Transcriptional activation of bovine mimecan by p53 through an intronic DNA-binding site.

Mimecan is a small leucine-rich proteoglycan that can occur as either keratan sulfate proteoglycan in the cornea or as glycoprotein in many connective tissues. As yet, there is no information on its transcriptional regulation. Recently we demonstrated the presence of eight mimecan mRNA transcripts generated by alternative transcription initiation, alternative polyadenylation, and differential splicing, all of which encode an identical protein. Here we report a conserved consensus p53-binding DNA sequence in the first intron of bovine and human mimecan genes and show that wild-type p53 binds to this sequence in vitro. Co-transfections of Saos-2, HeLa, NIH 3T3, and primary bovine corneal keratocytes with bovine mimecan promoter/luciferase reporter constructs in combination with p53 expression vectors activate the second mimecan promoter through the p53-binding sequence. In addition, we show absence of mimecan expression in different tumors and cancer cell lines, where p53 frequently is inactivated/mutated. Thus, this work provides novel information that links mimecan to the p53 network.

Specificity of corneal nerve positions during embryogenesis.

PURPOSE: Embryonic corneal innervation first involves pericorneal nerve ring formation, with nerves in specific positions, followed by innervation of the corneal stroma from the ring. Here we determine whether nerve bundles enter the cornea at specific locations along the ring and whether bundles enter the cornea at specific depths. METHODS: Whole mount embryonic quail corneas immunostained for nerves were scanned using confocal laser microscopy. Images were superimposed digitally in pseudo-colored pairs to detect similar positions of innervation, and then rotated stepwise to determine if degree of synchrony was decreased. Degrees of innervation of each corneal quadrant were quantified. Depths of stromal bundles innervating the cornea were determined by depth of focus analysis. RESULTS: Superimposition of images indicated many nerve entry points in similar locations, suggesting specificity. However, stepwise rotations of one image above the other revealed that degree of positional synchrony remained constant, suggesting that nerves do not occur in specific locations, but rather simply at approximately equal distances around the cornea. Corneas from both left and right sides are innervated by similar numbers of nerve bundles (Left, 44+/-0.4; Right, 44+/-1.0), with the same number/quadrant (Left, 11+/-0.2; Right, 11+/-0.2). Nerves entering the stroma closest to Descemet's layer innervate either the entire cornea along that radius or only the central-peripheral and central cornea; those entering nearer Bowman's layer innervate only peripheral cornea. CONCLUSIONS: Avian corneal nerve bundles enter along radii spaced at equal intervals along the pericorneal nerve ring, suggesting an innervation mechanism based on equal spacing between nerves. Nerve bundles from the nerve ring enter the stroma at depths correlated with their subsequent targets.

Molecular cloning and relative tissue expression of keratocan and mimecan in embryonic quail cornea.

We have cloned and sequenced the cDNAs for quail cornea keratan sulfate proteoglycan core proteins, keratocan and mimecan. The deduced quail keratocan protein contains a single conservative amino acid difference from the chick sequence, whereas quail mimecan protein contains a 58 amino acid-long avian-unique sequence that shares no homology with mammalian mimecan. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that keratocan and lumican are expressed at highest levels in cornea, whereas mimecan mRNA is expressed at a much lower level. Keratocan is expressed only in quail cornea, whereas mimecan is expressed in many different tissues as four transcripts of different sizes. Both lumican and mimecan are expressed at lowest levels in brain, liver and sternum.

Molecular cloning and relative tissue expression of decorin and lumican in embryonic quail cornea.

We have cloned and sequenced the cDNAs for quail cornea proteoglycan core proteins, decorin and lumican. Comparison of deduced amino acid sequences shows that two of five amino acid differences in the mature protein between quail and chick decorin, and two of three for lumican, are non-conservative. Ribonuclease protection assay of Day 16 embryonic quail tissues reveals that decorin and lumican are most highly expressed in cornea, and that both are also highly expressed at approximately equal levels in most other tissues. Decorin is highly expressed in sclera and sternum, whereas lumican is expressed in these tissues, as well as in liver, at very low levels. Both decorin and lumican are expressed at lowest levels in brain.

Developmental eye and neural tube defects in the eye blebs mouse.

In the mouse, eye blebs (eb) is a spontaneous mutation that presents a useful model for the study of abnormal eye development. Since its initial description three decades ago, little information has been generated regarding the developmental course of eb eyes. Although the gene for eb has not been identified, much can be learned from the developmental defects present in the eb mouse. First detected in the eye at embryonic day 11.5 (E11.5), the eb defect is observed as an increased vascularization throughout the developing eye and head region. As development proceeds, the embryonic eye fills with blood, and the resulting hematoma distorts the shape of the iris. The eyelids fail to close, and animals are born with open eyes. Lens degeneration and retinal folding are characteristic of eb, as are microphthalmia and thick, disorganized irises. A second presentation of the eb defect is disruption of neural tube closure in the anterior and hindbrain neuropores. These eb animals are born with open neural tubes but with apparently normal eyes.

Identification and characterization of conserved cis-regulatory elements in the human keratocan gene promoter.

Keratocan, along with lumican and mimecan, represent the keratan sulfate-containing proteoglycans of the vertebrate cornea that play a key role in development and maintenance of corneal transparency. In this study, we cloned 4.1 kb of the human Kera 5'-flanking region and characterized the promoter structure. Using primer extension and ribonuclease protection assay, we identify two major transcriptional start sites in the first exon. Using luciferase reporter gene transfection analysis of 5'-deletion and mutation constructs, we demonstrate positive and negative regulatory elements within a 1.3 kb upstream sequence. Comparison of human and bovine 5'-flanking sequences reveals three highly conserved regions: a 450 bp region in the first exon, a 92 bp promoter proximal conserved regulatory region identified as an enhancer in the natural context, and a 223 bp promoter distal conserved regulatory region identified as a silencer both in the natural context and in a heterologous promoter system. In addition, a conserved CArG-box residing 851 bp upstream of the first transcription start site also can lead to the repression of Kera expression in cultured corneal keratocytes. DNaseI footprinting and electrophoretic mobility shift assay demonstrate that cell type-specific factors bind to regulatory elements located in the conserved regions. Competition experiments indicate that the CTC factor and a protein that binds to the CAGA motif are likely to be among the multiple factors involved in the transcriptional regulation of the human Kera gene.

Embryonic quail eye development in microgravity.

The US-Russian joint quail embryo project was designed to study the effects of microgravity on development of Japanese quail embryos incubated aboard Mir. For this part of the project, eyes from embryonic days 14 and 16 (E14 and E16) flight embryos were compared with eyes from several groups of ground-based control embryos. Measurements were recorded for eye weights; eye, corneal, and scleral ring diameters; and numbers of bones in scleral ossicle rings. Transparency of E16 corneas was documented, and immunohistochemical staining was performed to observe corneal innervation. In addition, corneal ultrastructure was observed at the electron microscopic level. Except for corneal diameter of E16 flight embryos, compared with that of one of the sets of controls, results reported here indicate that eye development occurred normally in microgravity. Fixation by cracking the shell and placing the egg in paraformaldehyde solution did not adequately preserve corneal nerves or cellular ultrastructure.

Fibroblast growth factor-2 promotes keratan sulfate proteoglycan expression by keratocytes in vitro.

Keratocytes of the corneal stroma produce a specialized extracellular matrix responsible for corneal transparency. Corneal keratan sulfate proteoglycans (KSPG) are unique products of keratocytes that are down-regulated in corneal wounds and in vitro. This study used cultures of primary bovine keratocytes to define factors affecting KSPG expression in vitro. KSPG metabolically labeled with [(35)S]sulfate decreased during the initial 2-4 days of culture in quiescent cultures with low serum concentrations (0.1%). Addition of fetal bovine serum, fibroblast growth factor-2 (FGF-2), transforming growth factor beta, or platelet derived growth factor all stimulated cell division, but only FGF-2 stimulated KSPG secretion. Combined with serum, FGF-2 also prevented serum-induced KSPG down-regulation. KSPG secretion was lost during serial subculture with or without FGF-2. Expression of KSPG core proteins (lumican, mimecan, and keratocan) was stimulated by FGF-2, and steady state mRNA pools for these proteins, particularly keratocan, were significantly increased by FGF-2 treatment. KSPG expression therefore is supported by exogenous FGF-2 and eliminated by subculture of the cells in presence of serum. FGF-2 stimulates KSPG core protein expression primarily through an increase in mRNA pools.

Pretreatment methods to improve nerve immunostaining in corneas from long-term fixed embryonic quail eyes.

Pretreatment methods were used to improve neurofilament immunostaining in corneas from embryonic day 16 Japanese quail corneas that had been stored in fixative solution for several months. A sequential combination of the following three pretreatments: brief microwave heating in saline, followed by extraction with sodium dodecyl sulfate (SDS) at 37 degrees C, followed by digestion with hyaluronidase at 37 degrees C, produced significantly increased antibody staining of corneal neurofilament proteins, compared with embryonic corneas subjected to no prior pretreatments or to single or two-step protocols. After applying the sequence of all three pretreatments, darkest nerve staining and increased numbers of fine branches were observed, together with lower background staining. Thus, the result of applying the three-step pretreatment sequence is better than that of applying any of its component single pretreatments or even combinations of any two of them. These findings therefore suggest that each of these three pretreatments causes a unique effect, beneficial to immunostaining of neurofilament proteins, and that their individual effects are independent and additive. In addition to embryonic corneas, the three-step procedure also may be useful for immunostaining of nerves in other very delicate, highly-hydrated tissues containing an abundance of extracellular matrix.

Ag+ alters cell growth, neurite extension, cardiomyocyte beating, and fertilized egg constriction.

BACKGROUND: The Russian Space Agency uses electrochemically generated silver ions (Ag+) to purify drinking water for their space station, Mir, and their portion of the International Space Station. U.S. EPA guidelines allow 10.6 micromol x L(-1) Ag+ in human drinking water for up to 10 d. Studies correlate Ag+ exposure with tissue dysfunction in humans, rats, and mice, and with altered ion transport, skeletal muscle contraction, and embryonic cell constriction in other animal cells. Ag+ effects on cell shape change-related functions have not been assessed. METHODS: Immortalized embryonic human intestinal epithelial cells, freshly explanted embryonic avian nerve cells and cardiomyocytes, and marine fertilized eggs were grown in vitro in medium containing AgNO3. RESULTS: Intestinal cells detach from the substratum and viable cell number decreases by 5-6 d at 5 micromol x L(-1) AgNO3, and faster at higher concentrations. Microtubules appear unaltered in adherent cells. Detached cells are nonviable. Neurite outgrowth and glial cell migration from dorsal root ganglia are inhibited by 3 d at 15 micromol x L(-1) AgNO3 or greater. Contractions stop temporarily in most cardiomyocytes by 5 min at 5 micromol x L(-1) AgNO3 or more, but some cardiomyocytes beat 3 times faster than normal at 7.5-20 micromol x L(-1) AgNO3. Picomolar Ag+ increases marine egg polar lobe constriction within an hour, even in the absence of microtubules. CONCLUSION: Ag+ alters animal cell growth and shape changes by a MT-independent mechanism. This is the first report of Ag+ effects on vertebrate neurite outgrowth, glial cell migration, or cardiomyocyte beat rate.

Structure and sequence of the gene encoding human keratocan.

Keratocan is one of the three major keratan sulfate proteoglycans characteristically expressed in cornea. We have isolated cDNA and genomic clones and determined the sequence of the entire human keratocan (Kera) gene. The gene is spread over 7.65 kb of DNA and contains three exons. An open reading frame starting at the beginning of the second exon encodes a protein of 352 aa. The amino acid sequence of keratocan shows high identity among mammalian species. This evolutionary conservation between the keratocan proteins as well as the restricted expression of Kera gene in cornea suggests that this molecule might be important in developing and maintaining corneal transparency.

The bovine mimecan gene. Molecular cloning and characterization of two major RNA transcripts generated by alternative use of two splice acceptor sites in the third exon.

Mimecan is a proteoglycan expressed by many connective tissues. It was originally isolated in a truncated form as a bone-associated glycoprotein, osteoglycin, and was considered an osteoinductive factor. Recently, we demonstrated that the full-length translation product of the cDNA encoding mimecan is a corneal keratan sulfate proteoglycan present in other tissues without keratan sulfate chains. We also described multiple mimecan mRNA transcripts generated by differential splicing and alternative polyadenylation. In this study, we isolated genomic clones and determined the genomic organization of the bovine mimecan gene. The gene is spread over >33 kilobases of continuous DNA sequence and contains eight exons. The newly discovered first exon, identified by 5'-rapid amplification of cDNA ends, consists of a 5'-untranslated region and is enriched in C+G nucleotides. Two transcription initiation sites starting at the first and at the second exons were determined by primer extension. Molecular characterization shows that alternatively spliced RNA isoforms are generated by the use of two distinct splice acceptor sites in the third exon situated 278 base pairs apart. We determined a partial genomic structure of the human mimecan gene and demonstrated two alternatively spliced RNA transcripts that are generated likewise. Despite the diversity of mimecan transcripts, the primary structure of the core protein is encoded from exons 3 to 8 and remains unchanged, indicating its functional importance. Using ribonuclease protection assay, we analyzed the patterns of spliced RNA expressed in cultured bovine keratocytes. We demonstrated that their expression is differentially modulated in a temporal manner by basic fibroblast growth factor.

Optimal lipofection reagent varies with the molecular modifications of the DNA.

Cationic lipid reagents differ in their cytofection efficacy with different cell types. No evidence has addressed whether the same lipid reagent is best for different DNAs in a single cell line. Immortalized avian embryonic cardiomyocytes cultured in vitro were tested with 15 cationic lipid reagents using (A) a beta-gal expression plasmid, (B) a fluorescein-tagged, phosphorothioate-modified ODN B, (C) a fluorescein-tagged, ethoxy-modified ODN C with the same nucleotide sequence as ODN B, and (D) a fluorescein-tagged, phosphorothioate-modified ODN D with a different nucleotide sequence from ODNs B and C. Cytofection was scored as percent of cells expressing beta-gal activity or showing diffuse cellular fluorescence. The best lipid reagents for the phosphorothioate-modified ODNs were ODN-specific and markedly different from the best lipid reagents for the expression plasmid or for the ethoxy-modified ODN. These results suggest that the best cationic lipid reagent for a particular cell type varies with the physical and chemical form of the DNA being transfected into the cells.

Cloning, characterization and tissue-specific expression of the gene encoding bovine keratocan, a corneal keratan sulfate proteoglycan.

Keratocan is one of three major keratan sulfate proteoglycans characteristically expressed in cornea. We reported previously the sequence of bovine Kera cDNA. In this study, the complete bovine Kera gene was cloned and sequenced, and its expression pattern was determined. The Kera gene is composed of three exons and two introns that span 8.830kb of the bovine genome. The first exon contains 287 nucleotides of 5'-UTR sequence. Both of the two large introns of 1322 and 4178bp contain (CA)n repeats. The bovine Kera gene has a TATA box that is located 28bp upstream from tsp. Primer extension and S1 nuclease protection analyses were used to determine the major tsp. RPA indicate that cornea and sclera are the two tissues with the highest expression of Ktcn mRNA. This restricted expression in eye tissues, as well as the unique modification of keratocan with long keratan sulfate chains in cornea, suggests that this molecule may be important in developing and maintaining corneal transparency.

Decorin and biglycan of normal and pathologic human corneas.

PURPOSE: Corneas with scars and certain chronic pathologic conditions contain highly sulfated dermatan sulfate, but little is known of the core proteins that carry these atypical glycosaminoglycans. In this study the proteoglycan proteins attached to dermatan sulfate in normal and pathologic human corneas were examined to identify primary genes involved in the pathobiology of corneal scarring. METHODS: Proteoglycans from human corneas with chronic edema, bullous keratopathy, and keratoconus and from normal corneas were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), quantitative immunoblotting, and immunohistology with peptide antibodies to decorin and biglycan. RESULTS: Proteoglycans from pathologic corneas exhibit increased size heterogeneity and binding of the cationic dye alcian blue compared with those in normal corneas. Decorin and biglycan extracted from normal and diseased corneas exhibited similar molecular size distribution patterns. In approximately half of the pathologic corneas, the level of biglycan was elevated an average of seven times above normal, and decorin was elevated approximately three times above normal. The increases were associated with highly charged molecular forms of decorin and biglycan, indicating modification of the proteins with dermatan sulfate chains of increased sulfation. Immunostaining of corneal sections showed an abnormal stromal localization of biglycan in pathologic corneas. CONCLUSIONS: The increased dermatan sulfate associated with chronic corneal pathologic conditions results from stromal accumulation of decorin and particularly of biglycan in the affected corneas. These proteins bear dermatan sulfate chains with increased sulfation compared with normal stromal proteoglycans.

The cloning of mouse keratocan cDNA and genomic DNA and the characterization of its expression during eye development.

Keratan sulfate proteoglycans (KSPGs) play a pivotal role in the development and maintenance of corneal transparency. Keratocan, lumican, and mimecan (osteoglycin) are the major KSPGs in vertebrate corneas. To provide a better understanding of the structure/function relationship of keratocan, we have cloned both the mouse keratocan gene and its cDNA. We have also examined its expression during embryonic development. The mouse keratocan gene spans approximately 6.5 kilobases of the mouse genome and contains three exons and two introns. Northern blotting and in situ hybridization were employed to examine keratocan gene expression during mouse development. Unlike lumican gene, which is expressed by many tissues other than cornea, keratocan mRNA is more selectively expressed in the corneal tissue of the adult mouse. During embryonic development, keratocan mRNA was first detected in periocular mesenchymal cells migrating toward developing corneas on embryonic day 13.5 (E13.5). Its expression was gradually restricted to corneal stromal cells on E14. 5 approximately E18.5. Interestingly, keratocan mRNA can be detected in scleral cells of E15.5 embryos, but not in E18.5 embryos. In adult eyes, keratocan mRNA can be detected in corneal keratocytes, but not in scleral cells.

Species-specific immunostaining of embryonic corneal nerves: techniques for inactivating endogenous peroxidases and demonstration of lateral diffusion of antibodies in the plane of the corneal stroma.

Species-specific and species-common monoclonal antibodies (MAbs) to nerve-specific cell surface epitopes were used to compare pre-treatment techniques for nerve staining. Endogenous peroxidases were inactivated in four ways: (1) 0.3% hydrogen peroxide (H2O2); (2) 1% periodic acid (PA) (pH 1.85-1.95); (3) sodium meta-periodate (10-40 mM, pH 4.5); or (4) HCl (pH 1.80). Staining of chick and quail corneal nerves and dorsal root ganglia (DRG) nerves with the MAbs was species-specific. Staining of chick and quail corneal nerves was unaffected by pre-treatment with 0.3% H2O2, but was eliminated by pre-treatment with 1% PA. Chick and quail DRG nerve staining tolerated 0.3% H2O2, and at least one epitope also tolerated 1% PA. Corneal nerves of both chick and quail displayed concentration-dependent sensitivity to pre-treatment with sodium meta-periodate; DRG nerves were not sensitive to such pre-treatment. Corneal nerves tolerated pre-treatment with HCI (pH 1.80), whereas DRG nerves did not. These findings indicate sensitivity of corneal nerve epitopes to oxidation, in contrast with sensitivity of DRG nerve epitopes to low pH. Results also indicate that tissue trimming regulated whole-mount staining of corneal nerves, suggesting that antibodies cannot diffuse across corneal basement membranes, even after detergent extraction. However, antibodies are able to diffuse laterally into the stroma from any cut edge.