- Email: [email protected]
Effect of growth factors on proliferation and expression of growth factor receptors in a human lens epithelial cell line
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
Effect of growth factors on proliferation and expression of growth factor receptors in a human lens epithelial cell line Juergen Kampmeier, MD, Alicja Baldysiak-Figiel, Yvonne de Jong-Hesse, MD, Gerhard K. Lang, MD, Gabriele E. Lang, MD
PURPOSE: To investigate the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta 2 (TGFb2) on proliferation of a human lens epithelial cell line (HLEC-SRA 01/04); the effect of bFGF and TGFb2 on proliferation of human lens epithelial cells (HLECs); and the expression of bFGF, EGF, IGF-1, and TGFb2 receptors in an HLEC-SRA 01/04 cell line. SETTING: Department of Ophthalmology, University of Ulm, Ulm, Germany. METHODS: Both HLEC and HLEC-SRA 01/04 were treated with 1 to 50 ng/mL bFGF and TGFb2. Additionally, HLEC-SRA 01/04 were cultured with EGF and IGF-1 at a concentration of 1 to 50 ng/mL for 48 hours in the presence of [3H]-thymidine. In all experiments, untreated serum-free negative controls were used. (3H)-thymidine incorporation as a direct measure of lens epithelial cell proliferation was assessed by liquid scintillation counting. The expression of bFGF, EGF, IGF-1, and TGFb2 receptors in HLEC-SRA 01/04 were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was performed using the 2-sample t test for the means. RESULTS: Proliferation of HLECs was dose dependently induced by bFGF and TGFb2, showing maximum effects at 10 ng/mL (PZ .0003) and at 50 ng/mL (P<.0001), respectively. Proliferation of HLECSRA 01/04 was also induced by bFGF, showing slight but significant effects (P<.03). Additionally, HLEC-SRA 01/04 proliferation was dose-dependently induced by EGF with a maximum effect at 5 ng/mL (P<.01), IGF-1 with a maximum effect at 5 ng/mL (P<.0001), and TGFb2 with a maximum effect at 10 ng/mL (P<.0001) compared with the control. The RT-PCR analysis revealed bFGF, EGF, IGF-1, and TGFb2 receptor expression in the HLEC-SRA 01/04 cell line. CONCLUSIONS: The data showed that bFGF and TGFb2 are strong mitogens for HLEC. The HLEC-SRA 01/04 cell line derived from HLEC reacted to growth factors, with cell proliferation only to a lesser extent. Such quiescence of these cells, when compared with cells in primary culture, cannot be explained by the lack of respective receptors for growth factors. Further investigation of growth factor-induced responses of both cell types will provide new insight into the proliferative processes involved in postoperative secondary cataract formation. J Cataract Refract Surg 2006; 32:510–514 Q 2006 ASCRS and ESCRS
Posterior capsule opacification following extracapsular cataract extraction is a result of lens epithelial cell (LEC) proliferation on the posterior capsule, and its prevention is an important step toward improving the quality of the surgical outcome. Within the wide range of recent therapeutic targets, cytokines and receptors may play an important role because they are expressed by LECs and influence their postoperative proliferation in the capsular bag. They are also important in the inflammation and wound-healing Q 2006 ASCRS and ESCRS Published by Elsevier Inc.
510
process after cataract surgery.1,2 Cultured human lens epithelial cells (HLEC) express different growth factors and receptors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and transforming growth factor beta 2 (TGFb2), in the incubating medium.1,2 Primary cultures of HLEC would provide important information concerning the role of the epithelium in the healthy lens and in cataract formation. However, cells 0886-3350/06/$-see front matter doi:10.1016/j.jcrs.2005.08.063
LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
isolated from patients following cataract surgery do not grow well in cell culture conditions. Therefore, we used the HLEC line SRA 01/04 to determine whether it can be used as a model in cataract formation in vitro. This study examined the effects of bFGF, EGF, IGF-1, TGFb2 on proliferation of primary HLEC and HLEC-SRA 01/04 and illustrated differences in cell responses of both cell types. It also analyzed the expression of bFGF, EGF, and IGF-1, TGFb2 receptors in HLEC-SRA 01/04. MATERIALS AND METHODS
RESULTS
The experiments on HLEC revealed that cell proliferation is dose dependently induced by bFGF, showing a maximum increase of 57% at 10 ng/mL (PZ .0003), and by TGFb2, showing a maximum increase of 56% at 50 ng/mL (P!.0001) as compared with control (Figures 1 and 2). The stimulatory effect of bFGF on HLEC-SRA 01/04 was weak but significant (P!.03)(Figure 3). Epidermal growth factor stimulated HLEC-SRA 01/04 proliferation with maximal increase of 32% at 5 ng/mL as compared with control (P!.01) (Figure 4). Insulin-like growth factor 1 dose-independently stimulated HLEC-SRA 01/04 proliferation, with a maximal increase of 36% at 5 ng/mL as compared with control (P!.0001) (Figure 5). Finally, TGFb2 dose dependently stimulated HLEC-SRA 01/04 proliferation, with 1200
Accepted for publication August 12, 2005. From the Department of Ophthalmology, University of Ulm, Ulm, Germany. Supported by Rudolf and Clothilde Stiftung, Ulm, Germany. No author has a financial or proprietary interest in any material or method mentioned. The HLEC-SRA 01/04 was provided by Prof. V. N. Reddy, University of Kellog Eye Center, University of Michigan, Ann Arbor, Michigan, USA. Reprint requests to Dr. Juergen Kampmeier, MD, Universitaetsklinikum Ulm, Augenklinik Prittwitzstr. 43, D - 89075 Ulm, Germany. E-mail: [email protected].
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The HLEC were obtained by grasping the anterior capsule of a human lens with fine forceps. The HLECs were carefully detached from the lens capsule using a scalpel and placed on the culture plate without culture medium and allowed to attach for 1 hour. Subsequently, the minimum essential medium plus 10% fetal calf serum was added and cells were incubated in 5% CO2 at 37 C. The medium was renewed every third day. The HLEC-SRA 01-04 cell line was produced by transfecting cells with plasmid vector DNA containing the immortalizing gene (large T-antigen) of SV40.3 The HLEC-SRA 01/04 cells were cultivated in DMEM plus 10% fetal calf serum in 37 C in a humidified atmosphere of 5% CO2. To examine the effect of growth factors on cell proliferation, 5 104 cells/mL HLEC or HLEC-SRA 01/04 were plated on 96well flat-bottom plates (Falcon/Becton Dickinson) in the respective culture medium and grown to confluency. Subsequently the cells were incubated for 24 hours with serum-deprived medium supplemented for 48 hours with bFGF, EGF, IGF-1, and TGFb2 at a concentration of 1 to 50 ng/mL. For the last 24 hours of incubation, cells were additionally supplemented with 1 mCi/mL [3H]-thymidine. For scintillator counting, the cells were washed, dissociated in 0.25% trypsin-EDTA, and harvested on glass-fiber filters. The filters were dried and the incorporated radioactive material was measured in 10 mL scintillation fluid in a beta counter. For experiments, serum-deprived medium was used as a negative control. All experiments were repeated at least 3 times for each growth factor. Statistical analysis was performed using a 2-sample t test for the means. For reverse transcriptase polymerase chain reaction (RTPCR) analyses, total RNA was isolated from 35 mm culture dishes of HLEC-SRA 01/04 using PeqGold RNAPure (Peqlab) according to the manufacturer’s instructions. The RNA concentration was
calculated from spectrophotometric absorbance of RNA solution measured at 260 nm. A total of 10 mg RNA was reverse-transcribed at 37 C for 2 hours using 200 U M-MLV (Promega) and 2 mg random hexamer primers (Interactiva). Polymerase chain reaction was performed with 2 mL of the obtained templates in total volume of 50 mL in the presence of 20 pmol of the respective primers using recombinant Taq polymerase (Gibco). The PCR amplification included 35 cycles of denaturation at 95 C for 30 seconds, annealing at 60 C for 30 seconds, and elongation at 72 C for 30 seconds. The receptor cDNA was detected using the primer sequences of the human bFGF receptor (50 -GGTCGTTTCATCTGCCTGGT30 ; 50 -CCTTCCCGTTTTTCAGCCAC-30 ; 367 bp product), EGF receptor (50 -GAGAGGAGAACTGCCAGAA-30 ; 50 -GCATTTATGGAGAGTG-30 ; 454 bp product), IGF-1 receptor (50 -CACGAGGCTGAGAAGCT-30 ; 50 -AGGCATACAGCACTCCA-30 ; 502 bp product), and TGFb2 receptor (50 -AAGTCTTGCARGAGCAACTGC-30 ; 50 -CTATGGCAATCCCCAGCGGAG-30 ; 351 bp product). Reaction products were separated on 1.5% agarose gel in tris-acetat-EDTA puffer buffer and stained with ethidium bromide.
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bFGF (ng/ml) Figure 1. Basic fibroblast growth factor significantly stimulated the proliferation of HLEC at concentrations of 1 to 50 ng/mL, showing maximum effect at 10 ng/mL (increase of 57% versus control; ***P!.0001, **P Z .0003, *P!.002). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
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LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
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Figure 2. Transforming growth factor beta 2 significantly stimulated the proliferation of HLEC at concentrations of 1 to 50 ng/mL, with maximum effect at 50 ng/mL (increase of 56% versus control; *P!.0001). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
Figure 3. Basic fibroblast growth factor subtly stimulated the proliferation of HLEC-SRA 01/04 at concentrations of 1 to 50 ng/mL (*P!.03). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
a maximum increase of 48% at 10 ng/mL compared with the control (P!.0001) (Figure 6). The RT-PCR analysis showed expression of growth factor receptors in HLEC-SRA 01/04 and revealed a 454 bp product for human EGF receptor, a 502 bp product for human IGF-1 receptor, a 367 bp product for human bFGF receptor, and a 351 bp product for human TGFb2 receptor (Figure 7, a and b).
HLEC. These findings are similar to other observations made in normal HLEC and in animals cataract cell models.2,4,5 Cell proliferation of HLEC-SRA 01/04 was stimulated by bFGF, EGF, and IGF-1. However, in contrast to primary HLEC, this effect was neither potent nor concentration dependent. It seems that the human cell line was not responsive to growth factors. It is known that bFGF, EGF, and IGF-1 are mitogens for many cell types and may act as regulators of LEC growth and differentiation. Basic fibroblast growth factor also has an inhibitory effect on apoptosis through up-regulation of the inhibitor of apoptose.6 Our experiments show differences between responses of primary human epithelial cells and the human epithelial cell line to growth factors. Similar findings show
DISCUSSION
Our data show that bFGF and TGFb2 have a concentration-dependent stimulatory effect on the proliferation of
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Figure 4. Epidermal growth factor stimulated HLEC-SRA 01/04 proliferation, with maximum effect at 5 ng/mL (32% versus control; **P!.01, *P!.05). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
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Figure 5. Insulin-like growth factor 1 significantly stimulated HLEC-SRA 01/04 proliferation at concentrations of 1 to 50 ng/mL, with maximum effect at 5 ng/mL (increase of 36% versus control; ***P!.0001, **P!.001). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
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TGFß2 (ng/ml) Figure 6. Transforming growth factor beta 2 stimulated significantly dose-dependent HLEC-SRA 01/04 proliferation, with maximum effect at 10 ng/mL (increase of 48% compared with control) (***P!.0001, **P!.003, *P!.03). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
that immortalized cells have decreased responses to growth factors such as platelet-derived growth factor and bFGF.6,7,12 Very interesting is the effect of TGFb2 on the proliferation of both cell types: TGFb2 plays a major role in regulating the behavior of lens cells during lens repair after injury.8,9 According to our results, TGFb2 stimulates the proliferation of cells in a dose-dependent manner. The experiments of Richiert and Ireland10 also show a proliferation of LECs induced by TGFb2. This potent stimulation of lens cell growth by TGFb2 may be due to misregulated production of lens capsule proteins not normally found in great abundance. These findings differ from the results of other authors who found that TGFb down-regulates HLEC proliferation. One reason might be that the number of growth factor bindings and the number of growth factor receptors gradually decreases with number of passages. The
Figure 7. Expression of human EGF, IGF-1 (A), bFGF, and TGFb2 (B) receptors in HLEC-SRA 01/04. Reverse transcriptase PCR reactions revealed a 454 bp product from the human EGF receptor, a 502 bp product from the human IGF-1 receptor, a 367 bp product from the human bFGF receptor, and a 351 bp product from the human TGFb2 receptor.
loss of the effect of growth factors on fiber differentiation with an increasing number of passages correlates with the decrease in receptor number.11 In our second set of experiments, we examined the expression of growth factor receptors using RT-PCR. We were able to identify the presence of bFGF, EGF, FGF, and TGFb2 receptors on HLEC-SRA 01/04. Similar studies have shown that other epithelial cell lines generally express the full set of receptors for growth factors.12,13 In native HLEC, the expression of these growth factor receptors has already been proven.14,15 The immunocytochemical study of Shigemitsu et al.13 confirmed the presence of EGF, FGF, and TGFb2 receptors in cataractous lens epithelium before cataract surgery and in the lens epithelium of anterior capsules collected at the time of cataract surgery. Another study has shown that type 2 TGF receptor, in addition to being expressed in the epithelium, was also expressed in patterns consistent with a role in lens fiber differentiation.12,16 The low response of cells to growth factors is not likely to be attributed to the lack of growth factors receptors, but rather to differences in intracellular signal transduction cascades. Transforming growth factor belongs to serine protease inhibitor family and is secreted by cells as a ‘‘latent,’’ inactive form.17 Activation requires extracellular proteolytic digestion by serine proteases and subsequent dimerization, which results in a mature active TGFb. The active TGFb molecule can then bind the TGFb receptor, which is coupled to signaling pathways involving the Smad family of proteins, eventually leading to various proliferative responses.18 Transforming growth factor beta inhibits the growth of epithelial and endothelial cells in vitro.17 It is known that TGFb can inhibit primary LEC proliferation. In contrast, our results show that TGFb stimulates the proliferation of HLEC and a HLEC-SRA 01/04 cell line originated from primary LECs. Such discrepancies in proliferative cell responses to TGFb can be the result of changes in intracellular signaling pathways from TGFb receptor and changes in the regulation of TGFb receptor surface expression. Peterson et al.19 showed that TGFb receptor 2 expression was decreased in metastasis compared with primary tumor or normal cells. It is commonly known that primary cells also differ from transformed cell lines in terms of growth factor production and secretion. The presence of other growth factors such a EGF and IGF-1 can modulate proliferative responses exerted by TGFb on primary cells versus spontaneously immortalized cells.20 The development of cell lines resembling primary human epithelial cells is of great importance for cataract research. Although the HLEC-SRA 01/04 line shows features similar to HLEC, the differences in response to various growth factors can present a hindrance in the reproduction of conditions during HLEC proliferation.
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LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
REFERENCES 1. Nishi O. Posterior capsule opacification. J Cataract Refract Surg 1999; 25:106–117 2. Nishi O, Nishi K, Fujiwara T, et al. Effects of the cytokines on the proliferation of and collagen synthesis by human cataract lens epithelial cells. Br J Ophthalmol 1996; 80:63–68 3. Ibaraki N, Chen SC, Lin LR, et al. Human lens epithelial cell line. Exp Eye Res 1998; 67:577–585 4. Wu LN, Genge BR, Ishikawa Y, Wuthier RE. Modulation of cultured chicken growth plate chondrocytes by transforming growth factor beta 1 and basic fibroblast growth factor. J Cell Biochem 1992; 49:181–198 5. Power W, Neylan D, Collum L. Growth characteristics of human lens epithelial cells in culture: effect of media and donor age. Doc Ophthalmol 1993; 84:365–372 6. Wang Y, He H, Zigler JS Jr, et al. bFGF suppresses serum-deprivationinduced apoptosis in a human lens epithelial cell line. Exp Cell Res 1999; 249:123–130 7. Fleming TP, Song Z, Andley UP. Expression of growth control and differentiation genes in human lens epithelial cells with extended life span. Invest Ophthalmol Vis Sci 1998; 39:1387–1398 8. Saika S, Kono-Saika S, Ohnishi Y, et al. Smad3 signaling is required for epithelial-mesenchymal transition of lens epithelium after injury. Am J Pathol 2004; 164:651–663 9. Kurosaka D, Nagamoto T. Inhibitory effect of TGF-beta 2 in human aqueous humor on bovine lens epithelial cell proliferation. Invest Ophthalmol Vis Sci 1994; 35:3408–3412 10. Richiert DM, Ireland ME. TGF-beta elicits fibronectin secretion and proliferation in cultured chick lens epithelial cells. Curr Eye Res 1999; 18:62–71
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11. Ibaraki N, Lin LR, Reddy VN. A study of growth factor receptors in human lens epithelial cells and their relationship to fiber differentiation. Exp Eye Res 1996; 63:683–692 12. De Iongh RU, Lovicu FJ, Overbeek PA, et al. Requirement for TGF beta receptor signaling during terminal lens fiber differentiation. Development 2001; 128:3995–4010 13. Shigemitsu T, Ishiguro K, Shimizu Y, et al. Immunocytochemical features of lens after cataract tissue-signalling molecules (growth factors, cytokines, other signaling molecules), cytoskeleton proteins, cellular and extracellular matrix proteins. Int Ophthalmol 1999; 23: 137–144 14. De Longh RU, Gordon-Thomson C, Chamberlain CG, et al. TGFß receptor expression in lens: implications for differentiation and cataractogenesis. Exp Eye Res 2001; 72:649–659 15. Bhuyan DK, Reddy PG, Bhuyan KC. Growth factors gene and protein expressions in the human lens. Mech Ageing Dev 2000; 113:205–218 16. Jacobs DB, Ireland M, Pickett T, et al. Functional characterization of insulin and IGF-1 receptors in chicken lens epithelial and fiber cells. Curr Eye Res 1999; 11:1137–1145 17. Rifkin DB, Kojima S, Abe M, Harpel JG. TGF-b: structure, function, and formation. Thromb Haemost 1993; 70:177–179 18. Derynck R, Zhang Y, Feng XH. Smads: transcriptional activators of TGFbeta responses. Cell 1998; 95:737–740 19. Peterson IC, Matthews JB, Huntley S, et al. Decreased expression of TGF-beta cell surface receptors during progression of human oral squamous cell carcinoma. J Pathol 2001; 193:458–467 20. Lerner AA, Salamone DF, Chiappe ME, Baranao JL. Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: protein secretion, steroid metabolism, and responsiveness to growth factors. J Cell Physiol 1995; 164:395–403
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
Effect of growth factors on proliferation and expression of growth factor receptors in a human lens epithelial cell line Juergen Kampmeier, MD, Alicja Baldysiak-Figiel, Yvonne de Jong-Hesse, MD, Gerhard K. Lang, MD, Gabriele E. Lang, MD
PURPOSE: To investigate the effect of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor 1 (IGF-1), and transforming growth factor beta 2 (TGFb2) on proliferation of a human lens epithelial cell line (HLEC-SRA 01/04); the effect of bFGF and TGFb2 on proliferation of human lens epithelial cells (HLECs); and the expression of bFGF, EGF, IGF-1, and TGFb2 receptors in an HLEC-SRA 01/04 cell line. SETTING: Department of Ophthalmology, University of Ulm, Ulm, Germany. METHODS: Both HLEC and HLEC-SRA 01/04 were treated with 1 to 50 ng/mL bFGF and TGFb2. Additionally, HLEC-SRA 01/04 were cultured with EGF and IGF-1 at a concentration of 1 to 50 ng/mL for 48 hours in the presence of [3H]-thymidine. In all experiments, untreated serum-free negative controls were used. (3H)-thymidine incorporation as a direct measure of lens epithelial cell proliferation was assessed by liquid scintillation counting. The expression of bFGF, EGF, IGF-1, and TGFb2 receptors in HLEC-SRA 01/04 were analyzed by reverse transcriptase polymerase chain reaction (RT-PCR). Statistical analysis was performed using the 2-sample t test for the means. RESULTS: Proliferation of HLECs was dose dependently induced by bFGF and TGFb2, showing maximum effects at 10 ng/mL (PZ .0003) and at 50 ng/mL (P<.0001), respectively. Proliferation of HLECSRA 01/04 was also induced by bFGF, showing slight but significant effects (P<.03). Additionally, HLEC-SRA 01/04 proliferation was dose-dependently induced by EGF with a maximum effect at 5 ng/mL (P<.01), IGF-1 with a maximum effect at 5 ng/mL (P<.0001), and TGFb2 with a maximum effect at 10 ng/mL (P<.0001) compared with the control. The RT-PCR analysis revealed bFGF, EGF, IGF-1, and TGFb2 receptor expression in the HLEC-SRA 01/04 cell line. CONCLUSIONS: The data showed that bFGF and TGFb2 are strong mitogens for HLEC. The HLEC-SRA 01/04 cell line derived from HLEC reacted to growth factors, with cell proliferation only to a lesser extent. Such quiescence of these cells, when compared with cells in primary culture, cannot be explained by the lack of respective receptors for growth factors. Further investigation of growth factor-induced responses of both cell types will provide new insight into the proliferative processes involved in postoperative secondary cataract formation. J Cataract Refract Surg 2006; 32:510–514 Q 2006 ASCRS and ESCRS
Posterior capsule opacification following extracapsular cataract extraction is a result of lens epithelial cell (LEC) proliferation on the posterior capsule, and its prevention is an important step toward improving the quality of the surgical outcome. Within the wide range of recent therapeutic targets, cytokines and receptors may play an important role because they are expressed by LECs and influence their postoperative proliferation in the capsular bag. They are also important in the inflammation and wound-healing Q 2006 ASCRS and ESCRS Published by Elsevier Inc.
510
process after cataract surgery.1,2 Cultured human lens epithelial cells (HLEC) express different growth factors and receptors, such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and transforming growth factor beta 2 (TGFb2), in the incubating medium.1,2 Primary cultures of HLEC would provide important information concerning the role of the epithelium in the healthy lens and in cataract formation. However, cells 0886-3350/06/$-see front matter doi:10.1016/j.jcrs.2005.08.063
LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
isolated from patients following cataract surgery do not grow well in cell culture conditions. Therefore, we used the HLEC line SRA 01/04 to determine whether it can be used as a model in cataract formation in vitro. This study examined the effects of bFGF, EGF, IGF-1, TGFb2 on proliferation of primary HLEC and HLEC-SRA 01/04 and illustrated differences in cell responses of both cell types. It also analyzed the expression of bFGF, EGF, and IGF-1, TGFb2 receptors in HLEC-SRA 01/04. MATERIALS AND METHODS
RESULTS
The experiments on HLEC revealed that cell proliferation is dose dependently induced by bFGF, showing a maximum increase of 57% at 10 ng/mL (PZ .0003), and by TGFb2, showing a maximum increase of 56% at 50 ng/mL (P!.0001) as compared with control (Figures 1 and 2). The stimulatory effect of bFGF on HLEC-SRA 01/04 was weak but significant (P!.03)(Figure 3). Epidermal growth factor stimulated HLEC-SRA 01/04 proliferation with maximal increase of 32% at 5 ng/mL as compared with control (P!.01) (Figure 4). Insulin-like growth factor 1 dose-independently stimulated HLEC-SRA 01/04 proliferation, with a maximal increase of 36% at 5 ng/mL as compared with control (P!.0001) (Figure 5). Finally, TGFb2 dose dependently stimulated HLEC-SRA 01/04 proliferation, with 1200
Accepted for publication August 12, 2005. From the Department of Ophthalmology, University of Ulm, Ulm, Germany. Supported by Rudolf and Clothilde Stiftung, Ulm, Germany. No author has a financial or proprietary interest in any material or method mentioned. The HLEC-SRA 01/04 was provided by Prof. V. N. Reddy, University of Kellog Eye Center, University of Michigan, Ann Arbor, Michigan, USA. Reprint requests to Dr. Juergen Kampmeier, MD, Universitaetsklinikum Ulm, Augenklinik Prittwitzstr. 43, D - 89075 Ulm, Germany. E-mail: [email protected].
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The HLEC were obtained by grasping the anterior capsule of a human lens with fine forceps. The HLECs were carefully detached from the lens capsule using a scalpel and placed on the culture plate without culture medium and allowed to attach for 1 hour. Subsequently, the minimum essential medium plus 10% fetal calf serum was added and cells were incubated in 5% CO2 at 37 C. The medium was renewed every third day. The HLEC-SRA 01-04 cell line was produced by transfecting cells with plasmid vector DNA containing the immortalizing gene (large T-antigen) of SV40.3 The HLEC-SRA 01/04 cells were cultivated in DMEM plus 10% fetal calf serum in 37 C in a humidified atmosphere of 5% CO2. To examine the effect of growth factors on cell proliferation, 5 104 cells/mL HLEC or HLEC-SRA 01/04 were plated on 96well flat-bottom plates (Falcon/Becton Dickinson) in the respective culture medium and grown to confluency. Subsequently the cells were incubated for 24 hours with serum-deprived medium supplemented for 48 hours with bFGF, EGF, IGF-1, and TGFb2 at a concentration of 1 to 50 ng/mL. For the last 24 hours of incubation, cells were additionally supplemented with 1 mCi/mL [3H]-thymidine. For scintillator counting, the cells were washed, dissociated in 0.25% trypsin-EDTA, and harvested on glass-fiber filters. The filters were dried and the incorporated radioactive material was measured in 10 mL scintillation fluid in a beta counter. For experiments, serum-deprived medium was used as a negative control. All experiments were repeated at least 3 times for each growth factor. Statistical analysis was performed using a 2-sample t test for the means. For reverse transcriptase polymerase chain reaction (RTPCR) analyses, total RNA was isolated from 35 mm culture dishes of HLEC-SRA 01/04 using PeqGold RNAPure (Peqlab) according to the manufacturer’s instructions. The RNA concentration was
calculated from spectrophotometric absorbance of RNA solution measured at 260 nm. A total of 10 mg RNA was reverse-transcribed at 37 C for 2 hours using 200 U M-MLV (Promega) and 2 mg random hexamer primers (Interactiva). Polymerase chain reaction was performed with 2 mL of the obtained templates in total volume of 50 mL in the presence of 20 pmol of the respective primers using recombinant Taq polymerase (Gibco). The PCR amplification included 35 cycles of denaturation at 95 C for 30 seconds, annealing at 60 C for 30 seconds, and elongation at 72 C for 30 seconds. The receptor cDNA was detected using the primer sequences of the human bFGF receptor (50 -GGTCGTTTCATCTGCCTGGT30 ; 50 -CCTTCCCGTTTTTCAGCCAC-30 ; 367 bp product), EGF receptor (50 -GAGAGGAGAACTGCCAGAA-30 ; 50 -GCATTTATGGAGAGTG-30 ; 454 bp product), IGF-1 receptor (50 -CACGAGGCTGAGAAGCT-30 ; 50 -AGGCATACAGCACTCCA-30 ; 502 bp product), and TGFb2 receptor (50 -AAGTCTTGCARGAGCAACTGC-30 ; 50 -CTATGGCAATCCCCAGCGGAG-30 ; 351 bp product). Reaction products were separated on 1.5% agarose gel in tris-acetat-EDTA puffer buffer and stained with ethidium bromide.
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bFGF (ng/ml) Figure 1. Basic fibroblast growth factor significantly stimulated the proliferation of HLEC at concentrations of 1 to 50 ng/mL, showing maximum effect at 10 ng/mL (increase of 57% versus control; ***P!.0001, **P Z .0003, *P!.002). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
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LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
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Figure 2. Transforming growth factor beta 2 significantly stimulated the proliferation of HLEC at concentrations of 1 to 50 ng/mL, with maximum effect at 50 ng/mL (increase of 56% versus control; *P!.0001). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
Figure 3. Basic fibroblast growth factor subtly stimulated the proliferation of HLEC-SRA 01/04 at concentrations of 1 to 50 ng/mL (*P!.03). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
a maximum increase of 48% at 10 ng/mL compared with the control (P!.0001) (Figure 6). The RT-PCR analysis showed expression of growth factor receptors in HLEC-SRA 01/04 and revealed a 454 bp product for human EGF receptor, a 502 bp product for human IGF-1 receptor, a 367 bp product for human bFGF receptor, and a 351 bp product for human TGFb2 receptor (Figure 7, a and b).
HLEC. These findings are similar to other observations made in normal HLEC and in animals cataract cell models.2,4,5 Cell proliferation of HLEC-SRA 01/04 was stimulated by bFGF, EGF, and IGF-1. However, in contrast to primary HLEC, this effect was neither potent nor concentration dependent. It seems that the human cell line was not responsive to growth factors. It is known that bFGF, EGF, and IGF-1 are mitogens for many cell types and may act as regulators of LEC growth and differentiation. Basic fibroblast growth factor also has an inhibitory effect on apoptosis through up-regulation of the inhibitor of apoptose.6 Our experiments show differences between responses of primary human epithelial cells and the human epithelial cell line to growth factors. Similar findings show
DISCUSSION
Our data show that bFGF and TGFb2 have a concentration-dependent stimulatory effect on the proliferation of
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Figure 4. Epidermal growth factor stimulated HLEC-SRA 01/04 proliferation, with maximum effect at 5 ng/mL (32% versus control; **P!.01, *P!.05). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
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Figure 5. Insulin-like growth factor 1 significantly stimulated HLEC-SRA 01/04 proliferation at concentrations of 1 to 50 ng/mL, with maximum effect at 5 ng/mL (increase of 36% versus control; ***P!.0001, **P!.001). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
J CATARACT REFRACT SURG - VOL 32, MARCH 2006
LABORATORY SCIENCE: GROWTH FACTORS IN HUMAN EPITHELIAL CELLS
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TGFß2 (ng/ml) Figure 6. Transforming growth factor beta 2 stimulated significantly dose-dependent HLEC-SRA 01/04 proliferation, with maximum effect at 10 ng/mL (increase of 48% compared with control) (***P!.0001, **P!.003, *P!.03). The incorporation of [3H]-thymidine was determined by liquid scintillation counting.
that immortalized cells have decreased responses to growth factors such as platelet-derived growth factor and bFGF.6,7,12 Very interesting is the effect of TGFb2 on the proliferation of both cell types: TGFb2 plays a major role in regulating the behavior of lens cells during lens repair after injury.8,9 According to our results, TGFb2 stimulates the proliferation of cells in a dose-dependent manner. The experiments of Richiert and Ireland10 also show a proliferation of LECs induced by TGFb2. This potent stimulation of lens cell growth by TGFb2 may be due to misregulated production of lens capsule proteins not normally found in great abundance. These findings differ from the results of other authors who found that TGFb down-regulates HLEC proliferation. One reason might be that the number of growth factor bindings and the number of growth factor receptors gradually decreases with number of passages. The
Figure 7. Expression of human EGF, IGF-1 (A), bFGF, and TGFb2 (B) receptors in HLEC-SRA 01/04. Reverse transcriptase PCR reactions revealed a 454 bp product from the human EGF receptor, a 502 bp product from the human IGF-1 receptor, a 367 bp product from the human bFGF receptor, and a 351 bp product from the human TGFb2 receptor.
loss of the effect of growth factors on fiber differentiation with an increasing number of passages correlates with the decrease in receptor number.11 In our second set of experiments, we examined the expression of growth factor receptors using RT-PCR. We were able to identify the presence of bFGF, EGF, FGF, and TGFb2 receptors on HLEC-SRA 01/04. Similar studies have shown that other epithelial cell lines generally express the full set of receptors for growth factors.12,13 In native HLEC, the expression of these growth factor receptors has already been proven.14,15 The immunocytochemical study of Shigemitsu et al.13 confirmed the presence of EGF, FGF, and TGFb2 receptors in cataractous lens epithelium before cataract surgery and in the lens epithelium of anterior capsules collected at the time of cataract surgery. Another study has shown that type 2 TGF receptor, in addition to being expressed in the epithelium, was also expressed in patterns consistent with a role in lens fiber differentiation.12,16 The low response of cells to growth factors is not likely to be attributed to the lack of growth factors receptors, but rather to differences in intracellular signal transduction cascades. Transforming growth factor belongs to serine protease inhibitor family and is secreted by cells as a ‘‘latent,’’ inactive form.17 Activation requires extracellular proteolytic digestion by serine proteases and subsequent dimerization, which results in a mature active TGFb. The active TGFb molecule can then bind the TGFb receptor, which is coupled to signaling pathways involving the Smad family of proteins, eventually leading to various proliferative responses.18 Transforming growth factor beta inhibits the growth of epithelial and endothelial cells in vitro.17 It is known that TGFb can inhibit primary LEC proliferation. In contrast, our results show that TGFb stimulates the proliferation of HLEC and a HLEC-SRA 01/04 cell line originated from primary LECs. Such discrepancies in proliferative cell responses to TGFb can be the result of changes in intracellular signaling pathways from TGFb receptor and changes in the regulation of TGFb receptor surface expression. Peterson et al.19 showed that TGFb receptor 2 expression was decreased in metastasis compared with primary tumor or normal cells. It is commonly known that primary cells also differ from transformed cell lines in terms of growth factor production and secretion. The presence of other growth factors such a EGF and IGF-1 can modulate proliferative responses exerted by TGFb on primary cells versus spontaneously immortalized cells.20 The development of cell lines resembling primary human epithelial cells is of great importance for cataract research. Although the HLEC-SRA 01/04 line shows features similar to HLEC, the differences in response to various growth factors can present a hindrance in the reproduction of conditions during HLEC proliferation.
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11. Ibaraki N, Lin LR, Reddy VN. A study of growth factor receptors in human lens epithelial cells and their relationship to fiber differentiation. Exp Eye Res 1996; 63:683–692 12. De Iongh RU, Lovicu FJ, Overbeek PA, et al. Requirement for TGF beta receptor signaling during terminal lens fiber differentiation. Development 2001; 128:3995–4010 13. Shigemitsu T, Ishiguro K, Shimizu Y, et al. Immunocytochemical features of lens after cataract tissue-signalling molecules (growth factors, cytokines, other signaling molecules), cytoskeleton proteins, cellular and extracellular matrix proteins. Int Ophthalmol 1999; 23: 137–144 14. De Longh RU, Gordon-Thomson C, Chamberlain CG, et al. TGFß receptor expression in lens: implications for differentiation and cataractogenesis. Exp Eye Res 2001; 72:649–659 15. Bhuyan DK, Reddy PG, Bhuyan KC. Growth factors gene and protein expressions in the human lens. Mech Ageing Dev 2000; 113:205–218 16. Jacobs DB, Ireland M, Pickett T, et al. Functional characterization of insulin and IGF-1 receptors in chicken lens epithelial and fiber cells. Curr Eye Res 1999; 11:1137–1145 17. Rifkin DB, Kojima S, Abe M, Harpel JG. TGF-b: structure, function, and formation. Thromb Haemost 1993; 70:177–179 18. Derynck R, Zhang Y, Feng XH. Smads: transcriptional activators of TGFbeta responses. Cell 1998; 95:737–740 19. Peterson IC, Matthews JB, Huntley S, et al. Decreased expression of TGF-beta cell surface receptors during progression of human oral squamous cell carcinoma. J Pathol 2001; 193:458–467 20. Lerner AA, Salamone DF, Chiappe ME, Baranao JL. Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: protein secretion, steroid metabolism, and responsiveness to growth factors. J Cell Physiol 1995; 164:395–403
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