Hyunju Park

Activation of estrogen receptor β reduces blood–brain barrier breakdown following ischemic injury

Estrogen receptors (ERs) play important roles in estrogen-mediated neuroprotection. However, their effects on blood-brain barrier (BBB) disruption with vasogenic edema after ischemic stroke have not been determined. We evaluated a role for ERβ in the brain without effects in the peripheral reproductive organs for the amelioration of vasogenic edema following ischemic stroke. Transient focal ischemic stroke was induced in ovariectomized female C57BL/6 mice (age 10-11weeks) that were treated with the ERβ-selective agonist diarylpropionitrile (DPN). BBB breakdown as determined by the extravasation of endogenous immunoglobulin G (IgG), vasogenic edema, and the infarct volume was significantly reduced by DPN compared to vehicle. Protein expressions of endothelial tight junction proteins (occludin and claudin-5) and the water channel protein aquaporin 4 in the ischemic cortex were not changed by DPN. However, protein levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1α (HIF-1α), a transcription factor that increases VEGF expression, were significantly decreased in the ischemic cortex by DPN. These results suggest that ERβ contributes to the reduction of vasogenic edema caused by BBB breakdown via the inhibition of HIF-1α and VEGF following ischemic stroke.

SHP-2 binds to caveolin-1 and regulates Src activity via competitive inhibition of CSK in response to H2O2 in astrocytes.

Reactive oxygen species (ROS) regulate diverse cellular functions by triggering signal transduction events, such as Src and mitogen-activated protein (MAP) kinases. Here, we report the role of caveolin-1 and Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) in H2O2-induced signaling pathway in brain astrocytes. H2O2-mediated oxidative stress induced phosphorylation of caveolin-1 and association between p-caveolin-1 and SHP-2. SHP-2 specifically bound to wild-type caveolin-1 similarly to c-Src tyrosine kinase (CSK), but not to phosphorylation-deficient mutant of caveolin-1 (Y14A), and interfered with complex formation between caveolin-1 and CSK. In the presence of CSK siRNA, binding between caveolin-1 and SHP-2 was enhanced by H2O2 treatment, which led to reduced Src phosphorylation at tyrosine (Tyr) 530 and enhanced Src phosphorylation at Tyr 419. In contrast, siRNA targeting of SHP-2 facilitated H2O2-mediated interaction between caveolin-1 and CSK and enhanced Src phosphorylation at Tyr 530, leading to subsequent decrease in Src downstream signaling, such as focal adhesion kinase (FAK) and extracellular signal-related kinase (ERK). Our results collectively indicate that SHP-2 alters Src kinase activity by interfering with the complex formation between CSK and phosphotyrosine caveolin-1 in the presence of H2O2, thus functions as a positive regulator in Src signaling under oxidative stress in brain astrocytes.

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.

Necrotic cells influence migration and invasion of glioblastoma via NF-κB/AP-1-mediated IL-8 regulation.

Glioblastoma multiforme (GBM) is the most common primary intracranial tumor in adults and has poor prognosis. Diffuse infiltration into normal brain parenchyma, rapid growth, and the presence of necrosis are remarkable hallmarks of GBM. However, the effect of necrotic cells on GBM growth and metastasis is poorly understood at present. In this study, we examined the biological significance of necrotic tissues by exploring the molecular mechanisms underlying the signaling network between necrotic tissues and GBM cells. The migration and invasion of the GBM cell line CRT-MG was significantly enhanced by treatment with necrotic cells, as shown by assays for scratch wound healing and spheroid invasion. Incubation with necrotic cells induced IL-8 secretion in CRT-MG cells in a dose-dependent manner. In human GBM tissues, IL-8 positive cells were mainly distributed in the perinecrotic region, as seen in immunohistochemistry and immunofluorescence analysis. Necrotic cells induced NF-κB and AP-1 activation and their binding to the IL-8 promoter, leading to enhanced IL-8 production and secretion in GBM cells. Our data demonstrate that when GBM cells are exposed to and stimulated by necrotic cells, the migration and invasion of GBM cells are enhanced and facilitated via NF-κB/AP-1 mediated IL-8 upregulation.

Troglitazone Enhances the Apoptotic Response of DLD-1 Colon Cancer Cells to Photodynamic Therapy

Purpose The aim of this study was to investigate whether the peroxisomal proliferator-activated receptor gamma (PPARγ) ligand troglitazone in combination with photodynamic therapy (PDT) enhances the apoptotic response of DLD-1 colon cancer cells. Materials and Methods The effects of troglitazone, PDT, and troglitazone in combination with PDT on cell viability and apoptosis were assessed in DLD-1 cells. Cell viability and proliferation were evaluated using the tetrazolium-based MTT assay, and apoptosis was evaluated via cell staining with propidium iodide (PI) and annexin V-FITC. The levels of pro-caspase-3 were measured via Western blot analyses. Results Treatment of troglitazone and PDT induced the growth retardation and cell death of DLD-1 cells in a dose-dependent manner, respectively. The combination treatment significantly suppressed cell growth and increased the apoptotic response of DLD-1 and resulted in apoptosis rather than necrosis, as shown by PI/annexin V staining and degradation of procaspase-3. Conclusion Conclusion: These results document the anti-proliferative and apoptotic activities of PDT in combination with the PPARγ ligand troglitazone and provide a strong rationale for testing the therapeutic potential of combination treatment in colon cancer.

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 <unterline>M</unterline>onocyte <unterline>C</unterline>hemoattractant <unterline>P</unterline>rotein-1 (CCL2/MCP-1) and <unterline>M</unterline>acrophage <unterline>I</unterline>nflammatory <unterline>P</unterline>rotein-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 <unterline>N</unterline>uclear <unterline>F</unterline>actor (NF)-κB and <unterline>A</unterline>ctivator <unterline>P</unterline>rotein 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.

Leptin Suppresses Glutamate-Induced Apoptosis Through Regulation of ERK1/2 Signaling Pathways in Rat Primary Astrocytes

Background/Aims: Leptin is a hormone expressed by adipose tissue that regulates body energy homeostasis and weight loss by activating leptin receptors in the hypothalamus. Leptin receptors are also expressed in astrocytes. An anti-apoptosis effect of leptin in brain has recently been reported. However, the anti-apoptosis mechanism of leptin in the brain is unknown. Methods: To investigate whether leptin exerts protective effects against glutamate-induced apoptosis in astrocytes, we performed cell viability assays and apoptosis assays using rat primary astrocytes. Intracellular signaling pathways involved in anti-apoptosis effects of leptin were analyzed by immunoblotting together with a leptin mutant (S120A/T121A) with antagonist function and pharmacological inhibitors. Results: We found that glutamate-induced apoptosis in rat primary astrocytes was significantly decreased by treatment with leptin. Leptin inhibited glutamate-induced phosphorylation of ERK1/2 in astrocytes. The leptin S120A/T121A mutant did not inhibit glutamate-induced ERK1/2 phosphorylation and ERK1/2-mediated apoptosis. Conclusions: Collectively, our results provide initial evidence that leptin exerts an anti-apoptotic effect against glutamate toxicity through activation of intracellular signaling pathways which reverse glutamate-induced ERK1/2 phosphorylation in primary astrocytes. Therefore, our findings suggest that leptin might be considered a candidate for potential therapeutic applications in glutamate-induced brain excitotoxicity.