Defining α-synuclein species responsible for Parkinson disease phenotypes in mice
alpha-synuclein (a-synuclein); amyloid; cytotoxicity; fibril; Lewy Body; motor behavior defect; neurodegenerative disease; oligomer; Parkinson disease; protein aggregation
Parkinson disease (PD) is a neurodegenerative disorder characterized by fibrillar neuronal inclusions composed of aggregated α-synuclein. These inclusions are associated with behavioral and pathological PD phenotypes. One strategy for therapeutic interventions is to prevent the formation of these inclusions in order to halt disease progression. α-Synuclein exists in multiple structural forms, including disordered, non-amyloid oligomers, ordered amyloid oligomers, and fibrils. It is critical to understand which conformers contribute to specific PD phenotypes. Here, we utilized a mouse model to explore the pathological effects of stable amyloid β-sheet oligomers compared with those of fibrillar α-synuclein. We biophysically characterized these species with transmission EM, atomic-force microscopy, CD spectroscopy, FTIR spectroscopy, analytical ultracentrifugation, and thioflavin T assays. We then injected these different α-synuclein forms into the mouse striatum to determine their ability to induce PD-related phenotypes. We found that β-sheet oligomers produce a small but significant loss of dopamine neurons in the substantia nigra pars compacta (SNc). Injection of small β-sheet fibril fragments, however, produced the most robust phenotypes, including reduction of striatal dopamine terminals, SNc loss of dopamine neurons, and motor behavior defects. We conclude that although the β-sheet oligomers cause some toxicity, the potent effects of the short fibrillar fragments can be attributed to their ability to recruit monomeric α-synuclein and spread in vivo and hence contribute to the development of PD-like phenotypes. These results suggest that strategies to reduce the formation and propagation of β-sheet fibrillar species could be an important route for therapeutic intervention in PD and related disorders.
Froula Jessica M; Castellana-Cruz Marta; Anabtawi Nadia M; Camino José D; Chen Serene W; Thrasher Drake R; Freire Jennifer; Yazdi Allen A; Fleming Sheila; Dobson Christopher M; Kumita Janet R; Cremades Nunilo; Volpicelli-Daley Laura A
The Journal of Biological Chemistry
2019
2019-05
<a href="http://doi.org/10.1074/jbc.RA119.007743" target="_blank" rel="noreferrer noopener">10.1074/jbc.RA119.007743</a>
Orphan receptors chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) and retinoid X receptor (RXR) activate and bind the rat cholesterol 7alpha-hydroxylase gene (CYP7A).
Animals; Rats; *Gene Expression Regulation; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; Nuclear Proteins/*metabolism; Chickens; Transcription Factors/*metabolism; DNA-Binding Proteins/*metabolism; Retinoid X Receptors; COUP Transcription Factor II; COUP Transcription Factors; Circadian Rhythm; COUP Transcription Factor I; Recombinant Proteins/genetics/metabolism; Receptors; Models; Genetic; Enzymologic; Molecular; Electrophoresis; Polyacrylamide Gel; Promoter Regions; *Receptors; Steroid; Glucocorticoid/*genetics; Retinoic Acid/*metabolism
The cholesterol 7alpha-hydroxylase gene (CYP7A) is transcriptionally regulated by a number of factors, including hormones, bile acids, and diurnal rhythm. Previous studies have identified a region from nucleotides (nt) -74 to -55 of the rat CYP7A promoter that enhanced bile acid repression of the SV40 early promoter, as assayed with a luciferase reporter gene in transiently transfected HepG2 cells. The rat CYP7A promoter/reporter activity was strongly stimulated by cotransfection with an expression plasmid encoding the nuclear hormone receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) in a dose-dependent manner. Site-directed mutagenesis in the region of nt -74 to -55 altered this stimulation. Recombinant COUP-TFII expressed in HepG2 or COS-1 cells were found to bind to nt -74 -55 and nt -149 -128 probes by electrophoretic mobility shift assay (EMSA) and by supershifting the corresponding band with
Stroup D; Crestani M; Chiang J Y
The Journal of biological chemistry
1997
1997-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.
Female; Animals; Rats; Amino Acid Sequence; *Gene Expression Regulation; Kinetics; Base Sequence; Immunoblotting; Molecular Sequence Data; Steroid Hydroxylases/*genetics; Circadian Rhythm; DNA/genetics/isolation & purification; Restriction Mapping; Enzyme Induction; Liver/drug effects/*enzymology; Cell Fractionation; Cholesterol 7-alpha-Hydroxylase/biosynthesis/*genetics/immunology; Cholestyramine Resin/pharmacology; Cytochrome P-450 Enzyme System/genetics; Epitopes/analysis; Polyribosomes/metabolism/ultrastructure; Inbred Strains; RNA; Enzymologic; Sequence Homology; Cloning; Nucleic Acid; Messenger/*genetics; Centrifugation; Density Gradient; Molecular/methods
Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.
Li Y C; Wang D P; Chiang J Y
The Journal of biological chemistry
1990
1990-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
The expression of a catalytically active cholesterol 7 alpha-hydroxylase cytochrome P450 in Escherichia coli.
Gene Expression; Amino Acid Sequence; Base Sequence; Polymerase Chain Reaction; Liver/enzymology; Catalysis; Molecular Sequence Data; Substrate Specificity; Cholesterol/metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics; DNA/genetics; Codon; Escherichia coli/*enzymology; Plasmids; Chromatography; Blotting; Western; Electrophoresis; Polyacrylamide Gel; Liquid; Microsomes
We have recently cloned a full-length cDNA encoding the rat hepatic cholesterol 7 alpha-hydroxylase cytochrome P450 (P450c7) (Li, Y. C., Wang, D. P., and Chiang, J. Y. L. (1990) J. Biol. Chem. 265, 12012-12019), which catalyzes the rate-limiting reaction of bile acid synthesis in the liver. By using the polymerase chain reaction, we have designed two P450c7 cDNAs. One has the second Met codon deleted and the third Thr codon replaced with an Ala. The other lacks codons for the NH2-terminal hydrophobic sequence of amino acids 2-24 (P450c7 delta 2-24). The cDNAs were separately cloned into the expression vector pKK233-2 and transformed into Escherichia coli. After induction with isopropyl-beta-D-thiogalactopyranoside, bacteria harboring recombinant plasmids expressed a polypeptide which reacted with the antibody against cholesterol 7 alpha-hydroxylase in immunoblots. The slightly modified full-length enzyme was expressed to 0.2% of the total bacterial lysate and was located in the membrane fraction, whereas P450c7 delta 2-24 was expressed at a 10-fold higher level (2%), of which 85% was in the cytosol and the remaining associated with the membranes. We have purified P450c7 delta 2-24 which showed a typical reduced-CO difference spectrum of cytochrome P450 and reconstituted cholesterol 7 alpha-hydroxylase activity in the presence of NADPH-cytochrome P450 reductase. P450c7 delta 2-24 has a similar Km for cholesterol (24.6 microM) but a lower Vmax (0.10 nmol/min) and a lower turnover number (1.93 min-1) as compared with the enzyme isolated from rat liver microsomes. The purified P450c7 delta 2-24 has an unique hydrophilic NH2 terminus and contains monomers and dimers in equal amounts. This is the first report demonstrating that a genetically engineered cytochrome P450 enzyme lacking a typical NH2-terminal hydrophobic sequence is mainly cytosolic and catalytically active.
Li Y C; Chiang J Y
The Journal of biological chemistry
1991
1991-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Monoamine-activated alpha 2-macroglobulin binds trk receptor and inhibits nerve growth factor-stimulated trk phosphorylation and signal transduction.
Humans; Male; Animals; Mice; Signal Transduction/drug effects/*physiology; Phosphorylation; PC12 Cells; Protein-Serine-Threonine Kinases/metabolism; Neurites/drug effects/*physiology; Nerve Growth Factors/*pharmacology; Adrenal Gland Neoplasms; alpha-Macroglobulins/isolation & purification/*metabolism/pharmacology; ErbB Receptors/isolation & purification/metabolism; Fibrinolysin/metabolism; Mitogen-Activated Protein Kinase 1; Pheochromocytoma; Protein-Tyrosine Kinases/metabolism; Proto-Oncogene Proteins/drug effects/isolation & purification/*metabolism; Receptor Protein-Tyrosine Kinases/drug effects/isolation & purification/*metabolism; Serotonin/*metabolism/pharmacology; Receptors; Receptor; Nerve Growth Factor/drug effects/isolation & purification/*metabolism; Platelet-Derived Growth Factor/isolation & purification/metabolism; trkA
Monoamine-activated alpha 2-macroglobulin (alpha 2M) has been shown to inhibit beta-nerve growth factor (NGF)-promoted neurite outgrowth and the survival of embryonic sensory and forebrain neurons, whereas normal alpha 2M has little or no such activity. The objective of this study is to elucidate the mechanism of inhibition by monoamine-activated alpha 2M. Methylamine-activated alpha 2M (MA-alpha 2M) and serotonin-activated alpha 2M (5HT-alpha 2M) dose dependently inhibit NGF-promoted neurite outgrowth of the pheochromocytoma PC12 cell and its subline PC12(6-24) which overexpresses human trk protooncogene product, but have no effect on their viability, and this inhibition can be blocked by high concentrations of NGF. The binding of MA-alpha 2M to trk, which is a part of high-affinity NGF receptor, was studied with PC12(6-24) cells and NIH-3T3 fibroblasts expressing trk (trk-3T3). In each case MA-alpha 2M readily forms stable complexes with trk in vivo, whereas normal alpha 2M does not. Both
Koo P H; Qiu W S
The Journal of biological chemistry
1994
1994-02
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Regulation of cholesterol 7 alpha-hydroxylase in the liver. Purification of cholesterol 7 alpha-hydroxylase and the immunochemical evidence for the induction of cholesterol 7 alpha-hydroxylase by cholestyramine and circadian rhythm.
Animals; Rats; Organ Specificity; Kinetics; *Circadian Rhythm; Enzyme Induction; Liver/drug effects/*enzymology; Cholesterol 7-alpha-Hydroxylase/*biosynthesis/isolation & purification/metabolism; Cholestyramine Resin/*pharmacology; Chromatography; Durapatite; Hydroxyapatites; Isoenzymes/*biosynthesis/isolation & purification; Obesity/enzymology; Polyethylene Glycols; Steroid Hydroxylases/*biosynthesis; Inbred Strains; Zucker; Microsomes; Ion Exchange
Two cholesterol 7 alpha-hydroxylase isozymes were purified from liver microsomes of cholestyramine-treated female rats by using anion exchange high performance liquid chromatography. These two cytochrome P-450 isozymes were similar in electrophoretic mobility, immunocross-reactivity, and Vmax but differed in Km for cholesterol, turnover number, and charges. Antibody against the major isozyme was raised in rabbit. This antibody specifically inhibited microsomal cholesterol 7 alpha-hydroxylase activity. Immunoblot of microsomal polypeptides indicated that microsomal cholesterol 7 alpha-hydroxylase enzyme levels were increased in parallel with cholesterol 7 alpha-hydroxylase activity upon the treatment of rats with diet supplemented with cholestyramine. Both cholesterol 7 alpha-hydroxylase activity and enzyme levels were drastically reduced immediately after the removal of cholestyramine from the diet. Cholesterol 7 alpha-hydroxylase activity was also detected in the microsomes of kidney, heart, and lung in about 7-27% of the level found in the liver. 3-Methylcholanthrene treatment induced cholesterol 7 alpha-hydroxylase activity and enzyme level. In contrast, pregnenolone-16 alpha-carbonitrile or dexamethasone treatment greatly depressed enzyme and activity in rats. Cholesterol 7 alpha-hydroxylase enzyme level was 2-3-fold higher in liver microsomes of rats maintained under the reversed light cycle than under the normal light cycle. In genetically obese Zucker rats, cholesterol 7 alpha-hydroxylase activity and enzyme level did not respond to the change in the light cycle, however, were induced to the same levels as in the lean rats by cholestyramine treatment. This study provided the first direct evidence that the bile acid feedback regulation and circadian rhythm of microsomal cholesterol 7 alpha-hydroxylase activity involved the induction of cholesterol 7 alpha-hydroxylase enzyme level.
Chiang J Y; Miller W F; Lin G M
The Journal of biological chemistry
1990
1990-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Identification and characterization of a putative bile acid-responsive element in cholesterol 7 alpha-hydroxylase gene promoter.
Humans; Animals; Rats; Gene Expression Regulation; Base Sequence; Bile Acids and Salts/*metabolism; Cholesterol 7-alpha-Hydroxylase/*genetics/metabolism; Molecular Sequence Data; Recombinant Fusion Proteins/genetics/metabolism; Luciferases/genetics; DNA-Binding Proteins/metabolism; Deoxyribonuclease I; Nuclear Proteins/metabolism; Oligodeoxyribonucleotides; Thyroid Hormones/genetics; Sprague-Dawley; Genetic; Enzymologic; Cloning; Molecular; *Promoter Regions; Antigens; Polyomavirus Transforming/genetics
Nucleotide sequences of a 7997-base pair SacI fragment spanning 3643 base pairs of the upstream promoter region to exon 4 of the rat cholesterol 7 alpha-hydroxylase gene (CYP7) have been determined. DNase I footprinting and electrophoretic mobility shift assay of the proximal promoter from nucleotides -346 to +36 revealed two protected regions which specifically shifted proteins in rat liver nuclear extracts. Footprint A (nucleotides -81 to -35) contained a cluster of overlapping sequence motifs of TGT3, steroid/thyroid hormone response elements (7 alpha TRE), hepatocyte nuclear factors 1 and 4, and CAAT/enhancer-binding protein alpha and has been shown to confer bile acid repression of the CYP7 gene promoter activity. Footprint B (nucleotides -148 to -129) contained a sequence motif HNF4. When footprint A (-101 to -49) or 7 alpha TRE (-73 to -55) sequence was linked upstream to a heterologous SV40 promoter/luciferase plasmid and transiently transfected into HepG2 cells, taurodeoxycholate suppressed the SV40 promoter activity. Electrophoretic mobility shift assays revealed that one or two bands shifted by the 7 alpha TRE or by a direct repeat sequence in 7 alpha TRE were absent when liver nuclear extracts of deoxycholic acid-treated rats were used. Similar gel shift patterns were also observed when human 7 alpha TRE or human liver nuclear extracts were used. The rat direct repeat sequence interacted with two polypeptides (M(r) = 57,000 and 116,000) in both rat and human liver nuclear extracts. These results suggest that hydrophobic bile acids may suppress the CYP7 gene expression by binding to a bile acid receptor which interacts with and prevents the binding of liver nuclear protein(s) to a bile acid-responsive element and that the core of bile acid-responsive element is a direct repeat.
Chiang J Y; Stroup D
The Journal of biological chemistry
1994
1994-07
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Farnesoid X receptor responds to bile acids and represses cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription.
Humans; Animals; Cricetinae; Cholesterol 7-alpha-Hydroxylase/*genetics; Response Elements; Bile Acids and Salts/*pharmacology; Transcription Factors/genetics/*physiology; Retinoid X Receptors; DNA-Binding Proteins/*physiology; Repressor Proteins/*physiology; Cultured; Receptors; Genetic; Tumor Cells; Promoter Regions; *Transcription; Cytoplasmic and Nuclear; Retinoic Acid/genetics
Cholesterol 7alpha-hydroxylase gene (CYP7A1) transcription is repressed by bile acids. The goal of this study is to elucidate the mechanism of CYP7A1 transcription by bile acid-activated farnesoid X receptor (FXR) in its native promoter and cellular context and to identify FXR response elements in the gene. In Chinese hamster ovary cells transfected with retinoid X receptor alpha (RXRalpha)/FXR, only chenodeoxycholic acid (CDCA) and deoxycholic acid (DCA) were able to stimulate a heterologous promoter/reporter containing an ecdysone response element. In HepG2 cells, all bile acids (25 microM) were able to repress CYP7A1/luciferase reporter activity, and only CDCA and DCA further repressed reporter activity when cotransfected with RXRalpha/FXR. The concentration of CDCA required to inhibit 50% of reporter activity (IC(50)) was determined to be approximately 25 microM without FXR and 10 microM with FXR. Deletion analysis revealed that the bile acid response element located between nucleotides -148 and -128 was the FXR response element, but RXRalpha/FXR did not bind to this sequence. These results suggest that bile acid-activated FXR exerts its inhibitory effect on CYP7A1 transcription by an indirect mechanism, in contrast to the stimulation and binding of FXR to intestinal bile acid-binding protein gene promoter. Results also reveal that bile acid receptors other than FXR are present in HepG2 cells.
Chiang J Y; Kimmel R; Weinberger C; Stroup D
The Journal of biological chemistry
2000
2000-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Insulin regulation of cholesterol 7alpha-hydroxylase expression in human hepatocytes: roles of forkhead box O1 and sterol regulatory element-binding protein 1c.
*Gene Expression Regulation; *Transcriptional Activation; Adolescent; Adult; Animals; Child; Cholesterol 7-alpha-Hydroxylase/*biosynthesis/genetics; Enzymologic; Female; Forkhead Box Protein O1; Forkhead Transcription Factors/*physiology; Hepatocytes/*enzymology; Humans; Insulin/metabolism/*physiology; Male; Middle Aged; Preschool; Rats; Sterol Regulatory Element Binding Protein 1/*physiology; Transcription Factors/*physiology
Bile acid synthesis and pool size increases in diabetes, whereas insulin inhibits bile acid synthesis. The objective of this study is to elucidate the mechanism of insulin regulation of cholesterol 7alpha-hydroxylase gene expression in human hepatocytes. Real-time PCR assays showed that physiological concentrations of insulin rapidly stimulated cholesterol 7alpha-hydroxylase (CYP7A1) mRNA expression in primary human hepatocytes but inhibited CYP7A1 expression after extended treatment. The insulin-regulated forkhead box O1 (FoxO1) and steroid regulatory element-binding protein-1c (SREBP-1c) strongly inhibited hepatocyte nuclear factor 4alpha and peroxisome proliferator-activated receptor gamma coactivator-1alpha trans-activation of the CYP7A1 gene. FoxO1 binds to an insulin response element in the rat CYP7A1 promoter, which is not present in the human CYP7A1 gene. Insulin rapidly phosphorylates and inactivates FoxO1, whereas insulin induces nuclear SREBP-1c expression in human primary hepatocytes. Chromatin immunoprecipitation assay shows that insulin reduced FoxO1 and peroxisome proliferators-activated receptor gamma-coactivator-1alpha but increased SREBP-1c recruitment to CYP7A1 chromatin. We conclude that insulin has dual effects on human CYP7A1 gene transcription; physiological concentrations of insulin rapidly inhibit FoxO1 activity leading to stimulation of the human CYP7A1 gene, whereas prolonged insulin treatment induces SREBP-1c, which inhibits human CYP7A1 gene transcription. Insulin may play a major role in the regulation of bile acid synthesis and dyslipidemia in diabetes.
Li Tiangang; Kong Xiaoying; Owsley Erika; Ellis Ewa; Strom Stephen; Chiang John Y L
The Journal of biological chemistry
2006
2006-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M605815200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M605815200</a>
A Prospero-related homeodomain protein is a novel co-regulator of hepatocyte nuclear factor 4alpha that regulates the cholesterol 7alpha-hydroxylase gene.
*Gene Expression Regulation; Aged; Amino Acid Motifs; Bile Acids and Salts/metabolism; Cell Line; Cell Nucleus/metabolism; Cells; Cholesterol 7-alpha-Hydroxylase/*chemistry/*genetics; Cultured/metabolism; Enzymologic; Female; Genes; Genetic; Gluconeogenesis; Glutathione Transferase/metabolism; Hepatocyte Nuclear Factor 4/metabolism/*physiology; Hepatocytes/metabolism; Homeodomain Proteins/metabolism/*physiology; Humans; Immunoprecipitation; Liver/metabolism; Luciferases/metabolism; Male; Messenger/metabolism; Middle Aged; Phosphoenolpyruvate Carboxykinase (ATP)/metabolism; Plasmids/metabolism; Protein Structure; Reporter; Response Elements; Reverse Transcriptase Polymerase Chain Reaction; RNA; Small Interfering/metabolism; Tertiary; Time Factors; Transcription; Transcriptional Activation; Transfection; Tumor Suppressor Proteins; Two-Hybrid System Techniques
Prox1, an early specific marker for developing liver and pancreas in foregut endoderm has recently been shown to interact with alpha-fetoprotein transcription factor and repress cholesterol 7alpha-hydroxylase (CYP7A1) gene transcription. Using a yeast two-hybrid assay, we found that Prox1 strongly and specifically interacted with hepatocyte nuclear factor (HNF)4alpha, an important transactivator of the human CYP7A1 gene in bile acid synthesis and phosphoenolpyruvate carboxykinase (PEPCK) gene in gluconeogenesis. A real time PCR assay detected Prox1 mRNA expression in human primary hepatocytes and HepG2 cells. Reporter assay, GST pull-down, co-immunoprecipitation, and yeast two-hybrid assays identified a specific interaction between the N-terminal LXXLL motif of Prox1 and the activation function 2 domain of HNF4alpha. Prox1 strongly inhibited HNF4alpha and peroxisome proliferators-activated receptor gamma coactivator-1alpha co-activation of the CYP7A1 and PEPCK genes. Knock down of the endogenous Prox1 by small interfering RNA resulted in significant increase of CYP7A1 and PEPCK mRNA expression and the rate of bile acid synthesis in HepG2 cells. These results suggest that Prox1 is a novel co-regulator of HNF4alpha that may play a key role in the regulation of bile acid synthesis and gluconeogenesis in the liver.
Song Kwang-Hoon; Li Tiangang; Chiang John Y L
The Journal of biological chemistry
2006
2006-04
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M513420200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M513420200</a>
Bcl-2 positively regulates Sox9-dependent chondrocyte gene expression by suppressing the MEK-ERK1/2 signaling pathway.
*Gene Expression Regulation; Adenoviridae/genetics; Animals; Apoptosis; beta-Galactosidase/metabolism; Blotting; Butadienes/pharmacology; Caspase Inhibitors; Cell Differentiation; Cell Line; Chondrocytes/*metabolism; Collagen Type II/metabolism; Down-Regulation; Enzyme Inhibitors/pharmacology; Fibroblasts/metabolism; Fluorescence; Genetic; High Mobility Group Proteins/*metabolism; Lac Operon; Luciferases/metabolism; MAP Kinase Kinase Kinases/*metabolism; Messenger/metabolism; Microscopy; Mitogen-Activated Protein Kinase 1/*metabolism; Mitogen-Activated Protein Kinase 3/*metabolism; NF-kappa B/metabolism; Nitriles/pharmacology; Phenotype; Phosphorylation; Promoter Regions; Protein Kinase C-alpha; Protein Kinase C/antagonists & inhibitors; Proteoglycans/metabolism; Proto-Oncogene Proteins c-bcl-2/*metabolism; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA; Signal Transduction; Small Interfering/metabolism; SOX9 Transcription Factor; Sprague-Dawley; Time Factors; Transcription; Transcription Factors/*metabolism; Transfection; Western
Bcl-2 is an anti-apoptotic protein that has recently been shown to regulate other cellular functions. We previously reported that Bcl-2 regulates chondrocyte matrix gene expression, independent of its anti-apoptotic function. Here, we further investigate this novel function of Bcl-2 and examine three intracellular signaling pathways likely to be associated with this function. The present study demonstrates that the activity of Sox9, a master transcription factor that regulates the gene expression of chondrocyte matrix proteins, is suppressed by Bcl-2 small interference RNA in the presence of caspase inhibitors. This effect was attenuated by prior exposure of chondrocytes to an adenoviral vector expressing sense Bcl-2. In addition, the down-regulation of Bcl-2, Sox9, and chondrocyte-specific gene expression by serum withdrawal in primary chondrocytes was reversed by expressing Bcl-2. Inhibition of the protein kinase C alpha and NFkappaB pathways had no effect on the maintenance of Sox9-dependent gene expression by Bcl-2. In contrast, whereas the MEK-ERK1/2 pathway negatively regulated the differentiated phenotype in wild type chondrocytes, inhibition of this pathway reversed the loss of differentiation markers and fibroblastic phenotype in Bcl-2-deficient chondrocytes. In conclusion, the present study identifies a specific signaling pathway, namely, MEK-ERK1/2, that is downstream of Bcl-2 in the regulation of Sox9-dependent chondrocyte gene expression and phenotype.
Yagi Rieko; McBurney Denise; Horton Walter E Jr
The Journal of biological chemistry
2005
2005-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M502751200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M502751200</a>
Multiple signals induce endoplasmic reticulum stress in both primary and immortalized chondrocytes resulting in loss of differentiation, impaired cell growth, and apoptosis.
Animals; Annexin A5/metabolism; Anti-Bacterial Agents/metabolism; Apoptosis/*physiology; Biomarkers; Caspase 12; Caspases/metabolism; CCAAT-Enhancer-Binding Proteins/metabolism; Cell Differentiation/*physiology; Cells; Chondrocytes/cytology/*physiology; Collagen Type II/metabolism; Cultured; DNA Fragmentation; Endoplasmic Reticulum/*metabolism; Extracellular Matrix/metabolism; Gene Expression Regulation; Glucose/metabolism; Proliferating Cell Nuclear Antigen/metabolism; Rats; Signal Transduction/*physiology; Thapsigargin/metabolism; Transcription Factor CHOP; Transcription Factors/metabolism; Tunicamycin/metabolism
The endoplasmic reticulum is the site of synthesis and folding of secretory proteins and is sensitive to changes in the internal and external environment of the cell. Both physiological and pathological conditions may perturb the function of the endoplasmic reticulum, resulting in endoplasmic reticulum stress. The chondrocyte is the only resident cell found in cartilage and is responsible for synthesis and turnover of the abundant extracellular matrix and may be sensitive to endoplasmic reticulum stress. Here we report that glucose withdrawal, tunicamycin, and thapsigargin induce up-regulation of GADD153 and caspase-12, two markers of endoplasmic reticulum stress, in both primary chondrocytes and a chondrocyte cell line. Other agents such as interleukin-1beta or tumor necrosis factor alpha induced a minimal or no induction of GADD153, respectively. The endoplasmic reticulum stress resulted in decreased chondrocyte growth based on cell counts, up-regulation of p21, and decreased PCNA expression. In addition, perturbation of endoplasmic reticulum function resulted in decreased accumulation of an Alcian Blue positive matrix by chondrocytes and decreased expression of type II collagen at the protein level. Further, quantitative real-time PCR was used to demonstrate a down-regulation of steady state mRNA levels coding for aggrecan, collagen II, and link protein in chondrocytes exposed to endoplasmic reticulum stress-inducing conditions. Ultimately, endoplasmic reticulum stress resulted in chondrocyte apoptosis, as evidenced by DNA fragmentation and annexin V staining. These findings have potentially important implications regarding consequences of endoplasmic reticulum stress in cartilage biology.
Yang Ling; Carlson Sara G; McBurney Denise; Horton Walter E Jr
The Journal of biological chemistry
2005
2005-09
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M501069200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M501069200</a>
Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism.
*bile acid; *bile acid metabolism; *FXR; *Gene Expression Regulation; *GLP-1; *lipid metabolism; *liver metabolism; *non-alcoholic fatty liver disease; *obesity; *TGR5; *type 2 diabetes; Animals; Bile Acids and Salts/*biosynthesis/genetics; Cytoplasmic and Nuclear/genetics/*metabolism; Dietary Fats; G-Protein-Coupled/genetics/*metabolism; Glucagon-Like Peptide 1/genetics/metabolism; Glucose/metabolism; Knockout; Lipid Metabolism; Liver/*metabolism; Mice; Obesity/genetics/*metabolism/pathology; Receptors
The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr(-/-), and Tgr5(-/-) mice. INT-767 efficaciously stimulated intracellular Ca(2+) levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and
Pathak Preeti; Liu Hailiang; Boehme Shannon; Xie Cen; Krausz Kristopher W; Gonzalez Frank; Chiang John Y L
The Journal of biological chemistry
2017
2017-06
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M117.784322" target="_blank" rel="noreferrer noopener">10.1074/jbc.M117.784322</a>
MitoNEET Deficiency Alleviates Experimental Alcoholic Steatohepatitis in Mice by Stimulating Endocrine Adiponectin-Fgf15 Axis.
*adiponectin; *alcohol; *intestine; *iron; *liver injury; *Signal Transduction; Adiponectin/genetics/*metabolism; Alcoholic/genetics/*metabolism/pathology; Animals; Fatty Liver; Fibroblast Growth Factors/genetics/*metabolism; Humans; Iron-Binding Proteins/metabolism; Knockout; Male; Membrane Proteins/*deficiency/metabolism; Mice; NF-kappa B/genetics/metabolism; Sirtuin 1/genetics/metabolism
MitoNEET (mNT) (CDGSH iron-sulfur domain-containing protein 1 or CISD1) is an outer mitochondrial membrane protein that donates 2Fe-2S clusters to apo-acceptor proteins. In the present study, using a global mNT knock-out (mNTKO) mouse model, we investigated the in vivo functional role of mNT in the development of alcoholic steatohepatitis. Experimental alcoholic steatohepatitis was achieved by pair feeding wild-type (WT) and mNTKO mice with Lieber-DeCarli ethanol-containing diets for 4 weeks. Strikingly, chronically ethanol-fed mNTKO mice were completely resistant to ethanol-induced steatohepatitis as revealed by dramatically reduced hepatic triglycerides, decreased hepatic cholesterol level, diminished liver inflammatory response, and normalized serum ALT levels. Mechanistic studies demonstrated that ethanol administration to mNTKO mice induced two pivotal endocrine hormones, namely, adipose-derived adiponectin and gut-derived fibroblast growth factor 15 (Fgf15). The elevation in circulating levels of adiponectin and Fgf15 led to normalized hepatic and serum levels of bile acids, limited hepatic accumulation of toxic bile, attenuated inflammation, and amelioration of liver injury in the ethanol-fed mNTKO mice. Other potential mechanisms such as reduced oxidative stress, activated Sirt1 signaling, and diminished NF-kappaB activity also contribute to hepatic improvement in the ethanol-fed mNTKO mice. In conclusion, the present study identified adiponectin and Fgf15 as pivotal adipose-gut-liver metabolic coordinators in mediating the protective action of mNT deficiency against development of alcoholic steatohepatitis in mice. Our findings may help to establish mNT as a novel therapeutic target and pharmacological inhibition of mNT may be beneficial for the prevention and treatment of human alcoholic steatohepatitis.
Hu Xudong; Jogasuria Alvin; Wang Jiayou; Kim Chunki; Han Yoonhee; Shen Hong; Wu Jiashin; You Min
The Journal of biological chemistry
2016
2016-10
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M116.737015" target="_blank" rel="noreferrer noopener">10.1074/jbc.M116.737015</a>
Mutation in Osteoactivin Promotes Receptor Activator of NFkappaB Ligand (RANKL)-mediated Osteoclast Differentiation and Survival but Inhibits Osteoclast Function.
*Mutation; Akt; Animals; bone; bone marrow; Bone Remodeling; Cell Differentiation/*physiology; Cell Survival/*physiology; Eye Proteins/*genetics; Inbred DBA; MAP kinases (MAPKs); Membrane Glycoproteins/*genetics; Mice; osteoactivin; osteoclast; Osteoclasts/*cytology; osteopetrosis; RANK Ligand/metabolism/*physiology; Signal Transduction; X-Ray Microtomography
We previously reported on the importance of osteoactivin (OA/Gpnmb) in osteogenesis. In this study, we examined the role of OA in osteoclastogenesis, using mice with a nonsense mutation in the Gpnmb gene (D2J) and wild-type controls (D2J/Gpnmb(+)). In these D2J mice, micro-computed tomography and histomorphometric analyses revealed increased cortical thickness, whereas total porosity and eroded surface were significantly reduced in D2J mice compared with wild-type controls, and these results were corroborated by lower serum levels of
Abdelmagid Samir M; Sondag Gregory R; Moussa Fouad M; Belcher Joyce Y; Yu Bing; Stinnett Hilary; Novak Kimberly; Mbimba Thomas; Khol Matthew; Hankenson Kurt D; Malcuit Christopher; Safadi Fayez F
The Journal of biological chemistry
2015
2015-08
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M114.624270" target="_blank" rel="noreferrer noopener">10.1074/jbc.M114.624270</a>
Generation and characterization of ATP analog-specific protein kinase Cdelta.
Adenosine Triphosphate/*analogs & derivatives/*chemistry; Amino Acid; Amino Acid Sequence; Animals; ATP; Catalysis; Cercopithecus aethiops; Chemical Biology; COS Cells; Glutamine/chemistry; Humans; Inbred C57BL; Leucine/chemistry; Lysine/chemistry; Mice; Molecular Sequence Data; Neutrophils/metabolism; Phenylalanine/chemistry; Phosphorylation; Protein Binding; Protein Kinase C (PKC); Protein Kinase C-delta/*metabolism; Protein Phosphorylation; Purines/chemistry; Sequence Homology; Signal Transduction; Stroke; Substrate Specificity; Superoxides/chemistry; Transgenic
To better study the role of PKCdelta in normal function and disease, we developed an ATP analog-specific (AS) PKCdelta that is sensitive to specific kinase inhibitors and can be used to identify PKCdelta substrates. AS PKCdelta showed nearly 200 times higher affinity (Km) and 150 times higher efficiency (kcat/Km) than wild type (WT) PKCdelta toward N(6)-(benzyl)-ATP. AS PKCdelta was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and
Kumar Varun; Weng Yi-Chinn; Geldenhuys Werner J; Wang Dan; Han Xiqian; Messing Robert O; Chou Wen-Hai
The Journal of biological chemistry
2015
2015-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M114.598698" target="_blank" rel="noreferrer noopener">10.1074/jbc.M114.598698</a>
Modulation of ten-eleven translocation 1 (TET1), Isocitrate Dehydrogenase (IDH) expression, alpha-Ketoglutarate (alpha-KG), and DNA hydroxymethylation levels by interleukin-1beta in primary human chondrocytes.
*DNA Methylation; *Epigenesis; 5-hmC; 5-Methylcytosine/analogs & derivatives; Chondrocytes; Chondrocytes/chemistry/*metabolism; Cytokine; Cytosine/*analogs & derivatives/analysis/metabolism; Dioxygenase; DNA Methylation; DNA-Binding Proteins/*biosynthesis/genetics; Epigenetics; Genetic; Humans; IL-1; Interleukin-1beta/pharmacology/*physiology; Isocitrate Dehydrogenase/*biosynthesis/genetics; Ketoglutaric Acids/*metabolism; Messenger/biosynthesis/genetics; Mixed Function Oxygenases; Primary Cell Culture; Proto-Oncogene Proteins/*biosynthesis/genetics; RNA; TNF-alpha; Tumor Necrosis Factor-alpha/pharmacology/physiology
5-Hydroxymethylcytosine (5-hmC) generated by ten-eleven translocation 1-3 (TET1-3) enzymes is an epigenetic mark present in many tissues with different degrees of abundance. IL-1beta and TNF-alpha are the two major cytokines present in arthritic joints that modulate the expression of many genes associated with cartilage degradation in osteoarthritis. In the present study, we investigated the global 5-hmC content, the effects of IL-1beta and TNF-alpha on 5-hmC content, and the expression and activity of TETs and isocitrate dehydrogenases in primary human chondrocytes. The global 5-hmC content was found to be approximately 0.1% of the total genome. There was a significant decrease in the levels of 5-hmC and the TET enzyme activity upon treatment of chondrocytes with IL-1beta alone or in combination with TNF-alpha. We observed a dramatic (10-20-fold) decrease in the levels of TET1 mRNA expression and a small increase (2-3-fold) in TET3 expression in chondrocytes stimulated with IL-1beta and TNF-alpha. IL-1beta and TNF-alpha significantly suppressed the activity and expression of IDHs, which correlated with the reduced alpha-ketoglutarate levels. Whole genome profiling showed an erasure effect of IL-1beta and TNF-alpha, resulting in a significant decrease in hydroxymethylation in a myriad of genes including many genes that are important in chondrocyte physiology. Our data demonstrate that DNA hydroxymethylation is modulated by pro-inflammatory cytokines via suppression of the cytosine hydroxymethylation machinery. These data point to new mechanisms of epigenetic control of gene expression by pro-inflammatory cytokines in human chondrocytes.
Haseeb Abdul; Makki Mohammad Shahidul; Haqqi Tariq M
The Journal of biological chemistry
2014
2014-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M113.512269" target="_blank" rel="noreferrer noopener">10.1074/jbc.M113.512269</a>
Retinoic acid-related orphan receptor alpha regulates diurnal rhythm and fasting induction of sterol 12alpha-hydroxylase in bile acid synthesis.
Animals; Bile Acids and Salts/*biosynthesis; Cholesterol; Cholesterol/*biosynthesis/genetics; Circadian Rhythm/*physiology; Diabetes; Diabetes Mellitus; Enzyme Induction/physiology; Fasting/*metabolism; Fatty Liver/drug therapy/genetics/metabolism/pathology; Group F; Hep G2 Cells; Humans; Lipid Metabolism; Member 1/genetics/*metabolism; Mice; Non-alcoholic Fatty Liver Disease; Nuclear Receptor Subfamily 1; Nuclear Receptors; Obesity; Phosphorylation/physiology; Protein Kinases/genetics/metabolism; Response Elements/physiology; Steroid 12-alpha-Hydroxylase/*biosynthesis/genetics; Type 2/drug therapy/genetics/metabolism/pathology
Sterol 12alpha-hydroxylase (CYP8B1) is required for cholic acid synthesis and plays a critical role in intestinal cholesterol absorption and pathogenesis of cholesterol gallstone, dyslipidemia, and diabetes. In this study we investigated the underlying mechanism of fasting induction and circadian rhythm of CYP8B1 by a cholesterol-activated nuclear receptor and core clock gene retinoic acid-related orphan receptor alpha (RORalpha). Fasting stimulated, whereas restricted-feeding reduced expression of CYP8B1 mRNA and protein. However, fasting and feeding had little effect on the diurnal rhythm of RORalpha mRNA expression, but fasting increased RORalpha protein levels by cAMP-activated protein kinase A-mediated phosphorylation and stabilization of the protein. Adenovirus-mediated gene transduction of RORalpha to mice strongly induced CYP8B1 expression, and increased liver cholesterol and 12alpha-hydroxylated bile acids in the bile acid pool and serum. A reporter assay identified a functional RORalpha response element in the CYP8B1 promoter. RORalpha recruited cAMP response element-binding protein-binding protein (CBP) to stimulate histone acetylation on the CYP8B1 gene promoter. In conclusion, RORalpha is a key regulator of diurnal rhythm and fasting induction of CYP8B1, which regulates bile acid composition and serum and liver cholesterol levels. Antagonizing RORalpha activity may be a therapeutic strategy for treating inflammatory diseases such as non-alcoholic fatty liver disease and type 2 diabetes.
Pathak Preeti; Li Tiangang; Chiang John Y L
The Journal of biological chemistry
2013
2013-12
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M113.485987" target="_blank" rel="noreferrer noopener">10.1074/jbc.M113.485987</a>
An N-terminal arginine-rich cluster and a proline-alanine-threonine repeat region determine the cellular localization of the herpes simplex virus type 1 ICP34.5 protein and its ligand, protein phosphatase 1.
*Repetitive Sequences; Alanine/metabolism; Amino Acid; Amino Acid Sequence; Animals; Arginine/metabolism; Base Sequence; Cell Compartmentation; Cercopithecus aethiops; DNA Primers; Fluorescent Antibody Technique; Indirect; Ligands; Molecular Sequence Data; Phosphoprotein Phosphatases/*metabolism; Proline/metabolism; Protein Phosphatase 1; Recombinant Proteins/chemistry/metabolism; Subcellular Fractions/metabolism; Threonine/metabolism; Vero Cells; Viral Proteins/chemistry/*metabolism
The ICP34.5 protein facilitates herpes simplex virus replication by binding and activating protein phosphatase 1 (PP1) by means of a very conserved C-terminal
Mao Hanwen; Rosenthal Kenneth S
The Journal of biological chemistry
2002
2002-03
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M111553200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M111553200</a>
Glucose and insulin induction of bile acid synthesis: mechanisms and implication in diabetes and obesity.
*Gene Expression Regulation; Animals; Bile Acids and Salts/*biosynthesis; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism; Cytoplasmic and Nuclear/genetics/metabolism; Diabetes Mellitus; Dietary Fats/administration & dosage/adverse effects; Enzymologic; Epigenesis; Experimental/genetics/*metabolism; Fasting/metabolism; Genetic/genetics; Glucose/*metabolism/pharmacology; Insulin/*metabolism; Mice; Obesity/etiology/genetics/*metabolism; Postprandial Period/genetics; Receptors; Sweetening Agents/pharmacology; Transgenic
Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7alpha-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.
Li Tiangang; Francl Jessica M; Boehme Shannon; Ochoa Adrian; Zhang Youcai; Klaassen Curtis D; Erickson Sandra K; Chiang John Y L
The Journal of biological chemistry
2012
2012-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M111.305789" target="_blank" rel="noreferrer noopener">10.1074/jbc.M111.305789</a>
Identification of novel pathways that control farnesoid X receptor-mediated hypocholesterolemia.
Absorption; Animals; Biological; Cell Line; Cholesterol/*metabolism; Class B/*genetics/metabolism; Coronary Disease/metabolism; Cytoplasmic and Nuclear/*metabolism; Glucose/metabolism; Hepatocyte Nuclear Factor 4/metabolism; Homeostasis; Humans; Lipoproteins/metabolism; Liver/metabolism; Mice; Models; Receptors; Scavenger Receptors
Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr(-/-) mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an
Zhang Yanqiao; Yin Liya; Anderson Jody; Ma Huiyan; Gonzalez Frank J; Willson Timothy M; Edwards Peter A
The Journal of biological chemistry
2010
2010-01
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M109.083899" target="_blank" rel="noreferrer noopener">10.1074/jbc.M109.083899</a>
Transcriptional regulation of the human sterol 12alpha-hydroxylase gene (CYP8B1): roles of heaptocyte nuclear factor 4alpha in mediating bile acid repression.
*DNA-Binding Proteins; *Transcription; Base Sequence; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Bile Acids and Salts/*pharmacology; Binding Sites; Cholesterol 7-alpha-Hydroxylase/genetics; Cultured; Cytochrome P-450 Enzyme System/*genetics; Cytoplasmic and Nuclear/physiology; Genetic; Genetic/physiology; Hepatocyte Nuclear Factor 4; Humans; Molecular Sequence Data; Phosphoproteins/*physiology; Promoter Regions; Receptors; Repressor Proteins/*pharmacology; Steroid 12-alpha-Hydroxylase; Steroid Hydroxylases/*genetics; Transcription Factors/*physiology; Tumor Cells
Sterol 12alpha-hydroxylase catalyzes the synthesis of cholic acid and controls the ratio of cholic acid over chenodeoxycholic acid in the bile. Transcription of CYP8B1 is inhibited by bile acids, cholesterol, and insulin. To study the mechanism of CYP8B1 transcription by bile acids, we have cloned and determined 3389 base pairs of the 5'-upstream nucleotide sequences of the human CYP8B1. Deletion analysis of CYP8B1/luciferase reporter activity in HepG2 cells revealed that the sequences from -57 to +300 were important for basal and liver-specific promoter activities. Hepatocyte nuclear factor 4alpha (HNF4alpha) strongly activated human CYP8B1 promoter activities, whereas cholesterol 7alpha-hydroxylase promoter factor (CPF), an NR5A2 family of nuclear receptors, had much less effect. Electrophoretic mobility shift assay identified an overlapping HNF4alpha- and CPF-binding site in the +198/+227 region. The human CYP8B1 promoter activities were strongly repressed by bile acids, and the bile acid response element was localized between +137 and +220. Site-directed mutagenesis of the HNF4alpha-binding site markedly reduced promoter activity and its response to bile acid repression. On the other hand, mutation of the
Zhang M; Chiang J Y
The Journal of biological chemistry
2001
2001-11
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M105117200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M105117200</a>
Prostate apoptosis response-4 enhances secretion of amyloid beta peptide 1-42 in human neuroblastoma IMR-32 cells by a caspase-dependent pathway.
*Intracellular Signaling Peptides and Proteins; Amyloid beta-Peptides/*biosynthesis/metabolism; Apoptosis Regulatory Proteins; Apoptosis/*physiology; Carrier Proteins/genetics/*physiology; Caspases/*metabolism; Cultured; Enzyme Activation; Humans; Kinetics; Leucine Zippers; Neuroblastoma; Peptide Fragments/*biosynthesis/metabolism; Recombinant Proteins/metabolism; Time Factors; Transfection; Tumor Cells
Prostate apoptosis response-4 (Par-4) is a leucine zipper protein that promotes neuronal cell death in Alzheimer's disease (AD). Neuronal degeneration in AD may result from extracellular accumulation of amyloid beta peptide (Abeta) 1-42. To examine the effect of Par-4 on Abeta secretion and to reconcile amyloid/apoptosis hypotheses of AD, we generated IMR-32 cell lines that overexpress Par-4 and/or its leucine zipper domain. Overexpression of Par-4 did not significantly affect levels of the endogenously expressed beta amyloid precursor protein but drastically increased the Abeta(1-42)/Abeta(total) ratio in the conditioned media about 6-8 h after trophic factor withdrawal. Time course analysis of caspase activation reveals that Par-4 overexpression exacerbated caspase activation, which is detectable within 2 h after trophic factor withdrawal. Furthermore, inhibition of caspase activity by the broad spectrum caspase inhibitor BD-fmk significantly attenuated the Par-4-induced increase in Abeta 1-42 production. In addition, the effects of Par-4 on secretion of Abeta 1-42 were consistently blocked by co-expression of the leucine zipper domain, indicating that the effect of Par-4 on Abeta secretion may require its interaction with other protein(s). These results suggest that Par-4 increases secretion of Abeta 1-42 largely through a caspase-dependent pathway after apoptotic cascades are initiated.
Guo Q; Xie J; Chang X; Du H
The Journal of biological chemistry
2001
2001-05
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
<a href="http://doi.org/10.1074/jbc.M010996200" target="_blank" rel="noreferrer noopener">10.1074/jbc.M010996200</a>