Atypical induction of HIF-1α expression by pericellular Notch1 signaling suffices for the malignancy of glioblastoma multiforme cells.

Contact-based pericellular interactions play important roles in cancer progression via juxtacrine signaling pathways. The present study revealed that hypoxia-inducible factor-1α (HIF-1α), induced even in non-hypoxic conditions by cell-to-cell contact, was a critical cue responsible for the malignant characteristics of glioblastoma multiforme (GBM) cells through Notch1 signaling. Densely cultured GBM cells showed enhanced viability and resistance to temozolomide (TMZ) compared to GBM cells at a low density. Ablating Notch1 signaling by a γ-secretase inhibitor or siRNA transfection resensitized resistant GBM cells to TMZ treatment and decreased their viability under dense culture conditions. The expression of HIF-1α was significantly elevated in highly dense GBM cells even under non-hypoxic conditions. Atypical HIF-1α expression was associated with the Notch1 signaling pathway in both GBM and glioblastoma stem cells (GSC). Proteasomal degradation of HIF-1α was prevented by binding with Notch1 intracellular domain (NICD), which translocated to the nuclei of GBM cells. Silencing Notch1 signaling using a doxycycline-inducible Notch1 RNA-interfering system or treatment with chetomin, a HIF pathway inhibitor, retarded tumor development with a significant anti-cancer effect in a murine U251-xenograft model. Using GBM patient tissue microarray analysis, a significant increase in HIF-1α expression was identified in the group with Notch1 expression compared to the group without Notch1 expression among those with positive HIF-1α expression. Collectively, these findings highlight the critical role of cell-to-cell contact-dependent signaling in GBM progression. They provide a rationale for targeting HIF-1α signaling even in a non-hypoxic microenvironment.

Differential dependency of human glioblastoma cells on vascular endothelial growth factor­A signaling via neuropilin­1.

Despite the high expression of neuropilin­1 (NRP­1) in human glioblastoma (GB), the understanding of its function as a co­receptor of vascular endothelial growth factor receptors (VEGFRs) in angiogenesis is currently limited. Therefore, the aim of the present study was to elucidate the non­classical function of NRP­1 expression in human GB. Expression patterns of NRP­1 and VEGF­A were determined by sandwich ELISA, western blot analysis, or immunohistochemistry. Differential dependency of GB cells following ablation of VEGF­A signaling was validated in vitro and in vivo. Cellular mechanism responsible for distinct response to VEGF­A signaling was evaluated by western blotting and immunoprecipitation analysis. Prognostic implications were assessed using IHC analysis. GB cells exhibited differing sensitivity to silencing of vascular endothelial growth factor (VEGF)­A signaling, which resulted in a distinct expression pattern of wild­type or chondroitin­sulfated NRP­1. VEGF­A­sensitive GB exhibited the physical interaction between wild­type NRP­1 and FMS related receptor tyrosine kinase 1 (Flt­1) whereas VEGF­A­resistant GB exhibited chondroitin­sulfated NRP­1 without interaction with Flt­1. Eliminating the chondroitin sulfate modification in NRP­1 led to re­sensitization to VEGF­A signaling, and chondroitin sulfate modification was found to be associated with an adverse prognosis in patients with GB. The present study identified the distinct functions of NRP­1 in VEGF­A signaling in accordance with its unique expression type and interaction with Flt­1. The present research is expected to provide a strong basis for targeting VEGF­A signaling in patients with GB, with variable responses.

Strategies for Targeted Delivery of Exosomes to the Brain: Advantages and Challenges.

Delivering therapeutics to the central nervous system (CNS) is difficult because of the blood-brain barrier (BBB). Therapeutic delivery across the tight junctions of the BBB can be achieved through various endogenous transportation mechanisms. Receptor-mediated transcytosis (RMT) is one of the most widely investigated and used methods. Drugs can hijack RMT by expressing specific ligands that bind to receptors mediating transcytosis, such as the transferrin receptor (TfR), low-density lipoprotein receptor (LDLR), and insulin receptor (INSR). Cell-penetrating peptides and viral components originating from neurotropic viruses can also be utilized for the efficient BBB crossing of therapeutics. Exosomes, or small extracellular vesicles, have gained attention as natural nanoparticles for treating CNS diseases, owing to their potential for natural BBB crossing and broad surface engineering capability. RMT-mediated transport of exosomes expressing ligands such as LDLR-targeting apolipoprotein B has shown promising results. Although surface-modified exosomes possessing brain targetability have shown enhanced CNS delivery in preclinical studies, the successful development of clinically approved exosome therapeutics for CNS diseases requires the establishment of quantitative and qualitative methods for monitoring exosomal delivery to the brain parenchyma in vivo as well as elucidation of the mechanisms underlying the BBB crossing of surface-modified exosomes.

Exosomal delivery of NF-κB inhibitor delays LPS-induced preterm birth and modulates fetal immune cell profile in mouse models.

Accumulation of immune cells and activation of the pro-inflammatory transcription factor NF-κB in feto-maternal uterine tissues is a key feature of preterm birth (PTB) pathophysiology. Reduction of the fetal inflammatory response and NF-κB activation are key strategies to minimize infection-associated PTB. Therefore, we engineered extracellular vesicles (exosomes) to contain an NF-κB inhibitor, termed super-repressor (SR) IκBα. Treatment with SR exosomes (1 × 1010 per intraperitoneal injection) after lipopolysaccharide (LPS) challenge on gestation day 15 (E15) prolonged gestation by over 24 hours (PTB ≤ E18.5) and reduced maternal inflammation (n ≥ 4). Furthermore, using a transgenic model in which fetal tissues express the red fluorescent protein tdTomato while maternal tissues do not, we report that LPS-induced PTB in mice is associated with influx of fetal innate immune cells, not maternal, into feto-maternal uterine tissues. SR packaged in exosomes provides a stable and specific intervention for reducing the inflammatory response associated with PTB.

Exosome-based delivery of super-repressor IκBα relieves sepsis-associated organ damage and mortality.

As extracellular vesicles that play an active role in intercellular communication by transferring cellular materials to recipient cells, exosomes offer great potential as a natural therapeutic drug delivery vehicle. The inflammatory responses in various disease models can be attenuated through introduction of super-repressor IκB (srIκB), which is the dominant active form of IκBα and can inhibit translocation of nuclear factor κB into the nucleus. An optogenetically engineered exosome system (EXPLOR) that we previously developed was implemented for loading a large amount of srIκB into exosomes. We showed that intraperitoneal injection of purified srIκB-loaded exosomes (Exo-srIκBs) attenuates mortality and systemic inflammation in septic mouse models. In a biodistribution study, Exo-srIκBs were observed mainly in the neutrophils, and in monocytes to a lesser extent, in the spleens and livers of mice. Moreover, we found that Exo-srIκB alleviates inflammatory responses in monocytic THP-1 cells and human umbilical vein endothelial cells.

Cyclic-recombinase-reporter mouse model to determine exosome communication and function during pregnancy.

BACKGROUND: During pregnancy, feto-maternal communication can be mediated through extracellular vesicles, specifically exosomes, 30- to 150-nm particles released from each cell. Exosomes carry cellular signals, and traffic between fetal and maternal tissues to produce functional changes in recipient cells. Exosomes may function as a biomarker indicative of the physiologic status of their tissue of origin. These properties of exosomes during pregnancy are not well studied. OBJECTIVE: To test exosome trafficking and function, we used a transgenic mouse model containing membrane-targeted, red fluorescent protein tdTomato and enhanced green fluorescent protein cyclic recombinase-reporter construct expressed only in fetal tissues. This model allows fetal tissues and their exosomes to express tdTomato under normal conditions or green fluorescent protein if fetal tissues are exposed to cyclic recombinase that will excise tdTomato. As maternal tissue remains negative for this construct, tdTomato/green fluorescent protein expression and their switching can be used to determine fetal-specific cell and exosome trafficking. MATERIALS AND METHODS: tdTomato/green fluorescent protein-homozygous male mice were mated with wild-type females to have all fetal tissues express the tdTomato/green fluorescent protein allele. Red fluorescence due to tdTomato expression of the tdTomato/green fluorescent protein allele in fetal tissues (placenta, fetal membranes) was confirmed by confocal microscopy on embryonic day 16. Localization of fetal exosomes in maternal uterine tissues were performed by immunostaining for exosome marker CD81 and tdTomato expression followed by confocal microscopy. Fetal exosomes (tdTomato-positive) in maternal plasma were immunoprecipitated using anti-red fluorescent protein tdTomato, followed by confirmation with flow cytometry. To further illustrate the fidelity of fetal exosomes in maternal samples, exosomes bioengineered to contain cyclic recombinase (1.0 × 1010 exosomes) were injected intraperitoneally on embryonic day 13. On embryonic day 16, fetal (placenta and fetal membranes) tissues were imaged to show tdTomato-to-green fluorescent protein transition. The green fluorescent protein-expressing exomes were localized in maternal tissues (confocal microscopy) and plasma (flow cytometry). RESULTS: Mating between a male with the tdTomato/green fluorescent protein construct and a null female resulted in fetal tissues and their exosomes expressing tdTomato positivity. Total fetal exosomes in maternal plasma was about 35%. tdTomato-positive exosomes were isolated from maternal plasma and immunostaining localized tdTomato-positive exosomes in maternal uterine tissues. Maternal intraperitoneal injection of cyclic recombinase-enriched exosomes crossed placenta, excised tdTomato from the tdTomato/green fluorescent protein construct in the fetal tissues, and caused green fluorescent protein expression in fetal cells. Furthermore, green fluorescent protein-positive exosomes released from fetal cells were isolated from maternal blood. CONCLUSION: In this pilot study, we report feto-maternal and maternal-fetal trafficking of exosomes indicative of paracrine signaling during pregnancy. Exosomes from the maternal side can produce functional changes in fetal tissues. Trafficking of exosomes suggests their potential role in pregnancy as biomarkers of fetal functions and usefulness as a carrier of drugs and other cargo to the fetal side during pregnancy. Isolation and characterization of fetal exosomes can advance fetal research without performing invasive procedures.

MCP-1 and MIP-3α Secreted from Necrotic Cell-Treated Glioblastoma Cells Promote Migration/Infiltration of Microglia.

BACKGROUND/AIMS: Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults. The defining characteristics of GBM are diffuse infiltration of tumor cells into normal brain parenchyma, rapid growth, a high degree of infiltration of microglia and macrophages, and the presence of necrosis. Microglia/macrophages are frequently found in gliomas and they extensively infiltrate GBM tissue, up to 30% of total tumor mass. However, little is known about the effect of necrotic cells (NCs) on microglia infiltration in GBM and the tumor-infiltrating microglia-induced factors in GBMs. METHODS: In this study, to address whether necrosis or necrosis-exposed GBM cells affect the degree of microglia/macrophage infiltration, migration and invasion/infiltration assays were performed. Culture supernatants and nuclear extracts of CRT-MG cells treated or untreated with necrotic cells were analyzed using a chemokine array and electrophoretic mobility shift assay, respectively. RESULTS: The presence of NCs promoted the migration/infiltration of microglia, and GBM cell line CRT-MG cells exposed to NCs further enhanced the migration and infiltration of HMO6 microglial cells. Treatment with NCs induced mRNA and protein expression of chemokines such as Monocyte Chemoattractant Protein-1 (CCL2/MCP-1) and Macrophage Inflammatory Protein-3α (CCL20/MIP-3α) in CRT-MG cells. In particular, CCL2/MCP-1 and CCL20/MIP-3α were significantly increased in NC-treated CRT-MG cells. NCs induced DNA binding of the transcription factors Nuclear Factor (NF)-κB and Activator Protein 1 (AP-1) to the CCL2/MCP-1 and CCL20/MIP-3α promoters, leading to increased CCL2/MCP-1 and CCL20/MIP-3α mRNA and protein expression in CRT-MG cells. CONCLUSION: These results provide evidence that NCs induce the expression of CCL2/MCP-1 and CCL20/MIP-3α in glioblastoma cells through activation of NF-κB and AP-1 and facilitate the infiltration of microglia into tumor tissues.

Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

Ginsenoside 20(S)-Protopanaxadiol Suppresses Viability of Human Glioblastoma Cells via Down-regulation of Cell Adhesion Proteins and Cell-cycle Arrest.

BACKGROUND: Pharmacologically active components of ginseng, particularly protopanaxadiol (PPD)-type ginsenosides, have potent anticancer effects, although their effects on highly malignant glioblastoma multiforme (GBM) have not been systemically evaluated. Identification of effective anticancer ginsenosides and further delineation of their mechanisms of action may provide valuable information that aids in the development of alternative or adjuvant therapy for malignant cancer. MATERIALS AND METHODS: We examined the viability of human GBM U251-MG and U87-MG cells treated with structurally related PPD-type ginsenosides, including F2, Rh2, compound K (C-K), and PPD. RESULTS: Incubation with PPD, C-K, and Rh2 significantly reduced the viability of U251-MG and U87-MG cells in a dose- and time-dependent manner. The cytotoxic effect of PPD was accompanied by reduced expression of cell adhesion proteins, including N-cadherin and integrin ß1, which led to reduced phosphorylation of focal adhesion kinase. Furthermore, incubation with PPD reduced the expression of cyclin D1 and subsequently induced cell-cycle arrest at the G1 phase. CONCLUSION: These results collectively indicate that PPD might provide a new strategy for treating malignant GBM, which is quite resistant to conventional anticancer treatment.

Combined inhibition of vascular endothelial growth factor receptor signaling with temozolomide enhances cytotoxicity against human glioblastoma cells via downregulation of Neuropilin-1.

Glioblastoma multiforme (GBM) is the most common and aggressive type of primary brain tumor with grave prognosis. Despite the growing understanding of the complex signaling networks responsible for the initiation and progression of GBM, many experimental therapies have fallen short of their treatment goals. In the present study, we investigated the novel molecular mechanisms responsible for synergistic action of temozolomide (TMZ) and anti-VEGF therapy in GBM cells. We tested the combined effects of TMZ and VEGF blockade in four human GBM cell lines: TMZ-sensitive U251-MG and U373-MG cells, and TMZ-resistant CRT-MG and LN215-MG cells, which correlated with MGMT promoter methylation status. Treatment of TMZ along with a sublethal dosage range of SU1498, a chemical inhibitor of the VEGF receptor signaling, induced significant cell death in both TMZ-sensitive and TMZ-resistant GBM cells without changing the status of the MGMT promoter methylation. Treatment with TMZ specifically reduced the expression of NRP-1, a coreceptor of VEGF but not those of VEGF-R1 and VEGF-R2. We further confirmed the key role of NRP-1 by showing that the reduction of NRP-1 by siRNA also increased the SU1498-induced cytotoxicity of LN215-MG. These results collectively indicate that combined treatment of TMZ can sensitize GBM cells to blockade of autocrine VEGF signaling through specific down-regulation of NRP-1, which provide a rationale for further evaluation and a potential clinical trial of combinatorial therapy of TMZ and SU1498 or other VEGF inhibitors for intractable brain tumors.

Protopanaxatirol type ginsenoside Re promotes cyclic growth of hair follicles via inhibiting transforming growth factor ß signaling cascades.

Ginsenosides, the major bio-active ingredients included in Panax ginseng, have been known for the hair growth activity and used to treat patients who suffer from hair loss; however, the detailed mechanisms of this action are still largely unknown. This study was conducted to investigate the molecular and cellular mechanisms responsible for hair growth promoting effect of ginsenoside Re (GRe) in vitro and in vivo. Different doses of minoxidil and GRe were administered topically to the back regions of nude mice for up to 45 days, and hair shaft length and hair cycles were determined for hair promoting activities. Topical treatment of GRe significantly increased the hair shaft length and hair existent time, which was comparable to the action of minoxidil. We also demonstrated that GRe stimulated hair shaft elongation in the ex vivo cultures of vibrissa hair follicles isolated from C57BL/6 mouse. Systemic transcriptome analysis by next generation sequencing demonstrated that TGF-ß-pathway related genes were selectively down-regulated by treatment of GRe in vivo, and the same treatment suppressed TGF-ß-induced phosphorylation of ERK in HeLa cells. The results clearly indicated that GRe is the effective constituent in the ginseng on hair promotion via selective inhibition of the hair growth phase transition related signaling pathways, TGF-ß signaling cascades.

Vascular Proteomics Reveal Novel Proteins Involved in SMC Phenotypic Change: OLR1 as a SMC Receptor Regulating Proliferation and Inflammatory Response.

Neointimal hyperplasia of vascular smooth muscle cells (VSMC) plays a critical role in atherosclerotic plaque formation and in-stent restenosis, but the underlying mechanisms are still incompletely understood. We performed a proteomics study to identify novel signaling molecules organizing the VSMC hyperplasia. The differential proteomics analysis in a balloon-induced injury model of rat carotid artery revealed that the expressions of 44 proteins are changed within 3 days post injury. The combination of cellular function assays and a protein network analysis further demonstrated that 27 out of 44 proteins constitute key signaling networks orchestrating the phenotypic change of VSMC from contractile to epithelial-like synthetic. Among the list of proteins, the in vivo validation specifically revealed that six proteins (Rab15, ITR, OLR1, PDHß, PTPε) are positive regulators for VSMC hyperplasia. In particular, the OLR1 played dual roles in the VSMC hyperplasia by directly mediating oxidized LDL-induced monocyte adhesion via NF-κB activation and by assisting the PDGF-induced proliferation/migration. Importantly, OLR1 and PDGFRß were associated in close proximity in the plasma membrane. Thus, this study elicits the protein network organizing the phenotypic change of VSMC in the vascular injury diseases such as atherosclerosis and discovers OLR1 as a novel molecular link between the proliferative and inflammatory responses of VSMCs.

Low Plasma Proportion of Omega 3-Polyunsaturated Fatty Acids Predicts Poor Outcome in Acute Non-Cardiogenic Ischemic Stroke Patients.

BACKGROUND AND PURPOSE: Alterations in blood fatty acid (FA) composition are associated with cardiovascular diseases. We investigated whether plasma FA composition was related to stroke severity and functional outcome in acute ischemic stroke patients. METHODS: We prospectively enrolled 156 patients with first-episode cerebral infarction, within 7 days of symptom onset. The proportion of FAs was analyzed using gas chromatography, and the summation of the omega-3 polyunsaturated fatty acids (ω3-PUFA), 18:3 ω3 α-linolenic acid, 20:3 ω3 eicosatrienoic acid, 20:5 ω3 eicosapentaenoic acid (EPA), and 22:6 ω3 docosahexaenoic acid (DHA) was reported as Σω3-PUFAs. Stroke severity was assessed using the National Institutes of Health Stroke Scale (NIHSS) score on admission. Poor functional outcome was defined by modified Rankin scale (mRS) ≥3 at three months after the index stroke. RESULTS: Lower proportions of EPA (ß=-0.751), DHA (ß=-0.610), and Σω3-PUFAs (ß=-0.462) were independently associated with higher NIHSS score, after adjusting for stroke subtype, hemoglobin, high density lipoprotein, high sensitivity C-reactive protein, fasting glucose, 16:0 palmitic acid, and Σsaturated fatty acids. Moreover, a lower proportion of DHA (odds ratio [OR]: 0.20, 95% confidence interval [CI]: 0.04-0.88), and Σω3-PUFAs (OR: 0.22, 95% CI: 0.05-0.84) showed an independent relationship with poor functional outcome after adjusting for age, sex, smoking status, NIHSS score, stroke subtype, and 16:0 palmitic acid. CONCLUSIONS: Our results demonstrate that ω3-PUFAs correlated with stroke severity on admission and functional outcomes at 3 months. ω3-PUFAs are potential blood biomarkers for prognosis of acute non-cardiogenic ischemic stroke patients.

Transcriptional regulation of adrenomedullin by oncostatin M in human astroglioma cells: implications for tumor invasion and migration.

Adrenomedullin (ADM), a secretory peptide with multiple functions in physiological to pathological conditions, is upregulated in several human cancers, including brain, breast, colon, prostate, and lung cancer. However, the molecular mechanisms underlying the regulation of ADM expression in cancerous cells are not fully understood. Here, we report that oncostatin M (OSM), a cytokine belonging to the interleukin-6 family, induces ADM expression in astroglioma cells through induction of signal transducer and activator of transcription-3 (STAT-3) phosphorylation, nuclear translocation, and subsequent DNA binding to the ADM promoter. STAT-3 knockdown decreased OSM-mediated expression of ADM, indicating that ADM expression is regulated by STAT-3 in astroglioma cells. Lastly, scratch wound healing assay showed that astroglioma cell migration was significantly enhanced by ADM peptides. These data suggest that aberrant activation of STAT-3, which is observed in malignant brain tumors, may function as one of the key regulators for ADM expression and glioma invasion.

Mutant ubiquitin UBB+1 induces mitochondrial fusion by destabilizing mitochondrial fission-specific proteins and confers resistance to oxidative stress-induced cell death in astrocytic cells.

Mutant ubiquitin UBB+1 is observed in a variety of aging-related neurodegenerative diseases and acts as a potent inhibitor of the ubiquitin proteasome system (UPS). In the present study, we investigated the relationship between impaired UPS (using ectopic expression of UBB+1) and mitochondrial dynamics in astrocytes, which are the most abundant glial cells in the central nervous system. Immunocytochemistry and fluorescence recovery after photobleaching revealed that ectopic expression of UBB+1 induced mitochondrial elongation. We further demonstrated that overexpression of UBB+1 destabilized mitochondrial fission-specific proteins including Drp1, Fis1, and OPA3, but not the mitochondrial fusion-specific proteins Mfn1, Mfn2, and OPA1. The reduction in mitochondrial fission-specific proteins by UBB+1 was prevented by inhibiting the 26 S proteasome using chemical inhibitors, including MG132, lactacystin and epoxomicin. We then assessed the involvement of proteases that target mitochondrial proteins by using various protease inhibitors. Finally, we confirmed that either overexpression of UBB+1 or inhibiting the proteasome can protect astrocytic cells from H2O2-induced cell death compared with control cells. Our results suggest that UBB+1 destabilizes mitochondrial fission-specific proteins, leading to mitochondrial fusion and the subsequent resistance to oxidative stress. We therefore propose a protective role of UBB+1 overexpression or the proteasome inhibition in astrocytes in degenerative brains.

Phenotypic modulation of primary vascular smooth muscle cells by short-term culture on micropatterned substrate.

Loss of contractility and acquisition of an epithelial phenotype of vascular smooth muscle cells (VSMCs) are key events in proliferative vascular pathologies such as atherosclerosis and post-angioplastic restenosis. There is no proper cell culture system allowing differentiation of VSMCs so that it is difficult to delineate the molecular mechanism responsible for proliferative vasculopathy. We investigated whether a micropatterned substrate could restore the contractile phenotype of VSMCs in vitro. To induce and maintain the differentiated VSMC phenotype in vitro, we introduced a micropatterned groove substrate to modulate the morphology and function of VSMCs. Later than 7(th) passage of VSMCs showed typical synthetic phenotype characterized by epithelial morphology, increased proliferation rates and corresponding gene expression profiles; while short-term culture of these cells on a micropatterned groove induced a change to an intermediate phenotype characterized by low proliferation rates, increased migration, a spindle-like morphology associated with cytoskeletal rearrangement and expression of muscle-specific genes. Microarray analysis showed preferential expression of contractile and smooth muscle cell-specific genes in cells cultured on the micropatterned groove. Culture on a patterned groove may provide a valuable model for the study the role of VSMCs in normal vascular physiology and a variety of proliferative vascular diseases.

Mutant ubiquitin attenuates interleukin-1ß- and tumor necrosis factor-α-induced pro-inflammatory signaling in human astrocytic cells.

A frameshift mutation of ubiquitin called ubiquitin(+1) (UBB(+1)) was found in the aging and Alzheimer's disease brains and thought to be associated with neuronal dysfuction and degeneration. Even though ubiquitylation has been known to regulate vital cellular functions mainly through proteasome-dependent degradation of polyubiquitinated substrates, proteolysis-independent roles of ubiquitylation have emerged as key mechanisms in various signaling cascades. In this study, we have investigated the effect of UBB(+1) on proinflammatory signaling such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in human astrocytes. Treatment with TNF-α and IL-1ß induced expression of CCL2 and CXCL8 by human astrocytic cells; while ectopic expression of UBB(+1) significantly abrogated the proinflammatory cytokine-induced expression of chemokines. Ectopic expression of UBB(+1) suppressed TNF-α- and IL-1ß-induced activation of NF-κB and JNK signaling pathway. Furthermore, we have demonstrated that polyubiquitylation of TRAFs and subsequent phosphorylation of TAK1 were significantly inhibited by stable expression of UBB(+1). Collectively, these results suggest that UBB(+1) may affect proinflammatory signaling in the central nervous system via inhibitory mechanisms of ubiquitin-dependent signaling in human astrocytes.

Downregulation of OPA3 is responsible for transforming growth factor-ß-induced mitochondrial elongation and F-actin rearrangement in retinal pigment epithelial ARPE-19 cells.

Transforming growth factor-ß signaling is known to be a key signaling pathway in the induction of epithelial-mesenchymal transition. However, the mechanism of TGF-ß signaling in the modulation of EMT remains unclear. In this study, we found that TGF-ß treatment resulted in elongation of mitochondria accompanied by induction of N-cadherin, vimentin, and F-actin in retinal pigment epithelial cells. Moreover, OPA3, which plays a crucial role in mitochondrial dynamics, was downregulated following TGF-ß treatment. Suppression of TGF-ß signaling using Smad2 siRNA prevented loss of OPA3 induced by TGF-ß. Knockdown of OPA3 by siRNA and inducible shRNA significantly increased stress fiber levels, cell length, cell migration and mitochondrial elongation. In contrast, forced expression of OPA3 in ARPE-19 cells inhibited F-actin rearrangement and induced mitochondrial fragmentation. We also showed that Drp1 depletion increased cell length and induced rearrangement of F-actin. Depletion of Mfn1 blocked the increase in cell length during TGF-ß-mediated EMT. These results collectively substantiate the involvement of mitochondrial dynamics in TGF-ß-induced EMT.

Constitutive Expression of MAP Kinase Phosphatase-1 Confers Multi-drug Resistance in Human Glioblastoma Cells.

PURPOSE: Current treatment of glioblastoma after surgery consists of a combination of fractionated radiotherapy and temozolomide. However, it is difficult to completely remove glioblastoma because it has uncertain boundaries with surrounding tissues. Moreover, combination therapy is not always successful because glioblastoma has diverse resistances. To overcome these limitations, we examined the combined effects of chemotherapy and knockdown of mitogen-activated protein kinase phosphatase-1 (MKP-1). MATERIALS AND METHODS: We used ten different anti-cancer drugs (cisplatin, cyclophosphoamide, doxorubicin, epirubicin, etoposide, 5-fluorouracil, gemcitabine, irinotecan, mitomycin C, and vincristine) to treat glioblastoma multiforme (GBM) cells. Knockdown of MKP-1 was performed using siRNA and lipofectamine. The basal level of MKP-1 in GBM was analyzed based on cDNA microarray data obtained from the Gene Expression Omnibus (GEO) databases. RESULTS: Anti-cancer drug-induced cell death was significantly enhanced by knockdown of MKP-1, and this effect was most prominent in cells treated with irinotecan and etoposide. Treatment with these two drugs led to significantly increased phosphorylation of c-Jun N-terminal kinase (JNK) in a time-dependent manner, while pharmacological inhibition of JNK partially inhibited drug-induced cell death. Knockdown of MKP-1 also enhanced drug-induced phosphorylation of JNK. CONCLUSION: Increased MKP-1 expression levels could be the cause of the high resistance to conventional chemotherapeutics in human GBM. Therefore, MKP-1 is an attractive target for overcoming drug resistance in this highly refractory malignancy.

Reliable permeability assay system in a microfluidic device mimicking cerebral vasculatures.

Since most of the bioavailable drugs are impermeable through the blood-brain barrier (BBB), development of a rapid and reliable permeability assay system has been a challenge in drug discovery targeting central nervous system (CNS). Here, we designed a microfluidic device to monitor the drug permeability into the CNS. Human umbilical vein endothelial cells (HUVECs) were shortly (2 ~ 3 h) incubated with astrocyte-conditioned medium after being trapped on microholes in the microfluidic device and tested for chip-based permeability measurement of drugs. The measured permeability values were highly correlated with those measured by conventional in vitro methods and the brain uptake index representing the quantity of transported substances across the in vivo BBB of rats. Using the microfluidic device, we could easily monitor the effect of hydrogen peroxide on the trans-endothelial permeability, which are consistent with the finding that the same treatment disrupted the formation of tight junctions between endothelial cells. Considering relatively short period of time needed for endothelial cell culture and ability to monitor the BBB physiology continuously, we propose that this novel system can be used as an invaluable first-line tool for CNS-related drug development.