Ornithine-δ-Aminotransferase Inhibits Neurogenesis During Xenopus Embryonic Development.

PURPOSE: In humans, deficiency of ornithine-δ-aminotransferase (OAT) results in progressive degeneration of the neural retina (gyrate atrophy) with blindness in the fourth decade. In this study, we used the Xenopus embryonic developmental model to study functions of the OAT gene on embryonic development. METHODS: We cloned and sequenced full-length OAT cDNA from Xenopus oocytes (X-OAT) and determined X-OAT expression in various developmental stages of Xenopus embryos and in a variety of adult tissues. The phenotype, gene expression of neural developmental markers, and enzymatic activity were detected by gain-of-function and loss-of-function manipulations. RESULTS: We showed that X-OAT is essential for Xenopus embryonic development, and overexpression of X-OAT produces a ventralized phenotype characterized by a small head, lack of axial structure, and defective expression of neural developmental markers. Using X-OAT mutants based on mutations identified in humans, we found that substitution of both Arg 180 and Leu 402 abrogated both X-OAT enzymatic activity and ability to modulate the developmental phenotype. Neurogenesis is inhibited by X-OAT during Xenopus embryonic development. CONCLUSIONS: Neurogenesis is inhibited by X-OAT during Xenopus embryonic development, but it is essential for Xenopus embryonic development. The Arg 180 and Leu 402 are crucial for these effects of the OAT molecule in development.

Developmental cardiac hypertrophy in a mouse model of prolidase deficiency.

BACKGROUND: Hypertrophic cardiomyopathy, characterized by thickened ventricular walls and reduced ventricular chamber volume, is a common cause of sudden cardiac death in young people. Most inherited forms result from mutations in genes encoding sarcomeric proteins. METHODS: Histologic analysis identified embryonic cardiac hypertrophy in dark-like mutant mice. BrdU analysis was performed to measure proliferation and cardiomyocytes were isolated to measure cell size. The dark-like mutation was identified by positional cloning. RESULTS: The dark-like mutation causes cardiomyocyte hypertrophy due to loss-of-function of peptidase d (Pepd), which encodes prolidase, a cytosolic enzyme that recycles proline for collagen re-synthesis. Prolidase deficiency is a rare autosomal recessive disease in humans with a broad phenotypic spectrum not reported to include heart defects, but a conserved role for prolidase in heart development was confirmed by morpholino knockdown in zebrafish. We tested the hypothesis that loss of prolidase function disrupts collagen-mediated integrin signaling and determined that the levels of several key integrin transducers were reduced in the hearts of dark-like mutant embryos. CONCLUSIONS: This work identifies dark-like mice as a model of prolidase deficiency that will be valuable for studying the role of proline metabolism in normal physiology and disease processes, and suggests that integrin signaling may regulate the onset of hypertrophic cardiac growth.

The Nitric Oxide Prodrug V-PROLI/NO Inhibits Cellular Uptake of Proline.

V-PYRRO/NO is a well studied nitric oxide (NO) prodrug which has been shown to protect human liver cells from arsenic, acetaminophen, and other toxic assaults in vivo. Its proline-based analogue, V-PROLI/NO, was designed to be a more biocompatible form that decomposes to the naturally occurring metabolites of proline, NO, and glycolaldehyde. Like V-PYRRO/NO, this cytochrome P450-activated prodrug was previously assumed to passively diffuse through the cellular membrane. Using (14)C-labeled proline in a competition assay, we show that V-PROLI/NO is transported through proline transporters into multiple cell lines. A fluorescent NO-sensitive dye (DAF-FM diacetate) and nitrite excretion indicated elevated intracellular NO release after metabolism over V-PYRRO/NO. These results also allowed us to predict and design a more permeable analogue, V-SARCO/NO. We report a proline transporter-based strategy for the selective transport of NO prodrugs that may have enhanced efficacy and aid in development of further NO prodrugs with increased permeability.

Proline oxidase functions as a mitochondrial tumor suppressor in human cancers.

Tumor metabolism and bioenergetics have become important topics for cancer research and are promising targets for anticancer therapy. Although glucose serves as the main source of energy, proline, an alternative substrate, is important, especially during nutrient stress. Proline oxidase (POX), catalyzing the first step in proline catabolism, is induced by p53 and can regulate cell survival as well as mediate programmed cell death. In a mouse xenograft tumor model, we found that POX greatly reduced tumor formation by causing G2 cell cycle arrest. Furthermore, immunohistochemical staining showed decreased POX expression in tumor tissues. Importantly, HIF-1alpha signaling was impaired with POX expression due to the increased production of alpha-ketoglutarate, a critical substrate for prolyl hydroxylation and degradation of HIF-1alpha. Combined with previous in vitro findings and reported clinical genetic associations, these new findings lead us to propose POX as a mitochondrial tumor suppressor and a potential target for cancer therapy.

MnSOD inhibits proline oxidase-induced apoptosis in colorectal cancer cells.

Proline oxidase (POX), localized on inner mitochondrial membranes, is encoded by a p53-induced gene and metabolically participates in p53-induced apoptosis. Previously, we showed that POX catalyzed the generation of reactive oxygen species (ROS). We and others have demonstrated that overexpression of POX, independent of p53, causes apoptotic cell death in a variety of cancer cells. But a necessary role for ROS remains uncertain. Therefore, we asked whether superoxide dismutases (SOD) and catalase (CAT), important antioxidant enzymes, might interfere with the POX-dependent induction of apoptosis. In this study, we used DLD-1 colorectal cancer cells stably transfected with the POX gene under the control of a tetracycline-inducible promoter. When doxycycline was removed from the culture medium and the expression of POX was induced, apoptotic cell death was initiated. To examine the importance of the ROS-dependent component of the pathway, we infected DLD-1 POX cells with recombinant adenoviruses containing MnSOD, CuZnSOD, CAT or varying combinations of these adenoviruses followed by induced expression of POX. The expression of MnSOD inhibited POX-induced apoptosis, but others did not. Mechanistically, mitochondria-localized MnSOD dramatically reduced the release of cytochrome c to cytosol by POX. Compared with control cells, MnSOD-expressing DLD-1 POX cells generated a higher concentration of H2O2 owing to dismutation of superoxide radicals, which was elevated by POX. Thus, these data further suggest that the generation of superoxide radicals plays a crucial role in POX-induced apoptosis and the process is partially blocked by MnSOD.

Polyoma enhancer activator 3, an ets transcription factor, mediates the induction of cyclooxygenase-2 by nitric oxide in colorectal cancer cells.

Abundant evidence supports the role of cyclooxygenase-2 (COX-2) in colorectal cancer. Nitric oxide (NO), a pro-inflammatory signaling factor, may regulate COX-2 expression and activity thereby linking hyper-inflammatory states to cancer susceptibility. Previously we showed that NO induced COX-2 expression. Although NO also activated the beta-catenin.T-cell factor/lymphocyte enhancing factor transcriptional pathway, a direct causal link between this pathway and COX-2 expression was not demonstrated. In this current study, we focused on NO-induced transcriptional activity and elucidated its role in COX-2 expression. NO donors stimulated the expression of peroxisome proliferator-activated receptor-delta and c-myc, both downstream genes of beta-catenin. They also induced the expression of polyoma enhancer activator 3 (PEA3) and increased its DNA-binding activity. To establish a role for PEA3 to beta-catenin-induced COX-2, we transfected RKO cells with beta-catenin and found that beta-catenin increased PEA3 expression. Also, there was higher PEA3 in immortal mouse colon epithelium cells (Apc(Min/)(+)) compared with young adult mouse colon cells (Apc(+/+)). Luciferase reporter assays revealed that, although several transcription factors/coactivator, acting alone or in synergistic combination, induced COX-2 promoter activity, PEA3 was one of the most potent. Interestingly, NO from NO donors or generated endogenously from transfected inducible nitric-oxide synthase, increased PEA3/p300-induced COX-2 promoter activity. We also found that an ETS site (-75/-72) and the NF-IL6 site were responsible for COX-2 activity induced by PEA3, PEA3/p300, and NO. Taken together, our results demonstrated that NO through beta-catenin signaling stimulated PEA3 to increase COX-2 activity. In addition, NO augmented the synergistic interaction between PEA3 and CBP/p300.

Matrix metalloproteinase(s) mediate(s) NO-induced dissociation of beta-catenin from membrane bound E-cadherin and formation of nuclear beta-catenin/LEF-1 complex.

Modulation of the adenomatous polyposis coli (APC)-beta-catenin pathway by inflammatory mediators and extracellular matrix may be important in colon carcinogenesis. We have recently shown that nitric oxide (NO) induces the accumulation of cytosolic beta-catenin and subsequent formation of the nuclear beta-catenin/lymphocyte enhancing factor (LEF)-1 complex in conditionally immortalized young mouse colonic epithelial (YAMC) cells. In the present study, we explored the mechanism(s) through which NO exerts its effect on cytosolic beta-catenin accumulation and nuclear beta-catenin/LEF-1 complex formation. We found that NO-induced degradation of the membrane bound E-cadherin at tight junctions. Using an anti-E-cadherin antibody specific for its extracellular domain, we detected a 50kDa degradation fragment of E-cadherin (120 kDa) from the culture medium conditioned by YAMC cells exposed to the NO-releasing drug, NOR-1, for 4 and 24 h. As beta-catenin is normally bound to transmembrane E-cadherin and thus anchored to the cytoskeleton structure, the degradation of E-cadherin induced by NO may cause dissociation of beta-catenin from membrane bound E-cadherin. This was demonstrated by the detection of beta-catenin accumulation in the soluble cytosolic fractions in YAMC after exposure to NO-releasing drugs. Furthermore, the degradation of E-cadherin and the release of beta-catenin to cytosol were accompanied by the formation of nuclear beta-catenin/LEF-1 complex, demonstrating the dissociation of beta-catenin from E-cadherin may be responsible for the activation of beta-catenin/LEF-1 transcription complex. Co-treatment with NO donors and broad-spectrum matrix metalloproteinase (MMP) inhibitors TIMP-1 (100 ng/ml), GM6001 (10 micro M) and GM1489 (10 micro M) abolished the degradation of E-cadherin induced by NO as demonstrated by western blot analysis. These MMP inhibitors also blocked the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex. The sum effect of MMP inhibitors demonstrated that NO-induced activation of MMP may cause the degradation of E-cadherin and the subsequent dissociation of beta-catenin, thereby contributing to the cytosolic accumulation of beta-catenin and nuclear formation of beta-catenin/LEF-1 complex.