Pax-6 expression and activity are induced in the reepithelializing cornea and control activity of the transcriptional promoter for matrix metalloproteinase gelatinase B.

Recent evidence supports the idea that matrix metalloproteinases (MMPs) act as morphogenetic regulators in embryonic and adult events of tissue remodeling. MMP activity is controlled primarily at the level of gene expression. In a recent study we characterized the transcriptional promoter of the MMP gene, gelatinase B (gelB), in transgenic mice, demonstrating the requirement for DNA sequences between -522 and +19 for appropriate activity. In this study we investigated factors required for gelB promoter activity in the developing eye and reepithelializing adult cornea. Pax-6 is a homeobox and paired domain transcription factor that acts at the top of the hierarchy of genes controlling eye development. Pax-6 is also expressed in the adult eye. We show here that the tissue expression pattern of Pax-6 overlaps extensively with gelB promoter activity in the developing and adult eye. In addition Pax-6 is observed to be upregulated in repairing corneal epithelium, as is gelB promoter activity. In cell culture transfection experiments, we identified two promoter regions which mediate positive response to Pax-6. By electrophoretic mobility shift assay, we further pinpoint two Pax-6 binding sites within these response regions and demonstrate direct interaction of the Pax-6 paired domain with one of these sites. These data suggest a mechanism by which Pax-6 may direct gelB expression in an eye-specific manner.

Gelatinase B/lacZ transgenic mice, a model for mapping gelatinase B expression during developmental and injury-related tissue remodeling.

Matrix metalloproteinases (MMPs) drive normal tissue remodeling and are implicated in a wide range of pathologies. Although MMP activity is controlled at multiple levels, the primary regulation of MMP activity is transcriptional. The transcriptional promoter elements required for MMP gene expression in cultured cells have been defined, but this has not been extended to the in vivo situation. In this paper, we show that the DNA sequences between -522 and +19 of the rabbit gelatinase B gene (MMP-9) (as characterized in the transgenic mouse line 3445) constitute a minimal promoter that drives appropriate developmental and injury-induced reporter gene expression in transgenic mice. We further show that the expression and activity of three transcription factors (NF-kappaB, AP-2, and Sp1) that control the activity of the gelatinase B promoter are selectively induced in the epithelium migrating to heal a wound. Although promoter activity parallels expression of the endogenous gene in cell cultures, we show by several criteria that cell cultures cannot model many aspects of promoter regulation in vivo. This study reveals that the transgenic mouse line 3445 might be a useful model for investigating the regulation of gelatinase B expression in vivo and for identifying and characterizing new drugs that can control gelatinase B gene transcription.

Regulation of paracrine cytokine balance controlling collagenase synthesis by corneal cells.

PURPOSE: Classic studies have demonstrated that corneal epithelial cell density in culture can alter the balance of stimulatory and inhibitory cytokines controlling the elaboration of collagenolytic activity by co-cultured stromal cells. The current study attempts to bring the understanding of this mechanism to a molecular level. METHODS: A rabbit primary corneal cell culture model was used. RESULTS: Using molecular probes that bind to and neutralize specific cytokines, a major stimulator for stromal cell collagenase synthesis released by corneal epithelial cells into culture medium was identified as interleukin-1 alpha (IL-1 alpha), and a secondary stimulator was characterized as a heparin-binding cytokine. An inverse relationship between net collagenase stimulatory activity and epithelial cell plating density was demonstrated. In contrast, the release of inhibitory activity for IL-1-stimulated collagenase synthesis was not subject to the cell density effect. Direct measurement of IL-1 alpha protein levels revealed that this cytokine was released much more efficiently on a per cell basis when cells were plated at low density than when they were plated at high density. The effect was not caused by greater cell lysis at low cell density and was mediated only partially by changes at the IL-1 alpha synthesis level. CONCLUSIONS: These data provide evidence that epithelial cells release stimulatory cytokines for collagenase expression more efficiently when they have limited contact with their neighbors and that this has important consequences for the overall paracrine cytokine balance controlling collagenase synthesis. Alteration of the paracrine cytokine balance by changes in cell contact may be an important means for regulating epithelial-stromal interactions involved in corneal development and repair.

Role of serum amyloid A as an intermediate in the IL-1 and PMA-stimulated signaling pathways regulating expression of rabbit fibroblast collagenase.

The matrix metalloproteinase collagenase is expressed by resident tissue cells only when needed for biological remodeling. Exogenous addition of inflammatory and growth-promoting cytokines stimulates collagenase expression in early passage fibroblast cultures. In addition, the signal for collagenase expression in response to phorbol-12 myristate-13 acetate (PMA) or to agents which alter cell shape in early passage fibroblast cultures is routed extracellularly to an autocrine cytokine intermediate, IL-1 alpha. Importantly, fibroblasts, when freshly isolated from the tissue, are not competent for IL-1 alpha gene expression and, therefore, cannot produce collagenase in response to shape change agents. However, they do make a small amount of collagenase in response to PMA via an IL-1-independent pathway that has not been further characterized. In this paper, we investigate the role of a second autocrine, serum amyloid A3 (SAA3), in IL-1-dependent and -independent collagenase gene expression. We demonstrate that SAA3 is required for effective stimulation of collagenase expression by either exogenous or endogenous IL-1. Furthermore, while freshly isolated fibroblasts cannot express IL-1 alpha they can express SAA3, and this autocrine mediator acts independently of IL-1 alpha to control the low level of collagenase expression that can be stimulated by PMA. These results provide further evidence for a newly emerging paradigm of collagenase regulation which emphasizes the requirement for extracellular routing of signals. They also suggest that SAA3 might be utilized independently of IL-1 alpha to control tissue remodeling in vivo.

Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.

Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury.

A corneal epithelial inhibitor of stromal cell collagenase synthesis identified as TGF-beta 2.

PURPOSE: To determine the molecular mechanisms whereby substances released by corneal epithelial cells act to inhibit the elaboration of collagenolytic activity by corneal stromal cells and to determine whether inhibitory activity might be mediated by interleukin-1 receptor antagonist (IL-1ra) or transforming growth factor (TGF)-beta. METHODS: Conditioned media were generated from primary cultures of rabbit corneal epithelial cells, from passaged cultures of rabbit corneal stromal fibroblasts, and from cells of the rabbit corneal epithelial cell line, SIRC. Pure populations of stromal cells were isolated from rabbit cornea and used directly for bioassay of the conditioned media to detect substances that inhibit collagenase synthesis. The mink lung epithelial cell line, Mv1Lu, was used for bioassay of TGF-beta-like activity. The addition of specific neutralizing antisera to bioassays allowed an assessment of the contribution of each isoform to the net regulatory activity. Reverse transcription-polymerase chain reaction and Northern blot analysis were employed to detect the presence of IL-1ra or TGF-beta mRNA species in cells from cultures used to generate conditioned media. RESULTS: Both stimulatory and inhibitory substances that regulate the synthesis of stromal cell collagenase are released by corneal epithelial cells in primary culture. In contrast, only stimulatory activity is produced by corneal fibroblasts or SIRCs. MRNAs for a TGF-beta isoform, TGF-beta 2, and IL-1ra were identified in epithelial cells. Stromal fibroblasts also expressed TGF-beta 2 mRNA, but no evidence was found for expression of TGF-beta 3 mRNA in any of the three cell types. TGF-beta 2 is released by epithelial cells in both active and latent forms. This cytokine mediates the major portion of the net inhibitory activity against stromal cell collagenase synthesis produced by corneal epithelial cells. CONCLUSIONS: This study demonstrates the expression of TGF-beta 2 and IL-1ra by corneal epithelial cells in culture. It is the TGF-beta 2 that acts as the major inhibitor of collagenase synthesis by corneal stromal cells in culture. However, IL-1ra and TGF-beta 2 are likely to play important roles in epithelial-mesenchymal interactions regulating corneal development, homeostatic maintenance, and repair.

The rabbit gene for 92-kDa matrix metalloproteinase. Role of AP1 and AP2 in cell type-specific transcription.

We isolated the rabbit gene for the 92-kDa matrix metalloproteinase, gelatinase B, and sequenced 1802 contiguous bases covering the first three exons and 522 bases of DNA upstream of the start site for transcription. The DNA between bases -519 and +19 is sufficient to drive expression of a reporter gene in early passage cultures of corneal fibroblasts or primary cultures of corneal epithelial cells. Basal activity of the gelatinase B promoter in fibroblasts is lower than a collagenase promotor of 1800 base pairs, but activity of both promotors is similarly stimulated by treatment of transfected cells with phorbol 12-myristate 13-acetate, and stimulation is enhanced by co-treatment with transforming growth factor-beta. In contrast, basal activity of the gelatinase B promotor in epithelial cells is higher than the collagenase promotor. Deletion analysis demonstrated that sequences upstream of base -330 confer cell type-specific activity to the gelatinase B promotor. Site-directed mutagenesis revealed that an AP1-like element within this region is specifically utilized by fibroblasts. This region also contains elements that confer the capacity for activation by AP2, a transcription factor found to be expressed by corneal epithelial cells but not by corneal fibroblasts. In contrast, AP2 does not activate the collagenase promotor. These results provide a molecular basis for the unique cell type-specific expression pattern of gelatinase B as compared to other matrix metalloproteinases.

Interleukin 1 alpha mediates collagenase synthesis stimulated by phorbol 12-myristate 13-acetate.

Stimulation of collagenase expression in cultures of normal diploid fibroblasts by the tumor promotor phorbol 12-myristate 13-acetate (PMA) occurs secondarily to synthesis of unknown intermediary proteins. We have investigated the hypothesis that a form of the cytokine interleukin 1 (IL-1) is one intermediate controlling PMA-stimulated collagenase expression. Treatment with an IL-1 receptor antagonist inhibits the constitutive synthesis of collagenase in early passage fibroblast cultures from rabbit. Radioimmunoassay demonstrates that, of the two known IL-1 forms, IL-1 alpha and IL-1 beta, only IL-1 alpha is synthesized and released into the medium of corneal fibroblast cultures. PMA treatment of cells increases the level of IL-1 alpha mRNA; this occurs prior to the increase in collagenase mRNA and corresponds with increased synthesis and release of IL-1 alpha protein. Neutralizing antiserum to IL-1 alpha inhibits constitutive collagenase synthesis. Reagents that inhibit the activity of IL-1 alpha (IL-1 receptor antagonist or neutralizing antibody) also inhibit the PMA-mediated stimulation of collagenase synthesis. These results indicate that constitutive and PMA-stimulated expression of collagenase is regulated through an IL-1 alpha intermediate. In vivo, regulation of the lytic phase of tissue remodeling through the IL-1 alpha intermediate may ensure the recruitment of cells adjacent to the one that received the initial stimulus.

Norrie disease gene is distinct from the monoamine oxidase genes.

The genes for MAO-A and MAO-B appear to be very close to the Norrie disease gene, on the basis of loss and/or disruption of the MAO genes and activities in atypical Norrie disease patients deleted for the DXS7 locus; linkage among the MAO genes, the Norrie disease gene, and the DXS7 locus; and mapping of all these loci to the chromosomal region Xp11. The present study provides evidence that the MAO genes are not disrupted in "classic" Norrie disease patients. Genomic DNA from these "nondeletion" Norrie disease patients did not show rearrangements at the MAOA or DXS7 loci. Normal levels of MAO-A activities, as well as normal amounts and size of the MAO-A mRNA, were observed in cultured skin fibroblasts from these patients, and MAO-B activity in their platelets was normal. Catecholamine metabolites evaluated in plasma and urine were in the control range. Thus, although some atypical Norrie disease patients lack both MAO-A and MAO-B activities, MAO does not appear to be an etiologic factor in classic Norrie disease.

Monoamine oxidase deficiency in males with an X chromosome deletion.

Mapping of the human MAOA gene to chromosomal region Xp21-p11 prompted our study of two affected males in a family previously reported to have Norrie disease resulting from a submicroscopic deletion in this chromosomal region. In this investigation we demonstrate in these cousins deletion of the MAOA gene, undetectable levels of MAO-A and MAO-B activities in their fibroblasts and platelets, respectively, loss of mRNA for MAO-A in fibroblasts, and substantial alterations in urinary catecholamine metabolites. The present study documents that a marked deficiency of MAO activity is compatible with life and that genes for MAO-A and MAO-B are near each other in this Xp chromosomal region. Some of the clinical features of these MAO deletion patients may help to identify X-linked MAO deficiency diseases in humans.

Structural features of human monoamine oxidase A elucidated from cDNA and peptide sequences.

Monoamine oxidase (MAO), an important enzyme for the degradation of amine neurotransmitters, has been implicated in neuropsychiatric illness. The amino acid sequence for one form of the enzyme, MAO-A, has been deduced from human cDNA clones and verified against proteolytic peptides. The covalent binding site for the flavin adenine dinucleotide (FAD) cofactor is near the C-terminal region. The presence of features characteristic of the ADP-binding fold suggests that the N-terminal region is also involved in the binding of FAD. These cDNAs should facilitate the study of the structure, function, and intracellular targeting of MAO, as well as the analysis of its expression in normal and pathological states.