Basic fibroblast growth factor enhances proliferation and hepatocyte growth factor expression of feline mesenchymal stem cells.

INTRODUCTION: The objective of this study is to evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation and secretion activity of feline adipose-derived mesenchymal stem cells (MSC). METHODS: Feline MSC isolated from the subcutaneous adipose tissue of cats were cultured with or without bFGF. RESULTS: The bFGF addition enhanced the proliferation of feline MSC to a significant great extent compared with that without bFGF, although the cell proliferation tended to increase with the bFGF concentration. In addition, adipogenic and osteogenic staining assay demonstrated that the bFGF addition allowed MSC to maintain the differentiation ability even after the proliferation. Moreover, no change in the surface markers of MSC was observed between the cultures with or without bFGF. A quantitative RT-PCR assay revealed that the HGF and TSG-6 expression significantly increased by the bFGF addition. The highest mRNA expression of MMP-2 was observed for cells cultured in 1000 ng/ml bFGF concentration. CONCLUSIONS: The culture with bFGF is a promising way to enhance the proliferation, and HGF secretion ability of MSC as well as maintain their differentiation ability and immunophenotype nature.

SIRT1 Regulates Thyroid-Stimulating Hormone Release by Enhancing PIP5Kgamma Activity through Deacetylation of Specific Lysine Residues in Mammals.

BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.

Regulation of phosphatidylinositol 4-phosphate 5-kinase activity by partner proteins.

The remarkably versatile phospholipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P(2) is synthesized predominantly by phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kalpha, beta, and gamma, and three splicing variants of the gamma isozyme. Although the PIP5Kgamma splicing variant PIP5Kgamma661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.

The phosphoinositide kinase PIP5K that produces the versatile signaling phospholipid PI4,5P(2).

The phosphoinositide kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), produces the versatile phospholipid phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)), through which PIP5K plays crucial roles in a wide variety of cell functions. So far, three PIP5K isozymes and splicing variants have been identified. We speculate that each PIP5K isozyme or splicing variant is activated in a tempo-spatially different manner, due to the existence of activators or recruiters specific to each isozyme: this tempo-spatially different activation of PIP5K produces PI4,5P(2) at different compartments of the cell at different times, which phenomenon may be responsible for the apparent multifunction of PI4,5P(2)/PIP5K. Accumulating evidence supports this notion that each PIP5K isozyme is activated by its specific activator and plays a crucial role in a unique cell function. In this article, we describe recent advances regarding the PIP5K isozyme-specific activation mechanisms and physiological functions.

Syk-dependent actin dynamics regulate endocytic trafficking and processing of antigens internalized through the B-cell receptor.

Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.

Role of activation of PIP5Kgamma661 by AP-2 complex in synaptic vesicle endocytosis.

Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.

Crucial role of the small GTPase ARF6 in hepatic cord formation during liver development.

The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6-/- embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6-/- fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.

The small GTPase ADP-ribosylation factor 6 negatively regulates dendritic spine formation.

Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation.

Essential role for phospholipase D2 activation downstream of ERK MAP kinase in nerve growth factor-stimulated neurite outgrowth from PC12 cells.

The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.

B cell receptor-mediated Syk-independent activation of phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinase pathways.

The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.

Dynamics of major histocompatibility complex class II compartments during B cell receptor-mediated cell activation.

Antigen recognition by clonotypic B cell receptor (BcR) is the first step of B lymphocytes differentiation into plasmocytes. This B cell function is dependent on efficient major histocompatibility complex (MHC) class II-restricted presentation of BcR-bound antigens. In this work, we analyzed the subcellular mechanisms underlying antigen presentation after BcR engagement on B cells. In quiescent B cells, we found that MHC class II molecules mostly accumulated at the cell surface and in an intracellular pool of tubulovesicular structures, whereas H2-M molecules were mostly detected in distinct lysosomal compartments devoid of MHC class II. BcR stimulation induced the transient intracellular accumulation of MHC class II molecules in newly formed multivesicular bodies (MVBs), to which H2-M was recruited. The reversible downregulation of cathepsin S activity led to the transient accumulation of invariant chain-MHC class II complexes in MVBs. A few hours after BcR engagement, cathepsin S activity increased, the p10 invariant chain disappeared, and MHC class II-peptide complexes arrived at the plasma membrane. Thus, BcR engagement induced the transient formation of antigen-processing compartments, enabling antigen-specific B cells to become effective antigen-presenting cells.