Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism

Fibroblast growth factor-2 (FGF-2), the most abundant growth factor produced by melanoma cells but not by normal melanocytes, is an important regulator of cell proliferation, migration and differentiation. In this study we show that M5 human metastatic melanoma cells’ ability to migrate is significantly enhanced by exogenously added FGF-2 while, neutralization of endogenous FGF-2 stimulates their adhesion. Previously, we have demonstrated that FGF-2 distinctly modulates the synthesis of individual glycosaminoglycans/proteoglycans (GAGs/PGs) subclasses, changing both their amounts and distribution in M5 cells. Here, treatment with FGF-2 strongly reduces the expression levels of the heparan sulfate-containing proteoglycan, syndecan-4. Syndecan-4 is a focal adhesion component in a range of cell types, adherent to several different matrix molecules, including fibronectin (FN). The reduction in syndecan-4 expression by utilizing specific siRNA discriminately increased melanoma cell motility and decreased their attachment on FN, demonstrating a regulatory role of syndecan-4 on these cell functions. Syndecan-4 has previously been demonstrated to regulate focal adhesion kinase (FAK) phosphorylation. In this study FGF-2 was shown to downregulate FAK Y397-phosphorylation during FN-mediated M5 cell adhesion, promoting their migration. The observed decrease in FAK Y397 activation was correlated to syndecan-4 expression levels. Thus, a balance in syndecan-4 expression perpetrated by FGF-2 may be required for optimal M5 cell migration.These results suggest that essential in melanoma progression FGF-2, specifically regulates melanoma cell ability to migrate through a syndecan-4-dependent mechanism.     

Bradyrhizobial inoculation and P application effects on haricot and mung beans in the Ethiopian Rift Valley

Plant and Soil - Natural and managed soils have been identified as the largest sources of atmospheric nitrous oxide (N2O). However, the quantification of N2O emissions from soils under natural...     

Heparan sulfate proteoglycans and heparin regulate melanoma cell functions

Background

The solid melanoma tumor consists of transformed melanoma cells, and the associated stromal cells including fibroblasts, endothelial cells, immune cells, as well as, soluble macro- and micro-molecules of the extracellular matrix (ECM) forming the complex network of the tumor microenvironment. Heparan sulfate proteoglycans (HSPGs) are an important component of the melanoma tumor ECM. Importantly, there appears to be both a quantitative and a qualitative shift in the content of HSPGs, in parallel to the nevi–radial growth phase–vertical growth phase melanoma progression. Moreover, these changes in HSPG expression are correlated to modulations of key melanoma cell functions.

Scope of review

This review will critically discuss the roles of HSPGs/heparin in melanoma development and progression.

Major conclusions

We have correlated HSPGs' expression and distribution with melanoma cell signaling and functions as well as angiogenesis.

General significance

The current knowledge of HSPGs/heparin biology in melanoma provides a foundation we can utilize in the ongoing search for new approaches in designing anti-tumor therapy. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Influence of photoperiod,temperature and host plant on the production of diapause eggs inPetrobia (Tetranychina) harti (Acari: Tetranychidae)

Petrobia harti (Ewing) diapauses in the egg stage. Adult females lay either diapause or nondiapause eggs. On the University of Thessaloniki campus (41°N), the mite was found to develop on leaves ofOxalis corniculata L. throughout the year, while no mites were found on leaves ofOxalis articulata Savigny growing in the same area. In the laboratory the mite could be maintained equally well on detached leaves of both plant species, kept on wet cotton-wool.Forty to 90% females laying diapause eggs (dlf) were produced when the mites developed under LD 1212 and 19±1 °C, or LD 168 and 19±1 °C or 25±1 °C on leaves ofO. articulata detached from plants grown in the open in various seasons. Under the same conditions, a very low to zero percentage ofdlf was produced onO. corniculata. By rearing certain feeding stages on one of these twoOxalis hosts, and the other feeding stages on the other host, various percentages ofdlf were obtained. These percentages were the net effect of the antagonistic action of the twoOxalis species.By rearing the mites at LD 8.515.5, LD 1212 or LD 168 and a temperature of 19±1 °C onO. articulata leaves renewed every 3 days, or every 16–18 days, or not at all, it could be shown that diapause induction or aversion is caused by the direct effect of photoperiod on the mites, and not by an effect through the host leaves.When wholeO. articulata plants were grown under LD 168 and 19±1 °C in the laboratory, or developed in the open during April and May, flowers were produced, while under LD 1212 no flowering occurred. In the laboratory under diapause-inducing conditions, higher percentages ofdlf were produced on leaves detached from flowering plants than on leaves detached from plants not flowering.OnO. articulata leaves at 20 °C, photoperiods with photophases equal to or longer than 12 h induced from 70 to 80%dlf, while photoperiods with photophases equal to or shorter than 10.9 h induced very low to zero percentages. By transferring different chrysalis stages from a diapause-inducing (LD 1212) to a diapause-averting (LD 8.515.5) photoperiod, and vice versa, it was found that the nymphochrysalis through deutonymph stages were sensitive to photoperiod, the deutochrysalis and deutonymph being the most sensitive.Under an LD 1212 photoperiod, a temperature of 20 °C induced diapause, whereas 25 °C, 30 °C, or a daynight thermoperiod of 25 °C18 °C suppressed it.     

Inhibition of larval growth ofDacus oleae (Diptera: Tephritidae) by streptomycin

Olives of three varieties were oviposited in by females ofDacus oleae. A few hours or a day later, they were treated with streptomycin sulphate in tap water.Immersion for 20 and 120 min in 0.3 and 1% streptomycin sulphate of Koutsourelia olives inhibited larval growth in most fruits, while for 0.2 min it did not. Immersion at 30°C caused generally more inhibition than at 20°.In Koutsourelia and Megaritiki olives, the addition of 0.1, 0.3, and 1% K₂HPO₄ or 0.7 and 2% glycerol at 22°–23°, did not cause significantly more inhibition.Topical application of drops of 1% streptomycin sulphate to Megaritiki and Konservolia olives was inhibitory only when the drop covered the oviposition hole.

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Résumé Des olives de trois variétés ont été collectionnées à des dates différentes d'août à octobre 1972 et 1973, et ont reçu des ufs deDacus oleae (Gmelin). Quelques heures ou un jour après la ponte, les olives furent traitées à 0,3 et 1% de sulfate de streptomycine dans l'eau, avec ou sans Agral 90 (un produit dispersant-mouillant).Les olives de la variété Koutsourelia, recoltées au début d'octobre 1972, ont été immergées pendant 0,2, 20 et:120 minutes dans des solutions aqueuses de sulfate de streptomycine contenant 0, 0,3 et 1% de l'antibiotique, et cela à deux températures (20° et 30°). Une immersion brève de 0,2 minutes n'était pas assez longue pour inhiber le développement larvaire dans la plupart des fruits, sauf si on ajoutait l'Agral 90. Sans Agral 90 une immersion de 20 minutes fut approximativement aussi effective que celle de 120 minutes. A 30°, une solution à 0,3% de sulfate de streptomycine fut presque aussi effective que celle à 1%. En général, l'inhibition du développement larvaire à 30° fut plus grande qu' à 20°.Des fruits de la même variété furent immergés momentanément, où pendant 20 et 60 minutes dans des solutions de 0,3 et 1% de sulfate de streptomycine, avec ou sans K₂HPO₄ (0,1, 0,3 et 1%) ou 0,7 et 2% glycerine, à une température de 22°–23°. Le développement larvaire fut inhibé dans un grand pourcentage des fruits traités, sans différences significatives entre les traitements, à l'exception d'un cas d'immersion momentanée. Les résultats avec des olives d'une autre variété, Megaritiki, recoltées à mi-septembre, furent, en général, à peu près les mêmes.D'autres olives ont reçu des gouttes de 1% de sulfate de streptomycine. Le développement larvaire fut inhibé dans un pourcentage élevé des fruits, 1) quand une goutte de solution a été placé sur le trou de ponte et 2) quand le fruit entier a été immergé dans la solution qui contenait aussi l'Agral 90 (traitement de référence). Quand l'entrée du trou de ponte était paraffiné, quand les gouttes étaient placées quelques mm vens l'apex du fruit ou autour du trou et quand la moitié du fruit ne contenant pas le trou fut immergée dans la solution de streptomycine, le développement larvaire ne fut sensiblement pas inhibé. De même il n'y eut pas d'inhibition nette du développement larvaire quand les olives étaient en contact continu avec du coton saturé avec 1 et 5% de sulfate, de streptomycine.

     

bFGF induces changes in hyaluronan synthase and hyaluronidase isoform expression and modulates the migration capacity of fibrosarcoma cells

Background

Hyaluronan (HA) a glycosaminoglycan, is capable of transmitting extracellular matrix derived signals to regulate cellular functions. In this study, we investigated whether the changes in HT1080 and B6FS fibrosarcoma cell lines HA metabolism induced by basic fibroblast growth factor (bFGF) are correlated to their migration.

Methods

Real-time PCR, in vitro wound healing assay, siRNA transfection, enzyme digestions, western blotting and immunofluorescence were utilized.

Results

bFGF inhibited the degradation of HA by decreasing hyaluronidase-2 expression in HT1080 cells (p = 0.0028), increased HA-synthase-1 and -2 expression as we previously found and enhanced high molecular weight HA deposition in the pericellular matrix. Increased endogenous HA production (p = 0.0022) and treatment with exogenous high molecular weight HA (p = 0.0268) correlated with a significant decrease of HT1080 cell migration capacity. Transfection with siHAS2 and siHAS1 showed that mainly HAS1 synthesized high molecular weight HA regulates HT1080 cell motility. Induced degradation of the HA content by hyaluronidase treatment and addition of low molecular weight HA, resulted in a significant stimulation of HT1080 cells' motility (p < 0.01). In contrast, no effects on B6FS fibrosarcoma cell motility were observed.

Conclusions

bFGF regulates, in a cell-specific manner the migration capability of fibrosarcoma cells by modulating their HA metabolism.HA metabolism is suggested to be a potential therapeutic target in fibrosarcoma.     

Lumican regulates osteosarcoma cell adhesion by modulating TGFβ2 activity

Human osteosarcoma cell lines were recently shown to express and secrete the small leucine rich proteoglycan (SLRP) lumican, with the ability to regulate the growth and motility of these cells. In this study, lumican-deficient Saos 2 cells were demonstrated to have increased adhesive capability onto fibronectin (FN) (p≤0.01). Upon neutralization of endogenous transforming growth factor β2 (TGF-β2) activity, no difference in the ability of lumican siRNA-transfected and scramble siRNA-transfected Saos 2 cells to adhere onto FN was detected (p=NS). Exogenous TGF-β2 was shown to stimulate Saos 2 cell adhesion to FN (p≤0.01). These results therefore, suggest that the inverse correlation existing between lumican expression and Saos 2 cell adhesion is dependent on active TGF-β2 signaling. Furthermore, the significant increase in Smad 2 activation present in lumican-deficient cells (p≤0.01) was annulled in the presence of the anti-TGF-β2 peptide, demonstrating that lumican is an upstream regulator of the TGF-β2/Smad 2 signaling cascade. Crucial to FN-dependent adhesion, β1 integrin expression and pFAK activation were likewise identified as downstream TGF-β2 effectors regulated by lumican expression. In conclusion, this study demonstrates a novel out-in signaling circuit in human osteosarcoma cells: secreted to extracellular matrix lumican is an endogenous inhibitor of TGF-β2 activity, resulting in downstream effector modulation including pSmad 2, integrin β1 and pFAK to regulate osteosarcoma adhesion.     

Lumican, a small leucine-rich proteoglycan substituted with keratan sulfate chains is expressed and secreted by human melanoma cells and not normal melanocytes

Melanoma is a frequent and therapy-resistant human disease. Malignant melanocytes modulate their microenvironment in order to penetrate the dermal/epidermal junction and eventually invade the dermis. The small leucine-rich proteoglycans (SLRPs) constitute important constituents of the dermis extracellular matrix (ECM), participating in both the structural and the functional organization of the skin. The role of a keratan sulphate SLRP lumican, has recently been investigated in the growth and metastasis of several cancers. In this study, the expression of lumican was studied in two human melanoma cell lines (WM9, M5) as well as in normal neonatal human melanocytes (HEMN) using real time PCR, western blotting with antibodies against the protein core and keratan sulfate, and treatments with specific enzymes. Both human metastatic melanoma cell lines were found to express lumican mRNA and effectively secrete lumican in a proteoglycan form, characterized to be substituted mostly with keratan sulfate chains. Lumican mRNA was not detected in normal melanocytes. This is the first time that the synthesis and secretion of lumican in human melanoma cell lines is reported. The role of this proteoglycan in the development and progression of malignant melanoma has to be further investigated.     

Orthologous gene-expression profiling in multi-species models: search for candidate genes

Microarray-driven gene-expression profiles are generally produced and analyzed for a single specific experimental model. We have assessed an analytical approach that simultaneously evaluates multi-species experimental models within a particular biological condition using orthologous genes as linkers for the various Affymetrix microarray platforms on multi-species models of ventilator-associated lung injury. The results suggest that this approach may be a useful tool in the evaluation of biological processes of interest and selection of process-related candidate genes.