Endothelial basement membrane limits tip cell formation by inducing Dll4/Notch signalling in vivo

How individual components of the vascular basement membrane influence endothelial cell behaviour remains unclear. Here we show that laminin α4 (Lama4) regulates tip cell numbers and vascular density by inducing endothelial Dll4/Notch signalling in vivo. Lama4 deficiency leads to reduced Dll4 expression, excessive filopodia and tip cell formation in the mouse retina, phenocopying the effects of Dll4/Notch inhibition. Lama4-mediated Dll4 expression requires a combination of integrins in vitro and integrin β1 in vivo. We conclude that appropriate laminin/integrin-induced signalling is necessary to induce physiologically functional levels of Dll4 expression and regulate branching frequency during sprouting angiogenesis in vivo.     

α‐parvin controls vascular mural cell recruitment to vessel wall by regulating RhoA/ROCK signalling

During blood vessel development, vascular smooth muscle cells (vSMCs) and pericytes (PCs) are recruited to nascent vessels to stabilize them and to guide further vessel remodelling. Here, we show that loss of the focal adhesion (FA) protein α‐parvin (α‐pv) in mice leads to embryonic lethality due to severe cardiovascular defects. The vascular abnormalities are characterized by poor vessel remodelling, impaired coverage of endothelial tubes with vSMC/PCs and defective association of the recruited vSMC/PCs with endothelial cells (ECs). α‐pv‐deficient vSMCs are round and hypercontractile leading either to their accumulation in the tissue or to local vessel constrictions. Because of the high contractility, α‐pv‐deficient vSMCs fail to polarize their cytoskeleton resulting in loss of persistent and directed migration. Mechanistically, the absence of α‐pv leads to increased RhoA and Rho‐kinase (ROCK)‐mediated signalling, activation of myosin II and actomyosin hypercontraction in vSMCs. Our findings show that α‐pv represents an essential adhesion checkpoint that controls RhoA/ROCK‐mediated contractility in vSMCs.     

Hes binding to STAT3 mediates crosstalk between Notch and JAK-STAT signalling

Although the Notch and JAK-STAT signalling pathways fulfill overlapping roles in growth and differentiation regulation, no coordination mechanism has been proposed to explain their relationship. Here we show that STAT3 is activated in the presence of active Notch, as well as the Notch effectors Hes1 and Hes5. Hes proteins associate with JAK2 and STAT3, and facilitate complex formation between JAK2 and STAT3, thus promoting STAT3 phosphorylation and activation. Furthermore, suppression of endogenous Hes1 expression reduces growth factor induction of STAT3 phosphorylation. STAT3 seems to be essential for maintenance of radial glial cells and differentiation of astrocytes by Notch in the developing central nervous system. These results suggest that direct protein-protein interactions coordinate cross-talk between the Notch-Hes and JAK-STAT pathways.     

Suppressor of cytokine signalling protein SOCS3 expression is increased at sites of acute and chronic inflammation

Treatment of cells with cytokines and growth factors leads to the synthesis of Suppressor of Cytokine Signalling (SOCS) proteins that act as potent negative regulators of signalling via the Jak/STAT pathway. We used immunohistochemistry to identify cells and pathologies where SOCS3 expression might influence acute and chronic inflammatory responses in human tissues. Epitope and GFP tagged SOCS3 fusion proteins were localised predominantly in the nucleus of transfected cells and a validated anti SOCS3 antiserum revealed the expression of SOCS3 in the nucleus and cytoplasm of macrophages, endothelial and epithelial cells in a wide range of normal tissues in tissue microarrays (n = 31 different tissues). Nuclear SOCS3 was only seen in cells expressing a high level of the protein. Comparative immunostaining of acute, chronically and granulomatously inflamed human tissues revealed higher levels of nuclear and cytoplasmic SOCS3 expression in inflamed than in corresponding normal tissues, particularly in recruited leukocyte populations, but also in epithelia. The staining appeared more intense, suggesting higher expression levels, in areas where inflammation was more acute, consistent with the time course of SOCS3 induction described in vitro. Expression of SOCS3 protein by leucocytes and other cell types in tissue sections could be a useful marker of cells undergoing acute or chronic stimulation by cytokines in vivo.     

The hemifusion intermediate and its conversion to complete fusion: regulation by membrane composition.

To fuse, membranes must bend. The energy of each lipid monolayer with respect to bending is minimized at the spontaneous curvature of the monolayer. Two lipids known to promote opposite spontaneous curvatures, lysophosphatidylcholine and arachidonic acid, were added to different sides of planar phospholipid membranes. Lysophosphatidylcholine added to the contacting monolayers of fusing membranes inhibited the hemifusion we observed between lipid vesicles and planar membranes. In contrast, fusion pore formation depended upon the distal monolayer of the planar membrane; lysophosphatidylcholine promoted and arachidonic acid inhibited. Thus, the intermediates of hemifusion and fusion pores in phospholipid membranes involve different membrane monolayers and may have opposite net curvatures, Biological fusion may proceed through similar intermediates.     

Notch signal is sufficient to direct an endothelial conversion from non-endothelial somitic cells conveyed to the aortic region by CXCR4

During the early formation of the dorsal aorta, the first-forming embryonic vessel in amniotes, a subset of somitic cells selected as presumptive angioblasts, migrates toward the dorsal aorta, where they eventually differentiate into endothelial cells. We have recently shown that these processes are controlled by Notch signals (Sato, Y., Watanabe, T., Saito, D., Takahashi, T., Yoshida, S., Kohyama, J., Ohata, E., Okano, H., and Takahashi, Y., 2008. Notch mediates the segmental specification of angioblasts in somites and their directed migration toward the dorsal aorta in avian embryos. Dev. Cell 14, 890-901.). Here, we studied a possible link between Notch and chemokine signals, SDF1/CXCR4, the latter found to be dominantly expressed in developing aorta/somites. Although CXCR4 overexpression caused a directed migration of somitic cells to the aortic region in a manner similar to Notch, no positive epistatic relationships between Notch and SDF1/CXCR4 were detected. After reaching the aortic region, the CXCR4-electroporated cells exhibited no endothelial character. Importantly, however, once provided with Notch activity, they could successfully be incorporated into developing vessels as endothelial cells. These findings were obtained combining the tetracycline-inducible gene expression method with the transposon-mediated stable gene transfer technique. We conclude that Notch activation is sufficient to direct naïve mesenchymal cells to differentiate into endothelial cells once the cells are conveyed to the aortic region.     

NGF controls dendrite development in hippocampal neurons by binding to p75NTR and modulating the cellular targets of Notch

Notch and neurotrophins control neuronal shape, but it is not known whether their signaling pathways intersect. Here we report results from hippocampal neuronal cultures that are in support of this possibility. We found that low cell density or blockade of Notch signaling by a soluble Delta-Fc ligand decreased the mRNA levels of the nuclear targets of Notch, the homologues of enhancer-of-split 1 and 5 (Hes1/5). This effect was associated with enhanced sprouting of new dendrites or dendrite branches. In contrast, high cell density or exposure of low-density cultures to NGF increased the Hes1/5 mRNA, reduced the number of primary dendrites and promoted dendrite elongation. The NGF effects on both Hes1/5 expression and dendrite morphology were prevented by p75-antibody (a p75NTR-blocking antibody) or transfection with enhancer-of-split 6 (Hes6), a condition known to suppress Hes activity. Nuclear translocation of NF-kappaB was identified as a link between p75NTR and Hes1/5 because it was required for the up-regulation of these two genes. The convergence of the Notch and p75NTR signaling pathways at the level of Hes1/5 illuminates an unexpected mechanism through which a diffusible factor (NGF) could regulate dendrite growth when cell-cell interaction via Notch is not in action.     

The conversion of 3-deoxyarabinoheptulosonate 7-phosphate to 3-dehydroquinate by sorghum seedling preparations

Partially purified preparations from etiolated sorghum seedlings catalyzed the conversion of DAHP to DHQ. The reaction catalysed by DHQ synthetase was stimulated by 0.1 μM to 0.1 mM NAD in the presence O-0.5 mM Co²⁺. NADH at 1 μM stimulated the reaction as much as 50% but became inhibitory at 100μM. Co²⁺ at 0.5mM stimulated enzyme activity 3-fold; Mg²⁺, Mn²⁺, Cu²⁺, and Zn²⁺ were not stimulatory. EDTA at 5 mM inhibited the reaction 95% but its effects were reversed by equal concentrations of Co²⁺. Phe, Tyr, Trp, t-cinnamate, several hydroxylated cinnamates, DHS, quinate, and shikimate at 0.3 mM failed to affect enzyme activity but slight inhibition occurred with DHQ and protocatechuic acid at 0.3 mM, inhibition being 14 % and 22 %, respectively. DHQ synthetase activity also was detected in spinach leaves and potato tuber tissue. Synthetase activity appeared to increase in response to injury of potato tuber and sweet potato root tissues.     

Phosphorylation of transglutaminase 2 by PKA at Ser216 creates 14-3-3 binding sites

Transglutaminase 2 (TG2) is a multifunctional ubiquitous enzyme which is present in various cellular compartments and is subject to phosphorylation by PKA. To better understand the relevance of PKA induced phosphorylation of TG2, we performed pull-down assays using phosphorylated biotinylated-TG2(209-223) peptides spanning PKA induced phosphorylation sites as a bait. Subsequent analysis of pull-down protein by SDS-PAGE and LC/MS identified 14-3-3epsilon as the binding partner for TG2 which was further confirmed by immunoblotting with 14-3-3 specific antiserum. In contrast, non-phosphorylated and/or phosphorylation site substituted peptides fail to pull-down 14-3-3. Furthermore, we demonstrate that 14-3-3 co-immunoprecipitated with TG2 antiserum after activation of PKA from mouse embryonic fibroblasts (MEF)(TG2+/+) cells but not from MEF(TG2-/-) cells. In summary, we provide convincing evidence that phosphorylation of TG2 by PKA creates binding site(s) for 14-3-3 both in vitro and in vivo.     

High-rate conversion of methane to methanol by Methylosinus trichosporium OB3b

Methanol was produced from methane with a high conversion rate using a high cell density process with Methylosinus trichosporium OB3b in the presence of a high concentration of phosphate buffer. More than 1.1 g/L methanol accumulated in the reaction media under optimized reaction conditions (17 g dry cell/L, 400 mmol/L phosphate, and 10 mmol/L MgCl₂) in the presence of 20 mmol/L sodium formate. The conversion rate of methane was over 60%. About 0.95 g/L methanol was produced when the biotransformation was carried out in a membrane aerated reactor into which methane and oxygen were introduced via two separate dense silicone tubing. Our results provide an efficient method and a promising process for high-rate conversion of methane to methanol.     

Pseudolaric acid B ameliorates synovial inflammation and vessel formation by stabilizing PPARγ to inhibit NF‐κB signalling pathway

Synovial macrophage polarization and inflammation are essential for osteoarthritis (OA) development, yet the molecular mechanisms and regulation responsible for the pathogenesis are still poorly understood. Here, we report that pseudolaric acid B (PAB) attenuated articular cartilage degeneration and synovitis during OA. PAB, a diterpene acid, specifically inhibited NF‐κB signalling and reduced the production of pro‐inflammatory cytokines, which further decreased M1 polarization and vessel formation. We further provide in vivo and in vitro evidences that PAB suppressed NF‐κB signalling by stabilizing PPARγ. Using PPARγ antagonist could abolish anti‐inflammatory effect of PAB and rescue the activation of NF‐κB signalling during OA. Our findings identify a previously unrecognized role of PAB in the regulation of OA and provide mechanisms by which PAB regulates NF‐κB signalling through PPARγ, which further suggest targeting synovial inflammation or inhibiting vessel formation at early stage could be an effective preventive strategy for OA.     

Transdifferentiation of tadpole pancreatic acinar cells to duct cells mediated by Notch and stromelysin-3

The tadpole pancreas has differentiated acinar cells but an underdeveloped ductal system. At the climax of metamorphosis thyroid hormone (TH) induces the tadpole acinar cells to dedifferentiate to a progenitor state. After metamorphosis is complete the exocrine pancreas redifferentiates in the growing frog forming a typical vertebrate pancreas including a complex ductal system. A micro array analysis found that TH up regulates stromelysin 3 (ST3, matrix metalloproteinase 11) in the exocrine pancreas at metamorphic climax. Transgenic tadpoles were prepared with an elastase promoter driving either the ST3 gene or the constitutively active form of Notch (IC). Expression of the transgenes was controlled by the tetracycline system. A few days after either of these transgenes is activated by doxycycline the pancreatic acinar cells turn into duct-like cells. This transdetermination occurs without cell division since both acinar and ductal markers can be visualized transiently in the same cell. We propose that remodeling of the tadpole acinar cells is initiated when ST3 is up regulated by TH. Stromelysin-3 then cleaves and activates Notch.     

Engineering of TIMP‐3 as a LAP‐fusion protein for targeting to sites of inflammation

Tissue inhibitor of metalloproteinase (TIMP)‐3 is a natural inhibitor of a range of enzymes that degrade connective tissue and are involved in the pathogenesis of conditions such as arthritis and cancer. We describe here the engineering of TIMP‐3 using a novel drug‐delivery system known as the ‘LAP technology’. This involves creating therapeutic proteins in fusion with the latency‐associated peptide (LAP) from the cytokine TGF‐? to generate proteins that are biologically inactive until cleavage of the LAP to release the therapy. LAP‐TIMP‐3 was successfully expressed in mammalian cells and the presence of the LAP resulted in a 14‐fold increase in the quantity of recombinant TIMP‐3 produced. LAP‐TIMP‐3 was latent until release from the LAP by treatment with matrix metalloproteinase when it could inhibit proteases of the adamalysins and adamalysins with thrombospondin motifs families, but not matrix metalloproteinases, indicating that this version of TIMP‐3 is a more specific inhibitor than the native protein. There was sufficient protease activity in synovial fluid from human joints with osteoarthritis to release TIMP‐3 from the LAP fusion. These results demonstrate the potential for development of TIMP‐3 as a novel therapy for conditions where upregulation of catabolic enzymes are part of the pathology.     

Presenilin-dependent gamma-secretase processing of beta-amyloid precursor protein at a site corresponding to the S3 cleavage of Notch

The presenilin (PS)-dependent site 3 (S3) cleavage of Notch liberates its intracellular domain (NICD), which is required for Notch signaling. The similar γ-secretase cleavage of the β-amyloid precursor protein (βAPP) results in the secretion of amyloid β-peptide (Aβ). However, little is known about the corresponding C-terminal cleavage product (CTFγ). We have now identified CTFγ in brain tissue, in living cells, as well as in an in vitro system. Generation of CTFγ is facilitated by PSs, since a dominant-negative mutation of PS as well as a PS gene knock out prevents its production. Moreover, γ-secretase inhibitors, including one that is known to bind to PS, also block CTFγ generation. Sequence analysis revealed that CTFγ is produced by a novel γ-secretase cut, which occurs at a site corresponding to the S3 cleavage of Notch.     

Activated CRH receptors inhibit autophagy by repressing conversion of LC3BI to LC3BII

Clinical studies have elucidated the negative correlation between microtubules-associated protein 1 light chain 3-B (LC3B) protein expression and overall survival of breast cancer patients. Our previous data demonstrated corticortropin-releasing hormone family (CRHs) suppressed migration of breast cancer cells via CRH receptors (CRHRs). Here, we showed that the activation of CRHRs (CRHR1 and CRHR2) remarkably reduced the conversion of LC3BI to LC3BII and hence repressed macroautophagy/autophagy, resulting in migration inhibition. By means of RT-4 cells (expressing higher CRHR1) with stable CRHR1 silence which was constructed by lentivirus with short hairpin RNA, we further confirmed CRH-inhibited LC3BII conversion. Using CRHRs agonists and antagonists, we found CRHRs triggered a marked reduction in the number of LC3B dots in both RT-4 and Hela cells(expressing higher CRHR2) which stably express RFP-GFP-LC3B. Of note, this decreased amount of autophagosome was associated with activation of Phospholipase C β (PLCβ)-Inositol triphosphate (IP₃)-mTOR signaling. Earle's Balanced Salt Solution (EBSS) decreased the expression of the key focal adhesion protein, paxillin, which was recovered by CRHRs ligands (CRH and UCN2). The effect of CRHRs ligands on paxillin resulted in the suppression of cell migration. Altogether, these data reveal a new link between CRHRs signaling and autophagy, and may help to envisage therapeutic strategies in cancer cell invasion.