Anti-inflammatory role of microsomal prostaglandin E synthase-1 in a model of neuroinflammation

A major immunological response during neuroinflammation is the activation of microglia, which subsequently release proinflammatory mediators such as prostaglandin E(2) (PGE(2)). Besides its proinflammatory properties, cyclooxygenase-2 (COX-2)-derived PGE(2) has been shown to exhibit anti-inflammatory effects on innate immune responses. Here, we investigated the role of microsomal PGE(2) synthase-1 (mPGES-1), which is functionally coupled to COX-2, in immune responses using a model of lipopolysaccharide (LPS)-induced spinal neuroinflammation. Interestingly, we found that activation of E-prostanoid (EP)2 and EP4 receptors, but not EP1, EP3, PGI(2) receptor (IP), thromboxane A(2) receptor (TP), PGD(2) receptor (DP), and PGF(2) receptor (FP), efficiently blocked LPS-induced tumor necrosis factor α (TNFα) synthesis and COX-2 and mPGES-1 induction as well as prostaglandin synthesis in spinal cultures. In vivo, spinal EP2 receptors were up-regulated in microglia in response to intrathecally injected LPS. Accordingly, LPS priming reduced spinal synthesis of TNFα, interleukin 1β (IL-1β), and prostaglandins in response to a second intrathecal LPS injection. Importantly, this reduction was only seen in wild-type but not in mPGES-1-deficient mice. Furthermore, intrathecal application of EP2 and EP4 agonists as well as genetic deletion of EP2 significantly reduced spinal TNFα and IL-1β synthesis in mPGES-1 knock-out mice after LPS priming. These data suggest that initial inflammation prepares the spinal cord for a negative feedback regulation by mPGES-1-derived PGE(2) followed by EP2 activation, which limits the synthesis of inflammatory mediators during chronic inflammation. Thus, our data suggest a role of mPGES-1-derived PGE(2) in resolution of neuroinflammation.     

Control of megakaryocyte expansion and bone marrow fibrosis by lysyl oxidase

Lysyl oxidase (LOX), a matrix cross-linking protein, is known to be selectively expressed and to enhance a fibrotic phenotype. A recent study of ours showed that LOX oxidizes the PDGF receptor-β (PDGFR-β), leading to amplified downstream signaling. Here, we examined the expression and functions of LOX in megakaryocytes (MKs), the platelet precursors. Cells committed to the MK lineage undergo mitotic proliferation to yield diploid cells, followed by endomitosis and acquisition of polyploidy. Intriguingly, LOX expression is detected in diploid-tetraploid MKs, but scarce in polyploid MKs. PDGFR-BB is an inducer of mitotic proliferation in MKs. LOX inhibition with β-aminopropionitrile reduces PDGFR-BB binding to cells and downstream signaling, as well as its proliferative effect on the MK lineage. Inhibition of LOX activity has no influence on MK polyploidy. We next rationalized that, in a system with an abundance of low ploidy MKs, LOX could be highly expressed and with functional significance. Thus, we resorted to GATA-1(low) mice, where there is an increase in low ploidy MKs, augmented levels of PDGF-BB, and an extensive matrix of fibers. MKs from these mice display high expression of LOX, compared with control mice. Importantly, treatment of GATA-1(low) mice with β-aminopropionitrile significantly improves the bone marrow fibrotic phenotype, and MK number in the spleen. Thus, our in vitro and in vivo data support a novel role for LOX in regulating MK expansion by PDGF-BB and suggest LOX as a new potential therapeutic target for myelofibrosis.     

Insulin-stimulated GLUT4 protein translocation in adipocytes requires the Rab10 guanine nucleotide exchange factor Dennd4C

Insulin-stimulated translocation of the glucose transporter GLUT4 to the cell surface in fat and muscle cells is the basis for insulin-stimulated glucose transport. Studies in adipocytes strongly support the following molecular mechanism for this process. Insulin-elicited phosphorylation of the GTPase-activating protein TBC1D4 (AS160) suppresses its activity toward Rab10 and thereby leads to an increase in the GTP-bound form of Rab10, which in turn triggers movement of vesicles containing GLUT4 to the plasma membrane and their fusion with the membrane. This process is expected to require the participation of a guanine nucleotide exchange factor (GEF) to generate the GTP-bound form of Rab10, but this GEF has not hitherto been identified. The present study identifies Dennd4C, a recently described GEF for Rab10, as the primary GEF required for GLUT4 translocation. Knockdown of Dennd4C markedly inhibited GLUT4 translocation, and ectopic expression of Dennd4C slightly stimulated it. Dennd4C was found in isolated GLUT4 vesicles. This study thus identifies another key component in the machinery of GLUT4 translocation. Moreover, it provides a potential explanation for the moderate association of a variant in the Dennd4C gene with type 2 diabetes.     

Local elongation of endothelial cell-anchored von Willebrand factor strings precedes ADAMTS13 protein-mediated proteolysis

Platelet-decorated von Willebrand factor (VWF) strings anchored to the endothelial surface are rapidly cleaved by ADAMTS13. Individual VWF string characteristics such as number, location, and auxiliary features of the ADAMTS13 cleavage sites were explored here using imaging and computing software. By following changes in VWF string length, we demonstrated that VWF strings are cleaved multiple times, successively shortening string length in the function of time and generating fragments ranging in size from 5 to over 100 μm. These are larger than generally observed in normal plasma, indicating that further proteolysis takes place in circulation. Interestingly, in 89% of all cleavage events, VWF strings elongate precisely at the cleavage site before ADAMTS13 proteolysis. These local elongations are a general characteristic of VWF strings, independent of the presence of ADAMTS13. Furthermore, large elongations, ranging in size from 1.4 to 40 μm, occur at different sites in space and time. In conclusion, ADAMTS13-mediated proteolysis of VWF strings under flow is preceded by large elongations of the string at the cleavage site. These elongations may lead to the simultaneous exposure of many exosites, thereby facilitating ADAMTS13-mediated cleavage.     

Identification of novel interaction sites that determine specificity between fibroblast growth factor homologous factors and voltage-gated sodium channels

Fibroblast growth factor homologous factors (FHFs, FGF11-14) bind to the C termini (CTs) of specific voltage-gated sodium channels (VGSC) and thereby regulate their function. The effect of an individual FHF on a specific VGSC varies greatly depending upon the individual FHF isoform. How individual FHFs impart distinctive effects on specific VGSCs is not known and the specificity of these pairwise interactions is not understood. Using several biochemical approaches combined with functional analysis, we mapped the interaction site for FGF12B on the Na(V)1.5 C terminus and discovered previously unknown determinants necessary for FGF12 interaction. Also, we demonstrated that FGF12B binds to some, but not all Na(V)1 CTs, suggesting specificity of interaction. Exploiting a human single nucleotide polymorphism in the core domain of FGF12 (P149Q), we identified a surface proline that contributes a part of this pairwise specificity. This proline is conserved among all FHFs, and mutation of the homologous residue in FGF13 also leads to loss of interaction with a specific VGSC CT (Na(V)1.1) and loss of modulation of the resultant Na(+) channel function. We hypothesized that some of the specificity mediated by this proline may result from differences in the affinity of the binding partners. Consistent with this hypothesis, surface plasmon resonance data showed that the P149Q mutation decreased the binding affinity between FHFs and VGSC CTs. Moreover, immunocytochemistry revealed that the mutation prevented proper subcellular targeting of FGF12 to the axon initial segment in neurons. Together, these results give new insights into details of the interactions between FHFs and Na(V)1.x CTs, and the consequent regulation of Na(+) channels.     

Stem cell cultivation in bioreactors

Cell-based therapies have generated great interest in the scientific and medical communities, and stem cells in particular are very appealing for regenerative medicine, drug screening and other biomedical applications. These unspecialized cells have unlimited self-renewal capacity and the remarkable ability to produce mature cells with specialized functions, such as blood cells, nerve cells or cardiac muscle. However, the actual number of cells that can be obtained from available donors is very low. One possible solution for the generation of relevant numbers of cells for several applications is to scale-up the culture of these cells in vitro. This review describes recent developments in the cultivation of stem cells in bioreactors, particularly considerations regarding critical culture parameters, possible bioreactor configurations, and integration of novel technologies in the bioprocess development stage. We expect that this review will provide updated and detailed information focusing on the systematic production of stem cell products in compliance with regulatory guidelines, while using robust and cost-effective approaches.