Transcriptional regulation of genetic variants in the SLC40A1 promoter.

Solute carrier 40A1 (SLC40A1) encodes ferroportin, which is the only known transmembrane protein that exports elemental iron from mammalian cells and is essential for iron homeostasis. Mutations in SLC40A1 are associated with iron-overload disorders. In addition to ferroportin diseases, SLC40A1 expression is downregulated in various cancer types. Despite the clinical significance of the SLC40A1 transporter, only a few studies have investigated genetic variants in SLC40A1. The present study was performed to identify genetic variations in the SLC40A1 promoter and functionally characterize each variant using in vitro assays. We investigated four haplotypes and five variants in the SLC40A1 promoter. We observed that haplotype 3 (H3) had significantly lower promoter activity than H1, whereas the activity of H4 was significantly higher than that of H1. Luciferase activity of H2 was comparable to that of H1. In addition, four variants of SLC40A1, c.-1355G>C, c.-662C>T, c.-98G>C, and c.-8C>G, showed significantly increased luciferase activity compared to the wild type (WT), whereas c.-750G>A showed significantly decreased luciferase activity compared to the WT. Three transcription factors, cAMP response element-binding protein-1 (CREB-1), chicken ovalbumin upstream promoter transcription factor 1, and hepatic leukemia factor (HLF), were predicted to bind to the promoter regions of SLC40A1 near c.-662C>T, c.-98G>C, and c.-8C>G, respectively. Among these, CREB-1 and HLF bound more strongly to the variant sequences than to the WT and functioned as activators of SLC40A1 transcription. Collectively, our findings indicate that the two SLC40A1 promoter haplotypes affect SLC40A1 transcription, which is regulated by CREB-1 and HLF.

The effect of genetic variants of SLC22A18 on proliferation, migration, and invasion of colon cancer cells.

Solute carrier family (SLC) transporters are expressed in the digestive system and play important roles in maintaining physiological functions in the body. In addition, SLC transporters act as oncoproteins or tumor-suppressor proteins during the development, progression, and metastasis of various digestive system cancers. SLC22A18, a member of the SLC22 gene family, is an orphan transporter with an unknown endogenous substrate. Previous study revealed that SLC22A18 is downregulated in colorectal cancer tissues and that it acts as a suppressor in colorectal cancer, although the effects of SLC22A18 variants on colon cancer cell proliferation, migration, and invasion are unknown. Therefore, in this study, we identified SLC22A18 variants found in multiple populations by searching public databases and determined the in vitro effects of these missense variations on transporter expression and cancer progression. Our results indicated that three missense SLC22A18 variants-p.Ala6Thr, p.Arg12Gln, and p.Arg86His-had significantly lower cell expression than the wild type, possibly owing to intracellular degradation. Furthermore, these three variants caused significantly higher proliferation, migration, and invasion of colon cancer cells than the wild type. Our findings suggest that missense variants of SLC22A18 can potentially serve as biomarkers or prognostic tools that enable clinicians to predict colorectal cancer progression.

Functional characterization of ABCA4 genetic variants related to Stargardt disease.

The ATP-binding cassette subfamily 4 (ABCA4), a transporter, is localized within the photoreceptors of the retina, and its genetic variants cause retinal dystrophy. Despite the clinical importance of the ABCA4 transporter, a few studies have investigated the function of each variant. In this study, we functionally characterized ABCA4 variants found in Korean patients with Stargardt disease or variants of the ABCA4 promoter region. We observed that four missense variants-p.Arg290Gln, p.Thr1117Ala, p.Cys1140Trp, and p.Asn1588Tyr-significantly decreased ABCA4 expression on the plasma membrane, which could be due to intracellular degradation. There are four major haplotypes in the ABCA4 proximal promoter. We observed that the H1 haplotype (c.-761C>A) indicated significantly increased luciferase activity compared to that of the wild-type, whereas the H3 haplotype (c.-1086A>C) indicated significantly decreased luciferase activity (P < 0.01 and 0.001, respectively). In addition, c.-900A>T in the H2 haplotype exhibited significantly increased luciferase activity compared with that of the wild-type. Two transcription factors, GATA-2 and HLF, were found to function as enhancers of ABCA4 transcription. Our findings suggest that ABCA4 variants in patients with Stargardt disease affect ABCA4 expression. Furthermore, common variants of the ABCA4 proximal promoter alter the ABCA4 transcriptional activity, which is regulated by GATA-2 and HLF transcription factors.

Shrimp miR-965 transfers tumoricidal mitochondria.

BACKGROUND: Micro RNA of Marsupenaeus japonicas has been known to promote apoptosis of tumor cells. However, the detailed mechanisms are not well understood. RESULTS: Using tomographic microscope, which can detect the internal structure of cells, we observed breast tumor cells following treatment of the miRNA. Intriguingly, we found that mitochondria migrate to an adjacent tumor cells through a tunneling nanotube. To recapitulate this process, we engineered a microfluidic device through which mitochondria were transferred. We show that this mitochondrial transfer process released endonuclease G (Endo G) into tumor cells, which we referred to herein as unsealed mitochondria. Importantly, Endo G depleted mitochondria alone did not have tumoricidal effects. Moreover, unsealed mitochondria had synergistic apoptotic effects with subtoxic dose of doxorubicin thereby mitigating cardiotoxicity. CONCLUSIONS: Together, we show that the mitochondrial transfer through microfluidics can provide potential novel strategies towards tumor cell death.

STAT6 Signaling Mediates PPARγ Activation and Resolution of Acute Sterile Inflammation in Mice.

The signal transducer and activator of transcription 6 (STAT6) transcription factor promotes activation of the peroxisome proliferator-activated receptor gamma (PPARγ) pathway in macrophages. Little is known about the effect of proximal signal transduction leading to PPARγ activation for the resolution of acute inflammation. Here, we studied the role of STAT6 signaling in PPARγ activation and the resolution of acute sterile inflammation in a murine model of zymosan-induced peritonitis. First, we showed that STAT6 is aberrantly activated in peritoneal macrophages after zymosan injection. Utilizing STAT6-/- and wild-type (WT) mice, we found that STAT6 deficiency further enhanced zymosan-induced proinflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-6, and macrophage inflammatory protein-2 in peritoneal lavage fluid (PLF) and serum, neutrophil numbers and total protein amount in PLF, but reduced proresolving molecules, such as IL-10 and hepatocyte growth factor, in PLF. The peritoneal macrophages and spleens of STAT6-/- mice exhibited lower mRNA and protein levels of PPARγ and its target molecules over the course of inflammation than those of WT mice. The deficiency of STAT6 was shown to impair efferocytosis by peritoneal macrophages. Taken together, these results suggest that enhanced STAT6 signaling results in PPARγ-mediated macrophage programming, contributing to increased efferocytosis and inflammation resolution.

Interaction of promyelocytic leukemia/p53 affects signal transducer and activator of transcription-3 activity in response to oncostatin M.

Promyelocytic leukemia (PML) gene, through alternative splicing of its C-terminal region, generates several PML isoforms that interact with specific partners and perform distinct functions. The PML protein is a tumor suppressor that plays an important role by interacting with various proteins. Herein, we investigated the effect of the PML isoforms on oncostatin M (OSM)-induced signal transducer and activator of transcription-3 (STAT-3) transcriptional activity. PML influenced OSM-induced STAT-3 activity in a cell type-specific manner, which was dependent on the p53 status of the cells but regardless of PML isoform. Interestingly, overexpression of PML exerted opposite effects on OSM-induced STAT-3 activity in p53 wild-type and mutant cells. Specifically, overexpression of PML in the cell lines bearing wild-type p53 (NIH3T3 and U87-MG cells) decreased OSM-induced STAT-3 transcriptional activity, whereas overexpression of PML increased OSM-induced STAT-3 transcriptional activity in mutant p53-bearing cell lines (HEK293T and U251-MG cells). When wild-type p53 cells were co-transfected with PML-IV and R273H-p53 mutant, OSM-mediated STAT-3 transcriptional activity was significantly enhanced, compared to that of cells which were transfected with PML-IV alone; however, when cells bearing mutant p53 were co-transfected with PML-IV and wild-type p53, OSM-induced STAT-3 transcriptional activity was significantly decreased, compared to that of transfected cells with PML-IV alone. In conclusion, PML acts together with wild-type or mutant p53 and influences OSM-mediated STAT-3 activity in a negative or positive manner, resulting in the aberrant activation of STAT-3 in cancer cells bearing mutant p53 probably might occur through the interaction of mutant p53 with PML.

Motion microscopy for label-free detection of circulating breast tumor cells.

Circulating tumor cells (CTCs) are cancer cells that have been shed from a primary tumor and circulate in the bloodstream during progression of cancer. They may thus serve as circulating biomarkers that can predict, diagnose and guide therapy. Moreover, phenotypic and genotypic analysis of CTCs can facilitate prospective assessment of mutations and enable personalized treatment. A number of methodologies based on biological and physical differences between circulating tumor and non-tumor cells have been developed over the past few years. However, these methods did not have sufficient sensitivity or specificity. In this work, a remote analysis protocol was designed using motion microscopy that amplifies cellular micro motions in a captured video by re-rendering small motions to generate extreme magnified visuals to detect dynamic motions that are not otherwise visible by naked eye. Intriguingly, motion microscopy demonstrated fluctuations around breast tumor cells, which we referred to herein as cellular trail. Phenomena of cellular trail mostly emerged between 0.5 and 1.5 Hz on amplified video images. Interestingly, cellular trails were associated with cell surface proteins and flow rates rather than mitochondrial activity. Moreover, cellular trails were present only around circulating tumor cells from individuals with breast cancer under conditions of 20-30 µm/s and 0.5-1.5 Hz. Thus, motion microscopy based CTC detection method can offer a valuable supplementary diagnostic tool for assessment of drug efficacy and identifying physical characteristics of tumor cells for further research.

OCT3 promoter haplotype is associated with metformin pharmacokinetics in Koreans.

Organic cation transporter 3 (OCT3) is expressed in various organs in humans and plays an important role in the transport of organic cations and drugs including metformin. In this study, we identified genetic variations of the OCT3 promoter and functionally characterized each variant by in vitro assays. Next, the association between the functional haplotype of the OCT3 promoter and pharmacokinetics of metformin was evaluated. In our study population, 7 variations and 2 major haplotypes were identified, of which H2 haplotype yielded a significantly higher luciferase activity than did the wild type. Two variants of H2, c.-1603G > A and c.-1547T > G, yielded significantly lower luciferase activities, whereas the luciferase activity of another variant, c.-29G > A, was significantly higher. Two transcription factors, Sp1 and USF1, were involved in the regulation of OCT3 transcription. Analysis of clinical data revealed that 25 subjects, either homozygous or heterozygous for H2, showed increased AUCinf and Cmax by 17.2% and 15.9%, respectively [P = 0.016 and 0.031, GMR (90% CI) = 1.17 (1.06-1.29) and 1.17 (1.04-1.31), respectively], compared to the 20 subjects in the control group. Our study suggests that an OCT3 promoter haplotype affects the pharmacokinetics of metformin in Koreans as well as the OCT3 transcription rate.

Apoptotic cells trigger the ABCA1/STAT6 pathway leading to PPAR-γ expression and activation in macrophages.

The signal transducer and activator of transcription 6 (STAT6) transcription factor activates peroxisome proliferator-activated receptor gamma (PPAR-γ)-regulated gene expression in immune cells. We investigated proximal membrane signaling that was initiated in macrophages after exposure to apoptotic cells that led to enhanced PPAR-γ expression and activity, using specific siRNAs for ABCA1, STAT6, and PPAR-γ, or their antagonists. The interactions between mouse bone marrow-derived macrophages or RAW 264.7 cells and apoptotic Jurkat cells, but not viable cells, resulted in the induction of STAT6 phosphorylation as well as PPAR-γ expression and activation. Knockdown of ATP-binding cassette transporter A1 (ABCA1) after the transfection of macrophages with ABCA1-specific siRNAs reduced apoptotic cell-induced STAT6 phosphorylation as well as PPAR-γ mRNA and protein expression. ABCA1 knockdown also reduced apoptotic cell-induced liver X receptor α (LXR-α) mRNA and protein expression. Moreover, inhibition of STAT6 with specific siRNAs or the pharmacological inhibitor AS1517499AS reversed the induction of PPAR-γ, LXR-α, and ABCA1 by apoptotic Jurkat cells. PPAR-γ-specific siRNAs or the PPAR-γ antagonist GW9662 inhibited apoptotic cell-induced increases in LXR-α and ABCA1 mRNA and protein levels. Thus, these results indicate that apoptotic cells trigger the ABCA1/STAT6 pathway, leading to the activation of the PPAR-γ/LXR-α/ABCA1 pathway in macrophages.

Repression of adenosine triphosphate-binding cassette transporter ABCG2 by estrogen increases intracellular glutathione in brain endothelial cells following ischemic reperfusion injury.

The adenosine triphosphate-binding cassette efflux transporter ABCG2, which is located in the blood-brain barrier limits the entry of endogenous compounds and xenobiotics into the brain, and its expression and activity are regulated by estrogen. This study was aimed to define the role of ABCG2 in estrogen-mediated neuroprotection against ischemic injury. ABCG2 protein levels before and after ischemic stroke were increased in the brain of female mice by ovariectomy, which were reversed by estrogen replacement. In brain endothelial cell line bEnd.3, estrogen reduced the basal ABCG2 protein level and efflux activity and protected cells from ischemic injury without inducing ABCG2 expression. When bEnd.3 cells were transfected with ABCG2 small interfering RNA, ischemia-induced cell death was reduced, and the intracellular concentration of glutathione, an antioxidant that is transported by ABCG2, was increased. In addition, after ischemic stroke in ovariectomized mice, estrogen prevented the reduction of intracellular glutathione level in brain microvessels. These data suggested that the suppression of ABCG2 by estrogen is involved in neuroprotection against ischemic injury by increasing intracellular glutathione, and that the modulation of ABCG2 activity offers a therapeutic target for brain diseases in estrogen-deficient aged women.

Association between Genetic Variations of MERTK and Chronic Obstructive Pulmonary Disease in Koreans.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a debilitating lung disease. To date, a large number of clinical studies have been conducted to investigate the association between genetic variations and COPD. However, little is known regarding the genetic susceptibility of Koreans to this disease. MER receptor tyrosine kinase (MERTK) plays important roles in the inhibition of inflammation and in the clearance of apoptotic cells. Here, we investigated the association between genetic variations in MERTK and the development of COPD in Koreans. METHODS: We conducted genetic analysis of MERTK using genomic DNA samples from 87 patients with COPD and 88 healthy controls and compared the frequency of each variation or haplotype between the patient and control groups. Subsequently, the effect of each variation was evaluated using in vitro assays. RESULTS: Ten variations were identified in this study, four of them for the first time. In addition, we found that the frequency of each variation or haplotype was comparable between the patient and control groups. However, we observed that the frequency for the wild-type haplotype was higher in the control group, compared to that in the group of patients with COPD, in the subgroup analysis of current smokers, although the difference was not statistically significant (P = 0.080). In in vitro assays, we observed that none of the variations affected the activity of the promoter or the expression of MERTK. CONCLUSION: Our findings indicate that the susceptibility to COPD is not related to the genetic variations or haplotypes of MERTK in Koreans.

Identification and Functional Characterization of ST3GAL5 and ST8SIA1 Variants in Patients with Thyroid-Associated Ophthalmopathy.

PURPOSE: This study was conducted to identify and to functionally characterize genetic variants in ST3GAL5 and ST8SIA1 in Korean patients with thyroid-associated ophthalmopathy (TAO). MATERIALS AND METHODS: Genetic analyses were conducted using DNA samples from TAO patients (n=50) and healthy subjects (n=48) to identify TAO-specific genetic variants of ST3GAL5 or ST8SIA1. The effect of each genetic variant on the transcription or expression of these genes was examined. Additionally, correlations between functional haplotypes of ST3GAL5 or ST8SIA1 and clinical characteristics of the patients were investigated. RESULTS: Six promoter variants and one nonsynonymous variant of ST3GAL5 were identified, and four major promoter haplotypes were assembled. Additionally, three promoter variants and two major haplotypes of ST8SIA1 were identified. All ST3GAL5 and ST8SIA1 variants identified in TAO patients were also found in healthy controls. Promoter activity was significantly decreased in three promoter haplotypes of ST3GAL5 and increased in one promoter haplotype of ST8SIA1. Transcription factors activating protein-1, NKX3.1, and specificity protein 1 were revealed as having roles in transcriptional regulation of these haplotypes. The nonsynonymous variant of ST3GAL5, H104R, did not alter the expression of ST3GAL5. While no differences in clinical characteristics were detected in patients possessing the functional promoter haplotypes of ST3GAL5, exophthalmic values were significantly lower in patients with the ST8SIA1 haplotype, which showed a significant increase in promoter activity. CONCLUSION: These results from genotype-phenotype analysis might suggest a possible link between the ST8SIA1 functional promoter haplotype and the clinical severity of TAO. However, further studies with larger sample sizes are warranted.

Extracellular matrix-derived extracellular vesicles promote cardiomyocyte growth and electrical activity in engineered cardiac atria.

Extracellular matrix (ECM) plays a critical role in the provision of the necessary microenvironment for the proper regeneration of the cardiac tissue. However, specific mechanisms that lead to ECM-mediated cardiac regeneration are not well understood. To elucidate the potential mechanisms, we investigated ultra-structures of the cardiac ECM using electron microscopy. Intriguingly, we observed large quantities of micro-vesicles from decellularized right atria. RNA and protein analyses revealed that these contained exosomal proteins and microRNAs (miRNAs), which we referred to herein as ECM-derived extracellular vesicles (ECM-EVs). One particular miRNA from ECM-EVs, miR-199a-3p, promoted cell growth of isolated neonatal cardiomyocytes and sinus nodal cells by repressing homeodomain-only protein (HOPX) expression and increasing GATA-binding 4 (Gata4) acetylation. To determine the mechanisms, we knocked down Gata4 and showed that miR-199a-3p actions required Gata4 for cell proliferation in isolated neonatal cardiomyocytes and sinus nodal cells. To further explore the role of this miRNA, we isolated neonatal cardiac cells and recellularized into atrial ECM, referred here has engineered atria. Remarkably, miR-199a-3p mediated the enrichment of cardiomyocyte and sinus nodal cell population, and enhanced electrocardiographic signal activity of sinus nodal cells in the engineered atria. Importantly, antisense of miRNA (antagomir) against miR-199a-3p was capable of abolishing these actions of miR-199a-3p in the engineered atria. We further showed in Ang II-infused animal model of sinus nodal dysfunction that miR-199-3p-treated cardiac cells remarkably ameliorated and restored the electrical activity as shown by normalization of the ECG, in contrast to untreated cells, which did not show electrical recovery. In conclusion, these results provide clear evidence of the critical role of ECM, in not only providing a scaffold for cardiac tissue growth, but also in promoting atrial electrical function through ECM-derived miR-199a-3p.

Functional variation of SHP-2 promoter is associated with preterm birth and delayed myelination and motor development in preterm infants.

Src homology 2 domain-containing protein tyrosine phosphatase 2 (SHP-2) is a cytoplasmic tyrosine phosphatase that is highly expressed in hematopoietic cells and in the CNS and exerts opposite effects on signal transduction by exerting a neuroprotective or proapoptotic effect. Several mutations of SHP-2 have been found in children with myeloproliferative disorders or malignant leukemia, and some of these can affect brain development. In the present study, we aimed to identify and functionally characterize genetic variations in SHP-2 in 72 preterm and 58 full-term infants and to evaluate the effect of the variations on neurodevelopment in preterm infants. Twelve genetic variations were identified. Among them, two variations in the SHP-2 promoter, g.-317C > T and g.-273G > A, were found to significantly increase promoter activity, and the frequency of g.-273G > A was higher in preterm infants than in full-term infants. Two transcription factors, NF-κB and GABPα, were found to be involved in the transcriptional regulation of SHP-2 by the two above-mentioned variations. In particular, we found that g.-273G > A was significantly associated with delayed myelination and poor motor development in preterm infants. Our results suggest that a functional promoter variation in SHP-2 is associated with spontaneous preterm birth itself as well as white matter myelination and neurodevelopment.

Association study between OCTN1 functional haplotypes and Crohn's disease in a Korean population.

Crohn's disease (CD) is a chronic inflammatory bowel disease with multifactorial causes including environmental and genetic factors. Several studies have demonstrated that the organic cation/carnitine transporter 1 (OCTN1) non-synonymous variant L503F is associated with susceptibility to CD. However, it was reported that L503F is absent in Asian populations. Previously, we identified and functionally characterized genetic variants of the OCTN1 promoter region in Koreans. In that study, four variants demonstrated significant changes in promoter activity. In the present study, we determined whether four functional variants of the OCTN1 promoter play a role in the susceptibility to or clinical course of CD in Koreans. To examine it, the frequencies of the four variants of the OCTN1 promoter were determined by genotyping using DNA samples from 194 patients with CD and 287 healthy controls. Then, associations between genetic variants and the susceptibility to CD or clinical course of CD were evaluated. We found that susceptibility to CD was not associated with OCTN1 functional promoter variants or haplotypes showing altered promoter activities in in vitro assays. However, OCTN1 functional promoter haplotypes showing decreased promoter activities were significantly associated with a penetrating behavior in CD patients (HR=2.428, p=0.009). Our results suggest that the OCTN1 functional promoter haplotypes can influence the CD phenotype, although these might not be associated with susceptibility to this disease.

Functional characterization of ABCB4 mutations found in progressive familial intrahepatic cholestasis type 3.

Multidrug resistance 3 (MDR3), encoded by the ATP-binding cassette, subfamily B, member 4 gene (ABCB4), localizes to the canalicular membrane of hepatocytes and translocates phosphatidylcholine from the inner leaflet to the outer leaflet of the canalicular membrane. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic disease caused by genetic mutations of ABCB4. In this study, we characterized 8 ABCB4 mutations found in PFIC3 patients, using in vitro molecular assays. First, we examined the transport activity of each mutant by measuring its ATPase activity using paclitaxel or phosphatidylcholine. Then, the pathogenic mechanisms by which these mutations affect MDR3 were examined through immunoblotting, cell surface biotinylation, and immunofluorescence. As a result, three ABCB4 mutants showed significantly reduced transport activity. Among these mutants, one mutation A364V, located in intracellular domains, markedly decreased MDR3 expression on the plasma membrane, while the others did not affect the expression. The expression of MDR3 on the plasma membrane and transport activity of A364V was rescued by a pharmacological chaperone, cyclosporin A. Our study provides the molecular mechanisms of ABCB4 mutations and may contribute to the understanding of PFIC3 pathogenesis and the development of a mutation-specific targeted treatment for PFIC3.

Identification of OCTN2 variants and their association with phenotypes of Crohn's disease in a Korean population.

Crohn's disease (CD) is a chronic inflammatory bowel disease and a genetic variant in the OCTN2, g.-207G > C is significantly associated with CD susceptibility. This study was aimed to identify novel OCTN2 functional promoter variants and their roles in transcriptional regulation using various in vitro assays. In addition, we investigated the association between OCTN2 genotypes and CD through genetic analysis using DNA samples from 193 patients with CD and 281 healthy controls. Among the three major promoter haplotypes of OCTN2 identified, one haplotype, H3, showed a significant decrease in promoter activity: two polymorphisms in H3 were associated with a significant reduction in promoter activity. In particular, we found that the reduced transcriptional activity of those two polymorphisms results from a reduction in the binding affinity of the activators, NF-E2 and YY1, to the OCTN2 promoter. The functional haplotype of the OCTN2 promoter was associated with clinical course of CD such as the disease behavior and need for surgery. However, genetic variants or haplotypes of OCTN2 did not affect the susceptibility to CD. Our results suggest that a common promoter haplotype of OCTN2 regulates the transcriptional rate of OCTN2 and influences the clinical course of CD.

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.

Benzylideneacetophenone derivatives attenuate IFN-γ-induced IP-10/CXCL10 production in orbital fibroblasts of patients with thyroid-associated ophthalmopathy through STAT-1 inhibition.

The aim of the present study was to identify a new candidate anti-inflammatory compound for use in the active stage of thyroid-associated ophthalmopathy (TAO). Benzylideneacetophenone compound JC3 [(2E)-3-(4-hydroxy-3-methoxyphenyl)phenylpro-2-en-l-one] was synthesized based on a structural modification of yakuchinone B, a constituent of the seeds of Alpinia oxyphylla, which belongs to the ginger family (Zingiberaceae), has been widely used in folk medicine as an anti-inflammatory phytochemical. Orbital fibroblasts were primarily cultured from patients with TAO, and the potential of JC3 to suppress the interferon (IFN)-γ-induced protein (IP)-10/CXCL10 production in these cells was determined. IFN-γ strongly increased the level of IP-10/CXCL10 in orbital fibroblasts from patients with TAO. JC3 exerted a significant inhibitory effect on the IFN-γ-induced increase in IP-10/CXCL10 in a dose-dependent manner; its potency was greater than that of an identical concentration of yakuchinone B with no toxicity to cells at the concentration range used. Moreover, the constructed dimer and trimer polystructures of JC3, showed greater potency than JC3 in suppressing the IFN-γ-induced production of IP-10/CXCL10. JC3 significantly attenuated the IP-10/CXCL10 mRNA expression induced by IFN-γ, and a gel-shift assay showed that JC3 suppressed IFN-γ-induced DNA binding of signal transducer and activator of transcription-1 (STAT-1) in TAO orbital fibroblasts. Our results provide initial evidence that the JC3 compound reduces the levels of IP-10/CXCL10 protein and mRNA induced by IFN-γ in orbital fibroblasts of TAO patients. Therefore, JC3 might be considered as a future candidate for therapeutic application in TAO that exerts its effects by modulating the pathogenic mechanisms in orbital fibroblasts.

Identification and Functional Characterization of Novel Genetic Variations in the OCTN1 Promoter.

Human organic cation/carnitine transporter 1 (OCTN1) plays an important role in the transport of drugs and endogenous substances. It is known that a missense variant of OCTN1 is significantly associated with Crohn's disease susceptibility. This study was performed to identify genetic variants of the OCTN1 promoter in Korean individuals and to determine their functional effects. First, the promoter region of OCTN1 was directly sequenced using genomic DNA samples from 48 healthy Koreans. OCTN1 promoter activity was then measured using a luciferase reporter assay in HCT-116 cells. Seven variants of the OCTN1 promoter were identified, two of which were novel. There were also four major OCTN1 promoter haplotypes. Three haplotypes (H1, H3, and H4) showed decreased transcriptional activity, which was reduced by 22.9%, 23.0%, and 44.6%, respectively (p<0.001), compared with the reference haplotype (H2). Transcription factor binding site analyses and gel shift assays revealed that NF-Y could bind to the region containing g.-1875T>A, a variant present in H3, and that the binding affinity of NF-Y was higher for the g.-1875T allele than for the g.-1875A allele. NF-Y could also repress OCTN1 transcription. These data suggest that three OCTN1 promoter haplotypes could regulate OCTN1 transcription. To our knowledge, this is the first study to identify functional variants of the OCTN1 promoter.