ABSTRACT
We performed whole exome or genome sequencing in eight multiply affected families with ostensibly isolated congenital anosmia. Hypothesis-free analyses based on the assumption of fully penetrant recessive/dominant/X-linked models obtained no strong single candidate variant in any of these families. In total, these eight families showed 548 rare segregating variants that were predicted to be damaging, in 510 genes. Three Kallmann syndrome genes (FGFR1, SEMA3A, and CHD7) were identified. We performed permutation-based analysis to test for overall enrichment of these 510 genes carrying these 548 variants with genes mutated in Kallmann syndrome and with a control set of genes mutated in hypogonadotrophic hypogonadism without anosmia. The variants were found to be enriched for Kallmann syndrome genes (3 observed vs. 0.398 expected, p = 0.007), but not for the second set of genes. Among these three variants, two have been already reported in genes related to syndromic anosmia (FGFR1 (p.(R250W)), CHD7 (p.(L2806V))) and one was novel (SEMA3A (p.(T717I))). To replicate these findings, we performed targeted sequencing of 16 genes involved in Kallmann syndrome and hypogonadotrophic hypogonadism in 29 additional families, mostly singletons. This yielded an additional 6 variants in 5 Kallmann syndrome genes (PROKR2, SEMA3A, CHD7, PROK2, ANOS1), two of them already reported to cause Kallmann syndrome. In all, our study suggests involvement of 6 syndromic Kallmann genes in isolated anosmia. Further, we report a yet unreported appearance of di-genic inheritance in a family with congenital isolated anosmia. These results are consistent with a complex molecular basis of congenital anosmia.
Subject(s)
Kallmann Syndrome/genetics , Olfaction Disorders/congenital , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Extracellular Matrix Proteins/genetics , Female , Gastrointestinal Hormones/genetics , Humans , Male , Nerve Tissue Proteins/genetics , Neuropeptides/genetics , Olfaction Disorders/genetics , Olfaction Disorders/pathology , Pedigree , Receptor, Fibroblast Growth Factor, Type 1/genetics , Semaphorin-3A/genetics , Exome SequencingABSTRACT
The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor which represses transcription of the rat phosphoenolpyruvate carboxykinase gene. In this study, a regulatory mechanism of the SHARP-2 mRNA level by insulin was analyzed. Insulin rapidly induced the level of SHARP-2 mRNA. This induction was blocked by inhibitors for phosphoinositide 3-kinase (PI 3-K), protein kinase C (PKC), and mammalian target of rapamycin (mTOR), actinomycin D, and cycloheximide. Whereas an adenovirus infection expressing a dominant negative form of atypical PKC lambda (aPKCλ) blocked the insulin-induction of the SHARP-2 mRNA level, insulin rapidly activated the mTOR. Insulin did not enhance transcriptional activity from a 3.7 kb upstream region of the rat SHARP-2 gene. Thus, we conclude that insulin induces the expression of the rat SHARP-2 gene at the transcription level via both a PI 3-K/aPKCλ- and a PI 3-K/mTOR- pathways and that protein synthesis is required for this induction.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Homeodomain Proteins/genetics , Insulin/pharmacology , Signal Transduction/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/biosynthesis , Cell Line, Tumor , Homeodomain Proteins/biosynthesis , Isoenzymes/genetics , Protein Kinase C/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , TOR Serine-Threonine Kinases/metabolism , Transcription, Genetic/drug effectsABSTRACT
The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-ß-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is thePEPCKgene. In this study, we examined an issue of whether theSHARP-2gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKCλ). In addition, when a dominant negative form of aPKCλ was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKCλ. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of theSHARP-2gene is mediated at transcription level by a PI 3-K/aPKCλ pathway.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Aminoimidazole Carboxamide/analogs & derivatives , Basic Helix-Loop-Helix Transcription Factors/genetics , Gene Expression/drug effects , Homeodomain Proteins/genetics , Isoenzymes/metabolism , Protein Kinase C/metabolism , Ribonucleosides/pharmacology , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/genetics , Aminoimidazole Carboxamide/pharmacology , Animals , Calcium/metabolism , Cycloheximide/pharmacology , Dactinomycin/pharmacology , Enzyme Activation , HEK293 Cells , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Liver/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Phosphoenolpyruvate Carboxykinase (GTP)/metabolism , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Synthesis Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , RNA Polymerase II/antagonists & inhibitors , RNA, Messenger/metabolism , Rats , Signal Transduction , Transcription, Genetic/drug effectsABSTRACT
We previously reported that (-)-epigallocatechin-3-gallate (EGCG) increased the level of SHARP-1 mRNA via a phosphoinositide 3-kinase/atypical protein kinase C lambda signaling pathway in rat H4IIE hepatoma cells. In the present study, we investigated other signaling pathway(s). Treating with either compound-C, BAY11-7082, or both, partially blocked the up-regulation of the SHARP-1 gene by EGCG. This suggests that AMP-activated protein kinase (AMPK)- and nuclear factor-kappa B (NF-κB)-signaling pathways were additively involved in the induction mediated by EGCG. Indeed, an AMPK activator induced a level of SHARP-1 mRNA. Although actinomycin D partially blocked the EGCG-induction of that SHARP-1 mRNA level, the nucleotide sequence between -1501 and -1 in the rat SHARP-1 gene did not positively respond to EGCG and NF-κB, respectively. Thus, we conclude that EGCG stimulates multiple signaling pathways in the SHARP-1 gene expression at the transcriptional and post-transcriptional levels and that there is no regulatory region susceptible to EGCG and NF-κB in the examined region.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Catechin/analogs & derivatives , NF-kappa B/metabolism , Signal Transduction/drug effects , Up-Regulation/drug effects , AMP-Activated Protein Kinases/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Catechin/pharmacology , Cell Line, Tumor , NF-kappa B/genetics , RatsABSTRACT
The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor. In this study, we examined the mechanism(s) involved in the regulation of the rat SHARP-2 gene expression by (-)-epigallocatechin-3-gallate (EGCG). The induction of SHARP-2 mRNA by EGCG was repressed by pretreatments with inhibitors for either phosphoinositide 3-kinase (PI3K) or RNA polymerase II. Then, we examined a biological relationship between EGCG and transcription factor NF-κB interfering with the insulin action. The protein levels of the NF-κB were rapidly decreased by an EGCG treatment. Finally, the mechanism(s) of transcriptional activation of the rat SHARP-2 gene by both NF-κB and EGCG was analyzed. While overexpression of the NF-κB p65 protein decreased the promoter activity of the SHARP-2 gene, EGCG did not affect it. Thus, we conclude that EGCG induces the expression of the rat SHARP-2 gene via both the PI3K pathway and degradation of the NF-κB p65 protein.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Catechin/analogs & derivatives , Homeodomain Proteins/genetics , Insulin/metabolism , Up-Regulation/drug effects , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Catechin/pharmacology , Homeodomain Proteins/metabolism , NF-kappa B/genetics , NF-kappa B/metabolism , Rats , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolismABSTRACT
Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling type 2 diabetes mellitus. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcription factor that decreases expression of the phosphoenolpyruvate carboxykinase gene, a gluconeogenic enzyme gene. In this study, we screened for soybean isoflavones that can induce the rat SHARP-2 gene expression and analyzed their mechanism(s). Genistein and (S)-Equol, a metabolite of daidzein, induced rat SHARP-2 gene expression in H4IIE rat hepatoma cells. The (S)-Equol induction was mediated by both the phosphoinositide 3-kinase- and protein kinase C (PKC)-pathways. When a dominant negative form of atypical PKC lambda (aPKCλ) was expressed, the induction of SHARP-2 mRNA level by (S)-Equol was inhibited. In addition, Western blot analyses showed that (S)-Equol rapidly activated both aPKCλ and classical PKC alpha. Furthermore, the (S)-Equol induction was inhibited by treatment with a RNA polymerase inhibitor or a protein synthesis inhibitor. Finally, a reporter gene assay revealed that the transcriptional stimulation by (S)-Equol was mediated by nucleotide sequences located between -4687 and -4133 of the rat SHARP-2 gene. Thus, we conclude that (S)-Equol is an useful dietary supplement to control type 2 diabetes mellitus.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Equol/pharmacology , Homeodomain Proteins/genetics , Insulin/metabolism , Animals , Cell Line, Tumor , Equol/metabolism , Gene Expression Regulation/drug effects , Isoenzymes/metabolism , Isoflavones/metabolism , Protein Kinase C/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Signal Transduction/drug effects , Glycine max/chemistry , Transcription, Genetic/drug effectsABSTRACT
The rat enhancer of split- and hairy-related protein-1 (SHARP-1) is an insulin-inducible transcriptional repressor. In this study, we examined issues of whether (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol, regulates the expression of the rat SHARP-1 gene and which signaling pathway mediates the regulation. When H4IIE cells were treated with EGCG, SHARP-1 mRNA levels rapidly increased. Pretreatments with inhibitors for either phosphoinositide 3-kinase (PI 3-K) or protein kinase C partially blocked EGCG induction. Atypical protein kinase C lambda (aPKCλ) is known as a downstream target of PI 3-K in the liver. When a dominant-negative form of aPKCλ was expressed, the EGCG-induced SHARP-1 mRNAs was inhibited. Finally, Western blot analysis revealed that EGCG rapidly and temporarily stimulates aPKCλ phosphorylation. Thus, we conclude that EGCG induces SHARP-1 gene expression via a PI 3-K/aPKCλ signaling pathway.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Catechin/analogs & derivatives , Gene Expression/drug effects , Isoenzymes/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Kinase C/metabolism , Animals , Catechin/pharmacology , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Insulin/pharmacology , Isoenzymes/antagonists & inhibitors , Liver Neoplasms , Phosphoinositide-3 Kinase Inhibitors , Protein Kinase C/antagonists & inhibitors , RNA, Messenger/analysis , Rats , Signal Transduction/physiologyABSTRACT
Small compounds that activate the insulin-dependent signaling pathway have potential therapeutic applications in controlling insulin-independent diabetes mellitus. In this study, we investigated whether soybean isoflavones could induce the expression of SHARP-2, a downstream component of insulin-dependent signaling pathway, associated with the regulation of blood glucose. One such compound called genistein, rapidly and temporarily induced SHARP-2 mRNA levels in a dose-dependent manner in rat H4IIE hepatoma cells. This induction process was rapidly stimulated by a protein kinase C (PKC) activator and blocked by a PKC inhibitor, suggesting that SHARP-2 may be induced via PKC activation. Upon Western blot analysis, genistein showed a stimulation of PKC phosphorylation. Therefore, we concluded that genistein might transcriptionally induce SHARP-2 through the activation of PKC in H4IIE cells. Our results suggest that genistein might be a useful dietary supplement to control insulin-independent diabetes mellitus by inducing the SHARP-2 expression via a bypass of the insulin-dependent signaling pathway.
Subject(s)
Genistein/pharmacology , Insulin/metabolism , Signal Transduction/drug effects , Animals , Base Sequence , Blotting, Western , Cell Line, Tumor , DNA Primers , Enzyme Activation , Polymerase Chain Reaction , Protein Kinase C-alpha/metabolism , Rats , Transcription, Genetic/drug effectsABSTRACT
We found a novel hypofibrinogenemia designated as Matsumoto VII (M-VII), which is caused by a heterozygous nucleotide deletion at position g.7651 in FGG and a subsequent frameshift mutation in codon 387 of the gamma-chain. This frameshift results in 25 amino acid substitutions, late termination of translation with elongation by 15 amino acids, and the introduction of a canonical glycosylation site. Western blot analysis of the patient's plasma fibrinogen visualised with anti-gamma-chain antibody revealed the presence of two extra bands. To identify the extra bands and determine which of the above-mentioned alterations caused the assembly and/or secretion defects in the patient, 11 variant vectors that introduced mutations into the cDNA of the gamma-chain or gamma'-chain were transfected into Chinese hamster ovary cells. In vitro expression of transfectants containing gammaDelta7651A and gammaDelta7651A/399T (gammaDelta7651A with an amino acid substitution of 399Asn by Thr and a variant lacking the canonical glycosylation site) demonstrated a reduction in secretion to approximately 20% of the level seen in the transfectants carrying the normal gamma-chain. Furthermore, results from other transfectants demonstrated that eight aberrant residues between 391 and 398 of the M-VII variant, rather than the 15 amino acid extension or the additional glycosylation, are responsible for the reduced levels of assembly and secretion of M-VII variant fibrinogen. Finally, the results of this study and our previous reports demonstrate that the fibrinogen gamma-chain C-terminal tail (388-411) is not necessary for protein assembly or secretion, but the aberrant amino acid sequence observed in the M-VII variant (especially 391-398) disturbs these functions.
Subject(s)
Afibrinogenemia/genetics , Amino Acid Substitution , Blood Coagulation/genetics , Fibrinogens, Abnormal/genetics , Frameshift Mutation , Heterozygote , Afibrinogenemia/blood , Amino Acid Sequence , Animals , Blood Coagulation Tests , Blotting, Western , CHO Cells , Cricetinae , Cricetulus , DNA Mutational Analysis , Female , Fibrinogens, Abnormal/metabolism , Genetic Predisposition to Disease , Glycosylation , Humans , Japan , Middle Aged , Molecular Sequence Data , Phenotype , Protein Multimerization , Protein Processing, Post-Translational , Protein Structure, Tertiary , TransfectionABSTRACT
BACKGROUND: To study the functions of residues gamma326Cys and gamma339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring gamma326Tyr and gamma326Ser variants, along with gamma326Ala and gamma339Ala variants. METHODS: A fibrinogen gamma-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed. RESULTS: The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen. CONCLUSIONS: The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the gamma-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of gamma326Tyr and gamma326Ser suggest that heterozygous fibrinogen molecules containing variant gamma-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia.
Subject(s)
Fibrinogen/genetics , Fibrinogen/metabolism , Recombinant Proteins/metabolism , Amino Acid Substitution , Animals , Binding Sites , CHO Cells , Cricetinae , Cricetulus , Dimerization , Fibrinogen/chemistry , Genetic Variation , Mutagenesis, Site-Directed , Protein Binding , Recombinant Proteins/chemistry , Structure-Activity RelationshipABSTRACT
ZHX2 and ZHX3 are the members of the ZHX transcriptional repressor family. To investigate the regulatory role of the repressors in hepatocytes and their involvement in carcinogenesis, the expression levels of ZHX2 and ZHX3 mRNAs were examined. The dRLh-84 hepatoma cells considerably expressed cancer marker genes PKM and HK II that are expressed in developing fetal tissues and cancer cells but repressed in normal hepatocytes. In dRLh-84 cells, the expression levels of ZHX2 and ZHX3 were very low compared with rat hepatocytes. Upon the reporter gene analysis utilizing the promoter region of these genes, ZHX3 repressed the transcription of the reporter luciferase gene from both promoters while ZHX2 only repressed that from HK II promoter. The promoter activity of alpha-fetoprotein was also repressed by the expression of ZHX2 in HLE hepatoma cells in a dose-dependent manner. We concluded that ZHX2 and ZHX3 were involved in the transcriptional repression of the hepatocellular cacinoma markers in normal hepatocytes, suggesting that the failure of the ZHX2 and/or ZHX3 expression might be a critical factor in the hepatocellular carcinogenesis.
Subject(s)
Biomarkers, Tumor/metabolism , Hepatocytes/metabolism , Homeodomain Proteins/physiology , Transcription Factors/physiology , Animals , Base Sequence , Cell Line, Tumor , DNA Probes , Homeodomain Proteins/metabolism , Rats , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolismABSTRACT
BACKGROUND: Antibody-antigen complexes formed by IgG autoantibodies against citrullinated proteins and citrullinated forms of the alpha- and beta-chains of fibrin in rheumatoid synovial tissue play a key role in the pathophysiology of rheumatoid arthritis. METHODS: Recombinant fibrinogen was citrullinated by rabbit skeletal muscle peptidylarginine deiminase so that we could analyze the function of citrullinated fibrinogen. Namely, thrombin-catalyzed fibrin polymerization and fibrinopeptide release, protection against plasmin digestion, and factor XIIIa-catalyzed cross-linking of fibrin or fibrinogen were performed. RESULTS: Strong citrullination of the Aalpha- and Bbeta-chains and weak citrullination of the gamma-chain were detected by an anti-modified citrulline detection kit. Citrullinated fibrinogen did not release FPA or FPB by thrombin catalyzation and no thrombin-stimulated conversion of fibrinogen into fibrin occurred. The citrullination of fibrinogen did not affect the 3 functions of the C-terminal gamma-chain, "a-hole," low affinity Ca binding, and gamma-gamma cross-linking. CONCLUSION: Our functional analyses demonstrated that no thrombin-stimulated conversion of fibrinogen into fibrin occurred, because citrullinated fibrinogen did not release FPA or FPB after thrombin catalyzation. Our results and those of other reports suggest that citrullinated fibrin and fibrinogen are present in the synovium and might both be associated with the pathophysiology of RA.