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1.
Scand J Gastroenterol ; 36(3): 276-82, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11305515

ABSTRACT

BACKGROUND: Transforming growth factor-alpha (TGF-alpha) is a key mediator of colonic mucosal protection and/or repair mechanisms in orally induced acute dextran sodium sulphate (DSS) colitis. However, it also has been suggested that TGF-alpha may contribute to malignant transformation in the colon. The aim of the studies was to determine whether TGF-alpha is needed for malignant transformation in orally induced chronic DSS colitis using TGF-alpha deficient mice (wa-1) and Balb/c mice, a strain competent in TGF-alpha. METHODS: Chronic colitis was induced by oral administration of DSS (5%) for 7 days followed by drinking water for 10 days in wa-1 and Balb/c mice (n = 20, per group). In the two subsequent cycles (7 days DSS, 10 days water) 3% DSS-water was utilized due to a high mortality in the wa-1 group. Mucosal injury severity was assessed histologically and graded (three grades). A crypt damage score (CDS) reflecting all three grades of mucosal pathology was calculated. Mucosal dysplasia and cancerous lesions were noted. RESULTS: Seven per cent of the entire colonic mucosa was completely destroyed in wa-1 animals compared to 3% in Balb/c mice (P < 0.05). The CDS was 10.2 +/- 0.4 and 4.8 +/- 0.3 in wa-1 and Balb/c mice, respectively (P < 0.05). Fifteen incidences of mucosal dysplasia were found in the 10 surviving wa-1 animals and 31 incidences were found in 20 Balb/c animals. In both groups, one fully developed adenomatous cancerous lesion was present. CONCLUSIONS: The markedly increased severity of mucosal injury in chronic induced DSS colitis in TGF-alpha deficient wa-1 mice compared to Balb/c mice further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon. The appearance of dysplastic and cancerous lesions in TGF-alpha deficient animals suggests that TGF-alpha per se is not essential for malignant mucosal cell transformation in colitis.


Subject(s)
Cell Transformation, Neoplastic/pathology , Colitis/pathology , Intestinal Mucosa/pathology , Transforming Growth Factor alpha/analysis , Animals , Chronic Disease , Dextran Sulfate , Disease Models, Animal , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred Strains , Probability , Radioimmunoassay , Sensitivity and Specificity , Severity of Illness Index , Statistics, Nonparametric
2.
Circulation ; 99(24): 3103-9, 1999 Jun 22.
Article in English | MEDLINE | ID: mdl-10377072

ABSTRACT

BACKGROUND: Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques, where in their active form, they may contribute to vascular remodeling and plaque disruption. In this study, we tested the hypothesis that membrane type 1 MMP (MT1-MMP), a novel transmembrane MMP that activates pro-MMP-2 (gelatinase A), is expressed in human atherosclerotic plaques and that its expression is regulated by proinflammatory molecules. METHODS AND RESULTS: MT1-MMP expression was examined in normal and atherosclerotic human arteries by immunocytochemistry with specific antibodies. MT1-MMP expression in human saphenous vein-derived smooth muscle cells (SMCs) maintained in tissue culture was determined under basal conditions and in response to proinflammatory molecules (interleukin [IL]-1alpha, tumor necrosis factor [TNF]-alpha, and oxidized LDL [ox-LDL]) by use of Northern blot and ribonuclease protection assays for mRNA, Western blot and immunoprecipitation for protein, and gelatin zymography for catalytic activity. Medial SMCs of normal vessel wall expressed MT1-MMP. In atherosclerotic arteries, MT1-MMP expression was noted within the complex atheroma colocalizing with SMCs and macrophages (Mphi). Cultured SMCs constitutively expressed MT1-MMP mRNA and protein, which increased 2- to 4-fold over control in a time-dependent manner within 4 to 8 hours of exposure to IL-1alpha, TNF-alpha, and ox-LDL (thiobarbituric acid-reactive substances, 13.4 nmol/mg LDL protein), whereas native LDL had no effect. Flow cytometry revealed MT1-MMP expression by human monocyte-derived Mphi, which increased 3.8-fold over baseline within 6 hours after exposure to 10 ng/mL TNF-alpha. CONCLUSIONS: This study demonstrates that MT1-MMP, an activator of pro-MMP-2, is expressed by SMCs and Mphi in human atherosclerotic plaques. Furthermore, proinflammatory molecules upregulate MT1-MMP expression in vascular SMCs and Mphi. Thus, activation of SMCs and Mphi by proinflammatory molecules may influence extracellular matrix remodeling in atherosclerosis by regulating MT1-MMP expression.


Subject(s)
Arteriosclerosis/metabolism , Gene Expression Regulation, Enzymologic/immunology , Inflammation Mediators/metabolism , Metalloendopeptidases/genetics , Muscle, Smooth, Vascular/enzymology , Antibodies, Monoclonal , Arteriosclerosis/pathology , Blotting, Northern , Cells, Cultured , Coronary Vessels/chemistry , Coronary Vessels/enzymology , Enzyme Precursors/metabolism , Flow Cytometry , Gelatinases/analysis , Gelatinases/biosynthesis , Gelatinases/immunology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Interleukin-1/pharmacology , Lipoproteins/metabolism , Lipoproteins, LDL/pharmacology , Macrophages/chemistry , Macrophages/enzymology , Matrix Metalloproteinase 2 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/analysis , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/immunology , Metalloendopeptidases/metabolism , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/pathology , RNA, Messenger/analysis , Saphenous Vein/cytology , Tissue Inhibitor of Metalloproteinase-2/analysis , Tissue Inhibitor of Metalloproteinase-2/immunology , Tumor Necrosis Factor-alpha/pharmacology
3.
J Biol Chem ; 274(17): 11924-9, 1999 Apr 23.
Article in English | MEDLINE | ID: mdl-10207013

ABSTRACT

We investigated whether inflammatory cytokines or oxidized low density lipoproteins (Ox-LDL) present in human atheroma modulate extracellular matrix degradation by inducing membrane type 1-matrix metalloproteinase (MT1-MMP) expression. Cultured human endothelial cells (EC) constitutively expressed MT1-MMP mRNA and protein with enzymatic activity. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha, or interleukin-1beta caused a time-dependent increase in the steady-state MT1-MMP mRNA levels within 4 h of exposure, peaking about 4-fold by 6 h, and remaining elevated for 12 h. Increased MT1-MMP mRNA correlated with a 2.5-fold increase in MT1-MMP protein in EC membranes. Ox-LDL also increased MT1-MMP mRNA levels that varied with the duration of exposure and degree of LDL oxidation. The increase in MT1-MMP mRNA occurred within 6 h of exposure to Ox-LDL and peaked over 3-fold by 6 h. Ox-LDL, but not native LDL, increased MT1-MMP protein by 2-fold in EC membranes. A combination of TNF-alpha and Ox-LDL was additive in increasing MT1-MMP expression. Nuclear run-on assays showed that TNF-alpha or Ox-LDL augmented steady-state mRNA levels by increased transcription of the MT1-MMP gene. These findings indicate that activation of EC by inflammatory cytokines and/or Ox-LDL increase MT1-MMP expression. Since MT1-MMP promotes matrix degradation by activating pro-MMP-2, these results suggest a novel mechanism whereby cytokines or Ox-LDL may influence extracellular matrix remodeling.


Subject(s)
Endothelium, Vascular/metabolism , Interleukin-1/metabolism , Lipoproteins, LDL/metabolism , Metalloendopeptidases/metabolism , Tumor Necrosis Factor-alpha/metabolism , Base Sequence , Cells, Cultured , DNA Primers , Endothelium, Vascular/cytology , Enzyme Activation , Gelatinases/metabolism , Gene Expression Regulation , Humans , Matrix Metalloproteinase 2 , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Transcription, Genetic
4.
Gut ; 43(1): 64-70, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9771407

ABSTRACT

BACKGROUND: Transforming growth factor alpha (TGF-alpha) knockout mice have increased susceptibility to dextran sodium sulphate (DSS) induced colitis. AIM: To substantiate the findings that TGF-alpha is a key mediator of colonic mucosal protection and/or repair mechanisms by evaluating the susceptibility of mice overexpressing TGF-alpha to DSS induced colitis. METHODS: TGF-alpha overexpression was induced in transgenic mice by ZnSO4 administration in drinking water (TG+). Three groups were used as controls: one transgenic group without ZnSO4 administration (TG-), and two non-transgenic littermate groups receiving ZnSO4 (Non-TG+) or only water (Non-TG-). Acute colitis was induced in all groups by administration of DSS (5%, w/v) in drinking water for six days and libitum. RESULTS: About 35-39% of the entire colonic mucosa was destroyed in Non-TG-, Non-TG+, and TG- animals compared with 9% in TG+ mice. the crypt damage score was 18.7 (0.9), 18.2 (1.0), 18.9 (0.8), and 6.8 (1.5) (means (SEM)) in Non-TG-, Non-TG+, TG-, and TG+ mice respectively. Mucin and bromodeoxyuridine staining were markedly enhanced in colons of TG+ mice compared with controls, indicating increased mucosal protection and regeneration. CONCLUSIONS: The significantly reduced susceptibility of mice overexpressing TGF-alpha to DSS further substantiates that endogenous TGF-alpha is a pivotal mediator of protection and/or healing mechanisms in the colon.


Subject(s)
Colitis/metabolism , Transforming Growth Factor alpha/physiology , Acute Disease , Animals , Body Weight , Cell Division , Colitis/chemically induced , Colitis/pathology , Colon/metabolism , Colon/pathology , Dextran Sulfate , Disease Susceptibility , Intestinal Mucosa/metabolism , Intestinal Mucosa/pathology , Male , Mice , Mice, Transgenic , Radioimmunoassay , Statistics, Nonparametric , Transforming Growth Factor alpha/analysis , Zinc/administration & dosage
5.
Genomics ; 49(2): 283-9, 1998 Apr 15.
Article in English | MEDLINE | ID: mdl-9598316

ABSTRACT

The RBMY (RNA-binding motif, Y chromosome) gene family encodes a germ-cell-specific nuclear protein implicated in spermatogenesis. It consists of approximately 30 genes and pseudogenes, found on both arms of the Y chromosome. RBMY shares high homology with an autosomal hnRNPG gene that contains an RNA-binding motif and one of the four SRGY repeats found in RBMY. One proposal is that RBMY represents an ancestral hnRNPG gene, transposed to the Y chromosome and then amplified. We characterized seven RBMY genes in interval 6 of the Y chromosome long arm. Four have the normal structure with 12 exons spanning 15 kb, whereas one lacks the first 3 exons, therefore representing a pseudogene. The remaining two genes belong to a different subfamily, resembling the autosomal hnRNPG gene with only one SRGY repeat. We also found that most RBMY genes in interval 6 are arranged in tandem. The structure and organization of the Y-linked RBMY genes support the transposition-amplification hypothesis.


Subject(s)
DNA Transposable Elements/genetics , Gene Amplification/genetics , RNA, Heterogeneous Nuclear/genetics , RNA-Binding Proteins/genetics , Ribonucleoproteins/genetics , Y Chromosome/genetics , Amino Acid Sequence , Animals , Evolution, Molecular , Heterogeneous-Nuclear Ribonucleoproteins , Humans , Male , Mice , Molecular Sequence Data , Nuclear Proteins , Repetitive Sequences, Nucleic Acid
6.
Genomics ; 45(2): 355-61, 1997 Oct 15.
Article in English | MEDLINE | ID: mdl-9344660

ABSTRACT

The RBM (RNA-binding motif) gene family on the human Y chromosome encodes proteins with an RNA-binding domain. Its exclusive expression in germ cells and its partial deletion in some azoospermic or severely oligospermic males provide evidence of a role for RBM genes in spermatogenesis. There are approximately 30 RBM genes, found on both arms of the Y chromosome. Two RBM cDNA clones with slightly different sequences have been reported. To investigate the number of functional genes, we studied RBM expression by use of RT-PCR of RBM transcripts and by characterizing numerous RBM cDNA clones. A total of 27 RT-PCR and 19 cDNA clones were sequenced. Whereas the RT-PCR clones pointed to the existence of at least six RBM subfamilies (RBMI to RBMVI), the cDNA clones indicated that only RBMI is actively transcribed and encodes functional proteins. A total of six RBMI genes were identified, which produce four polypeptides due to some silent base substitutions. The transcripts of each gene are alternatively spliced to generate protein isoforms with three or four SRGY boxes, thus greatly increasing the complexity of the products of the RBM gene family. We also provide evidence suggesting that a 5-bp deletion in a previously reported RBM cDNA clone represents a processing irregularity.


Subject(s)
Multigene Family , RNA-Binding Proteins/genetics , Spermatogenesis/genetics , Y Chromosome/genetics , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Humans , Infertility, Male/genetics , Male , Mutation , Nuclear Proteins , Phenotype , Polymerase Chain Reaction
7.
Mamm Genome ; 8(10): 756-9, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9321470

ABSTRACT

The DAZ genes on the human Y Chromosome (Chr) are strong candidates for the azoospermia factor AZF. They are frequently deleted in azoospermic or severely oligospermic males and are expressed exclusively in germ cells. In addition, the DAZ genes share a high degree of similarity with a Drosophila male infertility gene, boule. The predicted DAZ proteins contain an RNA recognition motif (RRM), and multiple copies of a repeat (the DAZ repeat) in tandem array. To understand the DAZ gene family and its expression, the DAZ genomic structure and RNA transcripts in numerous males, as well as several DAZ cDNA clones were analyzed. The results of genomic Southern blot showed that each male contains multiple DAZ genes with varying numbers of DAZ repeats, and that the copy number of the DAZ repeats are polymorphic in the population. The presence of multiple species of DAZ transcripts with different copy number and arrangement of the DAZ repeats in an individual suggests that more than one DAZ gene are transcribed. The existence of multiple functional DAZ genes complicates the analysis of genotype/phenotype correlations among males with varying sperm counts.


Subject(s)
Infertility, Male/genetics , Oligospermia/genetics , RNA-Binding Proteins/genetics , Repetitive Sequences, Nucleic Acid , Y Chromosome/genetics , Alleles , DNA, Complementary/analysis , Deleted in Azoospermia 1 Protein , Humans , Male , Multigene Family , Polymerase Chain Reaction , Polymorphism, Genetic , RNA/analysis , Sequence Analysis, DNA , Testis/chemistry
8.
Mol Hum Reprod ; 3(8): 705-8, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9294855

ABSTRACT

The DAZLA (DAZ Like Autosomal) gene on human chromosome 3 shares a high degree of homology with the DAZ (Deleted in AZoospermia) gene family on the Y chromosome, a gene family frequently deleted in males with azoospermia or severe oligospermia. The involvement of both DAZ and DAZLA in spermatogenesis is suggested by their testis-specific expression and their homology with a Drosophila male infertility gene, boule. Whereas male infertility resulting from deletion of the DAZ genes on the Y chromosome occurs sporadically, that due to a defective DAZLA gene is expected to be inheritable. The fraction of males with idiopathic azoospermia or oligospermia that harbour mutations in the DAZLA gene remains unknown. As a prerequisite for mutation screening, the genomic structure of the DAZLA gene was elucidated and found to consist of 11 exons spanning 19 kh. The exon/intron boundaries are conserved between DAZ and DAZLA. The 5' end of both genes are hypomethylated in spermatozoa but not in leukocytes or placenta, consistent with the expression pattern of the genes. The genomic structure of DAZLA paves the way for mutation detection in families with autosomal recessive male infertility.


Subject(s)
Chromosomes, Human, Pair 3 , Infertility, Male/genetics , Proteins/genetics , RNA-Binding Proteins , Base Sequence , Chromosome Mapping , DNA Methylation , Exons , Genomic Library , Humans , Introns , Male , Molecular Sequence Data , Multigene Family , Restriction Mapping , Y Chromosome
9.
Hum Mol Genet ; 5(12): 2013-7, 1996 Dec.
Article in English | MEDLINE | ID: mdl-8968756

ABSTRACT

The DAZ (Deleted in AZoospermia) and DAZLA (DAZ-like autosomal) genes may be determinants of male infertility. The DAZ gene on the long arm of the human Y chromosome is a strong candidate for the 'azoospermia factor' (AZF). Its role in spermatogenesis is supported by its exclusive expression in testis, its deletion in a high percentage of males with azoospermia or severe oligospermia, and its homology with a Drosophila male infertility gene boule. No DAZ homologous sequences have been found on the mouse Y chromosome. Instead, a Dazla gene was isolated from mouse chromosome 17 and has been considered to be a murine homologue of DAZ. However, the homology between human DAZ and mouse Dazla is not strong, and Dazla contains only one of the seven DAZ repeats found in DAZ. We report the isolation of the human DAZLA gene by screening a human testis cDNA library with a DAZ cDNA clone. DAZLA encodes only one DAZ repeat and shares high homology with the mouse Dazla, indicating that these two genes are homologues. Using a panel of rodent-human somatic cell lines and fluorescence in situ hybridization, the DAZLA gene was mapped to 3p24, a region not known to share homology with mouse chromosome 17. The DAZLA gene may be involved in some familial cases of autosomal recessive male infertility.


Subject(s)
Infertility, Male/genetics , Proteins/genetics , RNA-Binding Proteins , Testis/metabolism , Y Chromosome , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , DNA, Complementary/genetics , DNA, Complementary/isolation & purification , Humans , Male , Mice , Molecular Sequence Data , Organ Specificity , Protein Biosynthesis , Sequence Alignment
10.
Artery ; 22(2): 61-79, 1996.
Article in English | MEDLINE | ID: mdl-8864249

ABSTRACT

The effects of the cholesterol oxides on low density lipoprotein receptor (LDLR) gene expression were investigated. Cultured rabbit aortic smooth muscle cells were incubated with 1, 2, and 5 micrograms/ml culture medium concentrations of pure cholesterol, 25-hydroxycholesterol (25-OH), 7-ketocholesterol (7-keto), cholestane-3 beta, 5 alpha, 6 beta-triol (triol) and cholesterol-5 alpha, 6 alpha-epoxide (epoxide) for 12 hours and with vehicle only as control. Total mRNAs were extracted and electrophoresed. Northern blot hybridization analyses were performed. The results showed mRNA expressions of LDLR gene were inhibited to 16.1 +/- 4.4%, 33.8 +/- 0.6%, 42.8 +/- 1.8% and 46.9 +/- 3.9% of control by 25-OH, 7-keto, epoxide and triol respectively. Pure cholesterol showed only minimal inhibition. The inhibitions were time dependent. Although cholesterol oxides have been shown to alter many membrane-related functions and the LDLR domain are located in the cell membrane. The findings of this study suggested that the cholesterol oxides exerted their repressive actions on LDLR function primarily by down-regulating LDLR gene expression rather than directly upon cell membrane.


Subject(s)
Cholesterol/pharmacology , Gene Expression Regulation/drug effects , Hydroxycholesterols/pharmacology , Muscle, Smooth, Vascular/metabolism , Receptors, Lipoprotein/genetics , Animals , Blotting, Northern , Cells, Cultured , Cholestanols/pharmacology , Cholesterol/analogs & derivatives , Ketocholesterols/pharmacology , RNA, Messenger/analysis , Rabbits , Receptors, Lipoprotein/metabolism
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