Levels Of Several Transcripts Research Articles

Expression Profiling Identified TRPM7 and HER2 as Potential Targets for the Combined Treatment of Cancer Cells.

TRPM7 is a divalent cation-permeable channel that is highly active in cancer cells. The pharmacological inhibitors of TRPM7 have been shown to suppress the proliferation of tumor cells, highlighting TRPM7 as a new anticancer drug target. However, the potential benefit of combining TRPM7 inhibitors with conventional anticancer therapies remains unexplored. Here, we used genome-wide transcriptome profiling of human leukemia HAP1 cells to examine cellular responses caused by the application of NS8593, the potent inhibitor of the TRPM7 channel, in comparison with two independent knockout mutations in the TRPM7 gene introduced by the CRISPR/Cas9 approach. This analysis revealed that TRPM7 regulates the expression levels of several transcripts, including HER2 (ERBB2). Consequently, we examined the TRPM7/HER2 axis in several non-hematopoietic cells to show that TRPM7 affects the expression of HER2 protein in a Zn2+-dependent fashion. Moreover, we found that co-administration of pharmacological inhibitors of HER2 and TRPM7 elicited a synergistic antiproliferative effect on HER2-overexpressing SKBR3 cells but not on HER2-deficient MDA-MB-231 breast cancer cells. Hence, our study proposes a new combinatorial strategy for treating HER2-positive breast cancer cells.

WHIRLY1 functions in the nucleus to regulate barley leaf development and associated metabolite profiles.

The WHIRLY (WHY) DNA/RNA binding proteins fulfil multiple but poorly characterised functions in leaf development. Here, we show that WHY1 transcript levels were highest in the bases of 7-day old barley leaves. Immunogold labelling revealed that the WHY1 protein was more abundant in the nuclei than the proplastids of the leaf bases. To identify transcripts associated with leaf development we conducted hierarchical clustering of differentially abundant transcripts along the developmental gradient of wild-type leaves. Similarly, metabolite profiling was employed to identify metabolites exhibiting a developmental gradient. A comparative analysis of transcripts and metabolites in barley lines (W1–1 and W1–7) lacking WHY1, which show delayed greening compared with the wild type revealed that the transcript profile of leaf development was largely unchanged in W1–1 and W1–7 leaves. However, there were differences in levels of several transcripts encoding transcription factors associated with chloroplast development. These include a barley homologue of the Arabidopsis GATA transcription factor that regulates stomatal development, greening and chloroplast development, NAC1; two transcripts with similarity to Arabidopsis GLK1 and two transcripts encoding ARF transcriptions factors with functions in leaf morphogenesis and development. Chloroplast proteins were less abundant in the W1–1 and W1–7 leaves than the wild type. The levels of tricarboxylic acid cycle metabolites and GABA were significantly lower in WHY1 knockdown leaves than the wild type. This study provides evidence that WHY1 is localised in the nuclei of leaf bases, contributing the regulation of nuclear-encoded transcripts that regulate chloroplast development.

The Pet127 protein is a mitochondrial 5'-to-3' exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts.

Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5′ end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5′-to-3′ exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady-state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans.

The Characterization of Sex Differences in Hypoglycemia-Induced Activation of HPA Axis on the Transcriptomic Level.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis using an insulin tolerance test (ITT) is a medical diagnostic procedure that is frequently used in humans to assess the HPA and growth-hormone (GH) axes. Whether sex differences exist in the response to ITT stress is unknown. Thus, investigations into the analysis of transcripts during activation of the HPA axis in response to hypoglycemia have revealed the underlying influences of sex in signaling pathways that stimulate the HPA axis. We assessed four time points of ITT application in Balb/c mice. After insulin injection, expression levels of 192 microRNAs and 41 mRNAs associated with the HPA, GHand hypothalamic-pituitary-gonadal (HPG) axes were determined by real-time RT-PCR in the hypothalamus, pituitary and adrenal tissues, as well as blood samples (Raw data accession: https://drive.google.com/drive/folders/10qI00NAtjxOepcNKxSJnQbJeBFa6zgHK?usp=sharing ). Although the ITT is commonly used as a gold standard for evaluating the HPA axis, we found completely different responses between males and females with respect to activation of the HPA axis. While activation of several transcripts in the hypothalamus and pituitary was observed after performing the ITT in males within 10min, females responded via the pituitary and adrenal immediately and durably over 40min. Additionally, we found that microRNA alterations precede mRNA responses in the HPA axis. Furthermore, robust changes in the levels of several transcripts including Avpr1b and Avpr2 observed at all time points strongly suggest that transcriptional control of these genes occurs mostly via differential signaling in pituitary and blood between males and females. Male and female HPA axis responses to ITT involve a number of sophisticated regulatory signaling pathways of miRNAs and mRNAs. Our results highlight the first robust markers in several layers of HPA, HPG and GH axis involved in ITT/hypoglycemia stress-induced dynamics.

MCPIP1 ribonuclease can bind and cleave AURKA mRNA in MYCN-amplified neuroblastoma cells

ABSTRACT The role of the inflammation-silencing ribonuclease, MCPIP1 (monocyte chemoattractant protein-induced protein 1), in neoplasia continuous to emerge. The ribonuclease can cleave not only inflammation-related transcripts but also some microRNAs (miRNAs) and viral RNAs. The suppressive effect of the protein has been hitherto suggested in breast cancer, clear cell renal cell carcinoma, osteosarcoma, and neuroblastoma. Our previous results have demonstrated a reduced levels of several oncogenes, as well as inhibited growth of neuroblastoma cells upon MCPIP1 overexpression. Here, we investigate the mechanisms underlying the suppression of MYCN proto-oncogene, bHLH transcription factor (MYCN)-amplified neuroblastoma cells overexpressing the MCPIP1 protein. We showed that the levels of several transcripts involved in cell cycle progression decreased in BE(2)-C and KELLY cells overexpressing MCPIP1 in a ribonucleolytic activity-dependent manner. However, RNA immunoprecipitation indicated that only AURKA mRNA (encoding for Aurora A kinase) interacts with the ribonuclease. Furthermore, the application of a luciferase assay suggested MCPIP1-dependent destabilization of the transcript. Further analyses demonstrated that the entire conserved region of AURKA seems to be indispensable for the interaction with the MCPIP1 protein. Additionally, we examined the effect of the ribonuclease overexpression on the miRNA expression profile in MYCN-amplified neuroblastoma cells. However, no significant alterations were observed. Our data indicate a key role of the binding and cleavage of the AURKA transcript in an MCPIP1-dependent suppressive effect on neuroblastoma cells.

Transcriptome alterations of prefrontal cortical parvalbumin neurons in schizophrenia.

Schizophrenia is associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC). This dysfunction is manifest as cognitive deficits that appear to arise from disturbances in gamma frequency oscillations. These oscillations are generated in DLPFC layer 3 via reciprocal connections between pyramidal cells and parvalbumin (PV)-containing interneurons. The density of cortical PV neurons is not altered in schizophrenia, but expression levels of several transcripts involved in PV cell function, including PV, are lower in the disease. However, the transcriptome of PV cells has not been comprehensively assessed in a large cohort of subjects with schizophrenia. In this study, we combined an immunohistochemical approach, laser microdissection, and microarray profiling to analyze the transcriptome of DLPFC layer 3 PV cells in 36 matched pairs of schizophrenia and unaffected comparison subjects. Over 800 transcripts in PV neurons were identified as differentially-expressed in schizophrenia subjects; most of these alterations have not previously been reported. The altered transcripts were enriched for pathways involved in mitochondrial function and tight junction signaling. Comparison with the transcriptome of layer 3 pyramidal cells from the same subjects revealed both shared and distinct disease-related effects on gene expression between cell types. Furthermore, network structures of gene pathways differed across cell types and subject groups. These findings provide new insights into cell type-specific molecular alterations in schizophrenia which may point toward novel strategies for identifying therapeutic targets.

Characterization of S. cerevisiae Phenotypes Associated with Cancer‐Causing Mutations in Hsh155p

The eukaryotic spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs) and many other associated protein splicing factors. The human U2 snRNP‐associated protein, SF3B1, allows the U2 snRNP to associate with and define the branchpoint region of precursor‐messenger RNA (pre‐mRNA). Recent evidence has shown that mutations to SF3B1 are linked to a variety of different myelodysplastic syndromes (MDS). These mutations are found in regions of high conservation between SF3B1 and the yeast homolog, Hsh155p. We have constructed a genetic system in S. cerevisiae to interrogate the effects of these mutations on splicing and cell proliferation. We used plasmid shuffling to introduce Hsh155p mutant proteins into a HSH155Δ yeast strain and growth assays to assess the impact of the mutations on cell proliferation. Several of the mutations surveyed thus far (H331D, K335N, P369E, D450G) have no obvious effect on yeast growth despite being correlated with MDS in humans. This may imply that the effects of these mutations are subtle and do not allow splicing to become limiting for yeast growth. To further understand the effects of these mutations, a Reverse Transcription‐Polymerase Chain Reaction (RT‐PCR) analysis will be used to compare pre‐mRNA and mRNA levels of several transcripts from Hsh155p mutant strains relative to the WT. The results from these experiments will be critical for establishing yeast as a model system for studying MDS. This research has been supported by startup funds from the U. Wisconsin‐Madison, WARF, and the Department of Biochemistry.

Genome-scale transcriptome analysis in response to nitric oxide in birch cells: implications of the triterpene biosynthetic pathway.

Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in Betula platyphylla cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10−5) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.

Transcriptome analysis of an endoparasitoid wasp Cotesia chilonis (Hymenoptera: Braconidae) reveals genes involved in successful parasitism.

For successful parasitization, parasitiods usually depend on the chemosensory cues for the selection of hosts, as well as a variety of virulence factors introduced into their hosts to overcome host immunity and prevent rejection of progeny development. In bracovirus-carrying wasps, the symbiotic polydnaviruses act in manipulating development and immunity of hosts. The endoparasitoid Cotesia chilonis carrying bracovirus as a key host immunosuppressive factor is a superior endoparasitoid of rice stem borer, Chilo suppressalis. So far, genomic information for C. chilonis is not available and transcriptomic data may provide valuable resources for global studying on physiological processes of C. chilonis, including chemosensation and parasitism at molecular level. Here, we performed RNA-seq to characterize the transcriptome of C. chilonis adults. We obtained 27,717,892 reads, assembled into 38,318 unigenes with a mean size of 690 bp. Approximately, 62.1% of the unigenes were annotated using NCBI databases. A large number of chemoreception-related genes encoding proteins including odorant receptors, gustatory receptors, odorant-binding proteins, chemosensory proteins, transient receptor potential ion channels, and sensory neuron membrane proteins were identified in silico. Totally, 72 transcripts possessing high identities with the bracovirus-related genes were identified. We investigated the mRNA expression levels of several transcripts at different developmental stages (including egg, larva, pupae, and adult) by quantitative real-time PCR analysis. The results revealed that some genes had adult-specific expression, indicating their potential significance for mating and parasitism. Overall, these results provide comprehensive insights into transcriptomic data of a polydnavirus-carrying parasitoid of a rice pest.

A Lotus japonicus mutant defective in nitrate uptake is also affected in the nitrate response to nodulation

A chlorate-resistant mutant (Ljclo1) of the model legume Lotus japonicus was identified that showed normal levels of nitrate reductase enzyme activity but had decreased uptake of nitrate, as determined from nitrate depletion and electrophysiological measurements. The data suggest that the mutant could be affected specifically in the low-affinity but not in the high-affinity nitrate transport system, and also showed decreased uptake of chlorate. Back-crosses of the mutant plant to the wild type indicated that it is affected in a single Mendelian recessive trait. Thus, the mutation produced in Ljclo1 may be related to some of the low-affinity nitrate transporters or to a regulatory mechanism associated with nitrate/chlorate uptake. Both size and chlorophyll content in young leaves of the mutant plants were significantly reduced compared to the wild type. In addition, nodulation performance of the mutant plants was similar to the wild type in the absence of any exogenous nitrate. However, the nodule:root biomass ratio in mutant plants was considerably reduced in the presence of 1-2mm nitrate. The levels of several transcripts for nitrate transport and assimilation genes were determined for the wild type and mutant plants and were slightly different. The results suggest interdependence between nitrate uptake, plant growth and nodulation in Ljclo1 mutant plants.

Differential Transcriptome Analysis between Paulownia fortunei and Its Synthesized Autopolyploid

Paulownia fortunei is an ecologically and economically important tree species that is widely used as timber and chemical pulp. Its autotetraploid, which carries a number of valuable traits, was successfully induced with colchicine. To identify differences in gene expression between P. fortunei and its synthesized autotetraploid, we performed transcriptome sequencing using an Illumina Genome Analyzer IIx (GAIIx). About 94.8 million reads were generated and assembled into 383,056 transcripts, including 18,984 transcripts with a complete open reading frame. A conducted Basic Local Alignment Search Tool (BLAST) search indicated that 16,004 complete transcripts had significant hits in the National Center for Biotechnology Information (NCBI) non-redundant database. The complete transcripts were given functional assignments using three public protein databases. One thousand one hundred fifty eight differentially expressed complete transcripts were screened through a digital abundance analysis, including transcripts involved in energy metabolism and epigenetic regulation. Finally, the expression levels of several transcripts were confirmed by quantitative real-time PCR. Our results suggested that polyploidization caused epigenetic-related changes, which subsequently resulted in gene expression variation between diploid and autotetraploid P. fortunei. This might be the main mechanism affected by the polyploidization. Our results represent an extensive survey of the P. fortunei transcriptome and will facilitate subsequent functional genomics research in P. fortunei. Moreover, the gene expression profiles of P. fortunei and its autopolyploid will provide a valuable resource for the study of polyploidization.

Biochemical, Cellular and Electrophysiological Characterization of HMCL-7304 a Human Skeletal Muscle-Derived Cell Line

In the past years a number of techniques have been developed to isolated single muscle fibers from small rodents allowing detailed investigations of the excitation contraction (EC) coupling mechanism at the ultrastructural, biochemical and cellular levels in normal and pathological conditions. Because of their high degree of differentiation and specialization however, it is difficult to maintain differentiated muscle fibers in culture for more than a few days and it is nearly impossible to obtain mature fibers starting from precursor satellite cells. However, starting from newborn mice one can obtain cultures of contracting and striated myotubes that can be used for a number of manipulations. As to human muscle cells, primary cultures can be obtained in vitro by culturing satellite cells from biopsies and differentiating them into myotubes, but there is a clear necessity to develop cell lines from control and diseased individuals which will develop into myotubes. In the present investigation we characterized for the first time the excitation contraction coupling machinery of HMCL-7304 an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor whereas super resolution structured illumination microscopy (3D-SIM) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in calcium homeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow twitch than fast twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in calcium homeostasis due to mutations in genes involved in calcium regulation.

Establishment of a human skeletal muscle-derived cell line: biochemical, cellular and electrophysiological characterization

Excitation-contraction coupling is the physiological mechanism occurring in muscle cells whereby an electrical signal sensed by the dihydropyridine receptor located on the transverse tubules is transformed into a chemical gradient (Ca2+ increase) by activation of the ryanodine receptor located on the sarcoplasmic reticulum membrane. In the present study, we characterized for the first time the excitation-contraction coupling machinery of an immortalized human skeletal muscle cell line. Intracellular Ca2+ measurements showed a normal response to pharmacological activation of the ryanodine receptor, whereas 3D-SIM (super-resolution structured illumination microscopy) revealed a low level of structural organization of ryanodine receptors and dihydropyridine receptors. Interestingly, the expression levels of several transcripts of proteins involved in Ca2+ homoeostasis and differentiation indicate that the cell line has a phenotype closer to that of slow-twitch than fast-twitch muscles. These results point to the potential application of such human muscle-derived cell lines to the study of neuromuscular disorders; in addition, they may serve as a platform for the development of therapeutic strategies aimed at correcting defects in Ca2+ homoeostasis due to mutations in genes involved in Ca2+ regulation.

The small molecule, genistein, increases hepcidin expression in human hepatocytes

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP6) and interleukin-6 (IL-6) by effects on mothers against decapentaplegic homolog (Smad)4 or signal transducer and activator of transcription (Stat)3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone, genistein, from 28-52 hours postfertilization in zebrafish embryos enhanced Hepcidin transcript levels, as assessed by whole-mount in situ hybridization and quantitative real-time reverse-transcriptase polymerase chain reaction. Genistein's stimulatory effect was conserved in human hepatocytes: Genistein treatment of HepG2 cells increased both Hepcidin transcript levels and promoter activity. We found that genistein's effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either BMP response elements or the Stat3-binding site in the Hepcidin promoter. RNA sequencing of transcripts from genistein-treated hepatocytes indicated that genistein up-regulated 68% of the transcripts that were up-regulated by BMP6; however, genistein raised levels of several transcripts involved in Stat3 signaling that were not up-regulated by BMP6. Chromatin immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. Conclusion: Genistein is the first small-molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes. (Hepatology 2013;58:1315–1325)

Increased ID2 Levels in Adult Precursor B Cells as Compared with Children Is Associated with Impaired Ig Locus Contraction and Decreased Bone Marrow Output

Precursor B cell production from bone marrow in mice and humans declines with age. Because the mechanisms behind are still unknown, we studied five precursor B cell subsets (ProB, PreBI, PreBII large, PreBII small, immature B) and their differentiation-stage characteristic gene expression profiles in healthy individual toddlers and middle-aged adults. Notably, the composition of the precursor B cell compartment did not change with age. The expression levels of several transcripts encoding V(D)J recombination factors were decreased in adults as compared with children: RAG1 expression was significantly reduced in ProB cells, and DNA-PKcs, Ku80, and XRCC4 were decreased in PreBI cells. In contrast, TdT was 3-fold upregulated in immature B cells of adults. Still, N-nucleotides, P-nucleotides, and deletions were similar for IGH and IGK junctions between children and adults. PreBII large cells in adults, but not in children, showed highly upregulated expression of the differentiation inhibitor, inhibitor of DNA binding 2 (ID2), in absence of changes in expression of the ID2-binding partner E2A. Further, we identified impaired Ig locus contraction in adult precursor B cells as a likely mechanism by which ID2-mediated blocking of E2A function results in reduced bone marrow B cell output in adults. The reduced B cell production was not compensated by increased proliferation in adult immature B cells, despite increased Ki67 expression. These findings demonstrate distinct regulatory mechanisms in B cell differentiation between adults and children with a central role for transcriptional regulation of ID2.

Bone marrow osteoblast vulnerability to chemotherapy

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.

Effects of nuclear receptor transactivation on boar taint metabolism and gene expression in porcine hepatocytes

The accumulation of the testicular steroid androstenone (AND) and tryptophan degradation product skatole (3MI) in fat results in boar taint, an off odor and flavor in boar meat. Increasing boar taint metabolism in the liver may help limit the deposition of AND and 3MI in fat, thereby improving meat quality. The effects of transactivation of the nuclear receptors constitutive androstane receptor (CAR), pregnane X receptor (PXR), and farnesoid X receptor (FXR) on the expression levels of several transcripts of interest and the metabolism of AND and 3MI in primary porcine hepatocytes were tested. Primary cells were isolated from mature boars, and transcript expression levels were assayed using real-time PCR. The transcripts of interest included porcine orthologs of common phase I and phase II metabolic enzymes and transcripts previously shown to be differentially expressed in boars with high boar taint levels. Transactivation of CAR, PXR, or FXR resulted in altered expression of several transcripts, including increased expression of cytochrome P450 (CYP) 2B22 by CAR, of CYP2A19, CYP2B22, CYP2C33, and CYP2C49 by PXR, of CYP2C33 and CYP2E1 by FXR, and of CYP19A2 by all three receptors. Only transactivation of PXR had a significant effect on AND metabolism, resulting in 7.5±1.5% of the initial level of AND remaining compared to 21.4±3.1% remaining with control dimethyl sulfoxide (DMSO) treatment. FXR had the greatest effect on 3MI metabolism, increasing the expression of CYP2E1 by 1.29-fold and increasing the production of the key metabolite 6-hydroxy-3-methylindole (6-OH-3MI), while decreasing 5-hydroxy-3-methylindole (5-OH-3MI) production. 3-Hydroxy-3-methyloxindole (HMOI) production was increased by CAR transactivation, while indol-3-carbinol (I3C) production was increased by PXR and FXR transactivation, and by treatment with 5β-dihydrotestosterone (5β-DHT). From this, it can be concluded that selective transactivation of PXR and FXR may be a viable means of decreasing boar taint by increasing the hepatic metabolism of AND and 3MI.

Mo2042 Identification of a Novel CFTR Interactome in Contributing Diarrhea in Inflammatory Bowel Disease

Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay,macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.

Mo2043 Protein Kinase D1 (Pkd1) Activation is an Early Event in Wounded Monolayers of Intestinal Epithelial Cells

Background: Inflammatory bowel disease (IBD) is a group of disorders in which the gut suffers from inflammation. Among the few predisposing genetic factors identified in IBD, a cGMP/cAMP-regulated chloride (Cl-) channel CFTR has emerged as one. Our lab and others have reported involvement of CFTR in formation of multiple protein complexes that can regulate the channel function in healthy and diseased states. In our current study, we aim to elucidate a molecular mechanism in the form of a signaling protein complex of CFTR that contributes to disease progression in IBD. Method: (a) Protein-protein interactions were studied using biochemical methods (protein pairwise binding assay,macromolecular complex assembly assay, and co-immunoprecipitation) and In Vitro methods for studying protein interaction in live cell (N-FRETc) in HEK293 cells. (b) Immunohistochemistry was done on mouse colon tissue for studying protein expression and distribution. Real time PCR was performed to monitor relative expression levels of several transcripts under inflammatory conditions. (c) CFTR dependent chloride secretion was monitored using iodide efflux, chloride efflux and short circuit measurements in colonic epithelial cell line HT29CL19A. (d) Dextran sodium sulfate (DSS) induced colitis mouse model was used in Nherf2+/+ and Nherf2-/C57BL/6 male mice 7-8 weeks of age. Results: (a) Upregulation of iNOS increased CFTR channel function by at least two folds in In Vivo and In Vitro models of inflammation. The phenomenon involves cGMP dependent signaling mechanism. (b) CFTR interacts with iNOS mediated by NHERF2 in a PDZ domain-dependent manner. (c) Diarrhea is attenuated in Nherf2-/compared to Nherf2+/+ mice in DSS-induced colitis model which simulates IBD. Conclusion: (a) A macromolecular complex of CFTR, NHERF2 and iNOS is integrative to CFTR dependent diarrhea in IBD. This compartmentalization of proteins and consequently of the signaling effectors enables a rapid and fine-tuned regulation of CFTR function. (b) Such signaling modules that are part of pathological profile can be approached as targets for improvement of IBD conditions.

Next-generation sequencing identifies TGF-β1-associated gene expression profiles in renal epithelial cells reiterated in human diabetic nephropathy

Transforming growth factor-beta (TGF-β1) is implicated in the onset and progression of renal fibrosis and diabetic nephropathy (DN), leading to a loss of epithelial characteristics of tubular cells. The transcriptional profile of renal tubular epithelial cells stimulated with TGF-β1 was assessed using RNA-Seq, with 2027 differentially expressed genes identified. Promoter analysis of transcription factor binding sites in the TGF-β1 responsive gene set predicted activation of multiple transcriptional networks, including NFκB. Comparison of RNA-Seq with microarray data from identical experimental conditions identified low abundance transcripts exclusive to RNA-Seq data. We compared these findings to human disease by analyzing transcriptomic data from renal biopsies of patients with DN versus control groups, identifying a shared subset of 179 regulated genes. ARK5, encoding an AMP-related kinase, and TGFBI — encoding transforming growth factor, beta-induced protein were induced by TGF-β1 and also upregulated in human DN. Suppression of ARK5 attenuated fibrotic responses of renal epithelia to TGF-β1 exposure; and silencing of TGFBI induced expression of the epithelial cell marker — E-cadherin. We identified low abundance transcripts in sequence data and validated expression levels of several transcripts (ANKRD56, ENTPD8) in tubular enriched kidney biopsies of DN patients versus living donors. In conclusion, we have defined a TGF-β1-driven pro-fibrotic signal in renal epithelial cells that is also evident in the DN renal transcriptome.