Rivka Hadar

Regulated IRE1-dependent decay participates in curtailing immunoglobulin secretion from plasma cells

Inositol‐requiring enzyme 1 (IRE1) is a kinase and ribonuclease that executes the splicing of X box binding protein 1 (XBP‐1) mRNA in response to the accumulation of unfolded protein in the ER, a signal cascade termed the unfolded protein response. Recently, IRE1 has been implicated in mRNA and miRNA cleavage and degradation, a pathway termed regulated IRE1‐dependent decay (RIDD). Deletion of XBP‐1 in the liver and pancreas strongly enhances RIDD by upregulating IRE1 protein levels and enhancing its ribo‐nuclease activity. Because XBP‐1 is essential for generating plasma cells with developed secretory capacity, we sought to evaluate the contribution of RIDD to this regulation. Mice were conditionally deleted for XBP‐1 and/or IRE1 in their B‐cell lineage. Similarly to the liver, deletion of XBP‐1 induces IRE1 expression in LPS‐treated B cells. In vitro, IRE1 cleaves the mRNA of secretory μ chains, which explains the reduction in secretory μ mRNA and its synthesis in XBP‐1 KO plasma cells. In accordance, the IgM response is partially restored in XBP‐1/IRE1 double KO mice relative to XBP‐1 KO mice. Interestingly, the IgG1 response is reduced to a similar level in XBP‐1 KO, IRE1 KO, and their double knockout animals. Our data demonstrate a specific contribution by RIDD in curtailing immunoglobulin synthesis and secretion.

Carbamoylphosphonate matrix metalloproteinase inhibitors 6: cis-2-aminocyclohexylcarbamoylphosphonic acid, a novel orally active antimetastatic matrix metalloproteinase-2 selective inhibitor--synthesis and pharmacodynamic and pharmacokinetic analysis.

cis-2-Aminocyclohexylcarbamoylphosphonic acid ( cis-ACCP) was evaluated in vitro and in two in vivo cancer metastasis models. It reduced metastasis formation in mice by approximately 90% when administered by a repetitive once daily dosing regimen of 50 mg/kg via oral or intraperitoneal routes and was nontoxic up to 500 mg/kg, following intraperitoneal administration daily for two weeks. Pharmacokinetic investigation of cis-ACCP in rats revealed distribution restricted into the extracellular fluid, which is the site of action for the antimetastatic activity and rapid elimination ( t 1/2 approximately 19 min) from blood. Sustained and prolonged absorption ( t 1/2 approximately 126 min) occurred via paracellular mechanism along the small and large intestine with overall bioavailability of 0.3%. The in vivo concentrations of cis-ACCP in the blood in rats was above the minimal concentration for antimetastatic/MMP-inhibitory activity, thus explaining the prolonged action following once daily administration. Finally, 84% of the intravenously administered cis-ACCP to rats was excreted intact in the urine.

Targeting the endocannabinoid/CB1 receptor system for treating obesity in Prader–Willi syndrome

Objective Extreme obesity is a core phenotypic feature of Prader–Willi syndrome (PWS). Among numerous metabolic regulators, the endocannabinoid (eCB) system is critically involved in controlling feeding, body weight, and energy metabolism, and a globally acting cannabinoid-1 receptor (CB1R) blockade reverses obesity both in animals and humans. The first-in-class CB1R antagonist rimonabant proved effective in inducing weight loss in adults with PWS. However, it is no longer available for clinical use because of its centrally mediated, neuropsychiatric, adverse effects. Methods We studied eCB ‘tone’ in individuals with PWS and in the Magel2-null mouse model that recapitulates the major metabolic phenotypes of PWS and determined the efficacy of a peripherally restricted CB1R antagonist, JD5037 in treating obesity in these mice. Results Individuals with PWS had elevated circulating levels of 2-arachidonoylglycerol and its endogenous precursor and breakdown ligand, arachidonic acid. Increased hypothalamic eCB ‘tone’, manifested by increased eCBs and upregulated CB1R, was associated with increased fat mass, reduced energy expenditure, and decreased voluntary activity in Magel2-null mice. Daily chronic treatment of obese Magel2-null mice and their littermate wild-type controls with JD5037 (3 mg/kg/d for 28 days) reduced body weight, reversed hyperphagia, and improved metabolic parameters related to their obese phenotype. Conclusions Dysregulation of the eCB/CB1R system may contribute to hyperphagia and obesity in Magel2-null mice and in individuals with PWS. Our results demonstrate that treatment with peripherally restricted CB1R antagonists may be an effective strategy for the management of severe obesity in PWS.

The appearance of the CD4+CD8+ phenotype on activated T cells: possible role of antigen transfer.

Stimulation of peripheral blood lymphocytes (PBL) with phytohemagglutinin (PHA) strikingly increased the proportion of CD4+CD8+ cells. Highly purified CD4+ and CD8+ lymphocyte populations cultured in the presence of PHA consistently failed to coexpress the CD8 and CD4 markers. Similarly, exposure of highly purified CD4+ cells to PHA and recombinant interleukin-2 resulted in augmented expression of CD25 but failed to induce the expression of CD8. When purified preparations of either CD4+ or CD8+ cells were activated separately for 3 days and incubated together for an additional 5 h, a considerable proportion of CD4+CD8+ cells was found in the mixture. Cycloheximide treatment did not prevent the appearance of the CD8 marker on CD4 cells. CD4+CD8+ cells isolated from PBL exposed for 3 days to PHA lost their CD8 antigenicity within 24-48 h in the absence of PHA. Increased levels of soluble CD4 and CD8 antigens were found in supernatant fluids of PHA-stimulated cells. T cells failed, however, to bind soluble markers even after prolonged incubation in the presence of supernatant fluids. Our studies show that activation of CD4+ cells per se does not elicit the CD4+CD8+ phenotype and that soluble T cell markers do not bind to T cells. Rather, it seems that direct cell-cell contact is required for the transfer of CD8 molecules from CD8+ cells to the membrane of CD4+ cells.

De Novo Ceramide Synthesis Is Required for N-Linked Glycosylation in Plasma Cells

Plasma cells (PCs) are terminally differentiated B lymphocytes responsible for the synthesis and secretion of Igs. The differentiation of B cells into PCs involves a remarkable expansion of both lipid and protein components of the endoplasmic reticulum. Despite their importance in many signal transduction pathways, the role of ceramides, and of complex sphingolipids that are derived from ceramide, in PC differentiation has never been directly studied. To assess their putative role in PC differentiation, we blocked ceramide synthesis with fumonisin B1, a specific inhibitor of ceramide synthase. Under fumonisin B1 treatment, N-linked glycosylation was severely impaired in LPS-activated, but not in naive, B cells. We also show that ceramide synthesis is strongly induced by XBP-1 (X box-binding protein-1). In the absence of ceramide synthesis, ER expansion was dramatically diminished. Our results underscore ceramide biosynthesis as a key metabolic pathway in the process of PC differentiation and reveal a previously unknown functional link between sphingolipids and N-linked glycosylation in PCs.

Carbamoylphosphonate Matrix Metalloproteinase Inhibitors 3: In vivo Evaluation of Cyclopentylcarbamoylphosphonic Acid in Experimental Metastasis and Angiogenesis

The spread of malignant tumor cells from a primary neoplasm to distant organs where they multiply and form new foci is the major cause of death from cancer. Despite the different modalities of cancer treatment, no effective curative therapy of metastatic lesions is available. To possess metastatic potential, a cell has to be able to invade the surrounding tissue, spread via lymphatics and/or the bloodstream, extravasate, and multiply at secondary sites. There is increasing evidence for a positive correlation between matrix metalloproteinase-2 (MMP-2) activity and tumor cell invasion. Agents blocking MMP-2 have been shown to prevent tumor cell invasion in vitro and in vivo. Inhibition of MMPs has, therefore, become the focus of considerable interest in connection with a variety of potential therapeutic applications. We have discovered a nontoxic MMP-2–selective inhibitor effective at nanomolar range on recombinant MMP. This compound, cyclopentylcarbamoylphosphonic acid, significantly inhibited cellular invasion and capillary formation in vitro. Further, i.p. or oral administration of the compound significantly reduced lung metastasis formation and s.c. tumor growth in a murine melanoma model. The effect of this novel compound on lung colonization, capillary formation, and s.c. tumor growth indicates that the compound might also be effective in treatment of primary tumor growth in reduction, or at least in prevention, of further tumor growth, thereby reducing the tumor burden of the patient by a nontoxic approach.

Expression of the peroxisome proliferator–activated receptors–α, –β, and –γ in ovarian carcinoma effusions is associated with poor chemoresponse and shorter survival

Peroxisome proliferator-activated receptors regulate lipid metabolism, affecting inflammation and cancer. The present study analyzed the anatomical site-related expression and prognostic role of peroxisome proliferator-activated receptors in ovarian carcinoma. Fresh-frozen effusions (n = 79), primary carcinomas (n = 44), and solid metastases (n = 16) were studied for peroxisome proliferator-activated receptor-alpha, -beta, and -gamma messenger RNA expression using reverse transcriptase polymerase chain reaction. Peroxisome proliferator-activated receptor-gamma messenger RNA expression was further assessed in 60 tumors (30 effusions, 20 primary carcinomas, 10 metastases) using in situ hybridization. Peroxisome proliferator-activated receptor-gamma protein expression was immunohistochemically analyzed in 160 effusions. All peroxisome proliferator-activated receptors were expressed in most tumors at all anatomical sites using reverse transcriptase polymerase chain reaction, but peroxisome proliferator-activated receptor-alpha (P = .004) and peroxisome proliferator-activated receptor-beta (P = .002) messenger RNA levels were higher in effusions compared with primary carcinomas and solid metastases. In situ hybridization localized peroxisome proliferator-activated receptor-gamma messenger RNA to carcinoma cells in both effusions and solid lesions. Peroxisome proliferator-activated receptor-gamma protein was detected in carcinoma cells in 102 of 160 (64%) effusions. Higher effusion messenger RNA levels of all peroxisome proliferator-activated receptors were associated with less favorable response to chemotherapy at diagnosis (P = .009). In univariate survival analysis, higher messenger RNA expression of all peroxisome proliferator-activated receptors was associated with poor progression-free (P = .045) and overall (P = .014) survival. Higher peroxisome proliferator-activated receptor-gamma protein expression was similarly associated with poor overall survival for the entire cohort (P = .046) and for patients with disease recurrence effusions (P = .009). Peroxisome proliferator-activated receptors were not independent predictors of survival in Cox multivariate analysis. Peroxisome proliferator-activated receptor members are frequently expressed in ovarian carcinoma, with upregulated expression in effusions. Peroxisome proliferator-activated receptor expression in effusions is associated with poor response to chemotherapy at disease recurrence and poor survival, suggesting a role in tumor biology at this unique microenvironment.

Farage, a Novel Early B Cell Lymphoma Cell Line with Trisomy 11

Farage, a new cell line established from a lymph node biopsy of a patient with non-Hodgkins lymphoma (NHL), constitutes a clonal expansion of cells at a distinct stage of B-cell differentiation. The cells lack both T and myeloid surface markers, express B cell surface antigens including CD19, CD20, CD22, HLA-DR, were positive for C3 receptors and EBNA and expressed BCL-2. No immunoglobulin determinants could be demonstrated on the cell surface. Intracellular IgM and kappa chains were detected, in an unusual but distinct localization and appeared to be localized to the nucleus or to the perinuclear area without any spread to the cytoplasm, as seen in the early B cells. Southern blot DNA analysis showed rearrangement of one of the IgJH alleles. The Farage cells were negative for B cell activation antigens including CD25, CD11b, HC2 and Bly-7. The cells were negative for two anti CD-10 (CALLA) reagents but weakly positive with one. Interestingly they were strongly positive for both nuclear and cytoplasmic T...

Proximal Tubular Cannabinoid-1 Receptor Regulates Obesity-Induced CKD

Obesity-related structural and functional changes in the kidney develop early in the course of obesity and occur independently of hypertension, diabetes, and dyslipidemia. Activating the renal cannabinoid-1 receptor (CB1R) induces nephropathy, whereas CB1R blockade improves kidney function. Whether these effects are mediated via a specific cell type within the kidney remains unknown. Here, we show that specific deletion of CB1R in the renal proximal tubule cells did not protect the mice from obesity, but markedly attenuated the obesity-induced lipid accumulation in the kidney and renal dysfunction, injury, inflammation, and fibrosis. These effects associated with increased activation of liver kinase B1 and the energy sensor AMP-activated protein kinase, as well as enhanced fatty acid β-oxidation. Collectively, these findings indicate that renal proximal tubule cell CB1R contributes to the pathogenesis of obesity-induced renal lipotoxicity and nephropathy by regulating the liver kinase B1/AMP-activated protein kinase signaling pathway.

SUMOylation of Blimp-1 promotes its proteasomal degradation

SUMO1 physically interacts with Blimp1 by anti tag coimmunoprecipitation (View interaction).