June 2000
Volume 41, Issue 7
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Overexpression of the Transforming Growth Factor-β–Inducible Gene βig-h3 in Anterior Polar Cataracts
Author Affiliations
  • Eunjoo H. Lee
    From the Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea; and the
  • Young Seomun
    From the Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea; and the
  • Kyung–Hoon Hwang
    From the Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea; and the
  • Jung–Eun Kim
    Department of Biochemistry, School of Medicine, Kyungpook National University, Korea.
  • In–San Kim
    Department of Biochemistry, School of Medicine, Kyungpook National University, Korea.
  • Jae Ho Kim
    From the Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea; and the
  • Choun–Ki Joo
    From the Department of Ophthalmology and Visual Science, College of Medicine, The Catholic University of Korea; and the
Investigative Ophthalmology & Visual Science June 2000, Vol.41, 1840-1845. doi:https://doi.org/
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      Eunjoo H. Lee, Young Seomun, Kyung–Hoon Hwang, Jung–Eun Kim, In–San Kim, Jae Ho Kim, Choun–Ki Joo; Overexpression of the Transforming Growth Factor-β–Inducible Gene βig-h3 in Anterior Polar Cataracts. Invest. Ophthalmol. Vis. Sci. 2000;41(7):1840-1845. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. In anterior polar cataracts and the fibrosis that can occur after cataract surgery, lens epithelial cells synthesize abundant extracellular matrix molecules and transdifferentiate into myofibroblast-like cells. Transforming growth factor (TGF)-β has been implicated as a key player in these cataractous changes. The purpose of this study was to determine whether the TGF-β–inducible gene h3 (βig-h3) is expressed in lens epithelial cells from patients with anterior polar cataracts and to test whether βig-h3 is induced by TGF-β in cultured lens epithelial cells.

methods. Lens epithelial cells attached to the anterior capsules of human cataractous lenses and noncataractous lenses were examined for the expression of βig-h3 mRNA and protein using reverse transcription–polymerase chain reaction and immunohistochemical analyses. The effect of TGF-β on βig-h3 gene expression was also tested in human lens epithelial B-3 cells using Northern and Western blot analyses.

results. βig-h3 mRNA was not detected in lens epithelial cells from patients with clear lenses or patients with nuclear cataracts. Significant expression of mRNA for βig-h3 was observed in lens epithelial cells from patients with anterior polar cataracts. Immunohistochemical analysis using anti–βig-h3 antiserum indicated that βig-h3 protein was present within the subcapsular plaques of anterior polar cataracts. Treatment of human lens epithelial B-3 cells with TGF-β1 led to an increase in βig-h3 mRNA and the secretion of βig-h3 protein into the culture medium.

conclusions. βig-h3 may serve as a marker for anterior polar cataracts in addition to previously known proteins, fibronectin, type I collagen, andα -smooth muscle actin. The functions of this protein in lens pathology need to be further investigated.

Transforming growth factor (TGF)-β plays a key role in wound healing processes by promoting the production and deposition of the extracellular matrix, which is essential to normal tissue repair after injury. However, sustained expression of TGF-β causes tissue fibrosis in a variety of pathologic conditions. 1 The role of TGF-β in lens pathology has been documented in association with anterior subcapsular cataracts and anterior polar cataracts. Rat lenses cultured with TGF-β form distinct anterior opacities. 2 In these cataracts, lens epithelial cells transdifferentiate into myofibroblast-like cells and form fibrotic plaques consisting of abnormal extracellular matrix components, such as type I collagen. 2 3 The posterior capsular opacification that often occurs after cataract surgery (after-cataract) can also be caused by a similar transdifferentiation of the lens epithelial cells remaining after surgery and the accompanying increase of extracellular matrix deposits. 4 5  
TGF-β–inducible gene h3 (βig-h3) was initially identified as a novel gene that was induced by TGF-β in a human lung adenocarcinoma cell line. 6 It was later shown that TGF-β induces the expression of βig-h3 mRNA and secretion of a 68-kDa βig-h3 protein in several other cell types. 6 7 8 βig-h3 transcripts were detected in several human tissues, suggesting that this protein may have an important function throughout the body. In the normal human eye, βig-h3 is expressed almost exclusively in the cornea. 9 Recently, mutations in this gene were found in 5q31-linked autosomal dominant corneal dystrophies. 10 β ig-h3 protein is thought to be a major constituent of the abnormal extracellular deposits found in these hereditary corneal diseases. 11 12 Although the biological function ofβ ig-h3 remains to be clarified, this protein has been shown to be associated with matrix molecules. 7 8 9 13  
We have previously shown that treatment of lens epithelial cells with TGF-β increases the synthesis of pathologic extracellular matrix proteins characteristic of fibroblasts. 14 Because βig-h3 is a TGF-β–inducible protein that plays a role in the development of pathologic extracellular deposits, we hypothesized that this protein might be implicated in the aberrant accumulation of extracellular matrix seen in anterior polar and secondary cataracts. To test this, we examined whether this protein and its mRNA were expressed in lens epithelial cells from patients with anterior polar cataracts. We also provide evidence that the expression of βig-h3 is increased by TGF-β in cultured lens epithelial cells. 
Methods
Human Specimens
This study was conducted according to the tenets of the Declaration of Helsinki. Lens capsules with adherent epithelial cells were obtained during cataract surgery from patients with the clinical diagnosis of nuclear and anterior polar cataracts. Lens capsules of noncataractous lenses were obtained during clear lens extraction for the correction of high myopia. The capsules were immediately placed in TRIzol reagent (GIBCO, Gaithersburg, MD) for RNA preparation or fixed in neutral-buffered formalin for immunohistochemical analysis. Corneal epithelial cells were obtained from patients undergoing excimer laser photorefractive keratectomy and immediately placed in TRIzol for RNA isolation or stored at −70°C for protein extraction. 
Cell Culture and Treatment
Human lens epithelial cell line HLE B-3 was kindly provided by Usha Andley, PhD, and maintained as described previously. 15 The cultures were treated with various cytokines at 10 ng/ml in serum-free medium. At the indicated time points, total cellular RNA and culture supernatants were collected for Northern and Western blot analyses, respectively. Human recombinant interleukin (IL)-1α was purchased from Pepro Tech (Rocky Hill, NJ); human recombinant interferon (IFN)-γ and tumor necrosis factor (TNF)-α were obtained from Upstate Biotechnology (Lake Placid, NY); and human recombinant TGF-β1 and fibroblast growth factor (FGF)-2 were from Sigma (St. Louis, MO). For the inhibition of new protein synthesis, cycloheximide (10 μg/ml; Sigma) was added to the cultures 1 hour before treatment with TGF-β1. 
Reverse Transcription–Polymerase Chain Reaction
Total cellular RNA was isolated and 1 μg was reverse–transcribed using the First Strand cDNA Synthesis Kit (Boehringer Mannheim, Indianapolis, IN). The resultant cDNA (0.2–1μ l) was amplified using gene-specific primers. DNA size markers were run in parallel to validate the predicted sizes of the amplified bands (D-15 DNA marker; Novex, San Diego, CA). The primer sequences specific for the genes examined and predicted product sizes are as follows: 5′-AGGCCAACCGCGAAGATTGACC-3′ (sense), 5′-GAAGTCCAGGGCGACGTAGCAC-3′ (antisense), 350 bp for β-actin, 16 and 5′-CCATCACCAACAACATCCAG-3′ (sense), 5′-GAGTTTCCAGGGTCTGTCCA-3′ (antisense), 660 bp for βig-h3. 
Immunohistochemical Analysis
Anterior lens capsules with attached lens epithelial cells were fixed in 10% neutral-buffered formalin and embedded in paraffin. The paraffin-embedded samples were sectioned on a microtome at a thickness of 5 μm, deparaffinized in xylene, and rehydrated in alcohol. The sections were incubated in 2% H2O2 for 5 minutes, 20% nonimmune horse serum (Biomeda, Foster City, CA) for 10 minutes, and 1:200 dilution of rabbit anti–βig-h3 antiserum or normal rabbit serum for 2 hours at room temperature. The sections were then incubated in biotinylated anti-rabbit IgG (Amersham, Cleveland, OH) for 10 minutes, and then visualized according to the manufacturer’s protocol using a detection kit (UltraTek HRP; ScyTek Laboratories, Logan, UT). The immunolabeled sections were counter-stained with 10% Mayer’s hematoxylin and examined under light microscope. 
Northern Blot Analysis
Samples of RNA (10 μg of total RNA per lane) were separated on 1% agarose formaldehyde gels 17 and transferred by downward alkaline transfer (Turboblotter; Schleicher & Schuell, Keene, NH) to nitrocellulose membranes (Optitran BA-S; Schleicher & Schuell). A βig-h3 cDNA probe and a probe for the housekeeping gene β-actin were labeled with random priming (Random primed DNA labeling kit; Boehringer Mannheim) and [32P]-dCTP (3000 Ci/mmol; Amersham). Blots were prehybridized and hybridized in 50% formamide, 5× SSPE, 10× Denhardt’s solution, and 0.5% sodium dodecyl sulfate (SDS) at 42°C. Blots were then washed and exposed to autoradiographic film. Equal loading was assessed by hybridization with a β-actin probe. 
Western Blot Analysis
Culture supernatants of B-3 cells were centrifuged to remove cell debris and were concentrated using a concentrator (Centricon-10; Amicon, Beverly, MA). Aliquots of 20 μl were subjected to SDS–polyacrylamide gel electrophoresis (PAGE) and electroblotting to nitrocellulose membrane. The membranes were blocked, incubated with rabbit anti–βig-h3 antiserum (1:1000 dilution), and then reacted with horseradish peroxidase–conjugated anti-rabbit antibody (Amersham; 1:1000 dilution). The blots were developed using chromogenic substrate solution (DAB substrate; Boehringer Mannheim). Prestained molecular weight standards (SeeBlue) were purchased from Novex. 
Preparation of Polyclonal βig-h3 Antiserum
A NdeI/BglII fragment corresponding to amino acids 175 to 653 of βig-h3 was inserted into pET-29b vector (Novagen; Madison, WI) and expressed in Escherichia coli. The His-tagged recombinant βig-h3 protein was purified using Ni-NTA resin (Qiagen; Valencia, CA) according to the manufacturer’s manual. All animal experiments were carried out in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Antibodies were then raised in rabbits by subcutaneous injection of the protein (200 μg) in Complete Freund’s adjuvant. Immunizations were repeated four times in Incomplete Freund’s adjuvant, every 3 weeks. Titer of the antibody was monitored by immunoblot analysis using recombinant βig-h3 protein and culture supernatant of COS cells overexpressing βig-h3. 
Overexpression of βig-h3 in COS Cells
The cDNA encoding amino acids 1 to 653 of βig-h3 was subcloned into pcDNA 3.1/Myc-His A (Invitrogen; Carlsbad, CA). For transient expression, the plasmid was transfected into COS cells using Lipofectamine (GIBCO) according to the manufacturer’s protocol. After cells were incubated overnight in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, the medium was replaced with serum-free DMEM. The cells were then incubated for 48 hours before supernatant collection. The supernatants were collected and analyzed by immunoblot analysis using βig-h3 antiserum and anti-Myc antibody. 
Results
Enhanced Expression of βig-h3 in Lens Epithelial Cells Obtained from Patients with Anterior Polar Cataracts
Because TGF-β serves as a potent regulator of transdifferentiation and fibrosis of lens epithelial cells, and becauseβ ig-h3 is inducible by TGF-β in other cell types, we examined whether βig-h3 is expressed in lens epithelial cells obtained from patients with anterior polar cataracts. First, we compared the levels of βig-h3 mRNA in lens epithelial cells from anterior polar cataracts with epithelial cells from nuclear cataracts and noncataractous lenses. As shown in Figure 1A , the expression of βig-h3 mRNA was readily detectable in lens epithelial cells of anterior polar cataracts but not detectable in lens epithelial cells from noncataractous lenses or in lens epithelial cells from lenses with nuclear cataracts. Corneal epithelial cells are known to express a high level of βig-h3 transcripts. 9 13 Therefore, RNA isolated from corneal epithelial cells obtained from the eyes of patients who underwent excimer laser photorefractive keratectomy was included as a positive control. The amount of β-actin product, an internal control for polymerase chain reaction (PCR) amplification, was similar among the samples. Sequence analysis of the amplified product confirmed that the nucleotide sequence of βig-h3 cDNA from lens epithelial cells was identical with that initially reported by Skonier et al. 6  
Next, we assayed for βig-h3 protein using immunohistochemical analysis. As shown in Figure 1B , these results correlated with the reverse transcription (RT)–PCR data, demonstrating abundant expression of βig-h3 protein within the subcapsular plaques of anterior polar cataracts. The protein appeared to be strongly expressed well below the capsule. In comparison, βig-h3 protein was not detected in lens epithelial cells obtained from nuclear cataracts. Sections incubated with normal rabbit serum in place of primary antiserum showed no reactivity (data not shown). 
Increased mRNA Expression for βig-h3 after TGF-β Treatment in Human Lens Epithelial Cells
We then determined whether the expression of mRNA for βig-h3 could be stimulated in cultured human lens epithelial cells by treatment with TGF-β. Northern blot analysis results showed that HLE B-3 cells produced βig-h3 under normal culture conditions and that treatment with TGF-β1 resulted in a time-dependent increase in a single βig-h3 transcript of approximately 3.4 kb (Fig. 2A ). Pretreatment of HLE B-3 cells with cycloheximide did not influence the stimulation of βig-h3 mRNA expression by TGF-β1 (Fig. 2B) , suggesting that the upregulation of βig-h3 mRNA expression by TGF-β1 does not require the de novo synthesis of transcription factors. 
Increased Secretion of βig-h3 Protein into the Culture Medium of HLE Cells after Treatment with TGF-β
Western blot analysis confirmed that treatment with TGF-β1 led to an increased accumulation of secreted proteins immunoreactive toβ ig-h3 antiserum in culture supernatants of HLE B-3 cells in a time- and dose-dependent manner (Figs. 3A few bands were detected around 68 kDa in culture supernatant of HLE B-3 cells and in corneal epithelial cells. The lower-molecular-weight bands may represent degradation products, possibly due to processing of the carboxyl-terminal amino acids as suggested by Skonier et al. 7 These bands may also represent posttranslationally modified forms of βig-h3 protein as pointed out by Escribano et al. 9 The antiserum appeared to be specific for βig-h3 because a single band with expected molecular weight around 69 kDa was detected in the culture supernatant of COS cells transfected with Myc-tagged βig-h3 expression plasmid (Fig. 3A) . The band coincided with a band detected by anti-Myc antibody. COS cells transfected with a plasmid that did not contain the βig-h3 cDNA showed no bands reactive to the βig-h3 antiserum (data not shown). In our observation, treatment of HLE B-3 cells with TGF-β1 for up to 72 hours caused few changes in cell morphology. Finally, we assessed whether the expression of βig-h3 can be stimulated by other cytokines in lens epithelial cells. Treatment with IL-1α, TNF-α, FGF-2, or IFN-γ did not increase the secretion of βig-h3 protein, suggesting that the stimulation of βig-h3 expression is specific for TGF-β (Fig. 3C)
Discussion
Expression of βig-h3 is increased by TGF-β in lung adenocarcinoma cells and some other cell types. 6 7 8 Most reports concerning the βig-h3 gene and protein to date have documented various aspects of the function of this protein in the cornea. Mutations of βig-h3 gene were identified in 5q31-linked autosomal dominant corneal dystrophies. 10 In addition to its role in the etiology of granular corneal dystrophies, βig-h3 is overexpressed in Fuchs’ corneal dystrophy and in the fibrous material of restenotic arterial plaques. 18 19 20 This suggests that the overexpression of wild-type βig-h3 protein may also be implicated in certain pathologic conditions. 
We have previously shown that proteins that are not normally produced by lens epithelial cells (including fibronectin, type I collagen, andα -smooth muscle actin) are prominently expressed in lens epithelial cells from patients with anterior polar cataracts. TGF-β mRNA was also overexpressed in lens epithelial cells of anterior polar cataracts. 14 We demonstrated in this study that significant expression of βig-h3 was detected within the fibrotic plaques formed in anterior polar cataracts. This result suggests thatβ ig-h3 protein provides an additional pathologic marker for anterior polar cataracts. We also observed that the expression for βig-h3 was augmented by TGF-β in lens epithelial cells in vitro. Based on previous results obtained by Skonier et al., 6 βig-h3 is not induced in all cell types tested. Our results add lens cells to the group of cells showing an increase in βig-h3 in response to TGF-β and suggest that βig-h3 plays a role in the cellular responses evoked by TGF-β in lens epithelial cells. 
According to Escribano et al., 9 βig-h3 transcripts are found in various tissues but not in normal human lens. In agreement with the report, βig-h3 message was not detected in cells of noncataractous lens specimens by RT–PCR analysis (Fig. 1A) . However, we observed constitutive βig-h3 expression in untreated HLE B-3 cells (Figs. 2 and 3) . LeBaron et al. found that βig-h3 was normally expressed at low levels in all cell lines they examined. 8 Similarly, ciliary epithelial cell lines but not normal ciliary epithelium showed prominent expression of βig-h3 mRNA. 9 Thus, the background level of βig-h3 transcripts in HLE B-3 cells could be attributed to the fact that B-3 cells are immortalized, or as a consequence of in vitro culture. This raises a general point that caution should be exercised when drawing conclusions about normal lens biology from work on cell lines. 
The biochemical functions of the βig-h3 gene product have not been precisely identified yet. βig-h3 protein is prominently expressed in the cornea, skin, and extracellular matrix of many connective tissues. 6 8 20 21 This protein contains a RGD (Arg-Gly-Asp) motif near the carboxyl-terminus, as found in various extracellular matrix molecules that interact with cell-surface integrins. 6 βig-h3 has also been proposed to have adhesive functions. 7 8 In the lens, βig-h3 protein may be important in mediating interaction between abnormal lens epithelial cells and the altered extracellular matrix. Further studies are required to elucidate the significance of the presence of βig-h3 protein in fibrotic plaques of anterior polar cataracts. 
The observation that mRNA level for βig-h3 increases in healing corneal tissues 11 22 suggests that βig-h3 protein might play a role in the formation of extracellular matrix during the healing process. Tissue fibrosis is caused by excessive extracellular matrix deposition due to unregulated wound healing. 1 Pathologic mechanisms underlying anterior polar cataracts and fibrosis type of after-cataract have been recognized to share many common points with those proposed for other fibrotic diseases in which TGF-β is implicated as a key cytokine. 23 In this regard, our data may give valuable insights into studies for the potential role ofβ ig-h3 protein in the fibrosis that often develops in other major organs, such as the liver, kidney, and lung. 
Further characterization of the role of βig-h3 in the lens may provide a better understanding of the mechanisms of TGF-β action with respect to cataract formation. Furthermore, it is expected that studies aimed at finding antagonists to the pathologic effects of TGF-β 24 could be used to prevent after-cataract. 
 
Figure 1.
 
Enhanced expression of βig-h3 in human anterior polar cataracts. (A) Total cellular RNA was isolated from lens epithelial cells of patients with noncataractous high myopia (lane 1), nuclear cataracts (lane 2), and anterior polar cataracts (lane 3). RNA was also isolated from corneal epithelial cells (lane 4) of patients who underwent excimer laser photorefractive keratectomy. mRNA levels for β-actin and βig-h3 were examined by RT–PCR. The data shown are from one of three independent assays that produced similar results. (B) Anterior lens capsules with adhering cells from patients with anterior polar or nuclear cataracts were examined by immunohistochemical staining for βig-h3 (magnification, ×400). The capsule in the anterior polar cataractous lens is not as thick as the capsule in the control lens. This appears to be another feature of anterior polar cataracts. The data presented are from one of five independent assays that produced similar results. M, molecular size standards (in base pairs); LC, lens capsule; MF, myofibroblast-like cells (open arrow); SP, subcapsular plaque; LE, epithelial cells (closed arrow).
Figure 1.
 
Enhanced expression of βig-h3 in human anterior polar cataracts. (A) Total cellular RNA was isolated from lens epithelial cells of patients with noncataractous high myopia (lane 1), nuclear cataracts (lane 2), and anterior polar cataracts (lane 3). RNA was also isolated from corneal epithelial cells (lane 4) of patients who underwent excimer laser photorefractive keratectomy. mRNA levels for β-actin and βig-h3 were examined by RT–PCR. The data shown are from one of three independent assays that produced similar results. (B) Anterior lens capsules with adhering cells from patients with anterior polar or nuclear cataracts were examined by immunohistochemical staining for βig-h3 (magnification, ×400). The capsule in the anterior polar cataractous lens is not as thick as the capsule in the control lens. This appears to be another feature of anterior polar cataracts. The data presented are from one of five independent assays that produced similar results. M, molecular size standards (in base pairs); LC, lens capsule; MF, myofibroblast-like cells (open arrow); SP, subcapsular plaque; LE, epithelial cells (closed arrow).
Figure 2.
 
Stimulation of βig-h3 mRNA levels by TGF-β1 in cultured lens epithelial cells. (A) HLE B-3 cells were seeded in 60-mm culture dishes coated with type IV collagen (10 μg/ml) at 1.0 × 106 cells/dish. After incubation for 18 hours in serum-free MEM, the cultures were treated with TGF-β1 (10 ng/ml) in fresh medium. After the indicated times, total cellular RNA was isolated and subjected to Northern blot analysis. Three separate experiments yielded similar results. (B) After incubation in serum-free MEM, cycloheximide (CHX, 10 μg/ml) was added to the cultures, followed by treatment with TGF-β1 (10 ng/ml) for 12 hours. Similar results were obtained from two independent experiments. HLE B-3 cells were maintained healthy for at least 3 to 4 days in serum-free medium when cultured on the plastic dishes coated with type IV collagen that is a major constituent of the lens capsule.
Figure 2.
 
Stimulation of βig-h3 mRNA levels by TGF-β1 in cultured lens epithelial cells. (A) HLE B-3 cells were seeded in 60-mm culture dishes coated with type IV collagen (10 μg/ml) at 1.0 × 106 cells/dish. After incubation for 18 hours in serum-free MEM, the cultures were treated with TGF-β1 (10 ng/ml) in fresh medium. After the indicated times, total cellular RNA was isolated and subjected to Northern blot analysis. Three separate experiments yielded similar results. (B) After incubation in serum-free MEM, cycloheximide (CHX, 10 μg/ml) was added to the cultures, followed by treatment with TGF-β1 (10 ng/ml) for 12 hours. Similar results were obtained from two independent experiments. HLE B-3 cells were maintained healthy for at least 3 to 4 days in serum-free medium when cultured on the plastic dishes coated with type IV collagen that is a major constituent of the lens capsule.
Figure 3.
 
Stimulation of βig-h3 protein secretion by TGF-β1 in lens epithelial cells. (A) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well and incubated for 18 hours in serum-free MEM. The culture medium of HLE B-3 cells subsequently incubated with TGF-β (10 ng/ml) for 0, 12, 24, 48, and 72 hours (lanes 1 through 5), corneal epithelial cell lysate (lane 6), and culture supernatant of COS cells overexpressing βig-h3 (lane 7) was loaded on SDS–10% polyacrylamide gel. Immunoreactive proteins were assessed by Western blot analysis using polyclonal βig-h3 antiserum. Three separate experiments yielded similar results. (B) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well. After incubation for 18 hours in serum-free MEM, the cultures were treated with various concentrations (0, 2.5, 5, 10, and 20 ng/ml; lanes 1 through 5) of TGF-β1 for 24 hours. Culture medium of HLE B-3 cells was then concentrated and subjected to Western blot analysis. Similar results were obtained from three independent experiments. Low levels ofβ ig-h3 were detected in culture supernatants of control cells incubated for a total 42 hours without TGF-β1 (B, lane 1). This low level of βig-h3 protein did not increase further without TGF-β treatment when the cultures were extended for a total 90 hours (data not shown). In (A), lane 1 cells were washed 18 hours before analysis. βig-h3 was not detectable in the supernatant after this shorter culture period. M, molecular size standards (in kilodaltons). (C) HLE B-3 cells were seeded at 3.0 × 105 cells/well in 6-well plates coated with type IV collagen. After incubation for 18 hours in serum-free MEM, the cultures were treated with medium only (lane 1), IL-1α (lane 2), TNF-α (lane 3), TGF-β (lane 4), FGF-2 (lane 5), or IFN-γ (lane 6). After 24 hours, culture medium of HLE B-3 cells was concentrated and then subjected to Western blot analysis.
Figure 3.
 
Stimulation of βig-h3 protein secretion by TGF-β1 in lens epithelial cells. (A) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well and incubated for 18 hours in serum-free MEM. The culture medium of HLE B-3 cells subsequently incubated with TGF-β (10 ng/ml) for 0, 12, 24, 48, and 72 hours (lanes 1 through 5), corneal epithelial cell lysate (lane 6), and culture supernatant of COS cells overexpressing βig-h3 (lane 7) was loaded on SDS–10% polyacrylamide gel. Immunoreactive proteins were assessed by Western blot analysis using polyclonal βig-h3 antiserum. Three separate experiments yielded similar results. (B) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well. After incubation for 18 hours in serum-free MEM, the cultures were treated with various concentrations (0, 2.5, 5, 10, and 20 ng/ml; lanes 1 through 5) of TGF-β1 for 24 hours. Culture medium of HLE B-3 cells was then concentrated and subjected to Western blot analysis. Similar results were obtained from three independent experiments. Low levels ofβ ig-h3 were detected in culture supernatants of control cells incubated for a total 42 hours without TGF-β1 (B, lane 1). This low level of βig-h3 protein did not increase further without TGF-β treatment when the cultures were extended for a total 90 hours (data not shown). In (A), lane 1 cells were washed 18 hours before analysis. βig-h3 was not detectable in the supernatant after this shorter culture period. M, molecular size standards (in kilodaltons). (C) HLE B-3 cells were seeded at 3.0 × 105 cells/well in 6-well plates coated with type IV collagen. After incubation for 18 hours in serum-free MEM, the cultures were treated with medium only (lane 1), IL-1α (lane 2), TNF-α (lane 3), TGF-β (lane 4), FGF-2 (lane 5), or IFN-γ (lane 6). After 24 hours, culture medium of HLE B-3 cells was concentrated and then subjected to Western blot analysis.
The authors thank Usha Andley for HLE B-3 cells and critical review of the manuscript; Hae-Suk Kim for DNA sequencing analysis; John McAvoy for valuable advice on immunohistochemical analysis and critical review of the manuscript; Jun–Sub Choi and Hong Lim Kim for assistance with immunohistochemical analysis; and, especially, David Beebe for insightful comments on this study and careful editing of the manuscript. 
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Figure 1.
 
Enhanced expression of βig-h3 in human anterior polar cataracts. (A) Total cellular RNA was isolated from lens epithelial cells of patients with noncataractous high myopia (lane 1), nuclear cataracts (lane 2), and anterior polar cataracts (lane 3). RNA was also isolated from corneal epithelial cells (lane 4) of patients who underwent excimer laser photorefractive keratectomy. mRNA levels for β-actin and βig-h3 were examined by RT–PCR. The data shown are from one of three independent assays that produced similar results. (B) Anterior lens capsules with adhering cells from patients with anterior polar or nuclear cataracts were examined by immunohistochemical staining for βig-h3 (magnification, ×400). The capsule in the anterior polar cataractous lens is not as thick as the capsule in the control lens. This appears to be another feature of anterior polar cataracts. The data presented are from one of five independent assays that produced similar results. M, molecular size standards (in base pairs); LC, lens capsule; MF, myofibroblast-like cells (open arrow); SP, subcapsular plaque; LE, epithelial cells (closed arrow).
Figure 1.
 
Enhanced expression of βig-h3 in human anterior polar cataracts. (A) Total cellular RNA was isolated from lens epithelial cells of patients with noncataractous high myopia (lane 1), nuclear cataracts (lane 2), and anterior polar cataracts (lane 3). RNA was also isolated from corneal epithelial cells (lane 4) of patients who underwent excimer laser photorefractive keratectomy. mRNA levels for β-actin and βig-h3 were examined by RT–PCR. The data shown are from one of three independent assays that produced similar results. (B) Anterior lens capsules with adhering cells from patients with anterior polar or nuclear cataracts were examined by immunohistochemical staining for βig-h3 (magnification, ×400). The capsule in the anterior polar cataractous lens is not as thick as the capsule in the control lens. This appears to be another feature of anterior polar cataracts. The data presented are from one of five independent assays that produced similar results. M, molecular size standards (in base pairs); LC, lens capsule; MF, myofibroblast-like cells (open arrow); SP, subcapsular plaque; LE, epithelial cells (closed arrow).
Figure 2.
 
Stimulation of βig-h3 mRNA levels by TGF-β1 in cultured lens epithelial cells. (A) HLE B-3 cells were seeded in 60-mm culture dishes coated with type IV collagen (10 μg/ml) at 1.0 × 106 cells/dish. After incubation for 18 hours in serum-free MEM, the cultures were treated with TGF-β1 (10 ng/ml) in fresh medium. After the indicated times, total cellular RNA was isolated and subjected to Northern blot analysis. Three separate experiments yielded similar results. (B) After incubation in serum-free MEM, cycloheximide (CHX, 10 μg/ml) was added to the cultures, followed by treatment with TGF-β1 (10 ng/ml) for 12 hours. Similar results were obtained from two independent experiments. HLE B-3 cells were maintained healthy for at least 3 to 4 days in serum-free medium when cultured on the plastic dishes coated with type IV collagen that is a major constituent of the lens capsule.
Figure 2.
 
Stimulation of βig-h3 mRNA levels by TGF-β1 in cultured lens epithelial cells. (A) HLE B-3 cells were seeded in 60-mm culture dishes coated with type IV collagen (10 μg/ml) at 1.0 × 106 cells/dish. After incubation for 18 hours in serum-free MEM, the cultures were treated with TGF-β1 (10 ng/ml) in fresh medium. After the indicated times, total cellular RNA was isolated and subjected to Northern blot analysis. Three separate experiments yielded similar results. (B) After incubation in serum-free MEM, cycloheximide (CHX, 10 μg/ml) was added to the cultures, followed by treatment with TGF-β1 (10 ng/ml) for 12 hours. Similar results were obtained from two independent experiments. HLE B-3 cells were maintained healthy for at least 3 to 4 days in serum-free medium when cultured on the plastic dishes coated with type IV collagen that is a major constituent of the lens capsule.
Figure 3.
 
Stimulation of βig-h3 protein secretion by TGF-β1 in lens epithelial cells. (A) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well and incubated for 18 hours in serum-free MEM. The culture medium of HLE B-3 cells subsequently incubated with TGF-β (10 ng/ml) for 0, 12, 24, 48, and 72 hours (lanes 1 through 5), corneal epithelial cell lysate (lane 6), and culture supernatant of COS cells overexpressing βig-h3 (lane 7) was loaded on SDS–10% polyacrylamide gel. Immunoreactive proteins were assessed by Western blot analysis using polyclonal βig-h3 antiserum. Three separate experiments yielded similar results. (B) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well. After incubation for 18 hours in serum-free MEM, the cultures were treated with various concentrations (0, 2.5, 5, 10, and 20 ng/ml; lanes 1 through 5) of TGF-β1 for 24 hours. Culture medium of HLE B-3 cells was then concentrated and subjected to Western blot analysis. Similar results were obtained from three independent experiments. Low levels ofβ ig-h3 were detected in culture supernatants of control cells incubated for a total 42 hours without TGF-β1 (B, lane 1). This low level of βig-h3 protein did not increase further without TGF-β treatment when the cultures were extended for a total 90 hours (data not shown). In (A), lane 1 cells were washed 18 hours before analysis. βig-h3 was not detectable in the supernatant after this shorter culture period. M, molecular size standards (in kilodaltons). (C) HLE B-3 cells were seeded at 3.0 × 105 cells/well in 6-well plates coated with type IV collagen. After incubation for 18 hours in serum-free MEM, the cultures were treated with medium only (lane 1), IL-1α (lane 2), TNF-α (lane 3), TGF-β (lane 4), FGF-2 (lane 5), or IFN-γ (lane 6). After 24 hours, culture medium of HLE B-3 cells was concentrated and then subjected to Western blot analysis.
Figure 3.
 
Stimulation of βig-h3 protein secretion by TGF-β1 in lens epithelial cells. (A) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well and incubated for 18 hours in serum-free MEM. The culture medium of HLE B-3 cells subsequently incubated with TGF-β (10 ng/ml) for 0, 12, 24, 48, and 72 hours (lanes 1 through 5), corneal epithelial cell lysate (lane 6), and culture supernatant of COS cells overexpressing βig-h3 (lane 7) was loaded on SDS–10% polyacrylamide gel. Immunoreactive proteins were assessed by Western blot analysis using polyclonal βig-h3 antiserum. Three separate experiments yielded similar results. (B) HLE B-3 cells were seeded in 6-well plates coated with type IV collagen at 3.0 × 105 cells/well. After incubation for 18 hours in serum-free MEM, the cultures were treated with various concentrations (0, 2.5, 5, 10, and 20 ng/ml; lanes 1 through 5) of TGF-β1 for 24 hours. Culture medium of HLE B-3 cells was then concentrated and subjected to Western blot analysis. Similar results were obtained from three independent experiments. Low levels ofβ ig-h3 were detected in culture supernatants of control cells incubated for a total 42 hours without TGF-β1 (B, lane 1). This low level of βig-h3 protein did not increase further without TGF-β treatment when the cultures were extended for a total 90 hours (data not shown). In (A), lane 1 cells were washed 18 hours before analysis. βig-h3 was not detectable in the supernatant after this shorter culture period. M, molecular size standards (in kilodaltons). (C) HLE B-3 cells were seeded at 3.0 × 105 cells/well in 6-well plates coated with type IV collagen. After incubation for 18 hours in serum-free MEM, the cultures were treated with medium only (lane 1), IL-1α (lane 2), TNF-α (lane 3), TGF-β (lane 4), FGF-2 (lane 5), or IFN-γ (lane 6). After 24 hours, culture medium of HLE B-3 cells was concentrated and then subjected to Western blot analysis.
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