Role of fibroblast growth factor receptor in regulation of membrane traffic

Several studies show that membrane transport mechanisms are regulated by signalling molecules. Recently, genome-wide screen analyses in C.elegans have enabled scientists to identify novel regulators in membrane trafficking and also signalling molecules which are found to couple with this machinery. Fibroblast growth factor (FGF) via binding to fibroblast growth factor receptor (FGFR) mediate signals which are essential in the development of an organism, patterning, cell migration and tissue homeostasis. Impaired FGFR-mediated signalling has been associated with various developmental, neoplastic, metabolic and neurological diseases and cancer. In this study, the potential role of FGFR-mediated signalling pathway as a regulator of membrane trafficking was investigated. The GFP-tagged yolk protein YP170-GFP trafficking was analysed in worms where 1) FGFR signalling cascade components were depleted by RNAi and 2) in mutant animals. From these results, it was found that the disruption of the genes egl-15 (FGFR), egl-17(FGF), let-756(FGF), sem-5, let-60, lin-45, mek-2, mpk-1 and plc-3 lead to abnormal localization of YP170-GFP, suggesting that signalling downstream of FGFR via activation of MAPK and PLC-γ pathway is regulating membrane transport. The route of trafficking was further investigated, to pinpoint which membrane step is regulated by worm FGFR, by analysing a number of GFP-tagged intracellular membrane markers in the intestine of Wild Type (WT) and FGFR mutant worms. FGFR mutant worms showed a significant difference in the localisation of several endosomal membrane markers, suggesting its regulatory role in early and recycling steps of endocytosis. Finally, the trafficking of transferrin in a mammalian NIH/3T3 cell line was investigated to identify the conservation of these membrane trafficking regulatory mechanisms between organisms. Results showed no significant changes in transferrin trafficking upon FGFR stimulation or inhibition.

Effect of a fish oil-enriched nutritional supplement on metabolic mediators in patients with pancreatic cancer cachexia

Weight loss in advanced cancer patients is refractory to conventional nutritional support. This may be due to metabolic changes mediated by proinflammatory cytokines, hormones, and tumor-derived products. We previously showed that a nutritional supplement enriched with fish oil will reverse weight loss in patients with pancreatic cancer cachexia. The present study examines the effect of this supplement on a number of mediators thought to play a role in cancer cachexia. Twenty weight-losing patients with pancreatic cancer were asked to consume a nutritional supplement providing 600 kcal and 2 g of eicosapentaenoic acid per day. At baseline and after 3 wk, patients were weighed and samples were collected to measure serum concentrations of interleukin (IL)-6 and its soluble receptor tumor necrosis factor receptors I and II, cortisol, insulin, and leptin, peripheral blood mononuclear cell production of IL-1 beta, IL-6, and tumor necrosis factor, and urinary excretion of proteolysis inducing factor. After 3 wk of consumption of the fish oil-enriched nutritional supplement, there was a significant fall in production of IL-6 (from median 16.5 to 13.7 ng/ml, P = 0.015), a rise in serum insulin concentration (from 3.3 to 5.0 mU/l, P = 0.0064), a fall in the cortisol-to-insulin ratio (P = 0.0084), and a fall in the proportion of patients excreting proteolysis inducing factor (from 88% to 40%, P = 0.008). These changes occurred in association with weight gain (median 1 kg, P = 0.024). Various mediators of catabolism in cachexia are modulated by administration of a fish oil-enriched nutritional supplement in pancreatic cancer patients. This may account for the reversal of weight loss in patients consuming this supplement.

Induction of placental heme oxygenase-1 is protective against TNFα-induced cytotoxicity and promotes vessel relaxation

Background: Pregnancy is characterized by an inflammatory-like process and this may be exacerbated in preeclampsia. The heme oxygenase (HO) enzymes generate carbon monoxide (CO) that induces blood vessel relaxation and biliverdin that acts as an endogenous antioxidant. Materials and Methods: We examined the expression and localization of HO-1 and HO-2 in normal and preeclamptic placenta using reverse transcription polymerase chain reaction (RT-PCR), RNase protection assay, immunoblotting and immunohistochemistry. In addition, the effect of HO activation on tumor necrosis factor-alpha (TNF) induced placental damage and on feto-placental circulation was studied. Results: We provide the first evidence for the role of HO as an endogenous placental factor involved with cytoprotection and placental blood vessel relaxation. HO-1 was significantly higher at term, compared with first trimester placentae indicating its role in placental vascular development and regulation. HO-1 predominantly localized in the extravascular connective tissue that forms the perivascular contractile sheath around the developing blood vessels. HO-2 was localized in the capillaries, as well as the villous stroma, with weak staining of trophoblast. Induction of HO-1 caused a significant attenuation of TNF-mediated cellular damage in placental villous explants, as assessed by lactate dehydrogenase leakage (p 0.01). HO-1 protein was significantly reduced in placentae from pregnancies complicated with preeclampsia, compared with gestationally matched normal pregnancies. This suggests that the impairment of HO-1 activation may compromise the compensatory mechanism and predispose the placenta to cellular injury and subsequent maternal endothelial cell activation. Isometric contractility studies showed that hemin reduced vascular tension by 61% in U46619-preconstricted placental arteries. Hemininduced vessel relaxation and CO production was inhibited by HO inhibitor, tin protoporphyrin IX. Conclusions: Our findings establish HO-1 as an endogenous system that offers protection against cytotoxic damage in the placenta, identifies the HO-CO pathway to regulate feto-placental circulation and provides a new approach to study the disease of preeclampsia.

Mechanism of attenuation of protein loss in murine C2C12 myotubes by d-myo-inositol 1,2,6-triphosphate

d-Myo-inositol 1,2,6-triphosphate (alpha trinositol, AT) has been shown to attenuate muscle atrophy in a murine cachexia model through an increase in protein synthesis and a decrease in degradation. The mechanism of this effect has been investigated in murine myotubes using a range of catabolic stimuli, including proteolysis-inducing factor (PIF), angiotensin II (Ang II), lipopolysaccharide, and tumor necrosis factor-α/interferon-γ. At a concentration of 100 μM AT was found to attenuate both the induction of protein degradation and depression of protein synthesis in response to all stimuli. The effect on protein degradation was accompanied by attenuation of the increased expression and activity of the ubiquitin-proteasome pathway. This suggests that AT inhibits a signalling step common to all four agents. This target has been shown to be activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) and the subsequent phosphorylation of eukaryotic initiation factor 2 on the α-subunit, together with downstream signalling pathways leading to protein degradation. AT also inhibited activation of caspase-3/-8, which is thought to lead to activation of PKR. The mechanism of this effect may be related to the ability of AT to chelate divalent metal ions, since the attenuation of the increased activity of the ubiquitin-proteasome pathway by PIF and Ang II, as well as the depression of protein synthesis by PIF, were reversed by increasing concentrations of Zn2+. The ability of AT to attenuate muscle atrophy by a range of stimuli suggests that it may be effective in several catabolic conditions. © 2009 Elsevier Inc. All rights reserved.