Small heterodimer partner (SHP) is an atypical member of nuclear receptor superfamily that lacks a DNA-binding domain. In previous study, we showed that SHP, c-jun, p65 of NF-kappaB subunits, and p21WAF1 expression was increased during monocytic differentiaton with the exposure of human leukemia cells to a differentiation agent, PMA. In this study, c-Jun and p65 were shown to mediate the transcriptional activation of the SHP promoter. In addition, SHP induced the cell cycle regulatory protein levels and cooperatively increased an induction of p21WAF1 expression with p65. Furthermore, SHP protected differentiated cells from etoposide-induced cellular apoptosis through the induction and cytoplasmic sequestration of p21WAF1. Complex formation between SHP and p21WAF1 was demonstrated by means of coimmunoprecipitation. These results suggest that SHP prolongs a cellular survival of differentiating monocytes through the transcriptional regulation of target genes of cell survival and differentiation.
*Apoptosis ; Cell Differentiation ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21/genetics/*metabolism ; Gene Expression Regulation ; Humans ; Monocytes/cytology ; Promoter Regions, Genetic ; Proto-Oncogene Proteins c-jun/genetics/metabolism ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Transcription Factor RelA/genetics/metabolism

Angiotensin II is a major effector molecule in the development of cardiovascular disease. In vascular smooth muscle cells (VSMCs), angiotensin II promotes cellular proliferation and extracellular matrix accumulation through the upregulation of plasminogen activator inhibitor-1 (PAI-1) expression. Previously, we demonstrated that small heterodimer partner (SHP) represses PAI-1 expression in the liver through the inhibition of TGF-beta signaling pathways. Here, we investigated whether SHP inhibited angiotensin II-stimulated PAI-1 expression in VSMCs. Adenovirus-mediated overexpression of SHP (Ad-SHP) in VSMCs inhibited angiotensin II- and TGF-beta-stimulated PAI-1 expression. Ad-SHP also inhibited angiotensin II-, TGF-beta- and Smad3-stimulated PAI-1 promoter activity, and angiotensin II-stimulated AP-1 activity. The level of PAI-1 expression was significantly higher in VSMCs of SHP-/- mice than wild type mice. Moreover, loss of SHP increased PAI-1 mRNA expression after angiotensin II treatment. These results suggest that SHP inhibits PAI-1 expression in VSMCs through the suppression of TGF-beta/Smad3 and AP-1 activity. Thus, agents that target the induction of SHP expression in VSMCs might help prevent the development and progression of atherosclerosis.
Adenoviridae/genetics ; Angiotensin II/*pharmacology ; Animals ; Blotting, Northern ; Cells, Cultured ; Electrophoretic Mobility Shift Assay ; Genetic Vectors/genetics ; Humans ; Mice ; Muscle, Smooth, Vascular/*cytology ; Myocytes, Smooth Muscle/*drug effects/*metabolism ; Plasminogen Activator Inhibitor 1/*genetics ; Promoter Regions, Genetic/genetics ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/*metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Smad3 Protein/genetics ; Transforming Growth Factor beta/pharmacology

TCR signaling leading to thymocyte apoptosis is mediated through the expression of the Nur77 family of orphan nuclear receptors. It has been shown that the Nur77 promoter is activated by at least two signaling pathways, one mediated by calcium and the other by protein kinase C (PKC). MEF2D has been known to regulate Nur77 expression in a calcium- dependent manner. The mechanism by which calcium regulates MEF2D is through dissociation of calcium-sensitive MEF2 corepressors (Cabin1/ HDACs, HDAC4/5) and the association with calcineurin-activated transcription factor NF-AT and the coactivator p300. However, little is known about how PKC activates the Nur77 promoter. Herein, we report that PKC theta targets AP-1 like response element in the Nur77 promoter where JunD constitutively binds. PKC theta triggers mitogen-activated protein kinase- inediated phosphorylation of JunD, and increases transcriptional activity of JunD, cooperatively with p300. Menin is identified as the transcriptional corepressor for JunD via recruitment of mSin3-istone deacetylases. In fact, Menin represses PKC theta/ p300-mediated transcriptional activity of JunD in T cell. Its dynamic regulation of histone modifiers with JunD is responsible for PKCq-synergistic effect on Nur77 expression in T cell.
Cell Line, Tumor ; DNA-Binding Proteins/*genetics ; Enzyme Activation ; *Gene Expression Regulation ; Humans ; Isoenzymes/*metabolism ; Mitogen-Activated Protein Kinases/metabolism ; *Multiple Endocrine Neoplasia Type 1 ; Promoter Regions (Genetics)/genetics ; Protein Kinase C/*metabolism ; Proto-Oncogene Proteins/genetics/*metabolism ; Proto-Oncogene Proteins c-jun/*antagonists & inhibitors/metabolism ; Receptors, Cytoplasmic and Nuclear/*genetics ; Receptors, Steroid/*genetics ; Research Support, Non-U.S. Gov't ; Response Elements ; Transcription Factors/*genetics ; Transcription, Genetic/*genetics

TNF-alpha, a trimeric cytokine, was known to inhibit differention of preadipocytes to adipocytes. In the present study, we investigated signal mediators working downstream of TNF-alpha using murine 3T3-L1 cells. TNF-alpha induced activation of both c-jun NH2-terminal kinase (JNK) and nuclear transcription factor-kappaB (NF-kappaB) in 3T3-L1 cells. Blockage of these two mediators activities by specific inhibitors, SP600125 and Ad-IkappaBalpha-SR restored adipogenesis differentiation suggesting their involvement in the inhibited differentiation of 3T3-L1 cells by TNF-alpha. Consistent with previous studies, peroxisome proliferator-activated receptor gamma (PPARgamma) a key transcriptional regulator was remarkably reduced by TNF-alpha treatment. Compared with adipogenesis, however, SP600125, a chemical JNK inhibitor hardly relieved TNF-alpha effect on PPARgamma expression whereas S32A/S36A mutant of IkappaBalpha considerably recovered PPARgamma expression, indicating that two signal mediators exploit separable main routes to achieve reduced adipogenesis. These results suggest that inhibition of 3T3-L1 cells differentiation by TNF-alpha is partly implemented through NF-kappaB and one of its downstream effectors be PPARgamma.
3T3-L1 Cells ; Adipocytes/cytology/drug effects ; Animals ; Cell Differentiation/*drug effects ; Gene Expression Regulation/*drug effects ; Mice ; NF-kappa B/*metabolism ; Promoter Regions (Genetics)/genetics ; RNA, Messenger/genetics/metabolism ; Receptors, Cytoplasmic and Nuclear/*genetics ; Support, Non-U.S. Gov't ; Transcription Factors/*genetics ; Tumor Necrosis Factor/*pharmacology

In this study, we investigated the role of Nur77, an orphan nuclear receptor, in HIF-alpha transcriptional activity. We found that Nur77 associates and stabilizes HIF-1alpha via indirect interaction. Nur77 was found to interact with pVHL in vivo via the alpha-domain of pVHL. By binding to pVHL, Nur77 competed with elongin C for pVHL binding. Moreover, Nur77-binding to pVHL inhibited the pVHL-mediated ubiquitination of HIF-1alpha and ultimately increased the stability and transcriptional activity of HIF-1alpha. The ligand-binding domain of Nur77 was found to interact with pVHL and the expression of this ligand-binding domain was sufficient to stabilize and transactivate HIF-1alpha. Under the conditions that cobalt chloride was treated or pVHL was knocked down, Nur77 could not stabilize HIF-alpha. Moreover, Nur77 could not further stabilize HIF-2alpha in A498/VHL stable cells, which is consistent with our finding that Nur77 indirectly stabilizes HIF-alpha by binding to pVHL. Thus, our results suggest that an orphan nuclear receptor Nur77 binds to pVHL, thereby stabilizes and increases HIF-alpha transcriptional activity under the non- hypoxic conditions.
Animals ; DNA-Binding Proteins/chemistry/*metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*genetics ; Models, Biological ; PC12 Cells ; Protein Binding ; *Protein Processing, Post-Translational ; Protein Structure, Tertiary ; Rats ; Receptors, Cytoplasmic and Nuclear/chemistry/*metabolism ; Receptors, Steroid/chemistry/*metabolism ; Thermodynamics ; Transcription Factors/chemistry/*metabolism ; Transcriptional Activation/genetics ; Ubiquitination ; Up-Regulation/*genetics ; Von Hippel-Lindau Tumor Suppressor Protein/*antagonists & inhibitors/chemistry/*metabolism

Information on precise effects of deflazacort on bone cell function, especially osteoclasts, is quite limited. Therefore, the present study was undertaken to test effects of deflazacort on osteoclast-like cell formation in mouse bone marrow cultures and on the regulation of osteoprotegerin (OPG) and its ligand (RANKL) mRNA expressions by RT-PCR in the ST2 marrow stromal cells. TRAP-positive mononuclear cells increased after the treatment of deflazacort at 10(-9) to 10(-7) M alone for 6 days in a dose-dependent manner. Number of TRAP-positive multi-nucleated cells (MNCs) increased significantly with combined treatment of deflazacort at 10(-7) M and 1,25-(OH)2D3 at 10(-9) M compared to that of cultures treated with 1,25-(OH)2D3 alone (p<0.05). Exposure to deflazacort at 10(-7) M in the presence of 1,25-(OH)2D3 at 10(-9) M in the last 3-day culture had greater stimulatory effect on osteoclast-like cell formation than that of the first 3-day culture did. Deflazacort at 10(-10) -10(-6) M downregulated OPG and upregulated RANKL in mRNA levels in a dose-dependent manner. These observations suggest that deflazacort stimulate osteoclast precursor in the absence of 1,25-(OH)2D3 and enhance differentiation of osteoclasts in the presence of 1,25-(OH)2D3. These effects are, in part, thought to be mediated by the regulation of the expression of OPG and RANKL mRNA in marrow stromal cells.
Animal ; Bone Marrow Cells/*cytology ; Calcitriol/pharmacology ; Calcium Channel Agonists/pharmacology ; Carrier Proteins/*genetics ; Cell Differentiation/drug effects ; Cells, Cultured ; Dexamethasone/pharmacology ; Gene Expression/drug effects ; Glucocorticoids, Synthetic/pharmacology ; Glycoproteins/*genetics ; Immunosuppressive Agents/*pharmacology ; Male ; Membrane Glycoproteins/*genetics ; Mice ; Mice, Inbred ICR ; Osteoclasts/*cytology ; Pregnenediones/*pharmacology ; RNA, Messenger/analysis ; Receptors, Cytoplasmic and Nuclear/*genetics ; Stromal Cells/cytology

Considerable controversy exists in determining the role of peroxisome proliferator-activated receptor-alpha(PPARalpha) on obesity. Previous reports demonstrated that PPARalpha is a critical modulator of lipid homeostasis, but the overt, obese phenotypic characterization in the strain of PPAR deficient (PPARalpha-/-) mice is influenced by other factors, including diet and genetics. Therefore, it is necessary to establish the phenotypic characterization of PPARalpha-/- mice prior to the obesity-related study. In this study, we observed phenotype of PPARalpha-/- mice on mixed genetic background (C57BL/6Nx129/Sv) fed a high fat diet for 16 weeks. PPARalpha-/- mice, regardless of sex, raised body growth rate significantly comparing with wild type and showed male-specific fatty change in the liver. They were shown to lack hepatic induction of PPARalpha target genes encoding enzymes for fatty acid beta-oxidation.
Adipose Tissue/metabolism ; Animals ; Body Weight ; Cholesterol/blood ; Crosses, Genetic ; Dietary Fats/*administration & dosage ; Female ; Histocytochemistry ; Liver/enzymology/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Knockout ; Obesity/genetics/*metabolism ; Phenotype ; RNA/chemistry/genetics ; Receptors, Cytoplasmic and Nuclear/*deficiency/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Specific Pathogen-Free Organisms ; Transcription Factors/*deficiency/genetics/metabolism ; Triglycerides/blood

Use of adenoviruses as vehicle for gene therapy requires that target cells express appropriate receptors such as coxsakievirus and adenovirus receptor (CAR). We show here that CAR-deficiency in cancer cells, that limits adenoviral gene delivery, can be overcome by using adenovirus complexed with the liposome, Ad-PEGPE [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(poly-ethylene glycol)-2000]. We first confirmed that CT-26 mouse colon cancer cells are deficient in CAR by RT-PCR, and then showed that CT-26 cells infected with Ad-GFP/PEGPE exhibited highly enhanced expression of green fluorescent protein (GFP), compared with those infected with Ad-GFP. GFP expression depends on the dose of liposome and adenovirus. Luciferase expression in livers treated with Ad-luc/PEGPE was about 1,000-fold less than those infected with Ad-luc. In a liver metastasis mouse tumor model developed by intrasplenic injection of CT-26 cells, luciferase expression following i.v. injection of Ad-luc/PEGPE was significantly higher in tumors than in adjacent non-neoplastic liver. Following systemic administration of Ad-GFP/PEGPE, GFP expression increased in tumors more than in adjacent liver while the reverse was true following administration of Ad-GFP. In the latter case, GFP expression was higher in liver than in tumors. This study demonstrates that systemic delivery of PEGPE-adenovirus complex is an effective tool of adenoviral delivery as it overcomes limitation due to CAR deficiency of target cells while reducing hepatic uptake and enhancing adenoviral gene expression in tumors.
*Adenoviridae/genetics/metabolism ; Animals ; Colonic Neoplasms/*genetics/metabolism/pathology/*therapy ; Dose-Response Relationship, Drug ; Gene Therapy ; *Gene Transfer Techniques ; Genetic Vectors ; Green Fluorescent Proteins/genetics ; Liposomes/administration & dosage/chemistry/pharmacokinetics/*therapeutic use ; Liver/drug effects/metabolism ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Transgenic ; NIH 3T3 Cells ; Phosphatidylethanolamines/administration & dosage ; Polyethylene Glycols/administration & dosage ; Receptors, Cytoplasmic and Nuclear/deficiency/genetics ; Receptors, Virus/deficiency/*genetics ; Transcription Factors/deficiency/genetics ; Tumor Cells, Cultured

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics

Cholesterol 7alpha-hydroxylase (CYP7A1) regulates the balance between cholesterol supply and metabolism by catalyzing the rate-limiting step of bile acid biosynthesis. The transcriptional activity of CYP7A1 is tightly controlled by various nuclear receptors. A forkhead transcription factor O1 (FOXO1) plays a critical role in metabolism, and insulin inactivates FOXO1 through Akt-dependent phosphorylation and nuclear exclusion. We investigated the role of insulin-Akt-FOXO1 signaling pathway in CYP7A1 transcriptional regulation since we found putative insulin-response elements, FOXO1 binding sequences, in both rat and human CYP7A1 promoters. However, ectopic expression of FOXO1 increased the rat CYP7A1-, but mildly reduced human CYP7A1-promoter activities in a dose-dependent manner. Similarly to bile acids, insulin treatment increased small heterodimer partner (SHP) mRNA rapidly and transiently, leading to the suppression of CYP7A1 transcription in both human and rodents. Chromatin immunoprecipitation showed that FOXO1 directly bound to rat CYP1A1 promoter in the absence of insulin. FOXO1 binding to the rat promoter was diminished by insulin treatment as well as by expression of SHP. Our results suggest that the stimulation of insulin- signaling pathway of Akt-FOXO1 and SHP expression may regulate cholesterol/bile acid metabolisms in liver, linking carbohydrate and cholesterol metabolic pathways. A prolonged exposure of insulin in hyperinsulinemic insulin resistance or diabetic status represses CYP7A1 transcription and bile acid biosynthesis through SHP induction and FOXO1 inactivation, leading to impairment of the hepatic cholesterol/bile acid metabolisms.
Animals ; Bile Acids and Salts/metabolism ; Cholesterol/*metabolism ; Cholesterol 7-alpha-Hydroxylase/genetics/*metabolism ; Forkhead Transcription Factors/genetics/*metabolism ; Gene Expression Regulation/drug effects ; Glucose/*metabolism ; Hep G2 Cells ; Humans ; Insulin/pharmacology ; Lipid Metabolism/drug effects ; Liver/*metabolism/pathology ; Mice ; Mice, Inbred C57BL ; Mutagenesis, Site-Directed ; Nerve Tissue Proteins/genetics/*metabolism ; Protein Binding/drug effects/genetics ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Receptors, Cytoplasmic and Nuclear/genetics/metabolism ; Sequence Deletion/genetics ; Signal Transduction/drug effects/genetics ; Transcriptional Activation/drug effects/genetics