Cytoplasmic pro-apoptotic function of the tumor suppressor p73 is mediated through a modified mode of recognition of the anti-apoptotic regulator Bcl-XL.

In response to genotoxic stress, the tumor suppressor protein p73 induces apoptosis and cell cycle arrest. Despite extensive studies on p73-mediated apoptosis, little is known about the cytoplasmic apoptotic function of p73. Here, using H1299 lung cancer cells and diverse biochemical approaches, including colony formation, DNA fragmentation, GST pulldown, and apoptosis assays along with NMR spectroscopy, we show that p73 induces transcription-independent apoptosis via its transactivation domain (TAD) through a mitochondrial pathway and that this apoptosis is mediated by the interaction between p73-TAD and the anti-apoptotic protein B-cell lymphoma-extra large (Bcl-XL or BCL2L1). This binding disrupted an interaction between Bcl-XL and the pro-apoptotic protein BH3-interacting domain death agonist (Bid). In particular, we found that a 16-mer p73-TAD peptide motif (p73-TAD16) mediates transcription-independent apoptosis, accompanied by cytochrome c release from the mitochondria, by interacting with Bcl-XL Interestingly, the structure of the Bcl-XL-p73-TAD16 peptide complex revealed a novel mechanism of Bcl-XL recognition by p73-TAD. We observed that the α-helical p73-TAD16 peptide binds to a noncanonical site in Bcl-XL, comprising the BH1, BH2, and BH3 domains in an orientation opposite to those of pro-apoptotic BH3 peptides. Taken together, our results indicate that the cytoplasmic apoptotic function of p73 is mediated through a noncanonical mode of Bcl-XL recognition. This finding sheds light on a critical transcription-independent, p73-mediated mechanism for apoptosis induction, which has potential implications for anticancer therapy.

Ionizing radiation-inducible miR-494 promotes glioma cell invasion through EGFR stabilization by targeting p190B rhoGAP.

MicroRNAs (miRNAs) play an important role in various stages of tumor progression. miR-494, which we had previously identified as a miRNA induced by ionizing radiation (IR) in the glioma cell line U-251, was observed to enhance invasion of U-251 cells by activating MMP-2. The miR-494-induced invasive potential was accompanied by, and dependent on, epidermal growth factor receptor (EGFR) upregulation and the activation of its downstream signaling constituents, Akt and ERK. The upregulation of EGFR by miR-494 involved the suppression of lysosomal protein turnover. Among the putative target proteins tested, p190B RhoGAP (p190B) was downregulated by miR-494, and its reduced expression was responsible for the increase in EGFR expression. A reporter assay using a luciferase construct containing p190B 3'-untranslated region (3'UTR) confirmed that p190B is a direct target of miR-494. Downregulation of p190B by small interfering RNA (siRNA) transfection closely mimicked the outcomes of miR-494 transfection, and showed increased EGFR expression, MMP-2 secretion, and invasion. Ectopic expression of p190B suppressed the miR-494-induced EGFR upregulation and invasion promotion, thereby suggesting that p190B depletion is critical for the invasion-promoting action of miR-494. Collectively, our results suggest a novel function for miR-494 and its potential application as a target to control invasiveness in cancer therapy.

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1.

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3'-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.

A ligand-independent Tie2-activating antibody reduces vascular leakage in models of Clarkson disease.

Vascular dysfunction resulting from endothelial hyperpermeability is a common and important feature of critical illness due to sepsis, trauma, and other conditions associated with acute systemic inflammation. Clarkson disease [monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome (ISCLS)] is a rare, orphan disorder marked by spontaneous and recurrent episodes of hypotensive shock and peripheral edema due to widespread vascular leakage in peripheral tissues. Mortality from acute flares approaches 30% due to lack of effective therapies. We evaluated a monoclonal antibody (4E2) specific for the endothelial receptor tyrosine kinase Tie2 in ISCLS models. 4E2 activated Tie2 in ISCLS patient-derived endothelial cells and reduced baseline and proinflammatory mediator-induced barrier dysfunction. 4E2 also reduced mortality and/or vascular leakage associated with systemic histamine challenge or influenza infection in the SJL/J mouse model of ISCLS. These findings support a critical role for Tie2 dysregulation in ISCLS and highlight a viable therapeutic approach to this catastrophic disorder.

Engineered Corynebacterium glutamicum as the Platform for the Production of Aromatic Aldehydes.

Aromatic aldehydes, including 4-hydroxybenzaldehyde (4-HB aldehyde), protocatechuic (PC) aldehyde, and vanillin, are used as important flavors, fragrances, and pharmaceutical precursors and have several biological and therapeutic effects. Production of aromatic aldehydes in microbial hosts poses a challenge due to its rapid and endogenous reduction to alcohols. To address this hurdle, prospecting of the genome of Corynebacterium glutamicum yielded 27 candidate proteins that were used in comprehensive screening with a 4-hydroxybenzyl (4-HB) alcohol-producing strain. We identified that NCgl0324 has aromatic aldehyde reductase activity and contributed to 4-HB aldehyde reduction in vivo since the NCgl0324 deletion strain HB-Δ0324 produced 1.36 g/L of 4-HB aldehyde, that is, about 188% more than its parental strain. To demonstrate that NCgl0324 knockout can also improve production of PC aldehyde and vanillin, first, a basal MA303 strain that produces protocatechuate was engineered from 4-hydroxybenzoate-synthesizing C. glutamicum APS963, followed by deletion of NCgl0324 to generate PV-Δ0324. The PC aldehyde/alcohol or vanillin/vanillyl alcohol biosynthetic pathways, respectively, were able to be expanded from protocatechuate upon introduction of carboxylic acid reductase (CAR) and catechol O-methyltransferase encoded by a mutated comt m gene. In shake flask culture, the resulting NCgl0324 deletion strains PV-IΔ0324 and PV-IYΔ0324 were shown to produce 1.18 g/L PC aldehyde and 0.31 g/L vanillin, respectively. Thus, modulation of the identified NCgl0324 gene was shown to have the potential to boost production of valuable aromatic aldehydes and alcohols.

Nur77 upregulates HIF-alpha by inhibiting pVHL-mediated degradation.

In this study, we investigated the role of Nur77, an orphan nuclear receptor, in HIF-alpha transcriptional activity. We found that Nur77 associates and stabilizes HIF-1alpha via indirect interaction. Nur77 was found to interact with pVHL in vivo via the alpha-domain of pVHL. By binding to pVHL, Nur77 competed with elongin C for pVHL binding. Moreover, Nur77-binding to pVHL inhibited the pVHL-mediated ubiquitination of HIF-1alpha and ultimately increased the stability and transcriptional activity of HIF-1alpha. The ligand-binding domain of Nur77 was found to interact with pVHL and the expression of this ligand-binding domain was sufficient to stabilize and transactivate HIF-1alpha. Under the conditions that cobalt chloride was treated or pVHL was knocked down, Nur77 could not stabilize HIF-alpha. Moreover, Nur77 could not further stabilize HIF-2alpha in A498/VHL stable cells, which is consistent with our finding that Nur77 indirectly stabilizes HIF-alpha by binding to pVHL. Thus, our results suggest that an orphan nuclear receptor Nur77 binds to pVHL, thereby stabilizes and increases HIF-alpha transcriptional activity under the non-hypoxic conditions.

Menin represses JunD transcriptional activity in protein kinase C theta-mediated Nur77 expression.

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. It has been shown that the Nur77 promoter is activated by at least two signaling pathways, one mediated by calcium and the other by protein kinase C (PKC). MEF2D has been known to regulate Nur77 expression in a calcium- dependent manner. The mechanism by which calcium regulates MEF2D is through dissociation of calcium-sensitive MEF2 corepressors (Cabin1/HDACs, HDAC4/5) and the association with calcineurin-activated transcription factor NF-AT and the coactivator p300. However, little is known about how PKC activates the Nur77 promoter. Herein, we report that PKCtheta targets AP-1 like response element in the Nur77 promoter where JunD constitutively binds. PKCtheta triggers mitogen-activated protein kinase-inediated phosphorylation of JunD, and increases transcriptional activity of JunD, cooperatively with p300. Menin is identified as the transcriptional corepressor for JunD via recruitment of mSin3-istone deacetylases. In fact, Menin represses PKCtheta/p300-mediated transcriptional activity of JunD in T cell. Its dynamic regulation of histone modifiers with JunD is responsible for PKCq-synergistic effect on Nur77 expression in T cell.

Ionizing radiation-inducible miR-30e promotes glioma cell invasion through EGFR stabilization by directly targeting CBL-B.

MicroRNAs (miRNAs) are small non-coding RNA molecules that regulate gene expression at the transcriptional and post-transcriptional levels. Here we show that miR-30e, which was previously identified as an ionizing radiation-inducible miRNA, enhances cellular invasion by promoting secretion of the matrix metalloproteinase MMP-2. The enhancement of cellular invasion by miR-30e involved up-regulation of the epidermal growth factor receptor (EGFR) and subsequent activation of its downstream signaling mediators, AKT and extracellular signal-regulated kinase. EGFR up-regulation by miR-30e occurred due to stabilization of the EGFR protein. The E3 ubiquitin ligase casitas B-lineage lymphoma B (CBL-B) was down-regulated by miR-30e, and this led to increased EGFR abundance. A 3' UTR reporter assay confirmed that CBL-B is a direct target of miR-30e. Knocking down CBL-B expression phenocopied the effects of miR-30e, whereas ectopic expression of CBL-B suppressed miR-30e-induced EGFR up-regulation and invasion. Collectively, our results suggest that targeting miR-30e may limit the invasiveness induced during glioma radiotherapy.

ß,ß-Dimethylacrylshikonin sensitizes human colon cancer cells to ionizing radiation through the upregulation of reactive oxygen species.

Shikonin, a naphthoquinone derivative, has been shown to possess antitumor activity. In the present study, the effects of shikonin and its analog, ß,ß-dimethylacrylshikonin, were investigated as radiosensitizers on the human colon cancer cell line, HCT-116. Shikonin and, to a greater extent, its analog-induced apoptosis of HCT-116 cells further synergistically potentiated the induction of apoptosis when combined with ionizing radiation (IR) treatment. Shikonins also stimulated an increase in reactive oxygen species (ROS) production and IR-induced DNA damage. Pre-treatment with the ROS scavenger, N-acetylcysteine, suppressed the enhancement of IR-induced DNA damage and apoptosis stimulated by shikonins, indicating that shikonins exert their radiosensitizing effects through ROS upregulation. The radiosensitizing effect of shikonins was also examined in vivo using the xenograft mouse model. Consistent with the in vitro results, injection of ß,ß-dimethylacrylshikonin combined with IR treatment significantly suppressed tumor growth of the HCT-116 xenograft. Taken together, the results show that ß,ß-dimethylacrylshikonin is a promising agent for developing an improved strategy for radiotherapy against tumors.

Phosphorylation and stabilization of c-Myc by NEMO renders cells resistant to ionizing radiation through up-regulation of γ-GCS.

The transcription factor c-Myc has been previously shown to be phosphorylated and stabilized by NEMO through direct interaction in the nucleus. Here, we show that NEMO induces up-regulation of the c-Myc target protein, γ-glutamyl-cysteine synthetase (γ-GCS), leading to an increase of intracellular glutathione (GSH) levels and simultaneous enhancement of redox-controlling capacity. NEMO enhanced c-Myc recruitment to γ-GCS promoters and c-Myc was essential for NEMO-mediated γ-GCS up-regulation. The phosphorylation and stabilization of c-Myc by NEMO rendered cells more resistant to ionizing radiation (IR). Thus, the interaction between NEMO and c-Myc may be targeted for the development of strategies to overcome the resistance to radiotherapy.

The cleavage fragment of retinoid X receptor-alpha ligand binding domain inhibits radiosensitization by retinoic acid.

Retinoid X receptor-alpha (RXR alpha) fragments are known to be produced in some cancer cells by proteolytic cleavage. Previous finding that ligand binding domain (LBD) fragment of RXR alpha specifically inhibits retinoic acid receptor-gamma (RAR gamma) activity led us to investigate the functional role of RXR alpha LBD fragment in radiosensitization by retinoic acid (RA). Ectopic expression of RXR alpha LBD fragment in cells that do not have a detectable endogenous RXR alpha LBD fragment, blocked synergistic radiosensitizing action of RA, as determined by growth inhibition, cell death and colony formation assays. However, H460 cell, which has an endogenous RXR alpha LBD fragment, was not radiosensitized by RA regardless of the ectopic RXR alpha LBD fragment expression. These results were paralleled with the pattern of p21 Waf1/Cip1 induction by the treatment of RA in combination with ionizing radiation (IR). Taken together, we hypothesize that the RXR alpha LBD fragment may act as a negative regulator of radiosensitizing effect of RA by restricting the RAR gamma-mediated biological response to RA.

NEMO stabilizes c-Myc through direct interaction in the nucleus.

The transcription factor c-Myc is a cellular oncoprotein generally upregulated in most of human cancers. NF-κB essential modulator (NEMO) caused phosphorylation and stabilization of c-Myc protein in the nucleus through direct interaction. The interaction caused reduced ubiquitination of c-Myc by inhibiting ubiquitinating activity of Fbw7 without blocking the interaction between c-Myc and Fbw7. As a consequence, NEMO enhanced the expression of several selected c-Myc targets. Compared to the classical role as an essential subunit for the activity of IKK complex, stabilization of c-Myc by direct interaction is a unique function of NEMO, representing a new mechanism to regulate c-Myc activity.

A novel function of Nur77: physical and functional association with protein kinase C.

Despite the involvement in diverse physiological process and pleiotropic expression profile, the molecular functions of Nur77 are not likely to be fully elucidated. From the effort to find a novel function of Nur77, we detected molecular interaction between Nur77 and PKC. Details of interaction revealed that C-terminal ligand binding domain (LBD) of Nur77 specifically interacted with highly conserved glycine-rich loop of PKC required for catalytic activity. This molecular interaction resulted in inhibition of catalytic activity of PKCtheta by Nur77. C-terminal LBD of Nur77 is sufficient for inhibiting the phosphorylation of substrate by PKCtheta. Ultimately, inhibition of catalytic activity by Nur77 is deeply associated with repression of PKC-mediated activation of AP-1 and NF-kappaB. Therefore, these findings demonstrate a novel function of Nur77 as a PKC inhibitor and give insights into molecular mechanisms of various Nur77-mediated physiological phenomena.