The Cloning of Mouse Keratocan cDNA and Genomic DNA and the Characterization of Its Expression during Eye Development

Liu, Chia-Yang; Shiraishi, Atsushi; Kao, Candace W.-C.; Converse, Richard; Funderburgh, James L.; Corpuz, Lolita M.; Conrad, Gary W.; Kao, Winston W.‐Y.

doi:10.1074/jbc.273.35.22584

Cited by 86 publications

(64 citation statements)

References 18 publications

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“…In Situ Hybridization of Lumican mRNA-Paraffin sections 5 m thick were deparaffinized and processed for in situ hybridization with sense and antisense riboprobes of mouse lumican and mouse keratocan as previously reported (12,31). Finally, the sections were counterstained with 0.5% neutral red and dehydrated through a series of ethanol, mounted, and observed under a light microscope.…”

Section: Methodsmentioning

confidence: 99%

“…Chimeric mice, identified by agouti coat color, were mated with C57BL/6J mice. Agouti coat-colored offspring were tested for the presence of the targeted locus by polymerase chain reaction (PCR) and Southern hybridization as described previously (12). To distinguish between individuals with none, one, or two copies of the mutant gene, we designed three primers in a PCR for detection of the wild type (primer 1, 5Ј-TACTTCAAGCGCTTCAC-3Ј; and primer 2, antisense 5Ј-CAAGTTCATTGACCTCCAGG-3Ј; 190 base pairs) and mutant (primers 2 and 3, antisense 5Ј-CGAGACTAGTGAGACGT-GCT-3Ј of the hprt minigene; 381 base pairs), respectively.…”

Section: Methodsmentioning

confidence: 99%

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Role of Lumican in the Corneal Epithelium during Wound Healing

Saika¹,

Shiraishi²,

Saika³

et al. 2000

Journal of Biological Chemistry

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208

204

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Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e.g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum ؊/؊ mice was significantly delayed compared with Lum ؉/؊ mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.Rapid re-epithelialization is essential for restoration of homeostasis in injured tissues; impaired healing of injured epithelium increases the risks of infection and further damage underlying tissues (1, 2). The cornea provides an ideal model to evaluate interactions of migrating epithelial cells and the extracellular matrix of the underlying basement membrane during wound healing because epithelial injuries of the avascular corneal tissue heal in a bloodless wound field. Various specific proteins such as vinculin (3), keratins (4), CD44 hyaluronan receptors (5), and gelatinases and metalloproteinase inhibitors (6, 7) are up-regulated during corneal epithelial wound healing. These proteins are believed to modulate cell adhesion or migration.Lumican belongs to the family of small leucine-rich proteoglycans (SLRPs) 1 that includes keratocan, mimecan, decorin, biglycan, fibromodulin, epiphycan, and osteoadherin. In the cornea, lumican, keratocan, and mimecan are modified with keratan sulfate glycosaminoglycan chains comprising the keratan sulfate proteoglycans (KSPG) of the stromal extracellular matrix (8 -13). In normal unwounded mouse cornea, lumican mRNA is expressed in stromal keratocytes (14). Lumican KSPG is a key regulator of collagen fibrillogenesis, a process critical to corneal transparency. Mice lacking lumican show an age-dependent corneal opacity and a high proportion of abnormally thick collagen fibers in the corneal stroma (15).Lumican is also widely present as a non-or low-sulfated glycoprotein in connective tissues of many other organ systems, e.g. skeleton, heart, kidney, and lung (14, 16 -18). During mouse embryonic ocular development, lumican is synthesized by keratocytes; detected as a glycoprotein, not as a KSPG (19); and also transiently expressed by the corneal epithelium, neural retina, and epidermis (14). These observations suggest that epithelial tissues possess the capacity to express lumican under certain conditions. Several studies have demonstrated that SLRP proteins can modulate cellular behaviors, i.e. cell migration and prolifera...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Role of Lumican in the Corneal Epithelium during Wound Healing

Saika¹,

Shiraishi²,

Saika³

et al. 2000

Journal of Biological Chemistry

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208

204

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“…The full-length zlum cDNA clone (ϳ1.9 kb) contains a 1032-bp ORF of 344 amino acids of zLum core protein. Like other SLRP core proteins such as bovine lumican (40), bovine mimecan (41), and mouse keratocan (42), zebrafish lumican consists of three distinct domains, a highly conserved central leucine-rich repeat region flanked by hypervariable N-and C-terminal regions. As shown in supplemental Fig.…”

Section: Primary Structure Of Zlum and Alignment Analysis Of The Zebrmentioning

confidence: 99%

Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats

Yeh

Liu

Kao

et al. 2010

Journal of Biological Chemistry

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The lumican gene (lum), which encodes one of the major keratan sulfate proteoglycans (KSPGs) in the vertebrate cornea and sclera, has been linked to axial myopia in humans. In this study, we chose zebrafish (Danio rerio) as an animal model to elucidate the role of lumican in the development of axial myopia. The zebrafish lumican gene (zlum) spans ϳ4.6 kb of the zebrafish genome. Like human (hLUM) and mouse (mlum), zlum consists of three exons, two introns, and a TATA box-less promoter at the 5-flanking region of the transcription initiation site. Sequence analysis of the cDNA predicts that zLum encodes 344 amino acids. zLum shares 51% amino acid sequence identity with human lumican. Similar to hLUM and mlum, zlum mRNA is expressed in the eye and many other tissues, such as brain, muscle, and liver as well. Transgenic zebrafish harboring an enhanced GFP reporter gene construct downstream of a 1.7-kb zlum 5-flanking region displayed enhanced GFP expression in the cornea and sclera, as well as throughout the body. Downregulation of zlum expression by antisense zlum morpholinos manifested ocular enlargement resembling axial myopia due to disruption of the collagen fibril arrangement in the sclera and resulted in scleral thinning. Administration of muscarinic receptor antagonists, e.g. atropine and pirenzepine, effectively subdued the ocular enlargement caused by morpholinos in in vivo zebrafish larvae assays. The observation suggests that zebrafish can be used as an in vivo model for screening compounds in treating myopia.Myopia is a very common ocular disorder, which is characterized by excessive elongation of the eyeball. In Taiwan, the prevalence of myopia is about 84% of schoolchildren aged 16 -18, and the prevalence of high myopia (less than Ϫ6.0 D) at 18 years of age is 24% in girls and 18% in boys (1). In contrast, the prevalence of high myopia is much lower in Western countries, about 1% of the general population (2). These studies imply that genetic susceptibility of ethnic differences may account for the high prevalence of myopia in Taiwanese.The sclera contains a collagen-rich extracellular matrix that undergoes significant biochemical and biomechanical remodeling during the development of myopia (3). Linkage studies of high myopia have identified potential loci MYP1 (Xq28) and MYP3 (12q21-23); these loci are within and/or near the loci of the human genome containing several genes that encode small leucine-rich proteoglycans (SLRP), 3 i.e. biglycan (Xq27ter), decorin (12q21-22), lumican (12q21.3-22), and DSPG3 (12q21) (4 -9). Our previous study showed that certain variations (rs3759223 (C3 T)) of single nucleotide polymorphism in the lumican regulatory region may influence the promoter activities of lumican and affect fibrillogenesis in myopic eyes (10). Furthermore, Majava et al. (11) found that a novel single nucleotide polymorphism (c.893-105G3 A) of the lumican gene was associated with high myopia. Recently, our prospective case control study also showed that genetic variation in the regulatory domai...

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“…2) that could putatively bind the same or similar transcription factors which bind XREs (2, 27) is also present in the rabbit Aldh1a1 promoter fragment. We identified several XREs (5Ј-TNGCGTG-3Ј) and E-boxes (5Ј-CACGTG-3Ј) using MatInspector Professional (50) in the gene regulatory regions of other highly expressed corneal genes, including mouse TKT (57), mouse keratocan (35), mouse keratin 12 (36), and human alpha-enolase (80). Members of the bHLH-PAS family of transcription factors can bind these XREs and E-boxes (23).…”

Section: Figmentioning

confidence: 99%

Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways

Hough

Piatigorsky

2004

Molecular and Cellular Biology

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Here we examine the molecular basis for the known preferential expression of rabbit aldehyde dehydrogenase class 1 (ALDH1A1) in the cornea. The rabbit Aldh1a1 promoter-firefly luciferase reporter transgene (؊3519 to ؉43) was expressed preferentially in corneal cells in transfection tests and in transgenic mice, with an expression pattern resembling that of rabbit Aldh1a1. The 5 flanking region of the rabbit Aldh1a1 gene resembled that in the human gene (60.2%) more closely than that in the mouse (46%) or rat (51.5%) genes. We detected three xenobiotic response elements (XREs) and one E-box consensus sequence in the rabbit Aldh1a1 upstream region; these elements are prevalent in other highly expressed corneal genes and can mediate stimulation by dioxin and repression by CoCl 2 , which simulates hypoxia. The rabbit Aldh1a1 promoter was stimulated fourfold by dioxin in human hepatoma cells and repressed threefold by CoCl 2 treatment in rabbit corneal stromal and epithelial cells. Cotransfection, mutagenesis, and gel retardation experiments implicated the hypoxia-inducible factor 3␣/aryl hydrocarbon nuclear translocator heterodimer for Aldh1a1 promoter activation via the XREs and stimulated by retinoic acid protein 13 for promoter repression via the E-box. These experiments suggest that XREs, E-boxes, and PAS domain/basic helix-loop-helix transcription factors (bHLH-PAS) contribute to preferential rabbit Aldh1a1 promoter activity in the cornea, implicating hypoxia-related pathways.Most mammalian corneas accumulate (5 to 40% depending on species) aldehyde dehydrogenase 3 (ALDH3A1) (unified nomenclature for the ALDH gene family [http://www.uchsc .edu/sp/sp/alcdbase/alcdbase.html]) mostly, but not exclusively, in their epithelium (47). While the function of the abundant ALDH3A1 in mammalian corneal epithelial cells is not established, detoxification of lipid peroxide radicals induced by UV light (26), absorption of UV (1), and a suspected structural role (47) are among the suggested possibilities. The fact that elimination of mouse ALDH3A1 (50% of corneal water-soluble protein) by homologous recombination in mice fails to show a corneal phenotype has increased the mystery of its function (44). Deficiencies in ALDH have also been implicated in keratoconus, which affects the corneal epithelium (18).Rabbits are exceptional in that they accumulate ALDH1A1 rather than ALDH3A1 in the cornea (29). ALDH1A1 comprises about 3% and ALDH3A1 comprises about 5% of the total soluble protein in human cornea (31). In contrast to the situation with ALDH3A1 in other mammals, rabbit ALDH1A1 is more abundant in the stromal cells of the cornea than in the epithelium. Wound healing experiments after freeze-induced injury suggested that the prevalence of ALDH1A1 may contribute to transparency of rabbit stromal cells (29), analogous to the role of crystallins in the lens. Indeed, -crystallin (ALDH1A8) is a lens crystallin, comprising 25% of the watersoluble protein of the elephant shrew lens (20). A protein equally similar to ALDH1A1 and ALDH2...

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The Cloning of Mouse Keratocan cDNA and Genomic DNA and the Characterization of Its Expression during Eye Development

Cited by 86 publications

References 18 publications

Role of Lumican in the Corneal Epithelium during Wound Healing

Role of Lumican in the Corneal Epithelium during Wound Healing

Knockdown of Zebrafish Lumican Gene (zlum) Causes Scleral Thinning and Increased Size of Scleral Coats

Preferential Transcription of Rabbit Aldh1a1 in the Cornea: Implication of Hypoxia-Related Pathways

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