Bone Morphogenic Protein (BMP) Signaling Up-regulates Neutral Sphingomyelinase 2 to Suppress Chondrocyte Maturation via the Akt Protein Signaling Pathway as a Negative Feedback Mechanism

Although bone morphogenic protein (BMP) signaling promotes chondrogenesis, it is not clear whether BMP-induced chondrocyte maturation is cell-autonomously terminated. Loss of function of Smpd3 in mice results in an increase in mature hypertrophic chondrocytes. Here, we report that in chondrocytes the Runx2-dependent expression of Smpd3 was increased by BMP-2 stimulation. Neutral sphingomyelinase 2 (nSMase2), encoded by the Smpd3 gene, was detected both in prehypertrophic and hypertrophic chondrocytes of mouse embryo bone cartilage. An siRNA for Smpd3, as well as the nSMase inhibitor GW4869, significantly enhanced BMP-2-induced differentiation and maturation of chondrocytes. Conversely, overexpression of Smpd3 or C₂-ceramide, which mimics the function of nSMase2, inhibited chondrogenesis. Upon induction of Smpd3 siRNA or GW4869, phosphorylation of both Akt and S6 proteins was increased. The accelerated chondrogenesis induced by Smpd3 silencing was negated by application of the Akt inhibitor MK2206 or the mammalian target of rapamycin inhibitor rapamycin. Importantly, in mouse bone culture, GW4869 treatment significantly promoted BMP-2-induced hypertrophic maturation and calcification of chondrocytes, which subsequently was eliminated by C₂-ceramide. Smpd3 knockdown decreased the apoptosis of terminally matured ATDC5 chondrocytes, probably as a result of decreased ceramide production. In addition, we found that expression of hyaluronan synthase 2 (Has2) was elevated by a loss of Smpd3, which was restored by MK2206. Indeed, expression of Has2 protein decreased in nSMase2-positive hypertrophic chondrocytes in the bones of mouse embryos. Our data suggest that the Smpd3/nSMase2-ceramide-Akt signaling axis negatively regulates BMP-induced chondrocyte maturation and Has2 expression to control the rate of endochondral ossification as a negative feedback mechanism.     

Molecular cloning and functional expression of a multifunctional triterpene synthase cDNA from a mangrove species Kandelia candel (L.) Druce

Homology based PCRs with degenerate primers designed from the conserved sequences among the known oxidosqualene cylases (OSCs) have resulted in cloning of a triterpene synthase (KcMS) from the young roots of Kandelia candel (L.) Druce (Rhizophoraceae). KcMS consists of a 2286 bp open reading frame, which codes for 761 amino acids. The deduced amino acid sequence showed 79% homology to a lupeol synthase from Ricinus communis suggesting it to be a lupeol synthase of K. candel. KcMS was expressed in a lanosterol synthase deficient yeast with the expression vector pYES2 under the control of GAL1 promoter. GC-MS analysis showed that the transformant accumulated a mixture of lupeol, beta-amyrin and alpha-amyrin in a 2:1:1 ratio, indicating that KcMS encodes a multifunctional triterpene synthase, although it showed high sequence homology to a R. communis lupeol synthase. This is the first OSC cloning from mangrove tree species.     

Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose biosensor

A novel FAD-dependent glucose dehydrogenase (FAD-GDH) was found and its enzymatic property for glucose sensing was characterized. FAD-GDH oxidized glucose in the presence of some artificial electron acceptors, except for O2, and exhibited thermostability, high substrate specificity and a large Michaelis constant for glucose. FAD-GDH was applied to an amperometric glucose sensor with Fe(CN)6(3-) as a soluble mediator. The use of a relatively high concentration of Fe(CN)6(3-) resulted in a good linearity between the current response and the glucose concentration, taking into account a large Michaelis constant for Fe(CN)6(3-). The glucose sensor was completely insensitive to O2 and responded linearly to glucose up to 30 mM. Compared to glucose, the response to other saccharides was negligible. The sensor can be stored at room temperature in a desiccator for at least one month without any change in the response or activity.     

Biosynthesis of branched-chain fatty acid in bacilli: FabD (malonyl-CoA:ACP transacylase) is not essential for in vitro biosynthesis of branched-chain fatty acids

It was found that the partially purified beta-ketoacyl-ACP synthase of Bacillus insolitus did not require the addition of FabD (malonyl-CoA:ACP transacylase, MAT) for the activity assay. This study therefore examined the necessity of FabD protein for in vitro branched-chain fatty acid (BCFA) biosynthesis by crude fatty acid synthetases (FAS) of Bacilli. To discover the involvement of FabD in the BCFA biosynthesis, the protein was removed from the crude FAS by immunoprecipitation. The His-tag fusion protein FabD of Bacillus subtilis was expressed in Escherichia coli and used for the preparation of antibody. The rabbit antibody raised against the expressed fusion protein specifically recognized the FabD in the crude FAS of B. subtilis. Evaluation of the efficacy of the immunoprecipitation showed that a trace of FabD protein was present in the antibody-treated crude FAS. However, this complete removal of FabD from the crude FAS did not abolish its BCFA biosynthesis, but only reduced the level to 50-60% of the control level for acyl-CoA primer and to 80% for alpha-keto-beta-methylvalerate primer. Furthermore, the FabD concentration did not necessarily correlate with the MAT specific activity in the enzyme fractions, suggesting the presence of another enzyme source of MAT activity. This study, therefore, suggests that FabD is not the sole enzyme source of MAT for in vitro BCFA biosynthesis, and implies the existence of a functional connection between fatty acid biosynthesis and another metabolic pathway.     

Role for Phospholipid Flippase Complex of ATP8A1 and CDC50A Proteins in Cell Migration

Type IV P-type ATPases (P4-ATPases) and CDC50 family proteins form a putative phospholipid flippase complex that mediates the translocation of aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to inner leaflets of the plasma membrane. In Chinese hamster ovary (CHO) cells, at least eight members of P4-ATPases were identified, but only a single CDC50 family protein, CDC50A, was expressed. We demonstrated that CDC50A associated with and recruited P4-ATPase ATP8A1 to the plasma membrane. Overexpression of CDC50A induced extensive cell spreading and greatly enhanced cell migration. Depletion of either CDC50A or ATP8A1 caused a severe defect in the formation of membrane ruffles, thereby inhibiting cell migration. Analyses of phospholipid translocation at the plasma membrane revealed that the depletion of CDC50A inhibited the inward translocation of both PS and PE, whereas the depletion of ATP8A1 inhibited the translocation of PE but not that of PS, suggesting that the inward translocation of cell-surface PE is involved in cell migration. This hypothesis was further examined by using a PE-binding peptide and a mutant cell line with defective PE synthesis; either cell-surface immobilization of PE by the PE-binding peptide or reduction in the cell-surface content of PE inhibited the formation of membrane ruffles, causing a severe defect in cell migration. These results indicate that the phospholipid flippase complex of ATP8A1 and CDC50A plays a major role in cell migration and suggest that the flippase-mediated translocation of PE at the plasma membrane is involved in the formation of membrane ruffles to promote cell migration.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

La autoantigen translocates to cytoplasm after cleavage during granzyme B-mediated cytotoxicity

Our recent report demonstrated that apoptosis-specific autoantibodies against granzyme B-induced cleavage fragments of SS-B (La) were found in the sera from patients with primary Sj?gren's syndrome. The objective of this study was identified by the intracellular redistribution of La autoantigen during granzyme B-induced apoptosis. We developed green fluorescence protein (GFP)-La and GFP-LaDelta220 (generation of granzyme B-specific cleavage of La protein) fusion proteins. GFP-La protein was localized in the nucleus, whereas the GFP-LaDelta220 protein predominantly existed in the cytoplasm in transformed A293T cells. Nuclear GFP-La protein was translocated to cytoplasm after granzyme B enriched YT cells incubation. La protein in human salivary grand HSG cells is cleaved and translocated from the nucleus to the cytoplasm after YT cell co-cultivation. These results suggest that La protein is cleaved by granzyme B and N-terminal La fragment (27 kD) translocated to the cytoplasm, thus leading to a novel autoantibody production during granzyme B-mediated cytotoxicity.     

Small molecule screening identifies targetable zebrafish pigmentation pathways

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types, including pigment cells, are conserved between zebrafish and other vertebrates, we present these chemicals as molecular tools to study developmental processes of pigment cells in living animals and emphasize the value of zebrafish as an in vivo system for testing the on- and off-target activities of clinically active drugs.     

Negative regulation of platelet clearance and of the macrophage phagocytic response by the transmembrane glycoprotein SHPS-1

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.