Loss of alpha3(IV) collagen expression associated with corneal keratocyte activation.
OBJECTIVE
To determine whether changes in the expression of type IV alpha1, alpha2, or alpha3 collagen isoforms are stringently associated with corneal stromal cell activation.


METHODS
Keratocytes isolated from rabbit corneal stroma by collagenase digestion were plated in serum-free or insulin-, bFGF/heparin sulfate (HS)-, TGF-beta1-, or fetal bovine serum (FBS)-supplemented DMEM/F12 medium. Expression of type IV collagen isoforms and keratan sulfate proteoglycans (KSPGs) was evaluated by immunocytochemical analysis, Western blot analysis, or both. Concentrations of mRNAs were estimated by quantitative RT-PCR using SYBR Green RT-PCR reagents.


RESULTS
Immunohistochemical analysis indicated that type IV alpha1, alpha2, and alpha3 collagens were expressed in normal rabbit corneal stroma and in keratocytes cultured in serum-free and insulin-supplemented media. However, alpha3(IV) collagen was not detectable in the regenerating stroma after photorefractive keratectomy (PRK) in rabbit or in corneal stromal cells cultured in media supplemented with FBS, bFGF/HS, or TGF-beta1. alpha3(IV) collagen mRNA levels were also diminished in the stromal cells cultured in these growth factor-supplemented media. KSPGs (lumican and keratocan) were expressed and secreted in serum-free medium. Although the expression of KSPGs was promoted by insulin, the expression and intracellular levels of lumican and keratocan mRNAs were downregulated by TGF-beta1 and FBS. bFGF/HS promoted the downregulation of intracellular keratocan but not lumican mRNA levels.


CONCLUSIONS
The loss in the expression of alpha3(IV) collagen is a stringent phenotypic change associated with activation of keratocytes in vivo and in vitro. This phenotypic change in activated corneal stromal cells is induced by bFGF/HS and by TGF-beta1, and it accompanies the downregulation of keratocan expression.
Guerriero
Emily
E
UPMC Eye Center, Ophthalmology and Visual Science Research Center, Eye and Ear Institute, Department of Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213, USA.
Chen
Jian
J
Sado
Yoshikazu
Y
Mohan
Rajiv R
RR
Wilson
Steven E
SE
Funderburgh
James L
JL
Sundarraj
Nirmala
N
D000818:Animals; D001324:Autoantigens / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D011508:Chondroitin Sulfate Proteoglycans / Q000235:genetics / Q000378:metabolism; D024141:Collagen Type IV / Q000235:genetics* / Q000378:metabolism; D003319:Corneal Stroma / Q000166:cytology / Q000502:physiology* / Q000601:surgery; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D007632:Keratan Sulfate / Q000235:genetics / Q000378:metabolism; D054018:Lasers, Excimer; D000071234:Lumican; D018903:Photorefractive Keratectomy; D020033:Protein Isoforms; D011509:Proteoglycans / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015533:Transcriptional Activation / Q000502:physiology*
D001324:Autoantigens; D011508:Chondroitin Sulfate Proteoglycans; D024141:Collagen Type IV; D005136:Eye Proteins; D000071234:Lumican; D020033:Protein Isoforms; D011509:Proteoglycans; D012333:RNA, Messenger; C057112:type IV collagen alpha3 chain; D007632:Keratan Sulfate