RIP3 blockade prevents immune-mediated hepatitis through a myeloid-derived suppressor cell dependent mechanism

Autoimmune hepatitis (AIH) is an immune-mediated chronic inflammatory liver disease, and its pathogenesis is not fully understood. Our previous study discovered that receptor interacting protein kinase 3 (RIP3) is correlated with serum transaminase levels in AIH patients. However, its role and underlying mechanism in AIH are poorly understood. Here, we detected the increased expression and activation of RIP3 in livers of patients and animal models with AIH. The inhibition of RIP3 kinase by GSK872 prevented concanavalin A (ConA)-induced immune-mediated hepatitis (IMH) by reduced hepatic proinflammatory cytokines and immune cells including Th17 cells and macrophages. Further experiments revealed that RIP3 inhibition resulted in an increase in CD11b⁺Gr1⁺ myeloid-derived suppressor cells (MDSCs) with immunoregulatory properties in the liver, spleen, and peripheral blood. Moreover, the depletion of Gr-1⁺ MDSCs abrogated the protective effect and immune suppression function of GSK872 in ConA-induced IMH. Altogether, our data demonstrate that RIP3 blockade prevents ConA-induced IMH through promoting MDSCs infiltration. Inhibition of RIP3 kinase may be a novel therapeutic avenue for AIH treatment.     

The complexities of G-protein-coupled receptor kinase function in Hedgehog signaling

Hedgehog (Hh) signaling is essential for proper tissue patterning and maintenance and has a substantial impact on human disease. While many of the main components and mechanisms involved in transduction of the Hh signal have been identified, the details of how the pathway functions are continually being refined. One aspect that has attracted much attention recently is the involvement of G-protein-coupled receptor kinases (GRKs) in the pathway. These regulators of G-protein-coupled receptor (GPCR) signaling have an evolutionarily-conserved function in promoting high-threshold Hh target gene expression through regulation of Smoothened (Smo), a GPCR family member that activates intracellular Hh signaling. Several models of how GRKs impact on Smo to increase downstream signaling have been proposed. Recently, we demonstrated that these kinases have surprisingly complex and conflicting roles, acting to limit signaling through the pathway while also promoting Smo activity. In addition to the previously described direct effects of Gprk2 on Smo activation, Gprk2 also indirectly affects Hh signaling by controlling production of the second messenger cyclic AMP to influence Protein kinase A activity.     

Entamoeba histolytica: lipid rafts are involved in adhesion of trophozoites to host extracellular matrix components

Adhesion is an important virulence function for Entamoeba histolytica, the causative agent of amoebic dysentery. Lipid rafts, cholesterol-rich domains, function in compartmentalization of cellular processes. In E. histolytica, rafts participate in parasite-host cell interactions; however, their role in parasite-host extracellular matrix (ECM) interactions has not been explored. Disruption of rafts with a cholesterol extracting agent, methyl-β-cyclodextrin (MβCD), resulted in inhibition of adhesion to collagen, and to a lesser extent, to fibronectin. Replenishment of cholesterol in MβCD-treated cells, using a lipoprotein-cholesterol concentrate, restored adhesion to collagen. Confocal microscopy revealed enrichment of rafts at parasite-ECM interfaces. A raft-resident adhesin, the galactose/N-acetylgalactosamine-inhibitable lectin, mediates interaction to host cells by binding to galactose or N-acetylgalactosamine moieties on host glycoproteins. In this study, galactose inhibited adhesion to collagen, but not to fibronectin. Together these data suggest that rafts participate in E. histolytica-ECM interactions and that raft-associated Gal/GalNAc lectin may serve as a collagen receptor.     

Transformed 3T3 cells have reduced levels and altered subcellular distribution of the major PKC substrate protein marcks

The MARCKS (myristylated alanine-rich C-kinase substrate) protein is an abundant calmodulin-binding protein that is a major and specific endogenous substrate of protein kinase C (PKC). Stimulation of cells with phorbol esters or other activators of PKC has been shown previously to result in rapid phosphorylation of MARCKS proteins and redistribution of these myristylated C-kinase substrates from membrane to cytosol. Here we show that NIH3T3 murine fibroblasts transformed by p21-HA-C-RAS or pp60-V-SRC oncoproteins have markedly reduced levels of p68-MARCKS and that most of the remaining MARCKS protein is found in the cytosol. 3T3 cells containing a nontransforming oncoprotein p26-BCL2, in contrast, exhibited normal levels and distribution of p68-MARCKS. When taken together with recent evidence that MARCKS proteins are involved in regulating organization of the membrane cytoskeleton, our findings suggest that oncoprotein-mediated alterations in MARCKS protein levels and subcellular distribution may contribute to the development or maintenance of the transformed phenotpe.     

Association Between the Rat Homologue of CO-029, a Metastasis-associated Tetraspanin Molecule and Consumption Coagulopathy

Recently, we have described a panel of metastasis-associated antigens in the rat, i.e., of molecules expressed on metastasizing, but not on nonmetastasizing tumor lines. One of these molecules, recognized by the monoclonal antibody D6.1 and named accordingly D6.1A, was found to be abundantly expressed predominantly on mesenchyme-derived cells. The DNA of the antigen has been isolated and cloned. Surprisingly, the gene product proved to interfere strongly with coagulation.

The 1.182-kb cDNA codes for a 235–amino acid long molecule with a 74.2% homology in the nucleotide and a 70% homology in the amino acid sequence to CO-029, a human tumor-associated molecule. According to the distribution of hydrophobic and hydrophilic amino acids, D6.1A belongs to the tetraspanin superfamily. Western blotting of D6.1A-positive metastasizing tumor lines revealed that the D6.1A, like many tetraspanin molecules, is linked to further membrane molecules, one of which could be identified as α6β1 integrin. Transfection of a low-metastasizing tumor cell line with D6.1A cDNA resulted in increased metastatic potential and provided a clue as to the functional role of D6.1A. We noted massive bleeding around the metastases and, possibly as a consequence, local infarctions predominantly in the mesenteric region and all signs of a consumption coagulopathy. By application of the D6.1 antibody the coagulopathy was counterregulated, though not prevented.

It has been known for many years that tumor growth and progression is frequently accompanied by thrombotic disorders. Our data suggest that the phenomenon could well be associated with the expression of tetraspanin molecules.

     

Genistein Inhibits the Activity of Kv1.3 Potassium Channels in Human T Lymphocytes

In the present study, the whole-cell patch-clamp technique was applied to follow the inhibitory effect of genistein — a tyrosine kinase inhibitor and a natural anticancer agent—on the activity of voltage-gated potassium channels Kv1.3 expressed in human T lymphocytes (TL). Obtained data provide evidence that genistein application in the concentration range of 1–80 μM reversibly decreased the whole-cell potassium currents in TL in a concentration-dependent manner to about 0.23 of the control value. The half-blocking concentration range of genistein was from 10 to 40 μM. The current inhibition was correlated in time with a significant decrease of the current activation rate. The steady-state activation of the currents was unchanged upon application of genistein, as was the inactivation rate. The inhibitory effect of genistein on the current amplitude and activation kinetics was voltage-independent. The current inhibition was not changed significantly in the presence of 1 mM of sodium orthovanadate, a tyrosine phosphatase inhibitor. Application of daidzein, an inactive genistein analogue, did not affect significantly either the current amplitudes or the activation kinetics. Possible mechanisms of the observed phenomena and their significance for genistein-induced inhibition of cancer cell proliferation are discussed.     

The effect of fructose 2,6-bisphosphate and AMP on the activity of phosphorylated and unphosphorylated fructose-1,6-bisphosphatase from rat liver

Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation.     

Drosophila p24 homologues eclair and baiser are necessary for the activity of the maternally expressed Tkv receptor during early embryogenesis

p24 proteins are assumed to play an important role in the transport of secreted and transmembrane proteins into membranes. However, only few cargo proteins are known that partially, but in no case completely require p24 proteins for membrane transport. Here, we show that two p24 proteins are essential for dorsoventral patterning of Drosophila melanogaster embryo. Mutations in the genes, eclair (eca) and baiser (bai), encoding two p24 proteins reduce signalling by the TGF-beta homologue, Dpp, in early embryos. This effect is strictly maternal and specific to early embryogenesis, as Dpp signalling in other contexts is not notably affected. We provide genetic evidence that in the absence of eca or bai function in the oocyte, the maternally expressed type I TGF-beta receptor Tkv is not active. We propose that during early embryogenesis eca and bai are specifically required for the activity of the maternal Tkv, while the zygotic Tkv is not affected in the mutant embryos. Mutations in either eca or bai are sufficient for the depletion of Tkv activity and no enhancement of the phenotypes was observed in embryos derived from oocytes mutant for both genes. The dependence of maternal Tkv protein on the products of p24 genes may serve as an in vivo model for studying p24 proteins.