Endocytosis,cadherins and tissue dynamics

By happy chance, the founding of Traffic in 1999 coincided with a clutch of reports that documented the endocytosis and recycling of classical cadherin adhesion receptors. This stimulated a concerted effort to elucidate the molecular regulation of cadherin endocytosis and to identify its functional implications. In particular, endocytosis provided new perspectives to understand how cadherins are modulated during tissue morphogenesis. In this short article, we consider some of what we have learnt about this problem and identify open questions for future research.     

A possible pivotal role of mitochondrial free calcium in neurotoxicity mediated by N-methyl-d-aspartate receptors in cultured rat hippocampal neurons

We have previously shown that mitochondrial membrane potential disruption is involved in mechanisms underlying differential vulnerabilities to the excitotoxicity mediated by N-methyl-d-aspartate (NMDA) receptors between primary cultured neurons prepared from rat cortex and hippocampus. To further elucidate the role of mitochondria in the excitotoxicity after activation of NMDA receptors, neurons were loaded with the fluorescent dye calcein diffusible in the cytoplasm and organelles for determination of the activity of mitochondrial permeability transition pore (mPTP) responsible for the leakage of different mitochondrial molecules. The addition of CoCl₂ similarly quenched the intracellular fluorescence except mitochondria in both cultured neurons, while further addition of NMDA led to a leakage of the dye into the cytoplasm in hippocampal neurons only. An mPTP inhibitor prevented the NMDA-induced loss of viability in hippocampal neurons, while an activator of mPTP induced a similarly potent loss of viability in cortical and hippocampal neurons. Although NMDA was more effective in increasing rhodamine-2 fluorescence as a mitochondrial calcium indicator in hippocampal than cortical neurons, a mitochondrial calcium uniporter inhibitor significantly prevented the NMDA-induced loss of viability in hippocampal neurons. Expression of mRNA was significantly higher for the putative uniporter uncoupling protein-2 in hippocampal than cortical neurons. These results suggest that mitochondrial calcium uniporter would be at least in part responsible for the NMDA neurotoxicity through a mechanism relevant to promotion of mPTP orchestration in hippocampal neurons.     

Insulin-like growth factor-I activation of Akt survival cascade in neuronal cells requires the presence of its cognate receptor in caveolae

Insulin-like growth factor-I (IGF-I) plays important roles in survival of neurons. Caveolae, cholesterol-rich microdomains of plasma membrane, act as platforms for some neurotrophic factors. In this study, we examined a possible role of caveolae in IGF-I signal transduction in pheochromocytoma PC12 cells. IGF-I treatment attenuated serum withdrawal-induced apoptosis, which was reversed by treatment with methyl-beta-cyclodextrin (CD) that removes cholesterol from plasma membrane. Immunocytochemical and subcellular fractionation analyses revealed that IGF-I receptor (IGF-IR) was colocalized with caveolin-1, a major protein component in caveolae, and that CD treatment reduced IGF-IR contents in caveolae. Consistent with these findings, IGF-I phosphorylation of insulin receptor substrate-1 and Akt was impaired, and cholesterol supply restored the IGF-I action. Furthermore, experiments using small interfering RNA revealed that the reduction of caveolin-1 expression impaired the IGF-I action. In addition, the colocalization of IGF-IR with caveolin-1, and the caveolae-dependent IGF-I action were duplicated in primary culture of rat cerebellar granule neurons. These results demonstrate that the presence of IGF-IR in caveolae is required for the neuroprotective action of IGF-I.     

Nitric oxide-mediated modulation of calcium/calmodulin-dependent protein kinase II

The mechanisms of NO inhibition of CaMK [Ca(2+)/CaM (calmodulin)-dependent protein kinase] II activity were studied. In rat pituitary tumour GH3 cells, TRH [thyrotrophin (TSH)-releasing hormone]-stimulated phosphorylation of nNOS [neuronal NOS (NO synthase)] at Ser(847) was sensitive to an inhibitor of CaMKs, KN-93, and was enhanced by inhibition of nNOS with 7NI (7-nitroindazole). Enzyme activity of CaMKII following in situ treatment with 7NI was also increased. The in vitro activity of CaMKII was inhibited by co-incubation either with nNOS and L-arginine or with NO donors SNAP (S-nitroso-N-acetyl-DL-penicillamine) and DEA-NONOate [diethylamine-NONOate (diazeniumdiolate)]. Once inhibited by these treatments, CaMKII was observed to undergo full reactivation on the addition of a reducing reagent, DTT (dithiothreitol). In transfected cells expressing CaMKII and nNOS, treatment with the calcium ionophore A23187 further revealed nNOS phosphorylation at Ser(847), which was enhanced by 7NI and CaMKII S-nitrosylation. Mutated CaMKII (C6A), in which Cys(6) was substituted with an alanine residue, was refractory to 7NI-induced enhancement of nNOS phosphorylation or to CaMKII S-nitrosylation. Furthermore, we could identify Cys(6) as a direct target for S-nitrosylation of CaMKII using MS. In addition, treatment with glutamate caused an increase in CaMKII S-nitrosylation in rat hippocampal slices. This glutamate-induced S-nitrosylation was blocked by 7NI. These results suggest that inactivation of CaMKII mediated by S-nitrosylation at Cys(6) may contribute to NO-induced neurotoxicity in the brain.     

Protection by exogenous pyruvate through a mechanism related to monocarboxylate transporters against cell death induced by hydrogen peroxide in cultured rat cortical neurons

In cortical neurons cultured for 3 or 9 days in vitro (DIV), exposure to hydrogen peroxide (H(2)O(2)) led to a marked decrease in cell viability in a concentration-dependent manner at a concentration range of 10 microm to 1 mm irrespective of the duration between 6 and 24 h. However, H(2)O(2) was more potent in decreasing cellular viability in cortical neurons cultured for 9 DIV than in those for 3 DIV. Pyruvate was effective in preventing the neuronal cell death at 1 mm even when added 1-3 h after the addition of H(2)O(2). Semi-quantitative RT-PCR and western blotting analyses revealed significantly higher expression of both mRNA and protein for a particular monocarboxylate transporter (MCT) in neurons cultured for 9 DIV than in those for 3 DIV. A specific inhibitor of MCT significantly attenuated the neuroprotection by pyruvate in neurons cultured for 9 DIV, without markedly affecting that in neurons cultured for 3 DIV. These results suggest that vulnerability to H(2)O(2) may at least in part involve expression of particular MCT isoforms responsible for the bi-directional transport of pyruvate across cell surfaces in cultured rat cortical neurons.     

Stem cell factor has a suppressive activity to IgE-mediated chemotaxis of mast cells

Stem cell factor (SCF), which is well known as a cytokine capable of amplifying development and functions of mast cells, is mainly released from fibroblasts in the peripheral tissue. To investigate whether SCF controlled chemotactic migration of mast cells induced by IgE-specific Ag, murine bone marrow-derived cultured mast cells (BMCMC) and human cord blood-derived cultured mast cells (HuCMC) were preincubated with SCF. Although BMCMC and HuCMC sensitized with IgE directly moved toward specific Ag, preincubation for even 1 h with an optimal dose of SCF suppressed the IgE-mediated chemotactic movement. No or little inhibitory effect of SCF was detected in BMCMC derived from c-kit receptor-defect WBB6F1-W/Wv mice. In contrast, preincubation of BMCMC and HuCMC with SCF enhanced beta-hexosaminidase release and Ca2+ mobilization in response to Ag after sensitization with IgE. Using the real-time record of chemotactic migration, BMCMC preincubated with SCF manifested motionless without degranulation. These results suggest that locally produced SCF may have an inhibitory effect on chemotaxis of mast cells, contributing to their accumulation and enhancement of functions at the peripheral site in allergic and nonallergic conditions.     

An additional carbohydrate chain in the variant thyroxine-binding globulin-Gary (TBGAsn-96) impairs its secretion.

The T4-binding globulin-Gary (TBG-G) variant has severely impaired T4 binding, is unstable at 37 C, and presents an apparent anodal shift of all isoforms when submitted to isoelectric focusing. Inheritance of this abnormal TBG produces a profound decrease in the serum levels of native TBG with reciprocal changes in its denatured form, causing thyroid hormone concentrations to be as low as those found in complete TBG deficiency. The TBG-G gene possesses a single nucleotide substitution replacing the normal IIe96 (ATC) with Asn (AAC), thus creating a new site for N-linked glycosylation. In order to determine whether TBG-G contains an additional carbohydrate chain as indirectly suggested by the isoelectric focusing results, cDNAs containing the normal TBG (TBG-N), and TBG-G were inserted in the appropriate vectors to allow their expression in mammalian cells (COS-1) and in amphibian (Xenopus) oocytes. In both systems, expression of TBG-G yielded a larger molecule than TBG-N when analyzed by polyacrylamide gel electrophoresis under denaturing conditions. However, both were identical in size when synthesized in COS-1 cells in the presence of tunicamycin or when deglycosylated after their synthesis in Xenopus oocytes. Pulse chase experiments revealed impaired secretion and excessive overall intracellular degradation of TBG-G relative to TBG-N. As expected from studies on serum from affected subjects, in vitro expressed TBG-G had a 10-fold lower affinity for T4. These studies prove that the new site for potential glycosylation created by the point mutation in TBG-G is indeed glycosylated.(ABSTRACT TRUNCATED AT 250 WORDS)