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In the previous study, we demonstrated that the nuclear isoform of T cell protein-tyrosine phosphatase (TC-PTP) dephosphorylated and deactivated signal transducer and activator of transcription 5a (STAT5a) and STAT5b, thereby negatively regulating prolactin (PRL)-mediated signaling pathway. In this study, we examined the involvement of the nuclear isoform of TC-PTP in Interleukin-6 (IL-6)-mediated signaling pathway. IL-6 is a multifunctional cytokine that plays important roles in the immune system, hematopoiesis, and acute phase reactions, and has also implicated in IL-6-related diseases. Here we demonstrate that IL-6-induced tyrosine-phosphorylation and activation of STAT3 were suppressed by overexpression of the nuclear isoform of TC-PTP in 293T cells.Tyrosine-phosphorylated STAT3 directly interacted with a substrate-trapping mutant of TC-PTP. Furthermore, retrovirus-mediated overexpression of the nuclear isoform of TC-PTP suppressed the IL-6-induced growth arrest of myeloid leukemia M1 cells.Endogenous TC-PTP complexed with STAT3 in the nucleus of M1 cells. These results strongly suggest that the nuclear isoform of TC-PTP may serve as a negative regulator of IL-6-mediated signaling pathway.

PC12 cells respond to a variety of external stimuli such as growth factors, neurotransmitters, and membrane depolarization by activating the Ras/mitogen-activated protein kinase pathway. Here we demonstrate that both depolarization-induced calcium influx and treatment with bradykinin stimulate tyrosine phosphorylation of the epidermal growth factor receptor (EGFR). Using a tetracycline-controlled expression system in conjunction with a dominant-negative EGFR mutant, we demonstrate that depolarization and bradykinin triggered signals involve EGFR function upstream of SHC and MAP kinase. Furthermore, bradykinin-stimulated EGFR transactivation is critically dependent on the presence of extracellular calcium, and when triggered by ionophore treatment, calcium influx is already sufficient to induce EGFR tyrosine phosphorylation. Taken together, our results establish calcium-dependent EGFR transactivation as a signaling mechanism mediating activation of the Ras/mitogen-activated protein kinase pathway in neuronal cell types.In neurons, the cytosolic calcium concentration is tightly regulated and represents a critical parameter for a variety of intracellular signaling processes. Intracellular calcium levels are modulated either by release of calcium from internal stores or by calcium entry across the plasma membrane through ligand-or voltage-gated calcium channels (1-3). Stimuli such as membrane depolarization result in activation of L-type voltage-sensitive calcium channels, leading to calcium-mediated induction of a specific set of genes and thereby contributing to physiological responses such as neuronal differentiation and survival (4) .Before altering gene expression, elevation of intracellular calcium levels can trigger various signaling events, among them the activation of the small G-protein Ras resulting in stimulation of the mitogen-activated protein kinase (MAPK) 1 pathway (5). In PC12 cells, a rat pheochromocytoma cell line widely used as a model system for neuronal differentiation, calcium influx rapidly induces tyrosine phosphorylation of the adaptor protein SHC and SHC-Grb2 complex formation, steps known to couple cell surface receptors such as receptor tyrosine kinases to Ras (6). Using a PC12 subline overexpressing a dominant-negative mutant of the cytoplasmatic tyrosine kinase Src, Rusanescu et al. found that inhibiton of membrane depolarization induced SHC tyrosine phosphorylation and MAPK activation (7). Moreover calcium influx following membrane depolarization was recently reported to mediate ligandindependent epidermal growth factor receptor (EGFR) tyrosine phosphorylation in this system (8). Although direct evidence is lacking regarding whether this represents an essential signaling event for activation of the MAPK pathway, this finding raises the possibility that in PC12 cells calcium may play a role in the EGFR transactivation mechanism as previously demonstrated for signaling through G-protein coupled receptor (GPCR) in Rat-1 fibroblasts (9). In addition to membrane depolarization-induced acti...

Adipocytes are now recognized as endocrine cells secreting adipocytokines, regulating multiple metabolic pathways. In this study, we addressed secretion of microvesicles by 3T3-L1 adipocytes. We found that MFG-E8, one of the exosomal proteins, was present in the microvesicles and was distributed in the sucrose density fractions with 1.13-1.20 g/ml, which has been reported for exosomes. Several integral, cytosolic, and nuclear proteins such as caveolin-1, c-Src kinase, and heat shock protein 70 were also found to be microvesicle components. Unexpectedly, adiponectin was also substantially distributed in the microvesicle fractions. Furthermore, proteomic analysis of the microvesicles revealed that many other proteins such as extracellular matrix-related proteins were also present. Microvesicles secreted by 3T3-L1 adipocytes exhibited heterogeneity in size and comprised both smaller exosome-like and larger membrane vesicles as revealed by electron microscopy. Milk fat globule-epidermal growth factor 8 (MFG-E8)-associated adiposomes exhibited binding activity toward phosphatidylserine and apoptotic cells. MFG-E8 in the microvesicles was reduced when cultured in the low-glucose medium or cultured in the high-glucose medium with antioxidant N-acetyl cysteine. Insulin and TNF-alpha also up-regulated MFG-E8 in the microvesicles. Moreover, MFG-E8 was strongly up-regulated in the hypertrophic adipose tissue, predominantly in adipocyte fractions, of diet-induced obese C57BL/6 mice, where increased oxidative stress is induced. Thus, it is suggested that microvesicles, especially MFG-E8-associated ones, modulate adipose functions under redox- and hormone-dependent regulation. Based on the above findings, the adipocyte-derived microvesicles were named adiposomes.

MFG-E8 (milk fat globule-EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two-repeated EGF-like domains, a mucin-like domain and two-repeated discoidinlike domains (C-domains), and contains an integrin-binding motif (RGD sequence) in the EGF-like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain-deletion mutants of MFG-E8 were expressed in COS-7 cells. The immunofluorescent staining of intracellular and cell-surface proteins and biochemical analyses of cell-surface-biotinylated and secreted proteins demonstrated that both of the two C-domains were required for the membrane association. During the course of these studies for domain functions, MFG-E8, but not C-domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size-exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high-molecular mass, low density and higher sedimentation velocity and to be detergentsensitive. Not only such a exogenously expressed MFG-E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA-1D, was secreted as the membrane vesicle-like complex. Scanning electron microscopic analyses revealed that MFG-E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG-E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG-E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).

The cAMP-response element-binding protein-binding protein (CBP) and p300 are common coactivators for several transcriptional factors. It has been reported that both CBP and p300 are significant for the activation of peroxisome proliferator-activated receptor ␥ (PPAR␥), which is a crucial nuclear receptor in adipogenesis. However, it remains unclear whether CBP and/or p300 is physiologically essential to the activation of PPAR␥ in adipocytes and adipocyte differentiation. In this study, we investigated the physiological significance of CBP/ p300 in NIH3T3 cells transiently expressing PPAR␥ and CBP and in 3T3-L1 preadipocytes stably expressing CBP-or p300-specific ribozymes. In PPAR␥-transfected NIH3T3 cells, induction of expression of PPAR␥ target genes such as adipocyte fatty acid-binding protein (aP2) and lipoprotein lipase (LPL) by adding thiazolidinedione was enhanced, depending on the amount of a CBP expression plasmid transfected. Expression of aP2 and LPL genes, as well as glycerol-3-phosphate dehydrogenase activity and triacylglyceride accumulation after adipogenic induction, was largely suppressed in 3T3-L1 adipocytes expressing either the CBP-or p300-specific active ribozyme, but not in inactive ribozyme-expressing cells. These data suggest that both CBP and p300 are indispensable for the full activation of PPAR␥ and adipocyte differentiation and that CBP and p300 do not mutually complement in the process.

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