Expression of Tenascin-C Splice Variants in

Expression of Tenascin-C Splice Variants in Normal and Bullous Keratopathy Human Corneas Alexander V. Ljubimov,1 Mehrnoosh Saghizadeh,l Konstantin S. Spirin,1 Htwe L Khin,l Sheryl L Lewin,1 Luciano Zardi,2 Mario A. Bourdon,5 and M. Cristina Kenney1 characterize the expression patterns of tenascin-C (TN-C) splice variants in normal corneas and in those affected by pseudophakic-aphakic bullous keratopathy (PBK-ABK).

PURPOSE. TO

METHODS. Alternatively spliced variants of TN-C mRNA from normal and age-matched human corneas with PBK-ABK were analyzed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot hybridization, using /32-microglobulin as a housekeeping gene to normalize the samples. Normal and PBK-ABK corneas were studied by immunofluorescence and western blot analysis with antibodies to specific fibronectin type Ill-like (FN-III) repeats of TN-C.

Tenascin-C mRNA expression was detected in epithelial, stromal, and endothelial cells of normal and PBK-ABK central corneas, although the protein was seen only in diseased corneas. Assessed by RT-PCR, PBK-ABK corneas expressed approximately three times more total TN-C mRNA than did normal corneas. Four major TN-C mRNA variants (with no FN-III insertional repeats or with retained insertional repeats D, Al, or Al+D) and three minor variants (with retained repeats A1+A2, A1+A2+D, or A1+A2+B+D) were much more abundant in PBK-ABK than in normal corneas. Repeat Al was more abundant in PBK-ABK TN-C protein than repeats A2, A3, B, or D. Major TN-C variants in PBK-ABK corneas were in the range of 190 kDa to 240 kDa. RESULTS.

CONCLUSIONS. Expression

of TN-C mRNA and protein is higher in PBK-ABK corneas than in normal corneas. This increase mainly concerns relatively small TN-C splice variants that may affect corneal cell adhesion and migration and contribute to the exacerbation of PBK-ABK. (Invest Ophthahnol VisSci. 1998;39:1135-H42)

I

n the past 30 years, there has been a significant increase in the number of corneal transplantations (penetrating keratoplasties) performed. Lensectomy-related corneal edema, or bullous keratopathy (pseudophakic and aphakic, PBKABK), remains the most common indication for penetrating keratoplasty.' Morphologically, PBK-ABK corneas have epithelial bullae (blisters) and a thickened Descemet's membrane (DM). Subepithelial nbrotic extracellular matrix (ECM) is often deposited in areas of bullae formation. New ECM accumulates to form the posterior collagenous layer, or retrocorneal fibrous membrane, between the endothelial cells and DM.2"4 The extracellular

From the 'Ophthalmology Research Laboratories, Bums and Allen Research Institute, Cedars-Sinai Medical Center, University of California Los Angeles Medical School Affiliate; 2Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy; and the 3La Jolla Institute of Experimental Medicine, California. The first two authors contributed equally to this study. Supported by grant EY10836 from the National Institutes of Health, Bethesda, Maryland (MCK); the Braille Transcribers Guild, San Diego, California (AVL, MAB); Skirball Program in Molecular Ophthalmology, Los Angeles, California (AVI.); the Discovery Fund for Eye Research, Los Angeles, California; and Associazione Italiana per la Ricerca sul Cancro & "Progetto finalizzato: Applicazioni cliniche della ricerca oncologica" of the Consiglio Nazionale delle Ricerche (LZ). Submitted for publication October 16, 1997; revised January 13, 1998; accepted February 27, 1998. Proprietary interest category: N. Reprint requests: Alexander V. Ljubimov, Burns and Allen Research Institute, Cedars-Sinai Medical Center, Davis-5069, 8700 Beverly Blvd., Los Angeles, CA 90048. Investigative Ophthalmology & Visual Science, June 1998, Vol. 39, No. 7 Copyright © Association for Research in Vision and Ophthalmology

matrix of subepithelial fibrosis areas and of posterior collagenous layer contains stromal and basement membrane components, including those that are not present in these locations in normal corneas.2'4"6 However, the most dramatic changes were observed for tenascin-C (TN-C), which was abnormally deposited in all PBK-ABK corneas.2 Tenascin (also known as cytotactin, hexabrachion, myotendinous antigen, Jl, and GMEM) is a large (more than 1 MDa) hexameric ECM glycoprotein.7"10 Four members of the tenascin family have been identified so far: TN-C (cytotactin), tenascin-R (TN-R; restrictin), tenascin-X (TN-X), and tenascin-Y (TNY).9 The TN-C monomer is composed of multiple domains (Fig. 1). The amino terminal knob serves to link six monomers to form a hexamer. It is followed by 14.5 (in human) epidermal growth' factor- (EGF) like repeats, 8 to 17 fibronectin type Ill-like (FN-III) repeats, and finally, a carboxyl-terminal globule homologous to fibrinogen.8'9 Eight FN-III repeats are constitutive and are never spliced out, and nine are insertional repeats that can be spliced out in various combinations (Fig. 1). Different FN-III repeats may mediate binding to cell surface receptors, modulate cell adhesion and migration, and affect neurite outgrowth.8"15 The alternative splicing of insertional FN-III repeats generates many TN-C variants, or isoforms (Fig. 1), that may serve different functions. 8 ' 9 " 16 " 18 Tenascins have been implicated in cell migration during development, cell proliferation, tissue repair, and oncogenesis, although knockout mice seem to develop normally without TN-C.8'14'19'20 Tenascin-C is adhesive for some cell types and antiadhesive for many others. In tissue culture, it can counteract cell adhesion to fibronectin.8 ' 9 Tenascin-C expression can be induced or upregulated by 1135

1136

Ljubimov et al.

Knob

IOVS, June 1998, Vol. 39, No. 7

EGF-like repeats

Fibronectin type Ill-like repeats 4

H,N

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

5

A1 A2 A3 A4

B AD2 AD1 C

Fbg-like

D

6

IW» 11 I

7 I

8

O

COOH

IFT5T1

cm

[=3

CZ1CZ3 •—IIBgl-

FIGURE 1. Diagram of human tenascin-C monomer with 22 known splice variants depicted. Insertional FN-III repeats are shaded. Variants in bold have been described in humans for the first time. EGF, epidermal growth factor; Fbg, fibrinogen. Reprinted by permission from Lippincott-Raven Publishers, with modification, from Saghizadeh M, Khin HL, Bourdon MA, Kenney MC, Ljubimov AV. Novel splice variants of human tenascin-C mRNA identified in normal and bullous keratopathy corneas. Cornea 1998. In press.

transforming growth factor-/3l, basic fibroblast growth factor-2, EGF, and platelet-derived growth factor-BB.9'21"23 In normal corneas, TN-C is found in the limbal basement membrane and stroma but is absent from adult central corneas.2'24 Corneas with PBK-ABK have prominent TN-C deposits in the stroma, posterior collagenous layer, and subepithelial fibrosis regions.2'25 We have recently described novel TN-C mRNA splice variants present in normal and PBK-ABK human corneas.26 In the present study, the expression of specific TN-C splice variants in PBK-ABK corneas was examined at the mRNA and protein levels. METHODS

Tissue Age-matched normal (n = 18; age range, 44-85 years; mean age, 693 ± 10.2 years) and PBK-ABK (n = 40; age range, 43-87 years; mean age, 76.8 ± 6.7 years) corneas were used. Normal autopsy corneas were obtained from the National Disease Research Interchange (Philadelphia, PA), and diseased corneas removed during penetrating keratoplasty were provided, preserved in chilled Optisol (Chiron Intraoptics, Irvine, CA), by collaborating surgeons. All tissues were embedded in OCT compound (Ted Pella, Redding, CA)2 within 24 hours after death or corneal transplantation. Blocks and sections were stored at —80°C. In diseased corneas, only a central part (where Bowman's layer lies beneath the epithelium) removed

during transplantation was available. Therefore, normal corneas were trephined and only central parts analyzed, to ensure adequate comparison with PBK-ABK corneas. Previously, we noted individual variations among PBK-ABK corneas in the expression of TN-C.2 Therefore, corneas were analyzed individually, and were not pooled. Because of this and because of the small amount of tissue available from a trephined cornea, a different set of normal and PBK-ABK corneas was analyzed for each of the three methods: semiquantitative RT-PCR, immunofluorescence, and western blot analysis. Semiquantitative Reverse Transcription—Polymerase Chain Reaction Total RNA was isolated from whole corneas (11 age-matched normal and 20 PBK-ABK) with Trizol reagent (Life Technologies, Gaithersburg, MD), according to the manufacturer's instructions. In some experiments, RNA was isolated from mechanically separated epithelium, stroma, and endothelium. cDNA was synthesized from 2 jag total RNA in 80 /xl reaction buffer comprised of 500 /LLM deoxyribonucleoside triphosphates (dNTP), 2.5 p,M random hexamer primers, 20 U RNase inhibitor, and 200 U reverse transcriptase (Superscript II; Life Technologies). The reaction was carried out for 10 minutes at 25°C, 30 minutes at 42°C, and 5 minutes at 95°C followed by cooling to 4°C. cDNA samples were subjected to PCR using specific primers for different FN-III repeats of TN-C and for /32-microglobulin (j32-MG) that served as an internal standard for sample normalization. Primers were designed using a soft-

IOVS, June 1998, Vol. 39, No. 7 ware program (Primer3 Internet software; The Whitehead Institute, Boston, MA) and their specificity was confirmed by a software-assisted search of a nonredundant nucleotide sequence database (BLAST27 Internet software; National Library of Medicine, Bethesda, MD). The primers for RT-PCR were the following: TN-C FN-HI repeat 4 forward, 5'-CTATTGA