Injury-Mediated Vascular Regeneration Requires Endothelial ER71/ETV2.

OBJECTIVE: Comprehensive understanding of the mechanisms regulating angiogenesis might provide new strategies for angiogenic therapies for treating diverse physiological and pathological ischemic conditions. The E-twenty six (ETS) factor Ets variant 2 (ETV2; aka Ets-related protein 71) is essential for the formation of hematopoietic and vascular systems. Despite its indispensable function in vessel development, ETV2 role in adult angiogenesis has not yet been addressed. We have therefore investigated the role of ETV2 in vascular regeneration. APPROACH AND RESULTS: We used endothelial Etv2 conditional knockout mice and ischemic injury models to assess the role of ETV2 in vascular regeneration. Although Etv2 expression was not detectable under steady-state conditions, its expression was readily observed in endothelial cells after injury. Mice lacking endothelial Etv2 displayed impaired neovascularization in response to eye injury, wounding, or hindlimb ischemic injury. Lentiviral Etv2 expression in ischemic hindlimbs led to improved recovery of blood perfusion with enhanced vessel formation. After injury, fetal liver kinase 1 (Flk1), aka VEGFR2, expression and neovascularization were significantly upregulated by Etv2, whereas Flk1 expression and vascular endothelial growth factor response were significantly blunted in Etv2-deficient endothelial cells. Conversely, enforced Etv2 expression enhanced vascular endothelial growth factor-mediated endothelial sprouting from embryoid bodies. Lentiviral Flk1 expression rescued angiogenesis defects in endothelial Etv2 conditional knockout mice after hindlimb ischemic injury. Furthermore, Etv2(+/-); Flk1(+/-) double heterozygous mice displayed a more severe hindlimb ischemic injury response compared with Etv2(+/-) or Flk1(+/-) heterozygous mice, revealing an epistatic interaction between ETV2 and FLK1 in vascular regeneration. CONCLUSIONS: Our study demonstrates a novel obligatory role for the ETV2 in postnatal vascular repair and regeneration.

Synthetic triterpenoids attenuate cytotoxic retinal injury: cross-talk between Nrf2 and PI3K/AKT signaling through inhibition of the lipid phosphatase PTEN.

PURPOSE: Evidence implicating oxidative stress in the pathogenesis of age-related macular degeneration suggests that antioxidant therapy could play a role in preventing its progression. The aim of this study was to determine whether derivatives of the triterpenoid (TP) 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO; CDDO-imidazolide [-Im], CDDO-ethylamide [-EA], and CDDO-trifluoroethylamide [-TFEA]) confer cytoprotection from oxidative- and photooxidative-induced cellular damage and to explore the molecular mechanisms of this cytoprotection. METHODS: Retinal pigment epithelial and retinal photoreceptor cell lines were treated with TP derivatives. Induction of Nrf2 signaling was measured by reporter assay. Cytoprotection was quantified by MTT assay. To determine whether TPs confer in vivo cytoprotection, BALB/c mice were pretreated with CDDO-TFEA, and retinal degeneration was induced by light exposure. To explore the association of TPs with PTEN, a biotinylated derivative of CDDO (CDDO-Bt) was used. RESULTS: Treatment with CDDO-Im-, -TFEA-, or -EA-induced Nrf2 signaling and TP pretreatment protected retinal cell lines from oxidant-induced cell death. The antioxidant and cytoprotective potential of these compounds was then examined in vivo. Treatment of BALB/c mice with CDDO-TFEA induced the Nrf2-regulated transcripts glcl and trx1 in retinal tissue and was protective from photooxidative retinal damage. Treatment with CDDO-Im leads to phosphorylation of AKT. CDDO-Bt directly binds cysteine 124 within PTEN's active site and inhibits PTEN's lipid phosphatase activity in vitro. Thus the stimulation of AKT activity is mediated by TP inhibition of PTEN activity. CONCLUSIONS: These studies highlight the potential of TPs in retinal cytoprotection and implicate PTEN inhibition as a target in cytoprotection.

UBE1L represses PML/RAR{alpha} by targeting the PML domain for ISG15ylation.

Acute promyelocytic leukemia (APL) is characterized by expression of promyelocytic leukemia (PML)/retinoic acid (RA) receptor alpha (RARalpha) protein and all-trans-RA-mediated clinical remissions. RA treatment can confer PML/RARalpha degradation, overcoming dominant-negative effects of this oncogenic protein. The present study uncovered independent retinoid degradation mechanisms, targeting different domains of PML/RARalpha. RA treatment is known to repress PML/RARalpha and augment ubiquitin-activating enzyme-E1-like (UBE1L) protein expression in NB4-S1 APL cells. We previously reported RA-induced UBE1L and the IFN-stimulated gene, 15-kDa protein ISG15ylation in APL cells. Whether the ubiquitin-like protein ISG15 directly conjugates with PML/RARalpha was not explored previously and is examined in this study. Transient transfection experiments with different PML/RARalpha domains revealed that RA treatment preferentially down-regulated the RARalpha domain, whereas UBE1L targeted the PML domain for repression. As expected, ubiquitin-specific protease 18 (UBP43/USP18), the ISG15 deconjugase, opposed UBE1L but not RA-dependent PML/RARalpha degradation. In contrast, the proteasomal inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal, inhibited both UBE1L- and RA-mediated PML/RARalpha degradation. Notably, UBE1L induced ISG15ylation of the PML domain of PML/RARalpha, causing its repression. These findings confirmed that RA triggers PML/RARalpha degradation through different domains and distinct mechanisms. Taken together, these findings advance prior work by establishing two pathways converge on the same oncogenic protein to cause its degradation and thereby promote antineoplastic effects. The molecular pharmacologic implications of these findings are discussed.

Viral defense, carcinogenesis and ISG15: novel roles for an old ISG.

Recent studies have established that type I interferon modulates expression of large number of cellular genes. While the proteins encoded by some of these genes have a direct antiviral activity, the functions of the majority of the others have not yet been determined. One of the first identified IFN stimulated gene, encodes ubiquitin like protein ISG15 that is also expressed in response to different stress stimuli. Although it was shown that ISG15 functions as protein modifier, it has been only recently that the targets of ISG15 conjugation were identified. Recent studies have also revealed mechanism of ISG15 conjugation and its interaction with the ubiquitin conjugation pathway. This review is focused on the possible role of ISG15 in the antiviral response, regulation of cell growth and carcinogenesis.

Gene profiling uncovers retinoid target genes.

Decades of hypothesis-driven research have identified candidate targets for cancer therapy and chemoprevention. Recently, genomic, proteomic, and tissue-based microarray approaches have made possible another scientific approach. This is one that interrogates comprehensively the complex profile of mRNA or protein expression present in normal, preneoplastic, or malignant cells and tissues. This in turn can uncover critical targets for cancer pharmacology and also lead to a better understanding of the known or novel networks of gene expression that play a rate-limiting role in carcinogenesis. This chapter addresses the use of mRNA expression profiling to uncover candidate target genes active in cancer pharmacology by citing as an example how this has already proven useful to reveal that retinoids (natural and synthetic derivatives of vitamin A) signal through pathways, which promote tumor cell differentiation, induce growth suppression, trigger apoptosis or affect other growth regulatory pathways. Pathways involved in the regulation of protein stability will be highlighted as these play a critical role in mediating pharmacological effects of the retinoids in cancer therapy or chemoprevention.

Innate antiviral response targets HIV-1 release by the induction of ubiquitin-like protein ISG15.

The goal of this study was to elucidate the molecular mechanism by which type I IFN inhibits assembly and release of HIV-1 virions. Our study revealed that the IFN-induced ubiquitin-like protein ISG15 mimics the IFN effect and inhibits release of HIV-1 virions without having any effect on the synthesis of HIV-1 proteins in the cells. ISG15 expression specifically inhibited ubiquitination of Gag and Tsg101 and disrupted the interaction of the Gag L domain with Tsg101, but conjugation of ISG15 to Gag or Tsg101 was not detected. The inhibition of Gag-Tsg101 interaction was also detected in HIV-1 infected, IFN-treated cells. Elimination of ISG15 expression by small interfering RNA reversed the IFN-mediated inhibition of HIV-1 replication and release of virions. These results indicated a critical role for ISG15 in the IFN-mediated inhibition of late stages of HIV-1 assembly and release and pointed to a mechanism by which the innate antiviral response targets the cellular endosomal trafficking pathway used by HIV-1 to exit the cell. Identification of ISG15 as the critical component in IFN-mediated inhibition of HIV-1 release advances the understanding of the IFN-mediated inhibition of HIV-1 replication and uncovers a target for the anti HIV-1 therapy.

Microarray analyses uncover UBE1L as a candidate target gene for lung cancer chemoprevention.

Retinoids, natural and synthetic derivatives of vitamin A, are active in cancer therapy and chemoprevention. We reported previously that all-trans-retinoic acid (RA) treatment prevented carcinogen-induced transformation of immortalized human bronchial epithelial (HBE) cells. To identify cancer chemopreventive mechanisms, immortalized (BEAS-2B), carcinogen-transformed (BEAS-2B(NNK)), and RA-chemoprevented (BEAS-2B(NNK/RA)) HBE cells were used to conduct microarray analyses independently. Species increased in chemoprevented as compared with immortalized HBE cells (group I) and those augmented in chemoprevented as compared with transformed HBE cells (group II) included known RA-target genes as well as previously unrecognized RA-target genes in HBE cells. Unexpectedly, both groups were also enriched for interferon-stimulated genes. One interferon-stimulated gene of particular interest was UBE1L, the ubiquitin-activating enzyme E1-like protein. UBE1L expression was also induced after prolonged RA-treatment of immortalized HBE cells. UBE1L mRNA was shown previously as repressed in certain lung cancer cell lines, directly implicating UBE1L in lung carcinogenesis. Notably, UBE1L immunoblot expression was reduced in a subset of malignant as compared with adjacent normal lung tissues that were examined. Immunohistochemical analyses were performed using a new assay developed to detect this species using rabbit polyclonal anti-UBE1L antibodies independently raised against the amino- or carboxyl-termini of UBE1L. Studies done on paraffin-embedded and fixed tissues revealed abundant UBE1L, but low levels of cyclin D1 expression in the normal human bronchial epithelium, indicating an inverse relationship existed between these species. To study this further, cotransfection into HBE cells of wild-type or mutant UBE1L species was accomplished. In a dose-dependent manner, wild-type but not mutant UBE1L species repressed cyclin D1 expression. This implicated UBE1L in a retinoid chemoprevention mechanism involving cyclin D1 repression described previously. Taken together, these findings directly implicate UBE1L as a candidate-pharmacologic target for lung cancer chemoprevention. These findings also provide a mechanistic basis for the tumor suppressive effects of UBE1L through cyclin D1 repression.

Specific chemopreventive agents trigger proteasomal degradation of G1 cyclins: implications for combination therapy.

PURPOSE: There is a need to identify cancer chemoprevention mechanisms. We reported previously that all-trans-retinoic acid (RA) prevented carcinogenic transformation of BEAS-2B immortalized human bronchial epithelial cells by causing G(1) arrest, permitting repair of genomic DNA damage. G(1) arrest was triggered by cyclin D1 proteolysis via ubiquitin-dependent degradation. This study investigated which chemopreventive agents activated this degradation program and whether cyclin E was also degraded. EXPERIMENTAL DESIGN: This study examined whether: (a) cyclin E protein was affected by RA treatment; (b) cyclin degradation occurred in derived BEAS-2B-R1 cells that were partially resistant to RA; and (c) other candidate chemopreventive agents caused cyclin degradation. RESULTS: RA treatment triggered degradation of cyclin E protein, and ALLN, a proteasomal inhibitor, inhibited this degradation. Induction of the retinoic acid receptor beta, growth suppression, and cyclin degradation were each inhibited in BEAS-2B-R1 cells. Transfection experiments in BEAS-2B cells indicated that RA treatment repressed expression of wild-type cyclin D1 and cyclin E, but ALLN inhibited this degradation. Mutation of threonine 286 stabilized transfected cyclin D1, and mutations of threonines 62 and 380 stabilized transfected cyclin E, despite RA treatment. Specific chemopreventive agents triggered cyclin degradation. Nonclassical retinoids (fenretinide and retinoid X receptor agonists) and a synthetic triterpenoid (2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid) each suppressed BEAS-2B growth and activated this degradation program. However, a vitamin D3 analog (RO-24-5531), a cyclooxygenase inhibitor (indomethacin), and a peroxisome proliferator-activated receptor gamma agonist (rosiglitazone) each suppressed BEAS-2B growth, but did not cause cyclin degradation. BEAS-2B-R1 cells remained responsive to nonclassical retinoids and to 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid. CONCLUSIONS: Specific chemopreventive agents activate cyclin proteolysis. Yet, broad resistance did not occur after acquired resistance to a single agent. This provides a therapeutic rationale for combination chemoprevention with agents activating non-cross-resistant pathways.

Expression profiling of mammalian microRNAs uncovers a subset of brain-expressed microRNAs with possible roles in murine and human neuronal differentiation.

BACKGROUND: The microRNAs (miRNAs) are an extensive class of small noncoding RNAs (18 to 25 nucleotides) with probable roles in the regulation of gene expression. In Caenorhabditis elegans, lin-4 and let-7 miRNAs control the timing of fate specification of neuronal and hypodermal cells during larval development. lin-4, let-7 and other miRNA genes are conserved in mammals, and their potential functions in mammalian development are under active study. RESULTS: In order to identify mammalian miRNAs that might function in development, we characterized the expression of 119 previously reported miRNAs in adult organs from mouse and human using northern blot analysis. Of these, 30 miRNAs were specifically expressed or greatly enriched in a particular organ (brain, lung, liver or skeletal muscle). This suggests organ- or tissue-specific functions for miRNAs. To test if any of the 66 brain-expressed miRNAs were present in neurons, embryonal carcinoma cells were treated with all-trans-retinoic acid to promote neuronal differentiation. A total of 19 brain-expressed miRNAs (including lin-4 and let-7 orthologs) were coordinately upregulated in both human and mouse embryonal carcinoma cells during neuronal differentiation. The mammalian ortholog of C. elegans lin-28, which is downregulated by lin-4 in worms via 3' untranslated region binding, was also repressed during neuronal differentiation of mammalian embryonal carcinoma cells. Mammalian lin-28 messenger RNAs contain conserved predicted binding sites in their 3' untranslated regions for neuron-expressed miR-125b (a lin-4 ortholog), let-7a, and miR-218. CONCLUSIONS: The identification of a subset of brain-expressed miRNAs whose expression behavior is conserved in both mouse and human differentiating neurons implicates these miRNAs in mammalian neuronal development or function.

Involvement of UBE1L in ISG15 conjugation during retinoid-induced differentiation of acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) cases expressing the t(15,17) product, promyelocytic leukemia (PML)/retinoic acid receptor alpha (RARalpha), have clinical remissions through leukemic cell differentiation after all-trans-retinoic acid (RA) treatment. This differentiation therapy propelled interest in uncovering molecular mechanisms for RA-dependent APL differentiation. We previously identified the ubiquitin-activating enzyme-E1-like protein (UBE1L) as an RA-regulated target gene in APL that triggers PML/RARalpha degradation and apoptosis. This study reports that conjugation of the ubiquitin-like species, interferon-stimulated gene, 15-kDa protein (ISG15), also occurs during RA-induced APL differentiation. Knock-down of UBE1L expression inhibited this conjugation. RA treatment of APL and other RA-responsive leukemic cells induced expression of UBE1L and ISG15 as well as intracellular ISG15 conjugates. Notably, ISG15 conjugation did not occur in RA-resistant NB4-R1 APL cells. Induction of UBE1L and ISG15 along with ISG15 conjugation in RA-sensitive NB4-S1 APL cells were detected following treatment with specific retinoids and type I interferon (IFN). UBE1L and ISG15 mRNAs were co-expressed in normal human tissues that were examined. In contrast, UBE1L mRNA expression was markedly repressed in several cancer cell lines. A physical association was found between UBE1L and ISG15 in vivo. This required the conserved diglycine motif in the carboxyl terminus of ISG15. Targeting UBE1L expression with small inhibitory RNA or small hairpin RNA inhibited IFN and RA-induced ISG15 conjugation. Formation of ISG15 conjugates through induction of an activating enzyme represents a novel pharmacologic mechanism for regulation of this ubiquitin-related species. Taken together, the observed rela tionship between expression of UBE1L and ISG15, their physical association and coordinate regulation, and induced ISG15 conjugation during leukemic cell differentiation implicate an important role for these proteins in retinoid response.

UBE1L is a retinoid target that triggers PML/RARalpha degradation and apoptosis in acute promyelocytic leukemia.

All-trans-retinoic acid (RA) treatment induces remissions in acute promyelocytic leukemia (APL) cases expressing the t(15;17) product, promyelocytic leukemia (PML)/RA receptor alpha (RARalpha). Microarray analyses previously revealed induction of UBE1L (ubiquitin-activating enzyme E1-like) after RA treatment of NB4 APL cells. We report here that this occurs within 3 h in RA-sensitive but not RA-resistant APL cells, implicating UBE1L as a direct retinoid target. A 1.3-kb fragment of the UBE1L promoter was capable of mediating transcriptional response to RA in a retinoid receptor-selective manner. PML/RARalpha, a repressor of RA target genes, abolished this UBE1L promoter activity. A hallmark of retinoid response in APL is the proteasome-dependent PML/RARalpha degradation. UBE1L transfection triggered PML/RARalpha degradation, but transfection of a truncated UBE1L or E1 did not cause this degradation. A tight link was shown between UBE1L induction and PML/RARalpha degradation. Notably, retroviral expression of UBE1L rapidly induced apoptosis in NB4 APL cells, but not in cells lacking PML/RARalpha expression. UBE1L has been implicated directly in retinoid effects in APL and may be targeted for repression by PML/RARalpha. UBE1L is proposed as a direct pharmacological target that overcomes oncogenic effects of PML/RARalpha by triggering its degradation and signaling apoptosis in APL cells.