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Significant differential expression genes from HUVECs induced by bg-CAT

ABSTRACT: In vertebrates, non-lens bg-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit) and trefoil factor (b-subunit), named bg-CAT, was identified from frog Bombina maxima skin secretions. Its a-subunit and b-subunit (containing three trefoil factor domains), with a non-covalently linked form of ab2, show significant sequence homology to ep37 proteins, a group of non-lens bg-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. The bg-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of bg-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. The bg-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membrane. However, what exactly target of bg-CAT act on HUVECs nuclear is still unknown. Primary cultured HUVECs treated with bg-CAT (25 nM, 2 h) were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genome wide level of significant differential gene expression induced by bg-CAT on HUVECs in order to get clues about bg-CAT action mechanisms. These findings illustrate novel cellular functions of non-lens bg-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals.

ORGANISM(S): Homo sapiens

PROVIDER: GSE10479 | GEO | 2008/04/30

SECONDARY ACCESSION(S): PRJNA107921

REPOSITORIES: GEO

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Project description:In vertebrates, non-lens bg-crystallins are widely expressed in various tissues, but their functions are unknown. The molecular mechanisms of trefoil factors, initiators of mucosal healing and being greatly involved in tumorigenesis, have remained elusive.A naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit) and trefoil factor (b-subunit), named bg-CAT, was identified from frog Bombina maxima skin secretions. Its a-subunit and b-subunit (containing three trefoil factor domains), with a non-covalently linked form of ab2, show significant sequence homology to ep37 proteins, a group of non-lens bg-crystallins identified in newt Cynops pyrrhogaster and mammalian trefoil factors, respectively. The bg-CAT showed multiple cellular effects on human umbilical vein endothelial cells. Low dosages of bg-CAT (25-50 pM) were able to stimulate cell migration and wound healing. At high concentrations, it induced cell detachment (EC50 10 nM) and apoptosis. The bg-CAT was rapidly endocytosed via intracellular vacuole formation. Under confocal microscope, some of the vacuoles were translocated to nucleus and partially fused with nuclear membraneï®. However, what exactly target of bg-CAT act on HUVECs nuclear is still unknown. Primary cultured HUVECs treated with bg-CAT (25 nM, 2 h) were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain the genome wide level of significant differential gene expression induced by bg-CAT on HUVECs in order to get clues about bg-CAT action mechanisms. These findings illustrate novel cellular functions of non-lens bg-cyrstallins and action mechanism via association with trefoil factors, serving as clues for investigating the possible occurrence of similar molecules and action mechanisms in mammals. Experiment Overall Design: We used microarrays to analysis genome wide level of significant differential gene expression induced by bg-CAT on HUVECs.Four independent biological replicates of HUVECs treated with bg-CAT (25 nM, 2 h) were compared with normal control by SAM. The significant differential expression of 123 genes (fold changeâ¥3, q value =0, FDR (false discovery rate)=0). 121 genes were up-regulated, among which members of nuclear receptor subfamily 4 (NR4A) were the most prominent. Only 2 genes were down-regulated (including collagen type I) (Supporting information Fig. S1 and Table S1 in accompanying publication). Members of NR4A exhibit differential roles in determining cell growth or cell death depending on its location in the nucleus or mitochondria. NR4A1 (also called Nur77, TR3) mediates cell apoptosis through translocation from nucleus to mitochondria and induction of cytochrome c releasing . Significant activation of NR4A members suggests these orphan nuclear receptors may be involved in the mediation of bg-CAT biological functions, like proapoptotic action of the protein at high concentrations. Up-regulated expression of a number of matrix metalloproteinases by bg-CAT could facilitate cell migration and detachment. The bg-CAT also induced the expression of a number of cytokines, including interleukin 1, interleukin 8 and interleukin 6, as well as various chemokines in HUVECs. These data provide clues for study in detail of molecular pathways through which bg-CAT exerts its biological functions.

Project description:Although majority of the genes linked to pediatric cataract exhibit lens fiber cell-enriched expression, our understanding of gene regulation in these cells is limited to function of just eight transcription factors and largely in the context of crystallins. Here, we identify small Maf transcription factors MafG and MafK as regulators of several non-crystallin human cataract genes in fiber cells and establish their significance to cataract. We applied a bioinformatics tool for cataract gene discovery iSyTE to identify MafG and its co-regulators in the lens, and generated various null-allelic combinations of MafG:MafK mouse mutants for phenotypic and molecular analysis. By age 4-months, MafG-/-:MafK+/- mutants exhibit lens defects that progressively develop into cataract. High-resolution phenotypic characterization of MafG-/-:MafK+/- lens reveals severe defects in fiber cells, while microarrays-based expression profiling identifies 97 differentially regulated genes (DRGs). Integrative analysis of MafG-/-:MafK+/- lens-DRGs with 1) binding-motifs and genomic targets of small Mafs and their regulatory partners, 2) iSyTE lens-expression data, and 3) interactions between DRGs in the String database, unravels a detailed small Maf regulatory network in the lens, several nodes of which are linked to human cataract. This analysis prioritizes 36 highly promising candidates from the original 97 DRGs. Significantly, 8/36 (22%) DRGs are associated with cataracts in human (GSTO1, MGST1, SC4MOL, UCHL1) or mouse (Aldh3a1, Crygf, Hspb1, Pcbd1), suggesting a multifactorial etiology that includes elevation of oxidative stress. These data identify MafG and MafK as new cataract-associated candidates and define their function in regulating largely non-crystallin genes linked to mouse and human cataract. Microarray comparision of lenses from mixed background (129Sv/J, C57BL/6J, and ICR) control (MafG+/-:MafK+/-; no-cataract) and compound (MafG-/-:MafK+/-; cataract) mouse mutants

Project description:Glutathione (GSH) is a critical endogenous antioxidant that protects against intracellular oxidative stress. As such, pathological alterations in GSH levels are linked to a myriad of diseases including cancer, neurodegeneration and cataract. The rate limiting step in GSH biosynthesis is catalyzed by the glutamate cysteine ligase catalytic subunit (GCLC). The high expression of GCLC in the lens supports the synthesis of millimolar concentrations of GSH in this tissue. Herein, we describe the morphological consequences of deleting (knocking out) Gclc from surface ectoderm-derived ocular tissues (using the Le-Cre transgene; Gclc KO) which includes an overt microphthalmia phenotype and severely disrupted formation of multiple ocular structures (i.e., cornea, iris, lens, retina). Controlling for the Le-Cre transgene revealed that the deletion of Gclc significantly exacerbated the microphthalmia phenotype in Le-Cre hemizygous mice and resulted in dysregulated gene expression that was unique to only the lenses of KO mice. We further characterized the impaired lens development by conducting an RNA-seq experiment on KO and Gclc control (CON) mouse lens at the day of birth. RNA-sequencing revealed significant differences between Gclc knockout (KO) and Gclc control (CON) lenses, including down-regulation of crystallins and lens fiber cell identity genes, and up-regulation of lens epithelial cell identity genes. In addition, genes related to the immune system (e.g., immune system process, inflammatory response, neutrophil chemotaxis) were upregulated, and genes related to eye/lens development were downregulated. TRANSFAC analysis of differentially expressed genes (DEGs) in the lens of Gclc KO mice implicated PAX6 as a key upstream regulator of Gclc KO sensitive genes. This was further supported by a strong positive correlation between the transcriptomes of the lenses of Gclc KO and Pax6 KO mice. Strikingly, the dysregulation of PAX6-regulated genes in Gclc KO mice was observed despite no change in the ocular localization of PAX6 or decrease in the expression of PAX6 in the lens. In vitro experiments demonstrated that suppression of intracellular GSH concentrations resulted in impairment of PAX6 transactivation activity. Taken together, the present results elucidate a novel mechanism wherein intracellular GSH concentrations may modulate PAX6 activity.

Project description:Methylation at cytosines (mCG) is a well-known regulator of gene expression but its requirements for cellular differentiation have yet to be fully elucidated. A well-studied cellular differentiation model system is the eye lens, consisting of a single anterior layer of epithelial cells that migrate laterally and differentiate into a core of fiber cells. Here, we explore the genome-wide relationships between mCG methylation, chromatin accessibility and gene expression during differentiation of eye lens epithelial cells into fiber cells. Whole genome bisulfite sequencing identified 7621 genomic loci exhibiting significant differences in mCG levels between lens epithelial and fiber cells. Changes in mCG levels were inversely correlated with the differentiation state-specific expression of 1285 genes preferentially expressed in either lens fiber or lens epithelial cells (Pearson correlation r = -0.37, p < 1x10-42). mCG levels were inversely correlated with chromatin accessibility determined by Assay for transposase-accessible sequencing (ATAC-seq) (Pearson correlation r = -0.86, p < 1x10-300). Many of the genes exhibiting altered regions of DNA methylation, chromatin accessibility and gene expression levels in fiber cells relative to epithelial cells are associated with lens fiber cell structure, homeostasis and transparency. These include lens crystallins (CRYBA4, CRYBB1, CRYGN, CRYBB2), lens beaded filament proteins (BFSP1, BFSP2), transcription factors (HSF4, SOX2, HIF1A), and Notch signaling pathway members (NOTCH1, NOTCH2, HEY1, HES5). Analysis of regions exhibiting cell-type specific alterations in DNA methylation revealed an overrepresentation of consensus sequences of multiple transcription factors known to play key roles in lens cell differentiation including HIF1A, SOX2, and the MAF family of transcription factors. Collectively, these results link DNA methylation with control of chromatin accessibility and gene expression changes required for eye lens differentiation. The results also point to a role for DNA methylation in the regulation of transcription factors previously identified to be important for lens cell differentiation.

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Significant differential expression genes from HUVECs induced by bg-CAT

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