EGF by the injection of propranolol, a -adrenoceptor antagonist, or the galactosyltransferase specific modifier protein, -lactalbumin. EGF-induced proliferation could, however, be prevented by treating the animals with monoclonal antibody to EGF receptor or galactosyltransferase modifier protein a-lactalbumin. These results for normal acinar cell proliferation suggest a direct role for cell surface 1–4 galactosyltransferase in signalling and maintaining active cell growth.     

S-nitrosoglutathione acts as a small molecule modulator of human fibrin clot architecture

Background

Altered fibrin clot architecture is increasingly associated with cardiovascular diseases; yet, little is known about how fibrin networks are affected by small molecules that alter fibrinogen structure. Based on previous evidence that S-nitrosoglutathione (GSNO) alters fibrinogen secondary structure and fibrin polymerization kinetics, we hypothesized that GSNO would alter fibrin microstructure.

Methodology/Principal Findings

Accordingly, we treated human platelet-poor plasma with GSNO (0.01–3.75 mM) and imaged thrombin induced fibrin networks using multiphoton microscopy. Using custom designed computer software, we analyzed fibrin microstructure for changes in structural features including fiber density, diameter, branch point density, crossing fibers and void area. We report for the first time that GSNO dose-dependently decreased fibrin density until complete network inhibition was achieved. At low dose GSNO, fiber diameter increased 25%, maintaining clot void volume at approximately 70%. However, at high dose GSNO, abnormal irregularly shaped fibrin clusters with high fluorescence intensity cores were detected and clot void volume increased dramatically. Notwithstanding fibrin clusters, the clot remained stable, as fiber branching was insensitive to GSNO and there was no evidence of fiber motion within the network. Moreover, at the highest GSNO dose tested, we observed for the first time, that GSNO induced formation of fibrin agglomerates.

Conclusions/Significance

Taken together, low dose GSNO modulated fibrin microstructure generating coarse fibrin networks with thicker fibers; however, higher doses of GSNO induced abnormal fibrin structures and fibrin agglomerates. Since GSNO maintained clot void volume, while altering fiber diameter it suggests that GSNO may modulate the remodeling or inhibition of fibrin networks over an optimal concentration range.     

p-Nonylphenol acts as a promoter in the BALB/3T3 cell transformation

p-Nonylphenol (NP) has attracted attention as an estrogenic contaminant, and the environmental pollution by NP has been found to be extensive. NP is classified as a phenolic antioxidant based on the chemical activity and structure. Some phenolic antioxidants are known to induce and/or enhance carcinogenesis. We examined the effects of NP on the two-stage transformation of BALB/3T3 cells, a model of two-stage carcinogenesis. The treatment by NP in the promotion phase markedly enhanced the transformation of the cells pre-treated with a subthreshold dose of a carcinogen, 3-methylcholanthrene (MCA), but not that of non-pretreated cells. The promoting activity of NP was approximately one hundredth of that of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, in the cell transformation. The treatment by NP in the initiation phase did not induce cell transformation with and without post-treatment by TPA. These results indicate that NP acts as a pure promoter of cell transformation implying that it may cause the enhancement of carcinogenesis in vivo. The enhancement by NP of MCA-initiated transformation was suggested not to be mediated by estrogen receptors in BALB/3T3 cells because 17 beta-estradiol did not promote cell transformation in our experiments, and it has been reported that BALB/3T3 cells do not express estrogen receptors at a detectable level.     

Rational design of a pirinixic acid derivative that acts as subtype-selective PPARγ modulator

Peroxisome proliferator-activated receptor γ (PPARγ) is involved in glucose and lipid homeostasis. PPARγ agonists are in clinical use for the treatment of type 2 diabetes. Lately, a new class of selective PPARγ modulators (SPPARγMs) was developed, which are believed to show less side effects than full PPARγ agonists. We have previously shown that α-substitution of pirinixic acid, a moderate agonist of PPARα and PPARγ, leads to low micromolar active balanced dual agonists of PPARα and PPARγ. Herein we present modifications of pirinixic acid leading to subtype-selective PPARγ agonists and furthermore the development of a selective PPARγ modulator guided by molecular docking studies.     

Neuropilin-1 binds to VEGF121 and regulates endothelial cell migration and sprouting

Neuropilin-1 (NRP1) was first described as a receptor for the axon guidance molecule, Semaphorin3A, regulating the development of the nervous system. It was later shown that NRP1 is an isoform-specific receptor for vascular endothelial growth factor (VEGF), specifically VEGF(165). Much interest has been placed on the role of the various VEGF isoforms in vascular biology. Here we report that blocking NRP1 function, using a recently described antibody that inhibits VEGF(165) binding to NRP1, surprisingly reduces VEGF(121)-induced migration and sprout formation of endothelial cells. Intrigued by this observation, direct binding studies of NRP1 to various VEGF isoforms were performed. We show that VEGF(121) binds directly to NRP1; however, unlike VEGF(165), VEGF(121) is not sufficient to bridge the NRP1.VEGFR2 complex. Additionally, we show that VEGFR2 enhances VEGF(165), but not VEGF(121) binding to NRP1. We propose a new model for NRP1 interactions with various VEGF isoforms.     

Pinin acts as a poor prognostic indicator for renal cell carcinoma by reducing apoptosis and promoting cell migration and invasion

Pinin (PNN) was originally characterized as a desmosome-associated molecule. Its function and the mechanism of its regulation in renal cell carcinoma (RCC) are still undefined. Data on PNN expression, clinicopathological features, and prognosis of patients with RCC were obtained from The Cancer Genome Atlas (TCGA) database. Immunohistochemistry revealed high PNN expression in tumour cells. PNN expression showed negative correlation with survival in patients with RCC, acting as an independent prognostic factor in RCC. PNN up-regulation might be attributed to epigenetic alterations in RCC. Immunofluorescence revealed PNN expression mainly in the nucleus of RCC cells. The transfection of siRNA targeting the PNN gene resulted in enhanced apoptosis, which was detected by flow cytometry, and reduced cell migration and invasion, which were assessed using wound healing and transwell migration assay. Gene set enrichment analysis revealed associations between PNN expression and several signalling pathways involved in cancer progression, as a potential mechanism underlying the carcinogenicity of PNN. The analyses of the Tumor Immune Estimation Resource platform showed significant positive associations between high PNN expression and tumour immune infiltrating cells. PNN may function as an oncogenic factor by reducing apoptosis and promoting cell migration and invasion in RCC.     

Developmental Upregulation of Ephrin-B1 Silences Sema3C/Neuropilin-1 Signaling during Post-crossing Navigation of Corpus Callosum Axons

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Endothelin-1 stimulates c-fos mRNA expression and acts as a modulator on cell proliferation of rat FRTL5 thyroid cells.

In FRTL5 thyroid cells, endothelin (ET)-1 alone had no effect on DNA synthesis but caused a transient increase in c-fos mRNA levels and stimulated IGF-I induced DNA synthesis and cell proliferation. By contrast, ET-1 inhibited the stimulatory effects of TSH actions on DNA synthesis, cell proliferation and c-AMP production. 8-Bromo-cAMP-induced DNA synthesis was also inhibited by ET-1, suggesting that ET-1 exerts its inhibitory effects at step(s) involving cAMP production and post cAMP pathway. ET-1-induced suppression of TSH actions were reversed by a C-kinase inhibitor, H-7. These results suggest that the effect of ET on functions of FRTL5 cells is, at least, in part mediated by C-kinase dependent pathway.     

Critical role of the FERM domain in Pyk2 stimulated glioma cell migration

The strong tendency of malignant glioma cells to invade locally into surrounding normal brain precludes effective surgical resection, reduces the efficacy of radiotherapy, and is associated with increased resistance to chemotherapy regimens. We report that the N-terminal FERM domain of Pyk2 regulates its promigratory function. A 3-dimensional model of the Pyk2 FERM domain was generated and mutagenesis studies identified residues essential for Pyk2 promigratory function. Model-based targeted mutations within the FERM domain decreased Pyk2 phosphorylation and reduced the capacity of Pyk2 to stimulate glioma cell migration but did not significantly alter the intracellular distribution of Pyk2. Expression of autonomous Pyk2 FERM domain fragments containing analogous mutations exhibited reduced capacity to inhibit glioma cell migration and Pyk2 phosphorylation relative to expression of an autonomous wild type FERM domain fragment. These results indicate that the FERM domain plays an important role in regulating the functional competency of Pyk2 as a promigratory factor in glioma.     

MMP-9 and uPAR regulated glioma cell migration

Integrin-dependent and -independent MMP-9 and uPAR signaling plays a key role in glioma cell migration and invasion. In this article, we comment on all the possible pathways and molecules associated with MMP-9- and uPAR-mediated glioma cell migration with a special emphasis on integrins, a family of cell adhesion molecules. Our recent research investigations highlighted the substantial benefit of silencing both MMP-9 and uPAR together compared with their individual treatments in glioma. Simultaneous knockdown of both MMP-9 and uPAR regulated a majority of the molecules associated with glioma cell migration and significantly reduced the migration potential of glioma cells. Our results point out that the bicistronic construct, which can simultaneously silence both MMP-9 and uPAR offers a great therapeutic potential and is worth developing as a new drug for treating GBM patients.