Cutting Edge: Lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals constitutive signals that promote cytokine secretion and cell survival

Engagement of the FcepsilonRI expressed on mast cells induces the production of phosphatidylinositol 3, 4, 5-trisphosphate by PI3K, which is essential for the functions of the cells. PTEN (phosphatase and tensin homologue deleted on chromosome ten) directly opposes PI3K by dephosphorylating phosphatidylinositol 3, 4, 5-trisphosphate at the 3' position. In this work we used a lentivirus-mediated short hairpin RNA gene knockdown method to study the role of PTEN in CD34(+) peripheral blood-derived human mast cells. Loss of PTEN caused constitutive phosphorylation of Akt, p38 MAPK, and JNK, as well as cytokine production and enhancement in cell survival, but not degranulation. FcepsilonRI engagement of PTEN-deficient cells augmented signaling downstream of Src kinases and increased calcium flux, degranulation, and further enhanced cytokine production. PTEN-deficient cells, but not control cells, were resistant to inhibition of cytokine production by wortmannin, a PI3K inhibitor. The findings demonstrate that PTEN functions as a key regulator of mast cell homeostasis and FcepsilonRI-responsiveness.     

The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.     

Recombination of the GFP gene to the BFP gene using a man-made site-selective DNA cutter

By using the recently developed man-made DNA cutter [a combination of Ce(IV)/EDTA and two DNA additives], green fluorescent protein (GFP) was converted to closely related blue fluorescent protein (BFP). The phosphodiester linkages at T196-A200 in the sense strand of GFP were hydrolyzed by the cutter, and the A1-T196 fragment in the product was selectively connected with the downstream fragment (C197-A720) of BFP by T4 DNA ligase. This recombination changed three codons in the GFP gene (TGC at 196–198, TAT at 199–201, and ACC at 502–504) to TCT, CAT, and ATC in BFP, and accordingly three amino acids in GFP (Cys65, Tyr66, and Thr167) were altered to Ser65, His66, and Ile167. The recombinant gene was successfully expressed in Escherichia coli and emitted blue fluorescence, confirming the absence of undesired side reactions (mutation, deletion, insertion, depurination, etc.) in the DNA manipulation. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Role of VAMP-2, VAMP-7, and VAMP-8 in constitutive exocytosis from HSY cells

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH–GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20°C for 2 h and then released by warming up to 37°C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH–GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.     

Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.     

Multiple roles of mesenchymal beta-catenin during murine limb patterning

Recently canonical Wnt signaling in the ectoderm has been shown to be required for maintenance of the apical ectodermal ridge (AER) and for dorsoventral signaling. Using conditional gain- and loss-of-function beta-catenin alleles, we have studied the role of mesenchymal beta-catenin activity during limb development. Here, we show that loss of beta-catenin results in limb truncations due to a defect in AER maintenance. Stabilization of beta-catenin also results in truncated limbs, caused by a premature regression of the AER. Concomitantly, in these limbs, the expression of Bmp2, Bmp4 and Bmp7, and of the Bmp target genes Msx1, Msx2 and gremlin, is expanded in the mesenchyme. Furthermore, we found that the expression of Lmx1b, a gene exclusively expressed in the dorsal limb mesenchyme and involved in dorsoventral patterning, is reduced upon loss of beta-catenin activity and is expanded ventrally in gain-of-function limbs. However, the known ectodermal regulators Wnt7a and engrailed 1 are expressed normally. This suggests that Lmx1b is also regulated, in part, by a beta-catenin-mediated Wnt signal, independent of the non-canoncial Wnt7a signaling pathway. In addition, loss of beta-catenin results in a severe agenesis of the scapula. Concurrently, the expression of two genes, Pax1 and Emx2, which have been implicated in scapula development, is lost in beta-catenin loss-of-function limbs; however, only Emx2 is upregulated in gain-of-function limbs. Mesenchymal beta-catenin activity is therefore required for AER maintenance, and for normal expression of Lmx1b and Emx2.     

Screening of siRNA target sequences by using fragmentized DNA

BACKGROUND: RNA interference (RNAi) has become a powerful tool in silencing target genes in various organisms. In mammals, RNAi can be induced by using short interfering RNA (siRNA). The efficacy of inducing RNAi in mammalian cells by using siRNA depends very much on the selection of the target sequences. METHODS: We developed an siRNA target sequence selection system by first constructing parallel-type siRNA expression vector libraries carrying siRNA expression fragments originating from fragmentized target genes, and then using a group selection system. For a model system, we constructed parallel-type siRNA expression vector libraries against DsRed and GFP reporter genes. RESULTS: We carried out the first screening of groups containing more than 100 random siRNA expression plasmids in total for each target gene, and successfully obtained target sequences with very strong efficacy. Furthermore, we also obtained some clones that express dsRNAs of various lengths that might induce cytotoxicity. CONCLUSIONS: This system should allow us to perform screening for powerful target sequences, by including all possible target sequences for any gene, even without knowing the whole sequence of the target gene in advance. At the same time, target sequences that should be avoided due to cytotoxicity can be identified.     

Selective delivery of rifampicin incorporated into poly(DL-lactic-co-glycolic) acid microspheres after phagocytotic uptake by alveolar macrophages, and the killing effect against intracellular Mycobacterium bovis Calmette-Guérin

Macrophages and their phagocytotic abilities play a dominant role for defense against infected organisms. However, Mycobacterium tuberculosis can survive in the phagosomes of macrophages. In this study, the effective delivery of a drug and the killing effect of tubercle bacilli within macrophages were investigated utilizing the phagocytotic uptake of rifampicin (RFP) that had been incorporated into poly(DL-lactic-co-glycolic) acid (PLGA) microspheres. The microspheres were composed of PLGA that had a monomer ratio (lactic acid/glycolic acid) of either 50/50 or 75/25. They had molecular weights from 5000 to 20,000, and diameters of 1.5, 3.5, 6.2 and 8.9 microm. The most significant factor for phagocytotic activity of macrophages was the diameter of the microspheres. By contrast, molecular weight and monomer ratio of PLGA did not influence phagocytosis. The amount of RFP delivered into cells was also investigated. RFP-PLGA microspheres composed of PLGA with a molecular weight of 20,000 and monomer ratio of 75/25 showed the highest amount of delivery (4 microg/1 x 10(6) cells). Fourteen days after infection, the survival rate of treated intracellular bacilli was 1% when compared with untreated cells. There was almost no killing effect of free RFP (4 or 15 microg/ml) on intracellular bacilli. In vivo efficacy of RFP-PLGA was also examined in rats infected with M. tuberculosis Kurono. Intratracheal administration of RFP-PLGA microspheres was shown to be superior to free RFP for killing of intracellular bacilli and preventing granuloma formation in some lobes. These results suggest that phagocytotic activity could be part of a new drug delivery system that selectively targeted macrophages.