Candace W.-C. Kao

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.

Lumican is an extracellular matrix glycoprotein widely distributed inmammalian connective tissues. Corneal lumican modified with keratan sulfateconstitutes one of the major proteoglycans of the stroma. Lumican-null miceexhibit altered collagen fibril organization and loss of corneal transparency.A closely related protein, keratocan, carries the remaining keratan sulfate ofthe cornea, but keratocan-null mice exhibit a less severe corneal phenotype.In the current study, we examined the effect of lumican overexpression incorneas of wild type mice. These mice showed no alteration in collagenorganization or transparency but had increased keratocan expression at bothprotein and mRNA levels. Corneas of lumican-null mice showed decreasedkeratocan. This coupling of keratocan expression with lumican also wasobserved after intrastromal injection of a lumican expression minigene intothe corneal stroma of Lum–/– mice. Smallinterfering RNA knockdown of lumican in vitro reduced keratocanexpression, whereas co-injection of a lumican-expressing minigene with aβ-galactosidase reporter driven by the keratocan promoter demonstrated anincrease of keratocan transcriptional activity in response to lumicanexpression in Lum–/– corneas invivo. These observations demonstrate that lumican has a novel regulatoryrole in keratocan expression at the transcriptional level. Such results helpprovide an explanation for the differences in severity of cornealmanifestation found in Lum–/– andKera–/– mice. The results also suggest acritical level of small proteoglycans to be essential for collagenorganization but that overabundance is not detrimental to extracellular matrixmorphogenesis. Lumican is an extracellular matrix glycoprotein widely distributed inmammalian connective tissues. Corneal lumican modified with keratan sulfateconstitutes one of the major proteoglycans of the stroma. Lumican-null miceexhibit altered collagen fibril organization and loss of corneal transparency.A closely related protein, keratocan, carries the remaining keratan sulfate ofthe cornea, but keratocan-null mice exhibit a less severe corneal phenotype.In the current study, we examined the effect of lumican overexpression incorneas of wild type mice. These mice showed no alteration in collagenorganization or transparency but had increased keratocan expression at bothprotein and mRNA levels. Corneas of lumican-null mice showed decreasedkeratocan. This coupling of keratocan expression with lumican also wasobserved after intrastromal injection of a lumican expression minigene intothe corneal stroma of Lum–/– mice. Smallinterfering RNA knockdown of lumican in vitro reduced keratocanexpression, whereas co-injection of a lumican-expressing minigene with aβ-galactosidase reporter driven by the keratocan promoter demonstrated anincrease of keratocan transcriptional activity in response to lumicanexpression in Lum–/– corneas invivo. These observations demonstrate that lumican has a novel regulatoryrole in keratocan expression at the transcriptional level. Such results helpprovide an explanation for the differences in severity of cornealmanifestation found in Lum–/– andKera–/– mice. The results also suggest acritical level of small proteoglycans to be essential for collagenorganization but that overabundance is not detrimental to extracellular matrixmorphogenesis. Lumican is a member of the small leucine-rich proteoglycan(SLRP) 1The abbreviations used are: SLRP, small leucine-rich proteoglycan; ECM,extracellular matrix; KSPG, keratan sulfate-containing proteoglycan; siRNA,small interfering RNA; RT, reverse transcription; CMTF, confocal microscopythrough focusing.1The abbreviations used are: SLRP, small leucine-rich proteoglycan; ECM,extracellular matrix; KSPG, keratan sulfate-containing proteoglycan; siRNA,small interfering RNA; RT, reverse transcription; CMTF, confocal microscopythrough focusing. family withexpression reported in cornea, sclera, aorta, cartilage, liver, skeletalmuscle, kidney, pancreas, brain, placenta, and lung(1Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. CellBiol. 1998; 141: 1277-1286Crossref PubMed Scopus (570) Google Scholar, 2Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar, 3Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, 4Grover J. Chen X.N. Korenberg J.R. Roughley P.J. J. Biol. Chem. 1995; 270: 21942-21949Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 5Krull N.B. Gressner A.M. FEBS Lett. 1992; 312: 47-52Crossref PubMed Scopus (32) Google Scholar, 6Ezura Y. Chakravarti S. Oldberg A. Chervoneva I. Birk D.E. J. Cell Biol. 2000; 151: 779-788Crossref PubMed Scopus (277) Google Scholar).Lumican co-localizes with collagen fibrils in the corneal stroma and has beenhypothesized to be involved in modulation of the highly organized collagenmatrix required for corneal transparency(7Chakravarti S. Petroll W.M. Hassell J.R. Jester J.V. Lass J.H. Paul J. Birk D.E. Invest.Ophthalmol. Vis. Sci. 2000; 41: 3365-3373PubMed Google Scholar,8Birk D.E. Trelstad R.L. J.Cell Biol. 1984; 99: 2024-2033Crossref PubMed Scopus (222) Google Scholar). These predictions weresubstantiated by findings of corneal opacity, skin fragility, and abnormallylarge collagen fibril diameters and disorganized interfibrillar spacingpresent in lumican-null mice(1Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. CellBiol. 1998; 141: 1277-1286Crossref PubMed Scopus (570) Google Scholar,7Chakravarti S. Petroll W.M. Hassell J.R. Jester J.V. Lass J.H. Paul J. Birk D.E. Invest.Ophthalmol. Vis. Sci. 2000; 41: 3365-3373PubMed Google Scholar,9Austin B.A. Coulon C. Liu C.Y. Kao W.W. Rada J.A. Invest Ophthalmol. Vis. Sci. 2002; 43: 1695-1701PubMed Google Scholar, 10Neame P.J. Kay C.J. McQuillan D.J. Beales M.P. Hassell J.R. Cell Mol. Life Sci. 2000; 57: 859-863Crossref PubMed Scopus (121) Google Scholar, 11Rada J.A. Cornuet P.K. Hassell J.R. Exp. Eye Res. 1993; 56: 635-648Crossref PubMed Scopus (286) Google Scholar).More recently, studies have showed lumican involvement in cell migration andproliferation during embryonic development and wound healing(12Saika S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar, 13Cornuet P.K. Blochberger T.C. Hassell J.R. Invest. Ophthalmol. Vis. Sci. 1994; 35: 870-877PubMed Google Scholar, 14Doane K.J. Ting W.H. McLaughlin J.S. Birk D.E. Exp. Eye Res. 1996; 62: 271-283Crossref PubMed Scopus (24) Google Scholar, 15Wilda M. Bachner D. Just W. Geerkens C. Kraus P. Vogel W. Hameister H. J. Bone Miner.Res. 2000; 15: 2187-2196Crossref PubMed Scopus (53) Google Scholar).The delayed epithelial wound healing phenotype inLum–/– mice is potentially due to theinvolvement of lumican in cellular migration, adhesion, and/or proliferation(12Saika S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar,16Vij N. Roberts L. Joyce S. Chakravarti S. Exp. Eye Res. 2004; 78: 957-971Crossref PubMed Scopus (90) Google Scholar). Under normal conditions,lumican is not expressed by epithelial cells, but transient expression isreported by migrating cells during wound healing and development(12Saika S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar,17Ying S. Shiraishi A. Kao C.W. Converse R.L. Funderburgh J.L. Swiergiel J. Roth M.R. Conrad G.W. Kao W.W. J. Biol. Chem. 1997; 272: 30306-30313Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). Impairment of cellmigration and proliferation may partially explain the delay of epithelialwound healing in lumican-null mice. Recent reports have also showed delayedepithelial-mesenchymal transition in lumican-null mice. Lumican-null mouselens epithelial cells showed decreased α-smooth muscle actin expressionand the delayed epithelial-mesenchymal transition induction by transforminggrowth factor β-2 in vitro(18Saika S. Miyamoto T. Tanaka S. Tanaka T. Ishida I. Ohnishi Y. Ooshima A. Ishiwata T. Asano G. Chikama T. Shiraishi A. Liu C.Y. Kao C.W. Kao W.W. InvestOphthalmol. Vis. Sci. 2003; 44: 2094-2102Crossref PubMed Scopus (120) Google Scholar). A role for lumican hasalso been suggested in growth and metastasis of breast, colon, and pancreaticcancer(19Leygue E. Snell L. Dotzlaw H. Troup S. Hiller-Hitchcock T. Murphy L.C. Roughley P.J. Watson P.H. J. Pathol. 2000; 192: 313-320Crossref PubMed Scopus (127) Google Scholar, 20Lu Y.P. Ishiwata T. Kawahara K. Watanabe M. Naito Z. Moriyama Y. Sugisaki Y. Asano G. Pathol. Int. 2002; 52: 519-526Crossref PubMed Scopus (58) Google Scholar, 21Ping L.Y. Ishiwata T. Asano G. J. Pathol. 2002; 196: 324-330Crossref PubMed Scopus (66) Google Scholar)and in cellular apoptosis(21Ping L.Y. Ishiwata T. Asano G. J. Pathol. 2002; 196: 324-330Crossref PubMed Scopus (66) Google Scholar). A cell surface receptorfor lumican has also been demonstrated(22Funderburgh J.L. Mitschler R.R. Funderburgh M.L. Roth M.R. Chapes S.K. Conrad G.W. InvestOphthalmol. Vis. Sci. 1997; 38: 1159-1167PubMed Google Scholar). These observationsdemonstrate lumican to have multiple functions, and it may serve as amatrikine in regulating cellular activities via interaction with integrinand/or growth factor receptors besides serving as a component of ECM, similarto what has been suggested for many other ECM components(23Swindle C.S. Tran K.T. Johnson T.D. Banerjee P. Mayes A.M. Griffith L. Wells A. J. CellBiol. 2001; 154: 459-468Crossref PubMed Scopus (219) Google Scholar, 24Duca L. Floquet N. Alix A.J. Haye B. Debelle L. Crit Rev. Oncol. Hematol. 2004; 49: 235-244Crossref PubMed Scopus (149) Google Scholar, 25Tran K.T. Griffith L. Wells A. Wound. Repair Regen. 2004; 12: 262-268Crossref PubMed Scopus (136) Google Scholar).The full extent of cellular functions mediated by lumican, however, remains tobe determined. Keratan sulfate-containing proteoglycans (KSPGs) are uniquely abundant inthe cornea and have long been thought to be essential for cornealtransparency. Lumican constitutes only about half of corneal KSPG. Most of theremaining corneal keratan sulfate modifies keratocan, a protein with highsequence similarity to lumican. In adult tissues, keratocan is limited tocorneal stroma, and keratocan expression is considered a phenotypic marker forkeratocytes. Lumican-null (Lum–/–) micemanifest corneal opacity, skin fragility, and impaired collagenfibrillogenesis (1Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. CellBiol. 1998; 141: 1277-1286Crossref PubMed Scopus (570) Google S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google however, of only results in a of cornea in of the collagen matrix C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google A.J. C. Liu C.Y. Kao W.W. Biol. 2003; PubMed Scopus Google Scholar). of the of similarity in and has been no explanation for the the and In the current study, we have the as to the ofthe in the cornea by lumican in the corneal stroma of This the of a corneal collagen KSPG. The results that the lumican not have corneal or corneal a lumican expression found to effect the expression of These findings a for the differences of in corneas andKera–/– mice and a novel of lumican. of a a by the of used to lumican of as a in K. Liu C.Y. R.L. Birk D.E. Funderburgh J.L. Kao W.W. 2003; PubMed Google Scholar). The by and and to promoter in the C. H. Kao C. Kao W.W. 2000; PubMed Scopus Google Scholar). The of minigene by in A of is minigene by and and injection of by the mice used for with a and a the for for for for and for cornea used in of of of reverse and of corneal RNA to the to The for for and for and A of the to of a of of of of and The for at for for and for for by a at at the of and corneas and and in of and to Corneas a for at and the at in a by for in of in The and with a at about of protein be with at as J.L. Funderburgh M.L. Liu C.Y. Kao W.W. Conrad G.W. Invest. Ophthalmol. Vis. Sci. 1995; Google and development to of to lumican keratocan used as S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In confocal microscopythrough used to corneal epithelial and corneal in the mice for by injection of and cornea a confocal with a surface J.V. L.Y. J. Chakravarti S. Paul J. Petroll W.M. 2001; 41: PubMed Scopus Google Scholar, J.V. Petroll W.M. PubMed Scopus Google Scholar, J. Jester J.V. T.D. Petroll W.M. Invest Ophthalmol. Vis. Sci. 2000; 41: Google to and a of the ofthe to serve as an a the cornea at that be to a of corneal and be in the to the of and for the stroma J.V. Petroll W.M. PubMed Scopus Google J. Jester J.V. T.D. Petroll W.M. Invest Ophthalmol. Vis. Sci. 2000; 41: Google Scholar). the the to the corneal stroma of J. Jester J.V. T.D. Petroll W.M. Invest Ophthalmol. Vis. Sci. 2000; 41: Google Petroll W.M. T. Jester J.V. 1997; PubMed Scopus Google Scholar). The and corneal the and the and mice by the in in with for D.E. Trelstad R.L. J.Cell Biol. 1984; 99: 2024-2033Crossref PubMed Scopus (222) Google Scholar).The corneas with and with in an by the corneas and a of and a and a and with cornea and the and stroma examined at a for the and fibril diameters in of the and stroma the at in a the and and diameters RNA corneas of RNA to keratocan as C.Y. Shiraishi A. Kao C.W. Converse R.L. Funderburgh J.L. Conrad G.W. Kao W.W. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). with a mice after for or S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). and to the for in and the of in by the and of the by and of to to as The mice to intrastromal Liu C.Y. M. B. J. Invest Sci. 2004; PubMed Scopus Google and during in a A.M. Y. J. C. Invest Ophthalmol. Vis. Sci. 2001; Google Scholar). A small in the corneal a to a the the stroma. The the stroma, and of K. Liu C.Y. R.L. Birk D.E. Funderburgh J.L. Kao W.W. 2003; PubMed Google corneal stroma injection and to and for lumican and and lumican in the and for lumican and keratocan as used as as S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). with The a and by Lumican of at for as C.J. Roth M.R. Funderburgh M. Conrad G.W. Funderburgh J.L. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). These or a of RNA lumican at a of in The the lumican a The and of the as after and cells in and proteoglycans after as the by and J.L. Mann M.M. Funderburgh M.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in at with Lumican and keratocan in the after a a to lumican by RNA at for of keratocan, and J.L. Mann M.M. Funderburgh M.L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). injection of in lumican-null mice as Liu C.Y. M. B. J. Invest Sci. 2004; PubMed Scopus Google Scholar). with of in a K. Liu C.Y. R.L. Birk D.E. Funderburgh J.L. Kao W.W. 2003; PubMed Google Scholar). corneas and activity reporter aβ-galactosidase to In corneal with a in of reporter in the a protein to the of used to the activity and to used to and The of to the role of lumican inthe corneal stroma by mice with a lumican minigene a protein the of the keratocan promoter C. H. Kao C. Kao W.W. 2000; PubMed Scopus Google of the minigene used to mice by with a and and the used for for the reporter The expression by and for a of mRNA the and used for and other also for the not by for the of mRNA of the by not not and mice the of protein in with S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar).Lumican in corneas of the that of corneas of of expression by the in the of the of protein, and mice showed a protein by in of lumican by the in the The in demonstrate that no differences and mice for epithelial and The epithelial and in mice and in the mice the level of in the mice with the not mice. mice of corneal with the of expression of the of of expression corneal epithelial and with the is for and and as by in a that the and the and and in normal differences and stroma. in the to be limited with less not in diameters the stroma and whereas stroma and for and diameters in the for lumican expression and corneal of keratocan expression to lumican overexpression corneal expression C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google keratocan expression in cornea of that of an by lumican. lumican keratocan proteoglycans corneas and mice. the results of a in corneal by expression decreased in the with wild type but a keratocan expression in the the lumican keratocan mRNA with corneal RNA and mice mRNA of that the keratocan mRNA mice decreased with and mice. the showed an in keratocan mRNA as with the These results the that lumican expression by the The results of to novel role of lumican in the to expression of a reporter in the A.M. Y. J. C. Invest Ophthalmol. Vis. Sci. 2001; Google Scholar). role of lumican in keratocan the corneal stroma of lumican-null mice to in lumican an in keratocan used in and with and for lumican. corneas exhibit the lumican Lumican in the increased expression by to keratocan in intrastromal of the Lum–/– of to the corneal stroma results in lumicanexpression by no lumican be the at a in keratocan expression by of as with the the This the keratocan for lumican and of corneal mice injection of or as intrastromal injection also after injection as with the In to the in keratocan examined in in vitro in been reduced by that lumican the during a a with in the by mRNA showed a of after lumican the other not altered in the of lumican to keratocan in and in vitro the by that lumican keratocan a keratocan promoter driven reporter mice with a or an corneas and determined. a of the of the or lumican minigene Lumican expression the the keratocan the of lumican to expression at the promoter level. and lumican are of collagen matrix organization in the corneal W.W. Liu C.Y. J. 2002; PubMed Scopus Google Scholar). lumican a role in as wound epithelial-mesenchymal and S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google S. Miyamoto T. Tanaka S. Tanaka T. Ishida I. Ohnishi Y. Ooshima A. Ishiwata T. Asano G. Chikama T. Shiraishi A. Liu C.Y. Kao C.W. Kao W.W. InvestOphthalmol. Vis. Sci. 2003; 44: 2094-2102Crossref PubMed Scopus (120) Google E. Snell L. Dotzlaw H. Troup S. Hiller-Hitchcock T. Murphy L.C. Roughley P.J. Watson P.H. J. Pathol. 2000; 192: 313-320Crossref PubMed Scopus (127) Google L.Y. Ishiwata T. Asano G. J. Pathol. 2002; 196: 324-330Crossref PubMed Scopus (66) Google L.Y. Ishiwata T. Asano G. J. Pathol. 2002; 196: 324-330Crossref PubMed Scopus (66) Google E. Snell L. Dotzlaw H. K. Hiller-Hitchcock T. Roughley P.J. Watson P.H. Murphy L.C. Res. 1998; Google Scholar). The results reported a novel role of lumican as a of keratocan by The of the lumican and keratocan-null reported (1Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. CellBiol. 1998; 141: 1277-1286Crossref PubMed Scopus (570) Google S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). the has a corneal and skin to altered collagen delayed corneal (1Chakravarti S. Magnuson T. Lass J.H. Jepsen K.J. LaMantia C. Carroll H. J. CellBiol. 1998; 141: 1277-1286Crossref PubMed Scopus (570) Google S. Shiraishi A. Saika S. Liu C-Y. Funderburgh J.L. Kao C.W.C. Converse R.L. Kao W.W.Y. J. Biol. Chem. 2000; 275: 2607-2612Abstract Full Text Full Text PDF PubMed Scopus (206) Google and delayedepithelial-mesenchymal transition of S. Miyamoto T. Tanaka S. Tanaka T. Ishida I. Ohnishi Y. Ooshima A. Ishiwata T. Asano G. Chikama T. Shiraishi A. Liu C.Y. Kao C.W. Kao W.W. InvestOphthalmol. Vis. Sci. 2003; 44: 2094-2102Crossref PubMed Scopus (120) Google the keratocan-null of but cornea and corneal as examined to C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google A.J. C. Liu C.Y. Kao W.W. Biol. 2003; PubMed Scopus Google Scholar). ofthe keratocan-null showed no alteration of other in the C.Y. Birk D.E. Hassell J.R. B. Kao W.W. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The the expression in the and a in the Lum–/– mice as type This that and in the lumican are not the of of lumican expression but also the of a expression by corneal stroma mice. is of to a of lumican expression in of mice suggested that of lumican in the phenotype K.J. J.H. Paul J. Roberts L. Y. Oldberg A. Birk D.E. Chakravarti S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The that the expression of of the family may be other in the The results of studies to a The of to the role of lumican inthe cornea lumican a wild collagen and collagen in an overexpression be corneal fibril and transparency of The of corneal of is not detrimental to The keratocan and mice have normal and only the Lum–/– mice current that the lumican lumican but also have reduced that corneal a and that a is not be This may be in by the of a of collagen L. I. Oldberg A. FEBS Lett. 2000; PubMed Scopus Google Scholar). a in lumican expression in intrastromal injection of lumican in lumican but anincrease in keratocan the lumican expression in the keratocan of regulating keratocan expression in the adult The lumican may serve as a for keratocan expression to the by lumican an in promoter the to keratocan promoter activity inLum–/– mice as observations are with the that of may have functions besides serving as of J. Full Text Full Text PDF PubMed Scopus Google Scholar). family member that functions with lumican. is an in ECM it has been be involved in cell proliferation and migration and protein to the found in lumican-null mice skin and impaired collagen J. 2002; PubMed Scopus Google Scholar). lumican are expressed in studies the of ECM to as factor receptors and the C.S. Tran K.T. Johnson T.D. Banerjee P. Mayes A.M. Griffith L. Wells A. J. CellBiol. 2001; 154: 459-468Crossref PubMed Scopus (219) Google Scholar). has as a and to the M. Mann McQuillan D.J. A.J. J. 1998; PubMed Scopus Google S. M. McQuillan D.J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google and a the ECM and S. E. M. N. C. J. CellBiol. 2003; PubMed Scopus Google Scholar, A. C. E. L. M. S. M.P. D.J. G. Mol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, W. T. Mol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, McQuillan D.J. I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). A the of lumican to serve as a in J.L. Mitschler R.R. Funderburgh M.L. Roth M.R. Chapes S.K. Conrad G.W. InvestOphthalmol. Vis. Sci. 1997; 38: 1159-1167PubMed Google Scholar). have that cells and C. T. Y. C. J. and W. are with the of cell surface The for has not been but may also the is that lumican the expression of keratocan and/or by A for lumican and/or the for extracellular matrix inthe corneal stroma, collagen in the cornea a highly organized to a that the involved is The that lumican involvement in is the in expression of lumican has been epithelial cells with K.J. J.H. Paul J. Roberts L. Y. Oldberg A. Birk D.E. Chakravarti S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google and E. Snell L. Dotzlaw H. Troup S. Hiller-Hitchcock T. Murphy L.C. Roughley P.J. Watson P.H. J. Pathol. 2000; 192: 313-320Crossref PubMed Scopus (127) Google E. Snell L. Dotzlaw H. K. Hiller-Hitchcock T. Roughley P.J. Watson P.H. Murphy L.C. Res. 1998; Google S. C. M. C. Chakravarti S. Roughley P.J. Murphy L.C. Watson P.H. Res. 2003; Google Scholar). the lumican involvement in remains of the of lumican expression during has in lumican expression in E. Snell L. Dotzlaw H. Troup S. Hiller-Hitchcock T. Murphy L.C. Roughley P.J. Watson P.H. J. Pathol. 2000; 192: 313-320Crossref PubMed Scopus (127) Google whereas lumicanexpression with S. C. M. C. Chakravarti S. Roughley P.J. Murphy L.C. Watson P.H. Res. 2003; Google Scholar). The of and multiple functions of lumican may an explanation for of lumican in The and are but the of the lumican and may to the of during results the that lumican protein for keratocan expression at the promoter level in corneal stroma. the of lumican has to be determined. lumican may as a a tobe are many of lumican as a that have the expression of are to and of

Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency. Lumican is one of the major keratan sulfate proteoglycans (KSPG) in vertebrate corneas. We previously cloned the murine lumican cDNA. This study determines the structure of murine lumican gene (Lum) and its expression during mouse embryonic developments. The mouse lumican gene was isolated from a bacterial artificial chromosome mouse genomic DNA library and characterized by polymerase chain reaction and Southern hybridization. The lumican gene spans 6.9 kilobase pairs of mouse genome. The gene consists of three exons and two introns. Exon 1 constitutes 88 bases (b) of untranslated sequence. Exon 2 is 883 b and contains most of the coding sequence of lumican mRNA, and exon 3 has 152 b of coding sequence and 659 b of 3′ noncoding sequence. The mouse lumican gene has a TATCA element, a presumptive TATA box, which locates 27 b 5′-upstream from the transcription initiation site. Northern hybridization and in situ hybridization indicate that in early stages of embryonic development, day 7 post coitus the embryo expresses little or no lumican. Thereafter, different levels of lumican mRNA can be detected in various organ systems, such as cornea stroma, dermis, cartilage, heart, lung, and kidney. The cornea and heart are the two tissues that have the highest expression in adult. Immunoblotting studies found that KSPG core proteins became abundant in the cornea and sclera by postnatal day 10 but that sulfated KSPG could not be detected until after the eyes open. These results indicate that lumican is widely distributed in most interstitial connective tissues. The modification of lumican with keratan sulfates in cornea is concurrent with eye opening and may contribute to corneal transparency. Corneal strength and transparency depend upon the development and maintenance of an organized extracellular matrix, including uniformly small diameter collagen fibrils with lamellae of consistent interfibrillar spacing. The collagen fibrils of adjacent lamella sheets are perpendicular to one another (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 143-160Crossref PubMed Scopus (72) Google Scholar, 2Hay E.D. Int. Rev. Cytol. 1980; 63: 263-322Crossref PubMed Scopus (295) Google Scholar). The mechanism that governs the formation of collagen lamellae in cornea stroma is not well understood. It has been suggested, however, that the ratios of different collagen types in making up the fibrillar corneal collagen and other extracellular specialized matrix components, e.g.proteoglycans and glycoprotein are essential for the development of a transparent cornea (1Linsenmayer T.F. Fitch J.M. Birk D.E. Ann. N. Y. Acad. Sci. 1990; 580: 143-160Crossref PubMed Scopus (72) Google Scholar, 3Hassell J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Scopus (176) Google Scholar, 4Linsenmayer T.F. Gibney E. Igoe F. Gordon M.K. Fitch J.M. Fessler L.I. Birk D.E. J. Cell Biol. 1993; 121: 1181-1189Crossref PubMed Scopus (247) Google Scholar, 5Hahn R.A. Birk D.E. Development. 1992; 115: 383-393PubMed Google Scholar, 6McLaughlin J.S. Linsenmayer T.F. Birk D.E. J. Cell Sci. 1989; 94: 371-379PubMed Google Scholar, 7Marchant J.K. Hahn R.A. Linsenmayer T.F. Birk D.E. J. Cell Biol. 1996; 135: 1415-1426Crossref PubMed Scopus (101) Google Scholar, 8Ruggiero F. Burillon C. Garrone R. Invest. Ophthalmol. Vis. Sci. 1996; 37: 1749-1760PubMed Google Scholar). In addition to interaction with collagen fibrils, proteoglycans in stroma also play a role in corneal hydration due to their high negative charge of sulfated carbohydrate moieties (9Rawe I.M. Tuft S.J. Meek K.M. Histochem. J. 1992; 24: 311-318Crossref PubMed Scopus (30) Google Scholar, 10Bettelheim F.A. Plessy B. Biochim. Biophys. Acta. 1975; 381: 203-214Crossref PubMed Scopus (92) Google Scholar, 11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar). The hydrophilic properties of the stroma result from stromal proteoglycans that constitute the second most abundant biological materials in stroma, after collagen (12Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 14226-14231Abstract Full Text PDF PubMed Google Scholar, 13Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar). The keratan sulfate proteoglycans (KSPGs) 1The abbreviations used are: KSPG, keratan sulfate proteoglycan; KS, keratan sulfate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PC, post coitus; SLRP, small leucine-rich proteoglycan; kb, kilobase pair(s); b, base(s); bp, base pair. are uniquely abundant in the cornea, constituting the major proteoglycans of the corneal stroma. Currently, three corneal KSPG core proteins have been identified, i.e. keratocan, lumican, and mimican (osteoglycin), which were previously designated 37A, 37B, and 25, respectively (13Funderburgh J.L. Conrad G.W. J. Biol. Chem. 1990; 265: 8297-8303Abstract Full Text PDF PubMed Google Scholar, 14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 17Grover J. Chen X.-N. Korenberg J.R. Roughley P.J. J. Biol. Chem. 1995; 270: 21942-21949Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar). These proteins are structurally and antigenically related, and each bears from one to threeN-linked keratan sulfate chains in addition to several nonsulfated oligosaccharides (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar). Lumican belongs to the family of small leucine-rich proteoglycans (SLRPs) that includes decorin, biglycan, fibromodulin, keratocan, epiphycan, and osteoglycin (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar). Each of these proteoglycans possesses 6–10 leucine-rich repeating units between the flanking cysteine-rich disulfide-bonded domains at the N and C termini of the core protein. The presence of a common structural motif implies that these proteoglycans may share common functional properties. Such a common function is thought to be the interaction with fibrillar collagen. The tissue distributions of each proteoglycan are distinct; therefore, it is likely that each family member fulfills a different role in connective tissues (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, 21Kresse H. Hausser H. Schonherr E. Exper. Suppl. (Basel). 1994; 70: 73-100PubMed Google Scholar, 22Scott J.E. Biochemistry. 1996; 35: 8795-8799Crossref PubMed Scopus (215) Google Scholar). For example, lumican only exists as a proteoglycan in cornea, it is a glycoprotein in the rest of connective tissues (11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar, 14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar, 23Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar, 24Funderburgh J.L. Conrad G.W. Greiling H. Scott J.E. Keratan Sulphate: Chemistry, Biology, and Chemical Pathology. Biochemical Society, London1989: 39-52Google Scholar, 25Funderburgh J.L. Caterson B. Conrad G.W. J. Biol. Chem. 1987; 262: 11634-11640Abstract Full Text PDF PubMed Google Scholar). The presence of sulfated lumican molecules in cornea suggests that in this tissue lumican may have unique functions, e.g. maintaining corneal transparency; however, its role serving in other noncorneal tissues remains elusive. Mouse lumican is a 338-amino acid protein with high sequence homology to bovine, human, and chicken lumican (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 17Grover J. Chen X.-N. Korenberg J.R. Roughley P.J. J. Biol. Chem. 1995; 270: 21942-21949Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, 26Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google Scholar). To examine the structure and function relationship of mouse lumican gene using transgenic mice and site-directed mutagenesis techniques; it is imperative to isolate and characterize the mouse lumican cDNA and genomic DNA and to determine the spatial-temporal expression of lumican gene during mouse development. In the present studies, we have cloned and determined the primary structure of mouse lumican gene (Lum). In situ and Northern hybridization were used to determine the temporospatial expression of Lum. Lumican isolated from eye shells (cornea plus sclera) at various developmental stages were also biochemically characterized. Our results indicate that lumican is widely expressed in a variety of connective tissues. Sulfation of the lumican in cornea occurs concomitantly with eye opening and therefore may be an essential step in providing corneal transparency. A pair of primers, sense 5′-CATGTATGGGCAAATATC and antisense 5′-TGTAGAAGGTTGTGGTCA (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar), derived from mouse lumican cDNA was used in polymerase chain reaction to screen a mouse bacterial artificial chromosome-genomic DNA library (Research Genetics, Inc., Huntsville, AL). A positive clone of 200 kb was isolated. The clone was characterized with restriction enzyme digestion and Southern blot hybridization with 32P-labeled lumican cDNA. A 6-kbSalI-XbaI fragment and an 8-kbXbaI-XbaI fragment together encoding the full-length cDNA were subcloned into pBSSK vector (Stratagene, La sequence of the lumican gene was determined with by the DNA core in the of at of A antisense to exon 1 and a sequence were used to a DNA fragment by polymerase chain reaction using a fragment of mouse lumican genomic DNA clone as the sense was with using a by the The antisense was with by Inc., The DNA was to of mouse mRNA from 1 day post This reaction was with units of R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The was used with the as a The was an To the transcription initiation a sequence reaction using the antisense and mouse lumican genomic DNA fragment as was with S. Biochemical A antisense to exon 1 was with by and to of mouse was as previously R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The reaction were the as the one used in the For of lumican mRNA in a blot 2 of from different developmental by a was from The blot was with 32P-labeled mouse lumican cDNA as previously (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). For tissue were from mouse tissues using as previously (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar). 10 of were in with The were to and with 32P-labeled lumican and mouse 3-phosphate cDNA in a hybridization at as previously (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, R. Kao H. J. Justice M.J. Stech M.E. Kao W.W.-Y. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar). The were by three with and at for The hybridization were detected with a The of lumican mRNA were with the mRNA in To the types that lumican, the mouse tissues were with and in as previously Kao Kao W.W.-Y. 1996; PubMed Scopus Google Scholar). and sense of lumican were and used in in situ hybridization To were to a in at and with of at for 1 by with at as previously Kao Kao W.W.-Y. 1996; PubMed Scopus Google Scholar). The hybridization were with using by were isolated from eye shells (cornea plus sclera) of day postnatal and and 1 of mice in of a and as by Funderburgh J.L. Corpuz L.M. Roty M.R. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1996; 37 (abstr.): S645PubMed Google Scholar). The tissue was by and for at The were to were a of in the The was with in the and proteoglycans were with proteoglycans were in for at and keratan proteoglycans were by the addition of to the to This isolated sulfated and of lumican J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar). The proteoglycans were by in in and in of 10 of protein of the corneal KSPG or with each of and for 2 at 37 was a 3 to The was in for 2 and in in acid and in the KSPG core proteins were by digestion of of KSPG protein with as and a The proteins were to and KSPG proteins were detected using KSPG as previously (14Corpuz L.M. Funderburgh J.L. Funderburgh M.L. Bottomley G.S. Prakash S. Conrad G.W. J. Biol. Chem. 1996; 271: 9759-9763Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The genomic DNA clone isolated from a mouse bacterial artificial chromosome genomic DNA library was characterized by Southern hybridization and 3′ of mouse lumican cDNA. The full-length mouse lumican gene spans 6.9 kb in mouse genome. 1 that gene has three exons and two introns. The genomic and were and the were determined in by the primary structure of lumican gene and the acid sequence from The not have a TATA box, it has a TATCA element, a presumptive TATA box, that is 27 bases 5′-upstream from the transcription initiation site. is a of at b to the transcription initiation site. The initiation is at the base from the of exon exon 1 the untranslated of lumican Exon 2 most of in lumican, and exon 3 acid and a 3′ untranslated 1 and 2 are and bp, of the and The is found from the in exon derived from and the and that the transcription initiation is the at b to the initiation and 27 b 3′ of the TATCA as with the sequence of lumican genomic DNA was determined by using the used in of the of 2 and To the of lumican it is to determine the and expression of mouse lumican during development. Northern hybridization and in indicate that in early stages of embryonic development day 7 post coitus the embryo not lumican or expresses only not A that the isolated from contains little lumican mRNA The levels of lumican mRNA in the at and at a high To the expression of lumican mRNA by various tissues during mouse development, Northern hybridization was with isolated from cornea, heart, lung, and kidney. that levels of lumican in to that of in cornea and are in embryonic and of postnatal and in heart levels of lumican expression are The lumican mRNA at a but and have of lumican expression the not To determine the types that lumican mRNA, tissue from mouse and mice were with as and The in situ hybridization that the stroma in mouse cornea to lumican at embryo day the lumican expression in cornea stroma at postnatal day 1 and situ hybridization also detected the expression of lumican mRNA in several other such as heart, lung, and kidney. of these to lumican mRNA at embryo day that the lumican mRNA is expressed by the and in the situ hybridization of mouse lumican mRNA in various tissues at antisense sense and C and and the expression of lumican in various tissues during mouse development. day 10 PC, the to mRNA in and day PC, the expresses lumican mRNA, but the the early development and also expresses lumican mRNA and It is of to that interstitial of various tissues lumican mRNA after of expression of lumican during mouse and of tissues from mouse at different developmental stages were to in situ hybridization with cDNA as and in a of tissues from mouse at different developmental stages were to in situ hybridization with cDNA as and KSPG core proteins were by with from of cornea and sclera and detected by using in at postnatal day little of KSPG core proteins was detected with this day 10 and were of the KSPG core proteoglycans were detected with a that the sulfated chains of the KSPG postnatal day 1 sulfated KSPG could not be detected but by day 10 of KSPG in that of mice was A in the and of KSPG between 10 and with the at day that of of the KSPG with an enzyme for sulfated moieties in the results in the of in These indicate that cornea and sclera not of sulfated KSPG to 10 after a in which the eyes are in The mouse lumican belongs to family and has the of a of leucine-rich by and domains with (16Funderburgh J.L. Funderburgh M.L. Hevelone N.D. Stech M.E. Justice M.J. Liu C.Y. Kao W.W.-Y. Conrad G.W. Invest. Ophthalmol. Visual Sci. 1995; 36: 2296-2303PubMed Google Scholar, 18Funderburgh J.L. Funderburgh M.L. Brown S.J. Vergnes J.P. Hassell J.R. Mann M.M. Conrad G.W. J. Biol. Chem. 1993; 268: 11874-11880Abstract Full Text PDF PubMed Google Scholar, R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, 26Blochberger T.C. Vergnes J.P. Hempel J. Hassell J.R. J. Biol. Chem. 1992; 267: 347-352Abstract Full Text PDF PubMed Google S. N. Hassell J.R. 1995; PubMed Scopus (101) Google Scholar). The structure of mouse gene is to that of fibromodulin, which has three with the second exon encoding leucine-rich Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar). are different from the other two in another of i.e. and biglycan, which are of exons and the sulfate chains (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, S. R. J.L. R.V. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, Y. J. S. R.V. 1995; PubMed Scopus Google Scholar). The mouse lumican gene not a TATA an TATCA is present at 27 b 5′-upstream to the transcription initiation site. of that have been characterized this TATA for of transcription initiation N. J.P. Mol. 1993; Google Scholar). In a is at b from the transcription It has been that the presence of the may the of a TATA box, by transcription For example, the and the TATA may these and the proteins to to transcription J. PubMed Scopus Google Scholar). It has been that are of tissue and in their to the of extracellular matrix in connective tissues (20Iozzo R.V. Crit. Rev. Biochem. Mol. Biol. 1997; 32: 141-174Crossref PubMed Scopus (453) Google Scholar, S. E.D. Biol. 35: PubMed Scopus Google Scholar, S. E.D. Acad. Sci. S. PubMed Scopus Google Scholar). The of a matrix with small and and interfibrillar is essential for the development and maintenance of transparent cornea R.A. J. Scopus Google Scholar). corneal and the of corneal it has been that are in cornea stromal proteoglycan that may for the formation of N. M.M. 1995; PubMed Scopus Google Scholar, J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Google Scholar, J.L. Cintron C. Conrad G.W. Invest. Ophthalmol. Visual Sci. Google Scholar, J.R. Newsome D.A. M.M. Acad. Sci. S. 1980; PubMed Scopus Google Scholar, M.E. Biophys. J. 1996; 70: Full Text PDF PubMed Scopus Google Scholar). In the corneal the of KSPG is and the of sulfate proteoglycan A to KSPG is upon of corneal transparency J.R. Cintron C. Kublin C. Newsome D.A. Arch. Biochem. Biophys. 1983; 222: 362-369Crossref PubMed Google Scholar). The corneal is characterized by the of the of sulfated due to a in the sulfate proteoglycan or the J.R. Newsome D.A. M.M. Acad. Sci. S. 1980; PubMed Scopus Google Scholar, Hassell J.R. Newsome D.A. J. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. N. Arch. Ophthalmol. PubMed Scopus Google Scholar). Our that is a of KSPG core proteins in the mouse cornea at postnatal day 10 with that of day Lumican with keratan sulfate chains at postnatal day This is consistent with that of studies that in a and sulfated keratan sulfate proteoglycans during the embryonic development of the cornea at day the cornea to transparent T.C. Hassell J.R. Invest. Ophthalmol. Visual Sci. 1994; 35: Google Scholar, J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar). of in KSPG during corneal development that the core protein of lumican and its keratan sulfate chains are to the development of corneal transparency T.C. Hassell J.R. Invest. Ophthalmol. Visual Sci. 1994; 35: Google Scholar, J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar, G.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Sulfation of the chain the core protein by 10 KSPG not to until day levels at day this the mouse eyes open. This is consistent with the study by Gibney E. Gordon M.K. J.K. Birk D.E. Linsenmayer T.F. 1996; 63: PubMed Scopus Google Scholar), a of mRNA of chicken an enzyme in the of the chain during embryonic the extracellular is detected and an in the stroma J.L. Caterson B. Conrad G.W. Biol. PubMed Scopus (92) Google Scholar). The chicken transparency during this and are consistent with the that lumican and other proteoglycans may play an role in the development and maintenance of corneal transparency. It is of to that the levels of lumican mRNA in in the mice and a in to as with of embryonic at day and the of lumican protein in the tissue not with the of lumican the and of lumican may be at to has been in collagen W.W.-Y. R.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, W.W.-Y. J. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar). the of lumican in tissues may be due to an by the of chains to the core protein. studies are to examine the Lumican the keratan sulfate chains may be only to the it is that it is also present in a variety of noncorneal e.g. cartilage, heart, lung, as a sulfated or nonsulfated glycoprotein (11Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. Biochem. Soc. Trans. 1991; 19: 871-876Crossref PubMed Scopus (48) Google Scholar, 23Funderburgh J.L. Funderburgh M.L. Mann M.M. Conrad G.W. J. Biol. Chem. 1991; 266: 24773-24777Abstract Full Text PDF PubMed Google Scholar, 24Funderburgh J.L. Conrad G.W. Greiling H. Scott J.E. Keratan Sulphate: Chemistry, Biology, and Chemical Pathology. Biochemical Society, London1989: 39-52Google Scholar, 25Funderburgh J.L. Caterson B. Conrad G.W. J. Biol. Chem. 1987; 262: 11634-11640Abstract Full Text PDF PubMed Google Scholar). This may play that are to be in the maintenance of tissue It likely that lumican in noncorneal tissues is a of collagen it to be in in addition to cornea the of lumican gene may have organ are in

PURPOSE: Expression of the K3-K12 keratin pair characterizes the corneal epithelial differentiation. To elucidate the role of keratin 12 in the maintenance of corneal epithelium integrity, the authors bred mice deficient in keratin 12 by gene-targeting techniques. METHODS: One allele of murine Krt1.12 gene was ablated in the embryonic stem cell line, E14.1, by homologous recombination with a DNA construct in which the DNA element between intron 2 and exon 8 of the keratin 12 gene was replaced by a neo-gene. The homologous recombinant embryonic stem cells were injected to mouse blastocysts, and germ lines of chimeras were obtained. The corneas of heterozygous and homozygous mice were characterized by clinical observations using stereomicroscopy, histology with light and electron microscopy, Western immunoblot analysis, immunohistochemistry, in situ hybridization, and Northern hybridization. RESULTS: The heterozygous mice (+/-) one allele of the Krt1.12 gene appear normal and do not develop any clinical manifestations (e.g., corneal epithelial defects). Homozygous mice (-/-) develop normally and suffer mild corneal epithelial erosion. Their corneal epithelia are fragile and can be removed by gentle rubbing of the eyes or brushing with a Microsponge. The corneal epithelium of the homozygote (-/-) does not express keratin 12 as judged by immunohistochemistry, Western immunoblot analysis with epitope-specific anti-keratin 12 antibodies, Northern hybridization with 32P-labeled keratin 12 cDNA, and in situ hybridization with an anti-sense keratin 12 riboprobe. Light and electron microscopy revealed subtle abnormalities in the corneal epithelia of -/- mice (i.e., a decrease in number of cell layers) and cytolysis of superficial cells, but the number of hemidesmosomes and desmosomes are normal in basal and suprabasal cells. The number of keratin intermediate filaments in basal and suprabasal corneal epithelial cells in -/- mice decreases, and they appear as dense bundles. This morphology is similar to that of keratin intermediate filaments in epidermal epithelial, cells but differs from that of normal corneal epithelial cells in which the keratins form fine filamentous networks. The superficial epithelial cells are devoid of keratin intermediate filaments and often detach from the corneal surface of -/- mice. CONCLUSIONS: The presence of cornea-specific K3-K12 keratin pairs is essential for the maintenance of corneal epithelium integrity.