Gene Expression In Cultures Research Articles

Simvastatin Abrogates TGF-β Induced Stimulation of Normal Human Dermal Fibroblast Collagen Gene Expression in Culture

A potent stimulation of fibroblast collagen production is one of the crucial pleotropic effects of transforming growth factor β (TGF-β) and has been considered to play a crucial role in the pathogenesis of fibrotic diseases including Systemic Sclerosis and Pulmonary Fibrosis. This complex process involves numerous intracellular reactions mediated by canonical Smad-dependent or non-canonical pathways that transduce the extracellular stimuli into the nucleus. Here, we demonstrated that Simvastatin, a widely used statin, induces a potent inhibition of TGF-β1 profibrotic effects in cultured normal human dermal fibroblasts, and studied the molecular mechanisms involved in these effects. We also examined Simvastatin modulation of TGF-β1 induced fibroblast to myofibroblast transition. Normal human dermal fibroblasts were cultured with various concentrations of Simvastatin in the presence or absence of TGF-β1 (10ng/ml) for 24, 48, and 72 h. The effects of Simvastatin on TGF-β1 stimulation of COL1A1 expression and type 1 collagen production were examined. Assessment of Smad2/3 and Erk1/2 phosphorylation, chromatin immunoprecipitation assays for Sp1 transcription factor binding to the COL1A1 proximal promoter, siRNA-mediated RhoA knockdown, and F-actin immunofluorescence microscopy was performed to examine the molecular mechanisms involved.

ANGI-08. EPIGENETIC REACTIVATION OF BAI1 SUPPRESSES TUMOR INVASION, RECURRENCE, AND SPINAL METASTASIS BY PREVENTING TGFΒ1-INDUCED MESENCHYMAL SWITCH IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells spread extensively in the brain and disseminate into the cerebrospinal fluid space, strongly restricting multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for developing effective treatments.Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in the normal brain, but its expression is lost in the majority of GBM through epigenetic silencing and restoration of its expression can inhibit glioma growth. However, whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM has not been investigated.Microarray analysis of the GBM TCGA dataset (restricted to IDHwt GBM; WHO 2021) revealed that low BAI1 mRNA expression correlates with elevated expression of many mesenchymal genes. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically suppresses cell invasion in the brain and reduces leptomeningeal dissemination of GBM cells to the spine in mouse xenografts.These experiments demonstrate that epigenetic silencing of BAI1 is important for activating the GBM invasive phenotype through TGFβ1 pathway activation. This new tumor-suppressive pathway can be epigenetically targeted to reactivate BAI1 expression in GBM patients.

SerpinE1 drives a cell-autonomous pathogenic signaling in Hutchinson–Gilford progeria syndrome

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, fatal disease caused by Lamin A mutation, leading to altered nuclear architecture, loss of peripheral heterochromatin and deregulated gene expression. HGPS patients eventually die by coronary artery disease and cardiovascular alterations. Yet, how deregulated transcriptional networks at the cellular level impact on the systemic disease phenotype is currently unclear. A genome-wide analysis of gene expression in cultures of primary HGPS fibroblasts identified SerpinE1, also known as Plasminogen Activator Inhibitor (PAI-1), as central gene that propels a cell-autonomous pathogenic signaling from the altered nuclear lamina. Indeed, siRNA-mediated downregulation and pharmacological inhibition of SerpinE1 by TM5441 could revert key pathological features of HGPS in patient-derived fibroblasts, including re-activation of cell cycle progression, reduced DNA damage signaling, decreased expression of pro-fibrotic genes and recovery of mitochondrial defects. These effects were accompanied by the correction of nuclear abnormalities. These data point to SerpinE1 as a novel potential effector and target for therapeutic interventions in HGPS pathogenesis.

CSIG-25. BAI1/ADGRB1 REGULATES TGFβ1-INDUCED MESENCHYMAL SWITCH IN MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant brain tumor in children. MB tends to metastasize to the brain meninges and subarachnoid space and the spinal cord. Leptomeningeal metastasis is frequently found at initial diagnosis and leads to tumor relapse after standard treatment. Leptomeningeal metastasis remains a major challenge and is related with poor outcome. Acquiring a better knowledge of molecular defects underlying metastatic disease is essential for the development of effective therapies. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in normal brain, but its expression is lost in the majority of medulloblastoma through epigenetic silencing. We reported that BAI1 protects p53 from Mdm2-mediated degradation and regulate tumor growth in medulloblastoma (Zhu D. et al, Cancer Cell, 2018). However, it is unclear whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in MB. Microarray analysis of the published MB dataset revealed that low BAI1 mRNA expression correlates with poor outcome and with expression of many key mesenchymal genes, including Fibronectin1, SLUG, and TWIST1. Restoration of BAI1 expression in human MB cells suppresses mesenchymal gene expression in culture, and dramatically decreases brain tumor invasion. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in MB cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically reduced tumor cell invasion of MB cells. These experiments demonstrate that epigenetic silencing of BAI1 is important for activation of the MB invasive phenotype through TGFβ1 pathway activation. Epigenetic targeting of this process by KCC-07 can reduce MB invasion.

Low folate induces abnormal neuronal maturation and DNA hypomethylation of neuronal differentiation-related genes in cultured mouse neural stem and progenitor cells

Folate deficiency in a fetus is well known to cause neurodevelopment defects and development disorders. A low level of folate is also thought to be a risk for depression in adults. We have previously shown that post-weaning low folate induces neuronal immaturity in the dentate gyrus in mice, which suggests that low folate causes neuropsychological disorders via inhibition of neuronal maturation. In this study, we examined the effects of low folate on expression and epigenetic modification of genes involved in neuronal differentiation and maturation in primary mouse neural stem/progenitor cells (NSPCs) in vitro. An increase in Nestin (NSPC marker)-positive cells was observed in cells differentiated in a low folate medium for 3 days. An increase in βIII-tubulin (Tuj1: immature neuron marker)-positive cells and a decrease in microtubule-associated protein 2 (MAP2: mature neuron marker)-positive cells were observed in cells differentiated in a low folate medium for 7 days. In these cells, mRNA levels for genes involved in neuronal differentiation and maturation were altered. Hypomethylation of DNA, but not of histone proteins, was also observed at some promoters of these neuronal genes. The level of S-adenosylmethionine (SAM), a methyl donor, was decreased in these cells. The abnormalities in neural maturation and changes in gene expression in culture under low folate conditions were partially normalized by addition of SAM (5 μM). Based on these results, decreased SAM may induce DNA hypomethylation at genes involved in neuronal differentiation and maturation under low folate conditions, and this hypomethylation may be associated with low folate-induced neuronal immaturity.

Fibroblast growth factor 2 differentially influences leptin gene expression in dispersed adipose tissue cell cultures

Re-establishing vasculature is an essential component to the wound healing process, as it is a mechanism for provision of substrates required for tissue repair. Due to the potent angiogenic properties of adipose tissue, the application of extract from ground subcutaneous fat has been utilized to facilitate angiogenesis to promote healing. Nearly all angiogenic processes are regulated through growth factors such as angiopoietin, vascular endothelial growth factor, and fibroblast growth factor (FGF). Fibroblast growth factor belongs to a family of 23 members that is involved in many physiological processes including cellular proliferation and morphological differentiation. Specifically, FGF2 facilitates vascular endothelial cell proliferation, migration, and differentiation required in vessel sprout formation. Interestingly, FGF2 is synthesized and secreted by adipocytes, a cell that also produces leptin, another potent angiogenic factor. Therefore, it is hypothesized that FGF2 stimulates leptin gene expression in adipose tissue cells. In exp 1, subcutaneous adipose tissue was collected from 4 prepubertal female pigs and enzymatically dispersed (collagenase type II) to separate mature adipocytes (MA) from all other cell types (AOC; vascular endothelial cells (VEC), preadipocytes, fibroblasts) in preparation for treatment. The MA and AOC cells were treated with porcine FGF2 (0, -11, -10, -9 M). In exp 2, subcutaneous adipose tissue was collected 6 prepubertal female pigs and processed as in exp. 1; however, following processing, MA and AOC were combined and treated with FGF2 (-11M) or FGF2 polyclonal antibody (1:100 dilution). Real time PCR was used to measure the expression of target genes and SAS was utilized to determine the effect of treatment on gene expression. In exp 1, FGF2 augmented leptin gene expression in cultured MA (P=0.03). In contrast, FGF2 did not appear to influence leptin gene expression in MA and AOC co-cultures, despite the detection of a significant decrease in the expression of FGF receptor 2 (P=0.05). In conclusion, FGF2 influences leptin gene expression in MA; however, this may be mitigated/altered through the interaction with different adipose tissue cells.

Patient-Derived Organotypic Epithelial Rafts Model Phenotypes in Juvenile-Onset Recurrent Respiratory Papillomatosis.

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the 3D tissue architecture and physiology of stratified, sequentially differentiated mucosal epithelium important in RRP disease pathogenesis. Herein we describe the establishment of JoRRP-derived primary cell populations that retain HPV genomes and viral gene expression in culture. These were directly compared to cells from matched adjacent non-diseased tissue, given the known RRP patient-to-patient variability. JoRRP papilloma versus control cells displayed decreased growth at subconfluency, with a switch to increased growth after reaching confluency, suggesting relative resistance to cell-cell contact and/or differentiation. The same papilloma cells grown as 3D organotypic rafts harbored hyperproliferation as compared to controls, with increased numbers of proliferating basal cells and inappropriately replicating suprabasal cells, mimicking phenotypes in the patient biopsies from which they were derived. These complementary model systems provide novel opportunities to elucidate disease mechanisms at distinct stages in JoRRP progression and to identify diagnostic, prognostic and therapeutic factors to personalize patient management and treatment.

Abstract 1996: Epigenetic reactivation of BAI1/ADGRB1 suppresses tumor invasion by preventing TGFβ1-induced mesenchymal switch in glioblastoma

Abstract Glioblastoma (GBM) is the most common and lethal type of malignant brain tumor in adults. GBM cells are highly invasive and diffusely infiltrate throughout the brain, which strongly restricts multimodal therapies. Acquiring a better knowledge of molecular defects underlying GBM invasion is essential for the development of effective therapies. Brain-specific Angiogenesis Inhibitor 1 (BAI1/ADGRB1) is a transmembrane receptor of the adhesion GPCR family widely expressed in normal brain, but its expression is lost in the majority of glioblastoma through epigenetic silencing and restoration of its expression can inhibit glioma growth (Zhu D. et al, Cancer Res, 2012). Recently, we reported that BAI1 protects p53 from Mdm2-mediated degradation and regulate tumor growth in medulloblastoma (Zhu D. et al, Cancer Cell, 2018). However, it is unclear whether BAI1 loss is important for tumor invasion and the mesenchymal phenotype in GBM. Microarray analysis of the GBM TCGA dataset revealed that low BAI1 mRNA expression correlates with poor outcome and with expression of many key mesenchymal genes, including Fibronectin1, SLUG, and TWIST1. Restoration of BAI1 expression in human GBM cells suppresses mesenchymal gene expression in culture, and dramatically decreases brain tumor invasion in mice xenografts. Mechanistically, we found that the N-terminal thrombospondin type 1 repeat (TSR#1) of BAI1 inhibits the maturation process of TGFβ1, a key growth factor involved in EMT. BAI1 is silenced epigenetically in GBM cells by methylated CpG-binding protein MBD2, and its expression can be reactivated by KCC-07, a blood-brain barrier permeable MBD2 inhibitor. We found that restoration of BAI1 expression by KCC-07 treatment dramatically reduced tumor cell invasion in orthotopic xenografts model of GBM cells. These experiments demonstrate that epigenetic silencing of BAI1 is important for activation of the GBM invasive phenotype through TGFβ1 pathway activation. Epigenetic targeting of this process by KCC-07 can reduce GBM invasion and improve patient survival. Citation Format: Satoru Osuka, Liquan Yang, Dan Zhu, Hideharu Hashimoto, Narra S. Devi, Erwin G. Van Meir. Epigenetic reactivation of BAI1/ADGRB1 suppresses tumor invasion by preventing TGFβ1-induced mesenchymal switch in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1996.

Direct inhibitory effect of caffeine on viability, synthesis activity and gene expression in cultures of chondrocytes extracted from the articular cartilage of rats

ABSTRACT The aim of this study was to evaluate the effect of concentrations of caffeine on the viability, synthesis activity and gene expression in cultures of chondrocytes. Extracted articular cartilage from the femurs and tibias of 15 Wistar rats at three days old to isolate chondrocytes. Chondrocytes were cultured in chondrogenic medium (control) or supplemented with caffeine (0.5, 1.0, 2.0mM). Cell viability, alkaline phosphatase activity and collagen synthesis were assessed using colorimetric assays at 7, 14, 21 days. The chondrocyte cultures of all groups grown under coverslips were stained with hematoxylin-eosin to determine the percentage of cells/field and with PAS, safranin O, alcian blue to determine the percentage of matrix chondrogenic/field at 21 days. The expressions of gene transcripts for aggrecan, collagen-II, Sox-9, Runx-2 and alkaline phosphatase were also evaluated by RT-PCR at 21 days. The means were compared using Student-Newman-Keuls. Caffeine significantly reduced the conversion of MTT to formazan, percentage of cells/field, collagen synthesis, alkaline phosphatase activity, synthesis of PAS+, safranin O+ and alcian blue+ chondrogenic matrix, and the expression of aggrecan, Sox-9 and II collagen. It is concluded that caffeine at concentrations of 0.5, 1.0, 2.0mM has a direct inhibitory effect on chondrogenesis in cultures of chondrocytes from rats.

Patterns of thaumarchaeal gene expression in culture and diverse marine environments.

Thaumarchaea are ubiquitous in marine habitats where they participate in carbon and nitrogen cycling. Although metatranscriptomes suggest thaumarchaea are active microbes in marine waters, we understand little about how thaumarchaeal gene expression patterns relate to substrate utilization and activity. Here, we report the global transcriptional response of the marine ammonia-oxidizing thaumarchaeon 'Candidatus Nitrosopelagicus brevis' str. CN25 to ammonia limitation using RNA-Seq. We further describe the genome and transcriptome of Ca. N. brevis str. U25, a new strain capable of urea utilization. Ammonia limitation in CN25 resulted in reduced expression of transcripts coding for ammonia oxidation proteins, and increased expression of a gene coding an Hsp20-like chaperone. Despite significantly different transcript abundances across treatments, two ammonia monooxygenase subunits (amoAB), a nitrite reductase (nirK) and both ammonium transporter genes were always among the most abundant transcripts, regardless of growth state. Ca. N. brevis str. U25 cells expressed a urea transporter 139-fold more than the urease catalytic subunit ureC. Gene coexpression networks derived from culture transcriptomes and 10 thaumarchaea-enriched metatranscriptomes revealed a high degree of correlated gene expression across disparate environmental conditions and identified a module of coexpressed genes, including amoABC and nirK, that we hypothesize to represent the core ammonia oxidation machinery.

DNA methylation defines regional identity of human intestinal epithelial organoids and undergoes dynamic changes during development

ObjectiveHuman intestinal epithelial organoids (IEOs) are increasingly being recognised as a highly promising translational research tool. However, our understanding of their epigenetic molecular characteristics and behaviour in culture remains limited.DesignWe...

Transcriptional Dynamics of Cultured Human Villous Cytotrophoblasts.

During human pregnancy, cytotrophoblasts (CTBs) play key roles in uterine invasion, vascular remodeling, and anchoring of the feto-placental unit. Due to the challenges associated with studying human placentation in utero, cultured primary villous CTBs are used as a model of the differentiation pathway that leads to invasion of the uterine wall. In vitro, CTBs emulate in vivo cell behaviors, such as migration, aggregation, and substrate penetration. Although some of the molecular features related to these cell behaviors have been described, the underlying mechanisms, at a global level, remain undefined at midgestation. Thus, in this study, we characterized second-trimester CTB differentiation/invasion in vitro, correlating the major morphological transitions with the transcriptional changes that occurred at these steps. After plating on Matrigel as individual cells, CTBs migrated toward each other and formed multicellular aggregates. In parallel, using a microarray approach, we observed differentially expressed (DE) genes across time, which were enriched for numerous functions, including cell migration, vascular remodeling, morphogenesis, cell communication, and inflammatory signaling. DE genes encoded several molecules that we and others previously linked to critical CTB function in vivo, suggesting that the novel DE molecules we discovered played important roles. Immunolocalization confirmed that CTBs in situ gave a signal for two of the most highly expressed genes in vitro. In summary, we characterized, at a global level, the temporal dynamics of primary human CTB gene expression in culture. These data will enable future analyses of various types of in vitro perturbations-for example, modeling disease processes and environmental exposures.

Cardiomyocyte Circadian Oscillations Are Cell-Autonomous, Amplified by β-Adrenergic Signaling, and Synchronized in Cardiac Ventricle Tissue.

Circadian clocks impact vital cardiac parameters such as blood pressure and heart rate, and adverse cardiac events such as myocardial infarction and sudden cardiac death. In mammals, the central circadian pacemaker, located in the suprachiasmatic nucleus of the hypothalamus, synchronizes cellular circadian clocks in the heart and many other tissues throughout the body. Cardiac ventricle explants maintain autonomous contractions and robust circadian oscillations of clock gene expression in culture. In the present study, we examined the relationship between intrinsic myocardial function and circadian rhythms in cultures from mouse heart. We cultured ventricular explants or dispersed cardiomyocytes from neonatal mice expressing a PER2::LUC bioluminescent reporter of circadian clock gene expression. We found that isoproterenol, a β-adrenoceptor agonist known to increase heart rate and contractility, also amplifies PER2 circadian rhythms in ventricular explants. We found robust, cell-autonomous PER2 circadian rhythms in dispersed cardiomyocytes. Single-cell rhythms were initially synchronized in ventricular explants but desynchronized in dispersed cells. In addition, we developed a method for long-term, simultaneous monitoring of clock gene expression, contraction rate, and basal intracellular Ca2+ level in cardiomyocytes using PER2::LUC in combination with GCaMP3, a genetically encoded fluorescent Ca2+ reporter. In contrast to robust PER2 circadian rhythms in cardiomyocytes, we detected no rhythms in contraction rate and only weak rhythms in basal Ca2+ level. In summary, we found that PER2 circadian rhythms of cardiomyocytes are cell-autonomous, amplified by adrenergic signaling, and synchronized by intercellular communication in ventricle explants, but we detected no robust circadian rhythms in contraction rate or basal Ca2+.

TAPP1 inhibits the differentiation of oligodendrocyte precursor cells via suppressing the Mek/Erk pathway.

Oligodendrocytes (OLs) are glial cells that form myelin sheaths around axons in the central nervous system (CNS). Loss of the myelin sheath in demyelinating and neurodegenerative diseases can lead to severe impairment of movement. Understanding the extracellular signals and intracellular factors that regulate OL differentiation and myelination during development can help to develop novel strategies for enhancing myelin repair in neurological disorders. Here, we report that TAPP1 was selectively expressed in differentiating OL precursor cells (OPCs). TAPP1 knockdown promoted OL differentiation and myelin gene expression in culture. Conversely, over-expression of TAPP1 in immature OPCs suppressed their differentiation. Moreover, TAPP1 inhibition in OPCs altered the expression of Erk1/2 but not AKT. Taken together, our results identify TAPP1 as an important negative regulator of OPC differentiation through the Mek/Erk signaling pathway.

593. Targeted Histidinylated Lipopolyplexes for siRNA Delivery in 4T1 Mammary Carcinoma Cells

Breast cancer is the leading cause of death among women aged 35 to 50 years. siRNA-based therapies aiming at reducing the overexpression of oncogenes and genes involved in tumor progression are developed for the treatment of cancer. However, clinical applications are delayed due to the absence of very efficient and safe delivery systems. For this purpose, we develop siRNA lipopolyplexes (LPRi) which are ternary complexes between cationic liposomes comprising imidazole/imidazolium lipophosphoramidates, histidinylated-lPEI cationic polymer and siRNA (1). Compared to commercial reagents, LPRi containing anti-eGFP or anti-Luc siRNA give in vitro higher specific inhibition (80% at 50nM siRNA) of reporter gene expression in cultures of eGFP-HeLa, Luc-B16F10 and Luc-4T1 cancer cells. Transfection of the 4T1 mammary carcinoma cells line with anti-survivin siRNA induces 30% reduction in cell viability. In vivo imaging after endotracheal administration of LPRi comprising polymer or liposomes labeled with a near-infrared fluorophore indicates a sustained delivery in the lung of the formulation until 24 hours post injection.For in vivo application, PEGylated LPRi has been armed with uPA peptide or folate which binds to the urokinase receptor (uPAR) and the folate receptor (FR), respectively. These targeted LPRi exhibit a 200 nm size and a reduced positive zeta potential. When tested for their uptake efficiency in 4T1 cells, targeted LPRi show a higher endocytosis compared to untargeted LPRi. When transfected with 50nM anti-Luc siRNA formulated with LPRi containing 2% Folate or 5% uPA LPRi, a better specific inhibition (54% and 40%, respectively) of luciferase expression in Luc-4T1 cells is obtained compared to untargeted LPRi (32%). Therapeutic applications of these new targeted siRNA formulations are now tested by using the murine orthotopic 4T1 breast cancer model.

Cytokine-induced sleep: Neurons respond to TNF with production of chemokines and increased expression of Homer1a in vitro.

Interactions of neurons with microglia may play a dominant role in sleep regulation. TNF may exert its somnogeneic effects by promoting attraction of microglia and their processes to the vicinity of dendrites and synapses. We found TNF to stimulate neurons (i) to produce CCL2, CCL7 and CXCL10, chemokines acting on mononuclear phagocytes and (ii) to stimulate the expression of the macrophage colony stimulating factor (M-CSF/Csf1), which leads to elongation of microglia processes. TNF may also act on neurons by affecting the expression of genes essential in sleep-wake behavior. The neuronal expression of Homer1a mRNA, increases during spontaneous and enforced periods of wakefulness. Mice with a deletion of Homer1a show a reduced wakefulness with increased non-rapid eye movement (NREM) sleep during the dark period. Recently the TNF-dependent increase of NREM sleep in the dark period of mice with CD40-induced immune activation was found to be associated with decreased expression of Homer1a. In the present study we investigated the effects of TNF and IL-1β on gene expression in cultures of the neuronal cell line HT22 and cortical neurons. TNF slightly increased the expression of Homer1a and IL-1β profoundly enhanced the expression of Early growth response 2 (Egr2). The data presented here indicate that the decreased expression of Homer1a, which was found in the dark period of mice with CD40-induced increase of NREM sleep is not due to inhibitory effects of TNF and IL-1β on the expression of Homer1a in neurons.

Triple Bioluminescence Imaging for In Vivo Monitoring of Cellular Processes

Bioluminescence imaging (BLI) has shown to be crucial for monitoring in vivo biological processes. So far, only dual bioluminescence imaging using firefly (Fluc) and Renilla or Gaussia (Gluc) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates. Here, we characterized a codon-optimized luciferase from Vargula hilgendorfii (Vluc) as a reporter for mammalian gene expression. We showed that Vluc can be multiplexed with Gluc and Fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin, coelenterazine, and D-luciferin substrates, respectively. We applied this triple imaging system to monitor the effect of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) delivered using an adeno-associated viral vector (AAV) on brain tumors in mice. Vluc imaging showed efficient sTRAIL gene delivery to the brain, while Fluc imaging revealed a robust antiglioma therapy. Further, nuclear factor-κB (NF-κB) activation in response to sTRAIL binding to glioma cells death receptors was monitored by Gluc imaging. This work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology, oncology, virology, and neuroscience.

298 Regulation and Function of BMP-4 Expression in the Inflamed Gastric Mucosa

BACKGROUND/AIMS: We previously reported that inhibition of Bone Morphogenetic Protein (BMP) signaling In Vivo, leads to enhancement of Helicobacter pylori (HP)-induced gastric inflammation suggesting that the BMPs can exert novel anti-inflammatory actions in the stomach. The aim of this study was to examine the function and the regulation of BMP-4 expression in gastric inflammation. METHODS: Inflammation was induced by infection of 3 month-old C57BL/6-BMP-4-β-gal/+ mice with HP. Animals were analyzed 2 months after inoculation. Expression of BMP-4 was assessed by X-Gal staining and QRT-PCR. Morphology of the gastric mucosa was examined in sections stained with H&E. Phosphorylation of Smad1-5-8 was determined by immunohistochemistry with anti-phospho-Smad1-5-8 antibodies (abs). Expression of INF-γ was measured by QRT-PCR. Gastric mesenchymal and mucus cells were isolated from the canine gastric mucosa and characterized by staining with anti-vimentin abs and the lectin GSII, respectively. Phosphorylation of STAT1 and ERK2 and expression of BMP-4 in the mesenchymal cells were assessed by western blots. IL-8 expression in the mucus cells was measured by QRT-PCR. INF-γ, IL-17-α, IL-12 and IL23 content in supernatants of cultures of mouse splenocytes and dendritic cells was measured by ELISA. RESULTS: Analysis of mucosal sections of C57BL/6-BMP-4-β-gal/+ mice stained with X-gal, demonstrated that BMP-4 is expressed in the mesenchymal layers of the gastric mucosa. HP induced inflammatory changes in the gastric mucosa and it stimulated the expression of both INF-γ and BMP-4. Phospho-Smad1-5-8 staining of the inflamed gastric mucosa indicated the presence of positively stained nuclei in gastric epithelial cells, but not in mesenchymal cells, or in cells of the inflammatory infiltrate. INF-γ induced the phosphorylation of both STAT-1 and ERK2 and it induced BMP-4 protein expression in the mesenchymal cells. Incubation of the cells with inhibitors of both STAT-1 and ERK2 phosphorylation abrogated the stimulatory effect of INF-γ. BMP-4 failed to affect LPS-induced release of INF-γ, IL-17-α, IL-12 and IL-23 in cultures of mouse splenocytes and dendritic cells. In contrast, BMP-4 potently inhibited both basal and TNF-α stimulated IL-8 gene expression in cultures of isolated mucus cells. CONCLUSIONS: Inflammatory stimuli induce the expression of BMP-4 both In Vivo and In Vitro, through mechanisms that appear to involve the activation of both ERK2 and STAT-1. BMP-4, in turn, inhibits the expression of inflammatory molecules in epithelial, but not in immune cells, suggesting that BMP-4 is a novel anti-inflammatoy peptide released by the mesenchyme to modulate the inflammatory response of the gastric epithelium.

Exogenous Fibroblast Growth Factor-10 Induces Cystic Lung Development with Altered Target Gene Expression in the Presence of Heparin in Cultures of Embryonic Rat Lung

Objectives: Signaling by fibroblast growth factor (FGF) receptor (FGFR) 2IIIb regulates branching morphogenesis in the mammalian lung. FGFR2IIIb is primarily expressed in epithelial cells, whereas its ligands, FGF-10 and keratinocyte growth factor (KGF; FGF-7), are expressed in mesenchymal cells. FGF-10 null mice lack lungs, whereas KGF null animals have normal lung development, indicating that FGF-10 regulates lung branching morphogenesis. In this study, we determined the effects of FGF-10 on lung branching morphogenesis and accompanying gene expression in cultures of embryonic rat lungs. Methods: Embryonic day 14 rat lungs were cultured with FGF-10 (0–250 ng/ml) in the absence or presence of heparin (30 ng/ml) for 4 days. Gene expression profiles were analyzed by Affymetrix microchip array including pathway analysis. Some of these genes, functionally important in FGF-10 signaling, were further analyzed by Northern blot, real-time PCR, in situ hybridization and immunohistochemistry. Results: Exogenous FGF-10 inhibited branching and induced cystic lung growth only in cultures containing heparin. In total, 252 upregulated genes and 164 downregulated genes were identified, and these included Spry1 (Sprouty-1), Spry2 (Sprouty-2), Spred-1, Bmp4 (bone morphogenetic protein-4, BMP-4), Shh (sonic hedgehog, SHH), Pthlh (parathyroid hormone-related protein, PTHrP), Dusp6 (MAP kinase phosphatase-3, MKP-3) and Clic4 (chloride intracellular channel-4, CLIC-4) among the upregulated genes and Igf1 (insulin-like growth factor-1, IGF-1), Tcf21 (POD), Gyg1 (glycogenin 1), Sparc (secreted protein acidic and rich in cysteine, SPARC), Pcolce (procollagen C-endopeptidase enhancer protein, Pro CEP) and Lox (lysyl oxidase) among the downregulated genes. Gsk3β and Wnt2, which are involved in canonical Wnt signaling, were up- and downregulated, respectively. Conclusions: Unlike FGF-7, FGF-10 effects on lung branching morphogenesis are heparin-dependent. Sprouty-2, BMP-4, SHH, IGF-1, SPARC and POD are known to regulate branching morphogenesis; however, potential roles of CLIC-4 and MKP-3 in lung branching morphogenesis remain to be investigated. FGF-10 may also function in regulating branching morphogenesis or inducing cystic lung growth by inhibiting Wnt2/β-catenin signaling.

Codon-OptimizedLuciola ItalicaLuciferase Variants for Mammalian Gene Expression in Culture and in Vivo

Luciferases have proven to be useful tools in advancing our understanding of biologic processes. Having a multitude of bioluminescent reporters with different properties is highly desirable. We characterized codon-optimized thermostable green- and red-emitting luciferase variants from the Italian firefly Luciola italica for mammalian gene expression in culture and in vivo. Using lentivirus vectors to deliver and stably express these luciferases in mammalian cells, we showed that both variants displayed similar levels of activity and protein half-lives as well as similar light emission kinetics and higher stability compared to the North American firefly luciferase. Further, we characterized the red-shifted variant for in vivo bioluminescence imaging. Intramuscular injection of tumor cells stably expressing this variant into nude mice yielded a robust luciferase activity. Light emission peaked at 10 minutes post-d-luciferin injection and retained > 60% of signal at 1 hour. Similarly, luciferase activity from intracranially injected glioma cells expressing the red-shifted variant was readily detected and used as a marker to monitor tumor growth over time. Overall, our characterization of these codon-optimized luciferases lays the groundwork for their further use as bioluminescent reporters in mammalian cells.