ABSTRACT
PURPOSE: Maspin, a tumor-suppressor protein that regulates cell migration, invasion, and adhesion, is synthesized by many normal epithelial cells, but downregulated in invasive epithelial tumor cells. The purpose of this study was to determine whether cells in the normal human cornea express maspin and whether maspin affects corneal stromal cell adhesion to extracellular matrix molecules. METHODS: Maspin expression was analyzed by immunodot blot, Western blot, and RT-PCR analyses in cells obtained directly from human corneas in situ. Maspin protein and mRNA were also studied in primary and passaged cultures of corneal stromal cells using Western blot analysis, RT-PCR, and immunofluorescence microscopy. Maspin cDNA was cloned and sequenced from human corneal epithelial cells and expressed in a yeast system. The recombinant maspin was used to study attachment of cultured human corneal stromal cells to extracellular matrices. RESULTS: Maspin mRNA and micromolar amounts of the protein were found in all three layers of the human cornea in situ, including the stroma. Maspin was also detected in primary and first-passage corneal stromal cells, but its expression was downregulated in subsequent passages. Late-passage stromal cells, which did not produce maspin, responded to exogenous recombinant maspin as measured by increased cell adhesion not only to fibronectin, similar to mammary gland tumor epithelial cells, but also to type I collagen, type IV collagen, and laminin. CONCLUSIONS: The corneal stromal cell is the first nonepithelial cell type shown to synthesize maspin. Loss of maspin expression in late-passage corneal stromal cells in culture and their biological response to exogenous maspin suggests a role for maspin on the stromal cells in the cornea. Maspin may function within the cornea to regulate cell adhesion to extracellular matrix molecules and perhaps to regulate the migration of activated fibroblasts during corneal stromal wound healing.
Subject(s)
Cornea/physiology , Extracellular Matrix/physiology , Proteins/physiology , Serine Proteinase Inhibitors/physiology , Serpins/physiology , Stromal Cells/physiology , Cell Adhesion/drug effects , Cell Adhesion/physiology , Cells, Cultured , Cornea/cytology , Genes, Tumor Suppressor , Humans , Protein Biosynthesis , Proteins/pharmacology , Reference Values , Serine Proteinase Inhibitors/biosynthesis , Serine Proteinase Inhibitors/pharmacology , Serpins/biosynthesis , Serpins/pharmacology , Tissue DistributionABSTRACT
Three N-terminal basic residues of Tn5 transposase, which are associated with proteolytic cleavages by Escherichia coli proteinases, were mutated to glutamine residues with the goal of producing more stable transposase molecules. Mutation of either arginine 30 or arginine 62 to glutamine produced transposase molecules that were more stable toward E. coli proteinases than the parent hyperactive Tn5 transposase, however, they were inactive in vivo. In vitro analysis revealed these mutants were inactive, because both Arg(30) and Arg(62) are required for formation of the paired ends complexes when the transposon is attached to the donor backbone. These results suggest Arg(30) and Arg(62) play critical roles in DNA binding and/or synaptic complex formation. Mutation of lysine 40 to glutamine did not increase the overall stability of the transposase to E. coli proteinases. This mutant transposase was only about 1% as active as the parent hyperactive transposase in vivo; however, it retained nearly full activity in vitro. These results suggest that lysine 40 is important for a step in the transposition mechanism that is bypassed in the in vitro assay system, such as the removal of the transposase molecule from DNA following strand transfer.
Subject(s)
Arginine/metabolism , Lysine/metabolism , Transposases/metabolism , Base Sequence , DNA/metabolism , DNA Primers , Hydrolysis , Models, Molecular , Transposases/chemistryABSTRACT
We have identified the key protein substrate of gelatinase B/MMP-9 (GB) that is cleaved in vivo during dermal-epidermal separation triggered by antibodies to the hemidesmosomal protein BP180 (collagen XVII, BPAG2). Mice deficient in either GB or neutrophil elastase (NE) are resistant to blister formation in response to these antibodies in a mouse model of the autoimmune disease bullous pemphigoid. Disease develops upon complementation of GB -/- mice with NE -/- neutrophils or NE -/- mice with GB -/- neutrophils. Only NE degrades BP180 and produces dermal-epidermal separation in vivo and in culture. Instead, GB acts upstream to regulates NE activity by inactivating alpha1-proteinase inhibitor (alpha1-PI). Excess NE produces lesions in GB -/- mice without cleaving alpha1-PI. Excess alpha1-PI phenocopies GB and NE deficiency in wild-type mice.
Subject(s)
Matrix Metalloproteinase 9/metabolism , Pemphigoid, Bullous/enzymology , alpha 1-Antitrypsin/metabolism , Animals , Animals, Newborn , Antibodies/immunology , Antibodies/pharmacology , Autoantigens/immunology , Autoantigens/metabolism , Blister/chemically induced , Blister/enzymology , Blister/immunology , Blister/pathology , Cell Adhesion , Dermis/drug effects , Dermis/enzymology , Dermis/immunology , Dermis/pathology , Disease Models, Animal , Epidermis/drug effects , Epidermis/enzymology , Epidermis/immunology , Epidermis/pathology , Leukocyte Elastase/deficiency , Leukocyte Elastase/genetics , Leukocyte Elastase/metabolism , Matrix Metalloproteinase 9/deficiency , Matrix Metalloproteinase 9/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophil Infiltration , Neutrophils/drug effects , Neutrophils/enzymology , Neutrophils/immunology , Non-Fibrillar Collagens , Pemphigoid, Bullous/chemically induced , Pemphigoid, Bullous/immunology , Pemphigoid, Bullous/pathology , Peroxidase/metabolism , Protein Processing, Post-Translational , Substrate Specificity , Collagen Type XVIIABSTRACT
BP180 is a homotrimeric transmembrane protein with a carboxy-terminal ectodomain that forms an interrupted collagen triple helix. Null type mutations in the BP180 gene produce a recessive subepidermal blistering disease, non-Herlitz junctional epidermolysis bullosa. Like the null mutations, a glycine substitution (G627V) within the longest BP180 collagenous domain (COL15) is also associated with the recessive skin disease; however, unlike the null mutations, this glycine substitution appears to act in a dominant fashion to give rise to a novel form of random pitting dental enamel hypoplasia. The dominant effects of this mutation were thought to be due to alterations in the assembly and/or stability of this BP180 collagenous region. To further investigate this issue, a structural analysis was performed on recombinant forms of the wild type and G627V mutant BP180 ectodomain. Both proteins were found to form collagen-like triple helices with very similar Stokes radii and melting temperatures and exhibited very similar rates of synthesis, secretion and turn-over. Tryptic digestion analysis revealed that the mutant G627V-sec180e contains an additional highly sensitive proteolytic site that maps within the region of the mutation. Thus, the disease-associated G627V mutation in BP180 does not grossly alter protein structure, but causes a local destabilization of the triple-helix that exposes sensitive residues to the in vitro effects of trypsin and possibly affects its structure-function in vivo.
Subject(s)
Autoantigens/metabolism , Carrier Proteins , Collagen/metabolism , Cytoskeletal Proteins , Glycine/metabolism , Nerve Tissue Proteins , Non-Fibrillar Collagens , Amino Acid Sequence , Amino Acid Substitution , Animals , Autoantigens/genetics , Cell Line, Transformed , Collagen/genetics , Dystonin , Gene Expression , Glycine/genetics , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , Rabbits , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Trypsin/metabolism , Collagen Type XVIISubject(s)
Blood Glucose/analysis , Diabetes Mellitus, Type 1/diet therapy , Diabetes Mellitus, Type 2/diet therapy , Adult , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/diagnosis , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/diagnosis , Dietetics , Eating/physiology , Education, Medical , Humans , Models, Educational , Nutritional Sciences/education , Patient Care Team , Physician's Role , WisconsinABSTRACT
Bullous pemphigoid (BP) is an autoimmune skin disease characterized by subepidermal blisters and autoantibodies against 2 hemidesmosome-associated proteins, BP180 and BP230. The immunopathologic features of BP can be reproduced in mice by passive transfer of anti-BP180 antibodies. Lesion formation in this animal model depends upon complement activation and neutrophil recruitment. In the present study, we investigated the role of neutrophil elastase (NE) in antibody-induced blister formation in experimental BP. Abnormally high levels of caseinolytic activity, consistent with NE, were detected in extracts of lesional skin and blister fluid of mice injected with anti-BP180 IgG. The pathogenic anti-BP180 IgG failed to induce subepidermal blistering in NE-null (NE(-/-)) mutant mice. NE(-/-) mice reconstituted with neutrophils from wild-type mice became susceptible to experimental BP. Wild-type mice given NE inhibitors (alpha1-proteinase inhibitor and Me-O-Suc-Ala-Ala-Pro-Val-CH(2)Cl), but not mice given cathepsin G/chymase inhibitors (alpha1-antichymotrypsin or Z-Gly-Leu-Phe-CH(2)Cl), were resistant to the pathogenic activity of anti-BP180 antibodies. Incubation of murine skin with NE induced BP-like epidermal-dermal detachment. Finally, NE cleaved BP180 in vitro and in vivo. These results implicate NE directly in the dermal-epidermal cleavage induced by anti-BP180 antibodies in the experimental BP model.
Subject(s)
Carrier Proteins , Collagen , Cytoskeletal Proteins , Leukocyte Elastase/physiology , Nerve Tissue Proteins , Non-Fibrillar Collagens , Pemphigoid, Bullous/etiology , Animals , Autoantigens/immunology , Autoantigens/physiology , Dystonin , Humans , Immunoglobulin G/toxicity , Mice , Mice, Inbred BALB C , Pemphigoid, Bullous/enzymology , Peroxidase/metabolism , Collagen Type XVIIABSTRACT
DNA transposition is an underlying process involved in the remodeling of genomes in all types of organisms. We analyze the multiple steps in cut-and-paste transposition using the bacterial transposon Tn5 as a model. This system is particularly illuminating because of the existence of structural, genetic, and biochemical information regarding the two participating specific macromolecules: the transposase and the 19-bp sequences that define the ends of the transposon. However, most of the insights should be of general interest because of similarities to other transposition-like systems such as HIV-1 DNA integration into the host genome.
Subject(s)
DNA Transposable Elements , Base Sequence , DNA/metabolism , Protein Binding , Transposases/chemistry , Transposases/metabolismABSTRACT
The avascular cornea has limited access to plasma proteins, including plasminogen, a protein that is synthesized by the liver and supplied to most tissues via the blood. Recent experiments by others using plasminogen-deficient mice revealed the importance of plasmin, the active form of plasminogen, for the maintenance of the normal cornea and for corneal wound healing [Kao, Kao, Bugge, Kaufman, Kombrinck, Converse, Good and Degan (1998) Invest. Ophthalmol. Vis. Sci. 39, 502-508; Drew, Kaufman, Kombrinck, Danton, Daugherty, Degen and Bugge (1998) Blood 91, 1616-1624]. In the present experiments, plasmin was identified as a major serine proteinase in the human cornea. The major plasminogen and plasmin forms on non-reducing zymograms and Western blots had Mr values of 76x10(3) and 85x10(3), with minor forms of Mr 200x10(3), 135x10(3), 68x10(3) and 45x10(3). Angiostatin-like peptides with Mrs of 48x10(3), 45x10(3) and 38x10(3) were observed which bound to lysine-Sepharose and reacted with anti-plasminogen monoclonal antibodies directed towards kringle domains 1-3 of plasminogen. The cornea contained 1.1+/-0.15 microgram of plasminogen+plasmin/cornea, or 0.54+/-0.05 microgram of plasminogen+plasmin/mg of protein. Cornea conditioned medium contained nine times the amount of plasminogen+plasmin that could be extracted from the cornea. These data suggested that corneal cells, unlike most extrahepatic cells, synthesize plasminogen. The synthesis of plasminogen by the cornea was confirmed by immunoprecipitation of metabolically labelled plasminogen, sequencing of its cDNA obtained by reverse transcriptase-PCR and inhibition of protein synthesis. Interleukins-1alpha and -1beta stimulated corneal plasminogen synthesis 2-3-fold; however, interleukin-6 decreased corneal plasminogen synthesis by approx. 40% at early times after addition of the cytokine. By 24 h of culture, no differences were noted in the presence and absence of interleukin-6. Thus the cornea can synthesize plasminogen and regulate its synthesis in response to its environment, including cytokines induced in the cornea by injury and inflammation. Therefore the cornea can control the amount of plasminogen, the precursor of both plasmin and angiostatin.
Subject(s)
ADP Ribose Transferases , Bacterial Toxins , Cornea/metabolism , Interleukin-1/pharmacology , Plasminogen/biosynthesis , Up-Regulation/drug effects , Virulence Factors , Blotting, Western , Caseins/metabolism , Cornea/drug effects , Cornea/enzymology , Culture Media, Conditioned/chemistry , Enzyme Activation/drug effects , Exotoxins/pharmacology , Fibrinolysin/analysis , Fibrinolysin/antagonists & inhibitors , Fibrinolysin/biosynthesis , Fibrinolysin/metabolism , Gelatinases/analysis , Gelatinases/biosynthesis , Humans , Interleukin-6/pharmacology , Molecular Weight , Organ Culture Techniques , Plasminogen/analysis , Plasminogen/antagonists & inhibitors , Plasminogen/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Serine Proteinase Inhibitors/pharmacology , Urokinase-Type Plasminogen Activator/pharmacology , Pseudomonas aeruginosa Exotoxin AABSTRACT
PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. Previous research showed that, in corneas obtained from patients with keratoconus, lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha1-proteinase inhibitor are reduced. This study was undertaken to examine further the expression of a spectrum of proteolytic enzymes and protease inhibitors. METHODS: Corneal buttons were collected from patients with keratoconus, healthy subjects, and patients with other corneal diseases. Immunohistochemical staining was performed on paraffin sections. Enzymatic assays and western blot analysis were carried out for cathepsins B and G. In addition, an in situ zymography procedure was used to examine the gelatin- and casein-digesting activities in corneas with keratoconus. RESULTS: An enhanced staining was found with antibodies to cathepsins B and G. Enzymatic assays and western blotting confirmed that the levels of these two enzymes were elevated in corneas with keratoconus. No alteration was noted with any of the matrix metalloproteinase (MMP) family members and other enzymes and inhibitors examined, although in situ zymography did indicate an increase in net gelatin- and casein-digesting activities in corneas with keratoconus. These activities were mostly abolished by inhibitors for serine and cysteine proteinases, but not by those for MMPs and aspartic proteinases. CONCLUSIONS: Levels of cathepsins B and G are increased in corneas with keratoconus. These enzymes may contribute to the heightened in situ gelatin- and casein-digesting activities, leading to abnormalities in keratoconus.
Subject(s)
Cornea/enzymology , Endopeptidases/metabolism , Keratoconus/enzymology , Protease Inhibitors/metabolism , Tissue Plasminogen Activator/metabolism , Adolescent , Adult , Aged , Aged, 80 and over , Blotting, Western , Child , Child, Preschool , Cornea/pathology , Corneal Diseases/enzymology , Fluorescent Antibody Technique, Indirect , Humans , Immunoenzyme Techniques , Keratoconus/pathology , Middle AgedABSTRACT
The role of quorum sensing by Pseudomonas aeruginosa in producing cytotoxicity has not been fully investigated. Strains of P. aeruginosa have been characterized as having an invasive or a cytotoxic phenotype (S. M. J. Fleiszig et al., Infect. Immun. 65:579-586, 1997). We noted that the application of a large inoculum of the invasive strain 6294 caused cytotoxicity of cultured epithelial cells. To investigate this dose-related cytotoxicity, we compared the behavior of 6294 to that of another invasive strain, PAO1, and determined whether the cytotoxicity could be related to quorum sensing. Both invasive strains, 6294 and PAO1, appear to have quorum-sensing systems that were operative when large doses of bacteria were applied to cultured lung epithelial cells or instilled into the lungs of animals. Nonetheless, only 6294 was cytotoxic. Cytotoxicity induced by 6294 correlated with increased elastase production. These experiments suggest that there are multiple mechanisms for the induction of cytotoxicity, pathology, and mortality in vivo. However, in vivo cytotoxicity and mortality, but not pathology, could be predicted by quantitative in vitro cellular damage experiments utilizing a range of bacteria-to-cell ratios. It appears that quorum sensing may inversely correlate with virulence in that strains that produced PAI [N-(3-oxododecanoyl) homoserine lactone] also appeared to attract more polymorphonuclear leukocytes in vivo and were possibly eliminated more quickly. In addition, exoproduct production in bacteriological medium in vitro may differ significantly from exoproduct expression from infections in vivo or during cocultivation of bacteria with tissue culture cells.
Subject(s)
Lung Diseases/pathology , Pseudomonas Infections/pathology , Pseudomonas aeruginosa/pathogenicity , 4-Butyrolactone/analogs & derivatives , 4-Butyrolactone/biosynthesis , Animals , Cell Line , Endopeptidases/biosynthesis , Homoserine/analogs & derivatives , Homoserine/biosynthesis , Humans , Lung/microbiology , Lung/pathology , Mice , VirulenceABSTRACT
Alpha 1-proteinase inhibitor is a major serine proteinase inhibitor in the human cornea involved in the protection of the avascular corneal tissue against proteolytic damage. This inhibitor is upregulated systemically during infection, inflammation and injury. Cytokines that mediate the acute phase response such as IL-1beta and IL-2 increased alpha1-proteinase inhibitor present in corneal organ culture media. This released inhibitor represented mainly newly synthesized protein. However, IL-6, a general inducer of the acute phase response that upregulates alpha1-proteinase inhibitor in all other tissues and cells tested, failed to alter corneal alpha1-proteinase inhibitor levels over the tested period of 24 h. In addition to IL-1beta and IL-2, alpha1-proteinase inhibitor levels in the corneal organ culture medium increased following the addition of FGF-2 and IGF-I. The effect of the above growth factors and cytokines was relatively fast with maximal induction observed within the first 5 h. Among the tested growth factors and cytokines, IL-1beta was the most potent and increased total corneal alpha1-proteinase inhibitor levels approximately 2.4-fold in the cornea organ culture medium. Newly, synthesized alpha1-proteinase secreted into the medium increased 3.9-fold. In addition to the effect on corneal alpha1-proteinase inhibitor, IL-1beta also increased the amount of alpha1-proteinase inhibitor released by monocytes and macrophages but not by HepG2, CaCo2, and MCF-7 cells within 24 h. These results suggest that the cornea can locally control levels of alpha1-proteinase inhibitor in response to an inflammatory insult.
Subject(s)
alpha 1-Antitrypsin/drug effects , alpha 1-Antitrypsin/metabolism , Caco-2 Cells/drug effects , Caco-2 Cells/metabolism , Cornea/cytology , Cornea/drug effects , Cornea/metabolism , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 2/pharmacology , Growth Substances/pharmacology , Humans , Insulin-Like Growth Factor I/pharmacology , Interleukin-1/pharmacology , Interleukin-2/pharmacology , Interleukin-6/pharmacology , Organ Culture Techniques , Transforming Growth Factor alpha/pharmacology , Tumor Cells, Cultured/drug effects , Tumor Cells, Cultured/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The transcripts of the alpha1-proteinase inhibitor in the cornea are different from those in hepatocytes and monocytes, suggesting that alpha1-proteinase inhibitor gene transcription may respond to different cell-specific regulatory mechanisms. Although information on alpha1-proteinase inhibitor gene structure has been obtained, little is known regarding the cis- and trans-acting factors that regulate its expression. In this study, we cloned and sequenced a 2. 7-kilobase 5'-flanking region upstream from the corneal transcription initiation site of the gene, demonstrated functional promoter activity, and identified the regulatory elements. Sequencing revealed that the 5'-flanking element was highly G/C-rich in regions proximal to the corneal transcription start site. DNase I footprinting located 10 potential Sp1-binding sites between nucleotides -1519 and +44. The putative promoter was functional in human corneal stromal cells, but not in human skin, scleral, and conjunctival fibroblasts, suggesting that the promoter may be corneal cell-specific. The promoter activity in the corneal cells was repressed when Sp1 was coexpressed. In the cornea-thinning disease keratoconus, down-regulation of the alpha1-proteinase inhibitor gene and increased Sp1 expression have both been demonstrated. The current results suggest that down-regulation of the inhibitor in keratoconus corneas may be related directly to overexpression of the Sp1 gene. This information may help elucidate the molecular pathways leading to the altered alpha1-proteinase inhibitor expression in keratoconus.
Subject(s)
Cornea/metabolism , Promoter Regions, Genetic , Sp1 Transcription Factor/metabolism , alpha 1-Antitrypsin/biosynthesis , alpha 1-Antitrypsin/genetics , Base Sequence , Cells, Cultured , Cloning, Molecular , Cornea/cytology , DNA Footprinting , Deoxyribonuclease I , Exons , Gene Expression Regulation , Humans , Liver/metabolism , Molecular Sequence Data , Skin/metabolism , Stromal Cells/metabolism , Transcription, GeneticABSTRACT
Previous studies showed a higher percentage of neutrophils from vitamin A deficient rats are hypersegmented and contain lower levels of cathepsin G than the neutrophils from control rats. In this study chemotaxis, phagocytosis and oxidant generation were studied using either isolated neutrophils or neutrophils in whole blood from four dietary groups of rats: 1) vitamin A deficient rats; 2) vitamin A deficient rats that received vitamin A for 16, 8, 4 or 2 d prior to killing; 3) weight-matched rats pair-fed a vitamin A-complete diet; and 4) rats fed nonrestricted, vitamin A complete diet. Chemotaxis towards P. aeruginosa conditioned medium and formylated methinyl leucinyl phenylalanine was significantly lower for neutrophils from vitamin A-deficient rats than for neutrophils from weight-matched pair-fed rats, nonrestricted vitamin A sufficient rats and vitamin A deficient rats that received vitamin A for 16 d prior to killing. No differences in chemotaxis towards activated rat serum were noted among the neutrophils from the four groups of rats. Adhesion of P. aeruginosa organisms, phagocytosis of these organisms and generation of active oxidative molecules were significantly lower in the neutrophils from the vitamin A-deficient rats relative to these functions in the neutrophils from the vitamin A deficient rats that received vitamin A for 16 d, weight-matched rats pair-fed a vitamin A complete diet; and rats fed nonrestricted, vitamin A-complete diet. Eight days after vitamin A administration to vitamin A deficient rats, the ability of the neutrophils to phagocytose P. aeruginosa organisms and to generate active oxidant molecules was restored to the levels observed for weight-matched, pair-fed rats and rats fed nonrestricted, vitamin A complete diet. The elucidated alterations in neutrophil function in vitamin A deficient rats probably contribute to the altered ability of vitamin A deficient rats to fight infections.
Subject(s)
Neutrophils/drug effects , Neutrophils/physiology , Vitamin A Deficiency/blood , Vitamin A/pharmacology , Animals , Chemotaxis/drug effects , Male , Phagocytosis/drug effects , Pseudomonas aeruginosa , Rats , Vitamin A/administration & dosageABSTRACT
alpha1-Proteinase inhibitor is a serpin and can inhibit most serine proteinases. The cornea is one of several extrahepatic tissues that synthesizes this inhibitor. In the presence of retinol, corneal alpha1-proteinase inhibitor levels were increased 3.8-fold. The maximal response was achieved 2 h after the addition of retinol (1 microM final concentration) to the culture medium. A similar increase in alpha1-proteinase inhibitor was observed with retinaldehyde (1 nM final concentration). Concentrations of alpha1-proteinase inhibitor in other tested cells (Hep G2, CaCo 2, MCF-7, monocytes and macrophages) remained unchanged in the presence of retinol. Retinoic acid did not affect alpha1-proteinase inhibitor levels in the cornea or the other cells tested. The acute-phase cytokine, interleukin-6, increased alpha1-proteinase inhibitor levels in all tested tissues/cells except the cornea. These results demonstrate that alpha1-proteinase inhibitor levels are controlled differently in the cornea compared with other tissues/cells. alpha1-Proteinase inhibitor is the first protein identified whose levels are regulated by a mechanism supported by retinol and retinaldehyde but not retinoic acid.
Subject(s)
Cornea/metabolism , Leukocyte Elastase , Pancreatic Elastase/metabolism , Retinaldehyde/pharmacology , Vitamin A/pharmacology , alpha 1-Antitrypsin/metabolism , Humans , Organ Culture Techniques , Organ SpecificityABSTRACT
PURPOSE: Vitamin A deficiency alters the transparency of the cornea due to epithelial cell keratinization and increases the susceptibility of the cornea to ulceration. The purpose of this study was to determine the effect of vitamin A deficiency on rat corneal matrix metalloproteinases and serine proteinases. METHODS: Four dietary groups of male WAG/RijMCW rats were prepared: (1) Vitamin A deficient rats were raised on a casein-based retinoid deficient diet; (2) Retinol repleted rats were raised on the retinoid deficient diet. On the eighty-sixth day on this diet, the rats were fed retinyl palmitate and then given free access to the retinyl palmitate-supplemented control diet; (3) The weight-matched, pair-fed rats were restricted in their intake of the retinyl palmitate-supplemented diet so that their weight gain matched that of the A-rats; (4) The non-restricted rats were given free access to the retinyl palmitate-supplemented diet. The animals were killed at the late plateau stage for weight of the deficiency (102-106 days). Zymography was used to study proteinases in the corneal extracts. RESULTS: Vitamin A deficient and control rat corneas contain multiple matrix metalloproteinases and serine proteinases. The matrix metalloproteinases at 90/92 kDa (gelatinase B) and 66/63/57 kDa (gelatinase A) were significantly decreased in the corneas of the vitamin A deficient rats relative to the control corneas. Corneas from the four groups of rats contained 76, 45, 38, 28 and 22 kDa proteinases that cleaved casein. Only the vitamin A deficient corneas contained a 50 kDa casein cleaving enzyme. The 76, 45, 38 and 28 kDa serine proteinases were significantly lower in the vitamin A deficient corneas. The major 22 kDa enzyme was not altered by the deficiency. All casein cleaving proteinases were inhibited by phenylmethylsulfonyl fluoride and chymostatin except for a minor 76 kDa band. The activity of this band was not altered by inhibitors for the other classes of proteinases, ethylenediaminetetraacetic acid, E-64 or pepstatin. The concentrations of the 61, 52 and 40 kDa plasminogen activators were not altered by the deficiency. CONCLUSIONS: Alterations in corneal proteinases under vitamin A deficiency conditions may be involved in the characteristic changes observed in the cornea under vitamin A deficiency conditions: decreased exfoliation of epithelial cells, increased levels of keratofibrils in the corneal keratocytes, increased stromal keratocyte degradation and increased susceptibility towards ulceration.
Subject(s)
Cornea/enzymology , Extracellular Matrix/enzymology , Metalloendopeptidases/metabolism , Serine Endopeptidases/metabolism , Vitamin A Deficiency/enzymology , Animals , Caseins/metabolism , Cornea/pathology , Gelatin/metabolism , Male , Oligopeptides/pharmacology , Phenylmethylsulfonyl Fluoride/pharmacology , Protease Inhibitors/pharmacology , Rats , Rats, Inbred Strains , Serine Proteinase Inhibitors/pharmacology , Vitamin A Deficiency/pathologyABSTRACT
PURPOSE: Keratoconus is characterized by thinning and scarring of the central region of the cornea. The authors have shown, in corneas obtained from patients with keratoconus, that lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha 1-proteinase inhibitor (alpha 1-PI) are reduced. This study was undertaken to examine further the gene expression of cathepsin G, acid phosphatase, and alpha 1-PI in keratoconus corneas. METHODS: Corneal buttons were collected from patients with keratoconus, normal subjects, and patients with other corneal diseases. In situ hybridization was performed on paraffin sections using a tritium-labeled probe for cathepsin G or alpha 1-PI. Competitive polymerase chain reaction (PCR) was used to determine the messenger RNA (mRNA) levels for lysosomal acid phosphatase and alpha 1-PI in epithelial and stromal cells of keratoconus corneas. RESULTS: Silver grains, indicative of positive in situ hybridization products, were observed in all three cell types of normal corneas for both DNA probes. Compared with normal and other diseased controls, the labeling was enhanced for cathepsin G but was diminished for alpha 1-PI in the epithelium of keratoconus corneas. Competitive PCR showed that the mRNA level for acid phosphatase was higher and that the mRNA level for alpha 1-PI was lower in keratoconus corneas. CONCLUSIONS: These results indicate that the mRNA level for degradative enzymes in increased and that for alpha 1-PI it is reduced in keratoconus corneas. This study provides the first evidence that the altered expression of multiple enzymes and inhibitors in keratoconus occurs at the gene level. Furthermore, it implicates a possible role of coordinated transcriptional regulation of gene expressions in keratoconus.
Subject(s)
Acid Phosphatase/genetics , Cathepsins/genetics , Cornea/metabolism , Gene Expression Regulation, Enzymologic , Keratoconus/metabolism , RNA, Messenger/metabolism , Serine Endopeptidases/genetics , Serine Proteinase Inhibitors/genetics , alpha 1-Antitrypsin/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Cathepsin G , Child , Child, Preschool , DNA Primers/chemistry , DNA Probes , Electrophoresis, Agar Gel , Humans , In Situ Hybridization , Middle Aged , Polymerase Chain ReactionABSTRACT
Vitamin A deficiency leads to an increased susceptibility to infections, increased severity of infections and increased mortality. Because the neutrophil is the first cell to respond to infection, this study explores the effect of vitamin A deficiency on neutrophil proteinases. We found that neutrophils from vitamin A-deficient rats had lower levels of two cathepsin G-like enzymes (28 and 24 kDa) when compared to neutrophils from weight-matched pair-fed rats, vitamin A-deficient rats which were repleted with retinyl palmitate and nonrestricted vitamin A complete diet rats. The 28 kDa cathepsin G-like enzyme, which migrated with the same mobility as elastase on SDS-polyacrylamide gels, was quantified using Western blots. The 24 kDa cathepsin G-like enzyme was quantified using zymogram gels. This activity was inhibited by chymostatin. Other neutrophil proteinases, elastase, plasminogen activators and gelatinase, were not altered significantly by vitamin A deficiency. The low levels of cathepsin G may contribute to differences in the inflammatory process observed under vitamin A deficiency.
Subject(s)
Cathepsins/blood , Neutrophils/enzymology , Vitamin A Deficiency/enzymology , Animals , Blotting, Western , Caseins/metabolism , Cathepsin G , Gelatinases/blood , Leukocyte Elastase/blood , Molecular Weight , Plasminogen Activators/blood , Rats , Serine EndopeptidasesABSTRACT
PURPOSE: Keratoconus is characterized by thinning and scarring of the central portion of the cornea. This study was performed on keratoconus corneas to examine the expression of proteins related to wound healing including vimentin, an intermediate filament protein, and tenascin, and extracellular matrix protein. The expression of stress-related cytokines, heat shock proteins and ubiquitin was also investigated. METHODS: Corneal buttons were collected from patients with keratoconus, normal subjects and patients with other corneal diseases such as pseudophakic bullous keratopathy. Immunofluorescence staining was performed on frozen sections for vimentin and tenascin, and immunoperoxidase staining was carried out on paraffin sections for cytokines, heat shock proteins and ubiquitin. RESULTS: To varying degrees, all proteins examined, except tenascin and heat shock protein 90, were found to be expressed in normal human corneas. The expression of vimentin, tenascin, transforming growth factor-beta, interleukin-1, heat shock protein 27, and ubiquitin was enhanced in keratoconus corneas. A similar enhancement however was also observed in other diseased corneas. CONCLUSIONS: Altered expression of several wound healing or stress-related proteins was noted in keratoconus corneas. The alterations appear to be nonspecific injury or wound responses in association with corneal diseases.
Subject(s)
Cornea/metabolism , Heat-Shock Proteins/metabolism , Keratoconus/metabolism , Tenascin/metabolism , Vimentin/metabolism , Wound Healing/physiology , Adolescent , Adult , Aged , Aged, 80 and over , Fluorescent Antibody Technique, Indirect , Humans , Immunoenzyme Techniques , Intracellular Fluid/metabolism , Keratoconus/pathology , Middle AgedABSTRACT
Retinoic acid bound to the nuclear retinoic acid receptor-alpha is required for the differentiation of promyelocytes to mature neutrophils. However, severely vitamin A-deficient rats have normal numbers of neutrophils in the blood and inflamed tissues. This paradox was explored using four dietary groups of rats: 1) vitamin A-deficient rats; 2) vitamin A-deficient rats subsequently receiving vitamin A; 3) weight-matched pair-fed rats; and 4) nonrestricted, vitamin A-complete diet-fed rats. Plasma and liver retinol concentrations of the vitamin A-deficient rats were < 1 % of those of the other three groups. In contrast, the bone marrow retinol concentrations of the vitamin A-deficient rats were fourfold higher than those in the other three groups. The distribution of myeloid-derived cells in the bone marrow was similar in all four groups of rats with the exception of a significantly greater (P < 0.05) occurrence of hypersegmented neutrophils (six or more lobes) in the vitamin A-deficient rats (2. 1 %) relative to the control groups (0-0.1%). The blood of the vitamin A-deficient rats also contained significantly higher numbers (P < 0.01) of hypersegmented neutrophils (67%) relative to those in the control groups (2-7%). The hypersegmentation of the neutrophils in this group of rats was not due to a concurrent deficiency of vitamin B-12 or folate. The importance of bone marrow-derived cells to the survival of the animal is suggested by retinol sequestration in the bone marrow of vitamin A-deficient rats, allowing the differentiation of myeloid cells to neutrophils.
Subject(s)
Bone Marrow/metabolism , Vitamin A Deficiency/metabolism , Vitamin A/metabolism , Animals , Bone Marrow/ultrastructure , Chromatography, High Pressure Liquid , Diet , Female , Folic Acid/blood , Liver/metabolism , Male , Neutrophils/ultrastructure , Rats , Vitamin A/administration & dosage , Vitamin A/blood , Vitamin A Deficiency/pathology , Vitamin B 12/bloodABSTRACT
PURPOSE: Vitamin A-deficient humans and animals are more susceptible to infections than are healthy humans and animals. This study compares the early corneal response (within 24 hours) to an experimental Pseudomonas aeruginosa infection between vitamin A deficient and control rats. METHODS: Male WAG/Rij/MCW rats were fed either a vitamin A- deficient diet (A-) or the same diet with retinyl palmitate added back in a nonrestricted manner (N) or under pair-fed conditions (A+) to yield weight-matched rats. Some A-rats were repleted wih retinyl palmitate 16 days before being killed and then given free access to the retinyl palmitate-supplemented diet (R). Twenty-four hours before being killed, the corneas of anesthetized rats were scratched and P. aeruginosa organisms were applied to the corneal surface. The rats were killed using an overdose of sodium pentobarbital. Corneas were either processed for light and electron microscopic examination or extracted for proteinase and myeloperoxidase determination. Corneal myeloperoxidase concentrations relative to neutrophil myeloperoxidase concentrations were used to determine the number of neutrophils in the cornea. Zymography was used to study caseinases, gelatinases, and plasminogen activators. Reverse zymography was used to detect proteinase inhibitors. Similar results were noted at early, mid, and late weight plateau stages of vitamin A deficiency. RESULTS: Ulceration occurred within 24 hours when low numbers of P. aeruginosa (10(4) cpu) were applied topically onto scratched A- corneas, whereas no ulceration was observed in the A+, R, and N corneas. When higher numbers of P. aeruginosa (10(7)-10(8)) were applied to the scratched corneas, all corneas became ulcerated within 24 hours. The extent of ulceration in the control corneas was greater than that in A- corneas by a factor of two. Only the A- corneas contained inflammatory cells with unusual striated deposits in phagolysosomes. The total number of neutrophils in the cornea and the concentrations of caseinases, plasminogen activators, and gelatinases in the infected corneal extracts were similar; however, the concentrations of cysteine proteinase inhibitors were elevated under A- conditions. CONCLUSIONS: Vitamin A deficiency alters the response of the cornea to a P. aeruginosa infection during the first 24 hours. The alterations observed are probably due to multiple factors: an insufficient tear film for bacterial clearance and migration of neutrophils, epithelial keratinization, alterations in corneal wound healing, and changes in polymorphonuclear function.