Cell surface protein disulfide isomerase regulates natriuretic peptide generation of cyclic guanosine monophosphate.

RATIONALE: The family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated. OBJECTIVE: We utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate. METHODS AND RESULTS: Inhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry. CONCLUSION: These data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.

Striatal cholinergic cell ablation attenuates L-DOPA induced dyskinesia in Parkinsonian mice.

3,4-Dihydroxyphenyl-L-alanine (L-DOPA)-induced dyskinesia (LID) is a debilitating side effect of long-term dopamine replacement therapy in Parkinson's Disease. At present, there are few therapeutic options for treatment of LID and mechanisms contributing to the development and maintenance of these drug-induced motor complications are not well understood. We have previously shown that pharmacological reduction of cholinergic tone attenuates the expression of LID in parkinsonian mice with established dyskinesia after chronic L-DOPA treatment. The present study was undertaken to provide anatomically specific evidence for the role of striatal cholinergic interneurons by ablating them before initiation of L-DOPA treatment and determining whether it decreases LID. We used a novel approach to ablate striatal cholinergic interneurons (ChIs) via Cre-dependent viral expression of the diphtheria toxin A subunit (DT-A) in hemiparkinsonian transgenic mice expressing Cre recombinase under control of the choline acetyltransferase promoter. We show that Cre recombinase-mediated DT-A ablation selectively eliminated ChIs when injected into striatum. The depletion of ChIs markedly attenuated LID without compromising the therapeutic efficacy of L-DOPA. These results provide evidence that ChIs play a key and selective role in LID and that strategies to reduce striatal cholinergic tone may represent a promising approach to decreasing L-DOPA-induced motor complications in Parkinson's disease.

Glutamatergic signaling from the parabrachial nucleus plays a critical role in hypercapnic arousal.

The mechanisms of arousal from apneas during sleep in patients suffering from obstructive sleep apnea are not well understood. However, we know that respiratory chemosensory pathways converge on the parabrachial nucleus (PB), which sends glutamatergic projections to a variety of forebrain structures critical to arousal, including the basal forebrain, lateral hypothalamus, midline thalamus, and cerebral cortex. We tested the role of glutamatergic signaling in this pathway by developing an animal model for repetitive CO2 arousals (RCAs) and investigating the effect of deleting the gene for the vesicular glutamate transporter 2 (Vglut2) from neurons in the PB. We used mice with lox P sequences flanking exon2 of the Vglut2 gene, in which adeno-associated viral vectors containing genes encoding Cre recombinase and green fluorescent protein were microinjected into the PB to permanently and selectively disrupt Vglut2 expression while labeling the affected neurons. We recorded sleep in these mice and then investigated the arousals during RCA. Vglut2 deletions that included the external lateral and lateral crescent subdivisions of the lateral PB more than doubled the latency to arousal and resulted in failure to arouse by 30 s in >30% of trials. By contrast, deletions that involved the medial PB subdivision had minimal effects on arousal during hypercapnia but instead increased non-rapid eye movement (NREM) sleep by ∼43% during the dark period, and increased delta power in the EEG during NREM sleep by ∼50%. Our results suggest that glutamatergic neurons in the lateral PB are necessary for arousals from sleep in response to CO2, while medial PB glutamatergic neurons play an important role in promoting spontaneous waking.

Characterization of variant diphtheria toxin-interleukin-3 fusion protein, DTIL3K116W, for phase I clinical trials.

We have produced clinical grade of DTIL3K116W, a variant diphtheria toxin-interleukin-3 fusion protein, for treatment of acute myeloid leukemia. The product was filter sterilized, aseptically vialed, and stored at -80 degrees C. It was characterized by Coomassie-stained SDS-PAGE, endotoxin assay, cytotoxicity assay, sterility, mass spectroscopy, receptor binding affinity, ADP-ribosylation, inhibition of normal human CFU-GM, disulfide bond analysis, immunoblots, stability, size exclusion chromatography-HPLC, sequencing, and immunohistochemistry. Vialed product was sterile in 0.25 M NaCl/5 mM Tris, pH 7.9, and had a protein concentration of 1.08 mg/ml. Purity by SDS-PAGE was >99%. Aggregates by HPLC were <1%. Endotoxin levels were 0.296EU/mg. Peptide mapping and mass spectroscopy confirmed its composition and molecular weight. The vialed drug kept reactivity with anti-IL3 and DT antibodies. Potency study revealed a 48-h EC(50) of 0.5 pM on TF1/H-ras cell. Its binding properties were confirmed by competitive experiments showing IC(50) of 1.4 nM. ADP-ribosylation activity was equivalent to DTGM-CSF. Drug did not react with tested frozen human tissue sections by immunohistochemistry. There was no evidence of loss of solubility, proteolysis aggregation, or loss of potency over 6 months at -80 degrees C. Further, the drug was stable at 4 and 25 degrees C in the plastic syringe and administration tubing for 48 h.

Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2.

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.

Diphtheria toxin-induced autophagic cardiomyocyte death plays a pathogenic role in mouse model of heart failure.

It is still not clear whether loss of cardiomyocytes through programmed cell death causes heart failure. To clarify the role of cell death in heart failure, we generated transgenic mice (TG) that express human diphtheria toxin receptor in the hearts. A mosaic expression pattern of the transgene was observed, and the transgene-expressing cardiomyocytes (17.3% of the total cardiomyocytes) were diffusely scattered throughout the ventricles. Intramuscular injection of diphtheria toxin induced complete elimination of the transgene-expressing cardiomyocytes within 7 days, and approximately 80% of TG showed pathophysiological features characteristic of heart failure and were dead within 14 days. Degenerated cardiomyocytes of the TG heart showed characteristic features indicative of autophagic cell death such as up-regulated lysosomal markers and abundant autophagosomes containing cytosolic organelles like cardiomyocytes of human dilated cardiomyopathy. The heart failure-inducible TG are a useful model for dilated cardiomyopathy, and provided evidence indicating that myocardial cell loss through autophagic cell death plays of a causal role in the pathogenesis heart failure.

Diphtheria toxin fused to variant interleukin-3 provides enhanced binding to the interleukin-3 receptor and more potent leukemia cell cytotoxicity.

Chemoresistance is a common cause of treatment failure in patients with acute myeloid leukemia (AML). We generated a diphtheria toxin (DT) fusion protein composed of the catalytic and translocation domains of DT (DT388) fused to interleukin-3 (IL-3). IL-3 receptors (IL-3R) are overexpressed on blasts from many AML patients. DT388IL-3 showed cytotoxicity to leukemic blasts in vitro and in vivo and minimal damage to normal tissues in nonhuman primate models. However, only a fraction of patient leukemic samples were sensitive to the agent. To enhance the potency and specificity of the DT388IL-3 molecule, we constructed variants with altered residues in the IL-3 moiety. Two of these variants, DT388IL-3[K116W] and DT388IL-3[Delta125-133], were produced and partially purified from Escherichia coli with excellent yields. They showed enhanced binding to the human IL-3R and greater cytotoxicity to human leukemia cell lines relative to wild-type DT388IL-3. Interestingly, the results support a previously hypothesized model for interaction of the C-terminal residues of IL-3 with a hydrophobic patch on the alpha-subunit of IL-3R. Rational modification of the targeting domain based on structural analysis can produce a fusion toxin with increased ability to kill tumor cells. One or both of these variant fusion proteins merit further development for therapy of chemotherapy refractory AML.

Malignant mesothelioma growth inhibition by agents that target the VEGF and VEGF-C autocrine loops.

Malignant mesothelioma (MM) is a locally aggressive tumor that originates from the mesothelial cells of the pleural and sometimes peritoneal surface. Conventional treatments for MM, consisting of chemotherapy or surgery give little survival benefit to patients, who generally die within 1 year of diagnosis. Hence, there is an urgent need for the development of alternative therapies. Vascular endothelial growth factor (VEGF) is an autocrine growth factor for MM. The closely related molecule, VEGF-C, is also implicated in malignant mesothelioma growth. VEGF-C and its cognate receptor VEGFR-3 are co-expressed in mesothelioma cell lines. A functional VEGF-C autocrine growth loop was demonstrated in mesothelioma cells by targeting VEGF-C expression and binding to VEGFR-3. The ability of novel agents that reduce the levels of VEGF and VEGF-C to inhibit mesothelioma cell growth in vitro was assessed. Antisense oligonucleotide (ODN) complementary to VEGF that inhibited VEGF and VEGF-C expression simultaneously specifically inhibited mesothelioma cell growth. Similarly, antibodies to VEGF receptor (VEGFR-2) and VEGF-C receptor (VEGFR-3) were synergistic in inhibiting mesothelioma cell growth. In addition, a diphtheria toxin-VEGF fusion protein (DT-VEGF), which is toxic to cells that express VEGF receptors was very effective in inhibiting mesothelioma cell growth in vitro. These results indicate that targeting VEGF and VEGF-C simultaneously may be an effective therapeutic approach for malignant mesothelioma.

Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.

The adult mouse intestine contains an intricate vascular network. The factors that control development of this network are poorly understood. Quantitative three-dimensional imaging studies revealed that a plexus of branched interconnected vessels developed in small intestinal villi during the period of postnatal development that coincides with assembly of a complex society of indigenous gut microorganisms (microbiota). To investigate the impact of this environmental transition on vascular development, we compared the capillary networks of germ-free mice with those of ex-germ-free animals colonized during or after completion of postnatal gut development. Adult germ-free mice had arrested capillary network formation. The developmental program can be restarted and completed within 10 days after colonization with a complete microbiota harvested from conventionally raised mice, or with Bacteroides thetaiotaomicron, a prominent inhabitant of the normal mouse/human gut. Paneth cells in the intestinal epithelium secrete antibacterial peptides that affect luminal microbial ecology. Comparisons of germ-free and B. thetaiotaomicron-colonized transgenic mice lacking Paneth cells established that microbial regulation of angiogenesis depends on this lineage. These findings reveal a previously unappreciated mechanism of postnatal animal development, where microbes colonizing a mucosal surface are assigned responsibility for regulating elaboration of the underlying microvasculature by signaling through a bacteria-sensing epithelial cell.

In vitro-translated diphtheria toxin A chain inhibits translation in wheat germ extracts: analysis of biologically active, caspase-3-resistant diphtheria toxin mutants.

The diphtheria toxin A chain (DTA) is a potent cytocidal agent that inactivates elongation factor 2. This activity of DTA inhibits protein synthesis and rapidly leads to cell death through apoptosis. In this paper, we have developed a simple in vitro assay for DTA activity in which in vitro-translated DTA is used to inhibit the translation of proteins in wheat germ extracts. Inhibition of translation by DTA is dependent on cofactor NAD+, and the analysis of an attenuated DTA mutant indicates that this in vitro assay accurately reflects the in vivo activity of DTA. We have also identified aspartic acid at residue 8 (Asp-8) of DTA as a site of cleavage by the cell-death protease caspase-3. Cleavage of DTA by caspase-3 inactivates its ability to inhibit translation in wheat germ extracts. Conservative mutations at Asp-8 render DTA resistant to cleavage by caspase-3, but only slightly affect the ability of DTA to inhibit translation in vitro. Moreover, caspase-3-resistant DTA mutants are toxic in cells in tissue culture. The in vitro assay that we describe here will be useful for the rapid analysis of DTA activity and the development of DTA mutants with altered biological properties that may be of therapeutic value. Lastly, these studies serve as a prototype for the creation of caspase-resistant effector molecules.

Anti-idiotypic antibodies that protect cells against the action of diphtheria toxin.

An anti-idiotypic serum prepared against the combining site (idiotype) of specific anti-diphtheria toxoid antibodies was characterized with respect to its interaction with highly diphtheria toxin-sensitive Vero cells. Although the anti-idiotypic serum protected Vero cells against the cytotoxic action of diphtheria toxin, it did not prevent the binding of 125I-labeled diphtheria toxin to the cells but did inhibit the internalization and degradation of 125I-labeled toxin. This anti-idiotypic serum immunoprecipitated a cell-surface protein from radiolabeled Vero cells with an apparent Mr of approximately 15,000. These results are consistent with the hypothesis that the anti-idiotypic serum contains antibodies that carry an internal image of an internalization site on the toxin and that a cell-surface protein involved in toxin internalization possesses a complementary site recognized by both the toxin and the anti-idiotypic antibodies.

Cytotoxic and mutagenic properties of shale oil byproducts II. Comparison of mutagenic effects at five genetic markers induced by retort process water plus near ultraviolet light in Chinese hamster ovary cells.

A chinese hamster ovary (CHO) cell line heterozygous at the adenine phosphoribosyl transferase (APRT) locus was used for selection of induced mutants resistant to 8-azaadenine (8AA), 6-thioguanine (6TG), ouabain (OUA), emetine (EMT) and diphtheria toxin (DIP). The expression times necessary for optimizing the number of mutants recovered at the different loci have been determined using the know direct acting mutagen, far ultraviolet light (FUV), and a complex aqueous organic mixture (shale oil process water) activated with near ultraviolet light (NUV). Our results indicate that optimal expression times following treatment with either mutagen was between 2 and 8 days (depending on the genetic marker examined). For CHO cells treated with shale oil process water and subsequently exposed to NUV a linear dose response for mutant induction was observed for all five genetic loci. At 10% surviving fraction of cells, between 35- and 130-fold increases above background mutation frequencies were observed for the various markers examined. Among the five genetic loci tested, OUAR was the most sensitive marker tested.

Inhibitors of polypeptide elongation on yeast polysomes.

Yeast polysomes are very active for amino acid incorporation when supplemented with elongation factors and the different components required for elongation of the polypeptide chain. This polysomal system is suitable for the study of the individual streps of the elongation cycle and to test the effect of different inhibitors. Anisomycin, trichodermin, trichodermol, trichothecin, fusarenon X, sparsomycin and blasticidin S inhibit peptide bond formation on these polysomes, whereas diphtheria toxin, pederine, cycloheximide and cryptopleurine block translocation.