Molecular Vision 1999; 5:3 <http://www.molvis.org/molvis/v5/p3/> Received 4 September 1998 | Accepted 19 March 1999 | Published 8 April 1999

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Identification of a novel gene product preferentially expressed in rat lens epithelial cells

Huicong Cai,¹ Richard D. Howells,¹ B. J. Wagner^1,2
 
 

Departments of ¹Biochemistry and Molecular Biology, and ²Ophthalmology, UMDNJ-New Jersey Medical School and UMDNJ-Graduate School of Biomedical Sciences, Newark, NJ, 07103

Correspondence to: B. J. Wagner, Department of Biochemistry and Molecular Biology, UMDNJ-NJ Medical School, 185 South Orange Avenue, Newark, NJ, 07103; Phone: (973) 972-4486; FAX: (973) 972-5594; email: wagner@umdnj.edu
 
Dr. Cai is now at Bausch and Lomb Pharmaceuticals, 401 North Middletown Road, Pearl River, NY, 10965; email: hui_cai@bausch.com

Abstract

Purpose: We searched for an mRNA that is differentially expressed in rat lens epithelial and fiber cells to use as a marker for differentiation.

Methods: An mRNA differential display method was used to identify genes with altered expression during differentiation of lens epithelial cells.

Results: One gene (partial sequence) was identified and confirmed to be expressed preferentially in undifferentiated rat lens epithelial cells. Among non-lens tissues, a trace amount was detected in cornea, but not in retina or eleven non-ocular tissues tested. This cloned partial gene sequence (322 base pairs) is at least 93% identical to overlapping rat and mouse EST sequences, and extends the sequence of both ESTs at the 5' end.

Conclusions: A clone has been isolated that is preferentially expressed in lens epithelial cells. Expression of this gene was down-regulated when rat pup lens epithelial cell explants were induced to differentiate. The unusual pattern of expression suggests a novel function for this gene.

Introduction

The mammalian ocular lens is composed of two cell types: epithelial cells, which form a monolayer on the anterior surface, and differentiated fiber cells, which make up the bulk of the lens. During differentiation to fiber cells, epithelial cells exit from the cell cycle and undergo dramatic morphological changes (cell elongation, loss of subcellular organelles) [1,2]. Along with these morphological changes, changes in expression of different genes are expected. Identification of these genes and their functions may provide insight into the process of differentiation and/or the specific functions of the epithelial and fiber cells. For example, ß- and [gamma]-crystallin are expressed as the cells elongate and differentiate [3], as are other genes such as c-myc [4], c-fos, c-jun [5] and HSP70 [6]. In bovine lens, changes in phospholipid and sphingomyelin content also appear to be related to differentiation [7]. In this study, we used an mRNA differential display method [8] to identify a novel gene product preferentially expressed in undifferentiated lens epithelial cells. Induction of differentiation in lens epithelial explants resulted in down regulation of this transcript. Among thirteen other tissues tested, only the cornea expressed trace amounts.

Methods

Induction of differentiation in cultured explants

Animal care procedures published by the US Public Health Service in "Public Health Service Policy on Humane Care and Use of Laboratory Animals", were followed. Three-day old Sprague Dawley rat pup (Taconic Farms, Germantown, NY) lenses were dissected and the epithelial cells cultured as explants as previously described [9,10]. The capsules with adherent epithelial cells were cultured with medium 199 and bovine serum albumin (BSA, 1 mg/ml, Gibco BRL, Gaithersburg, MD) without antibiotics at 37 °C, 5% CO₂, 80% relative humidity. To induce differentiation, basic FGF (bFGF, 40 ng/ml, Sigma Chemical Co., St. Louis, MO) was added to the culture medium containing lens epithelial explants after 3 h of initial culture. Explants were incubated at 37 °C for 10 days.

Differential display RT-PCR

Total RNA from rat tissues was isolated using RNAzol^TM (Tel-Test, Inc., Friendswood, TX), as described previously [11]. mRNA differential display RT-PCR was performed using the RNAmp^TM Kit (GenHunter Corporation, Brookline, MA), which provides arbitrary primers and a degenerate anchored 3'-primer. The reaction was performed according to the protocol from the manufacturer. Total RNA (0.2 µg) from cultured (with or without bFGF) rat lens epithelial cell samples was used to generate first strand cDNA using 5'-TTTTTTTTTTTTMC-3' (T₁₂MC, 20 nM) as an anchored 3'-primer in 20 µl final volume containing 4 µl of 5X reverse-transcription buffer (125 mM Tris-Cl, pH 8.3, 188 mM KCl, 7.5 mM MgCl₂ and 25 mM DTT) and 1.6 µl dNTP (250 µM). The mixture was incubated in a DNA Thermal Cycler (Perkin-Elmer Cetus) at 65 °C for 5 min, then 37 °C for 10 min. Then 1 µl MMLV reverse transcriptase was added to each tube and the tubes were incubated at 37 °C for another 50 min. The reverse transcription reaction was stopped by heating at 95 °C for 5 min. Then 2 µl of RT-mix was transferred into a fresh PCR tube and 10X PCR buffer (2 µl), dNTP (25 µM, 1.6 µl), 0.2 µl AmpliTaq (Perkin-Elmer) and 1 µl of [alpha]-³⁵S-dATP (1200 Ci/mM) was added. Further PCR was performed on this product using arbitrary decamer primers AP-1 (200 µM) and T₁₂MC (1 µM) for the 5'- and 3'-ends, respectively. The PCR conditions were denaturing at 94 °C for 1 min; annealing at 45 °C for 1 min; extension at 72 °C for 1 min, for 40 cycles. The PCR products were resolved by urea polyacrylamide gel electrophoresis. Samples treated with or without bFGF were loaded side by side to compare band migration differences. The gel was dried under vacuum and then exposed to X-ray film (Type XAR; Eastman Kodak Co., Rochester, NY) for 14 h.

Analysis of differentially displayed bands

The bands of interest were located on the gel and cut out with a razor blade. The gel slice was soaked in 100 µl distilled water for 10 min and boiled for 15 min. The sample was spun by microcentrifugation and eluted DNA was pelleted by sodium acetate/glycogen/ethanol precipitation. The pellet was dissolved in 10 µl of distilled water and 4 µl was taken for re-amplification. Re-amplification was done using the same primer set and PCR conditions. PCR products were then cloned into the TA-vector using a TA Cloning^TM Kit (Invitrogen, Carlsbad, CA). The plasmid DNA with insert was purified using a Wizard^TM Miniprep (Promega, Madison, WI). The nucleotide sequences were determined by the dideoxy chain termination method [12] in both the forward and reverse directions, and the GenBank and EST databases were searched with the sequence in both directions.

Standard northern blot analysis was performed [13] with total RNA (10 or 15 µg) isolated from 3-day old rat lens epithelial cells, lens fibers, retina, lung, heart and muscle, and probed with ³²P-dCTP labeled cDNAs from differentially displayed clones. Other rat tissues (cornea, brain, intestine, kidney, liver, muscle, ovary, spleen and testis) were analyzed for expression of clone 2 by RT-PCR. The methods for isolating total RNA and carrying out RT-PCR on 1 µg total RNA have been described elsewhere [10,11]. PCR primers (0.15 µM) for clone 2 were 5'-cactttcctcgttgcggtatttgt in the forward direction and 5'-ttttcacgttaataccgaggtcac in the reverse direction. ß-actin primers were from Stratagene (La Jolla, CA, Cat. No. 302010). Annealing temperature was 55 °C and 30 cycles of PCR were carried out. The RT-PCR products were separated by 4-20% gradient gel electrophoresis (Novex, San Diego, CA), and visualized by staining with ethidium bromide.

Results & Discussion

Most cellular and developmental processes are characterized by changes in gene expression. mRNA differential display analysis allows the identification of altered mRNA expression in various tissues, and can lead to the identification of new genes and functions [14,15]. Our mRNA differential display analysis of undifferentiated and differentiated lens epithelial cells revealed that several mRNA species were present only in the bFGF minus group (Figure 1), suggesting that they are synthesized only in undifferentiated cells. After five days of culture with bFGF, ßB1-crystallin mRNA expression was up-regulated in rat lens explants (data shown in [10]), indicating that bFGF had induced differentiation, consistent with other reports using the same experimental system [16].

Clones labeled 1, 2, and 3 were isolated and analyzed. Northern blot analysis was used to confirm that the transcript from which clone 2 originated was expressed preferentially in epithelial cells (Figure 2). Clones 1 and 3 were false positives, expressed in both epithelial and fiber cells (Figure 2 and data not shown). Clone 2 RNA was found to be approximately 2000 nucleotides in length by comparison with RNA standards (data not shown).

Differentiation-induced cultured lens epithelial explants were compared with non-induced cells for the expression of clone 2 mRNA by northern blot analysis. While ß-actin mRNA remained the same during differentiation, the expression of clone 2 mRNA was down-regulated (Figure 3). It remains to be determined whether the down-regulation induced by bFGF is at the transcriptional, or post-transcriptional, level.

The nucleotide sequence of clone 2 is shown in Figure 4 (AF070940). Since the cDNA sequence of 322 bp is considerably shorter than the length of the 2000 nucleotide transcript detected by northern blot analysis, it is clear that the cDNA clone represents only 15% of the full-length sequence. Sequence analysis indicated that neither the forward nor the reverse strand of the sequence shown in Figure 4 contained a complete open reading frame (data not shown). Frame +1 of the clone 2 sequence, as shown in Figure 4, begins with the longest possible open reading frame. The open reading frame would encode a polypeptide fragment of 65 amino acids, and is followed by the stop codon, TAA, located at nucleotides 152-154. Additional studies are required to confirm whether that is the actual reading frame of clone 2 mRNA. A search of the GenBank database (release 105.0) with both the forward and reverse sequences of clone 2 revealed no substantial homology (less than 9%) with any other DNA sequence. A search of the human, mouse and other organism EST databases resulted in identification of two sequences with significant similarity with clone 2. One is derived from rat eye (minus lens) RNA (AI072904) and one from whole mouse embryo RNA (AA198287). The rat and mouse EST sequences are aligned with the clone 2 sequence in Figure 4. In certain instances, gaps were introduced into sequences in order to maximize the nucleotide alignments. These gaps may be due to sequence mismatches or, more likely, sequencing artifacts. Further sequencing of cDNA clones will be necessary to sort out possible sequence artifacts (insertions or deletions) or mismatches that give rise to the gaps. Clone 2 and the rat EST sequences differ in only 1/107 nucleotides (99% identical assuming sequence artifacts, 93% identical assuming mismatched nucleotides). The mouse EST sequence differs from clone 2 in just 3/230 nucleotides (99% identical assuming sequence artifacts, 93% identical assuming mismatched nucleotides). The sequence analysis strongly suggests that the three cDNAs are products of the same gene. It also appears that, as expected, clone 2 is derived from a polyA-containing mRNA. If the sequences of clone 2 and the rat EST are joined into a contiguous sequence, the result is a 574 nucleotide cDNA sequence, containing a polyadenylation signal 15 nucleotides upstream of the polyA tail.

Tissue distribution studies using northern blot analysis revealed that clone 2 mRNA is not expressed in retina, lung, heart or muscle (Figure 5). A trace amount is expressed in lens fibers. This may arise from the outer layer of fibers, which are not fully differentiated. The fact that the expression level of this gene is down regulated during differentiation (Figure 3) suggests that this gene is predominantly expressed in lens epithelial cells. In addition to lens cells, RT-PCR analysis (Figure 6) showed a trace amount of clone 2 mRNA in the cornea. This may explain the appearance of an homologous sequence in the EST library, AI072904. No positive bands were found in brain, intestine, kidney, liver, muscle, ovary, spleen or testis. This tissue expression survey, together with the absence of significant homologous cDNA sequences from specific tissues in EST databases, indicates that this gene is preferentially, if not specifically, expressed in undifferentiated lens epithelial cells. Its expression pattern suggests that it has a role in lens development, and the identification of its 5' regulatory element would provide a lens specific promoter which could be used for transgenic mouse studies. Experiments to clone the full-length cDNA and 5' regulatory region will reveal information about the coding region and regulatory mechanisms of gene expression.

Acknowledgements

This work was supported in part by NIH grant EY02299.

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