Molecular Vision 2003; 9:723-729 <http://www.molvis.org/molvis/v9/a86/> Received 20 October 2003 | Accepted 17 December 2003 | Published 18 December 2003

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Influence of hormones and growth factors on lens protein composition: The effect of dexamethasone and PDGF-AA

Laura Marin Vinader, Siebe T. van Genesen, Wilfried W. de Jong, Nicolette H. Lubsen
 
 

Department of Biochemistry, University of Nijmegen, The Netherlands

Correspondence to: Nicolette H. Lubsen, Department of Biochemistry 161, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands; email: nhl@sci.kun.nl

Abstract

Purpose: To investigate the effect of hormones and ocular growth factors on the expression of α-, β-, and γ-crystallins in rat lens epithelial and fiber cells.

Methods: PDGF-AA, EGF, NGF, M-CSF, BMP-2, BMP-4, dexamethasone, and estrogen were tested for their ability to alter the spectrum of crystallins in explanted newborn rat lens epithelial cells or in vitro differentiating newborn rat lens fiber cells. The accumulation of αA-, αB-, βA3/1-, βB2-, and γ-crystallin was measured by western blot and dot blot analysis. The morphology of the rat lens explants after culture was examined by hematoxylin-eosin staining, while crystallins were localized by immunofluoresence.

Results: Only dexamethasone and PDGF-AA showed an effect on relative crystallin levels. In the presence of dexamethasone the amount of αB-crystallin was increased in lens epithelial cells, but dexamethasone did not affect the crystallin spectrum in fiber cells. In rat lens epithelial explants cultured with PDGF-AA an increase in β- and γ-crystallin expression was seen. The spectrum of β- and γ-crystallins synthesized differed from that present in lens fiber cells. The cells expressing β- and γ-crystallin after culture with PDGF-AA were scattered in the epithelial cell layer and retained an epithelial morphology. PDGF-AA did not change the spectrum of crystallins synthesized in lens fiber cells but did enhance the rate of fiber cell differentiation, in agreement with results of others.

Conclusions: Both dexamethasone and PDGF-AA influence crystallin gene expression in cultured rat lens epithelial cells. Dexamethasone enhances the expression of αB-crystallin while culturing in the presence of PDGF-AA caused an increase in β- as well as γ-crystallin synthesis. Since at least the γ-crystallin genes are known to be silenced in epithelial cells by DNA methylation, PDGF-AA may be able to induce one of the steps towards fiber cell differentiation in some epithelial cells.

Introduction

Transparency of the lens is the result of its unusual cellular architecture and its unique protein content. Any disturbance of either the architecture or cell content is likely to lead to loss of lens transparency, i.e. cataract. The bulk of the lens is made up of lens fiber cells, while the anterior portion is covered by a monolayer of epithelial cells. The lens grows by division of epithelial cells and the progeny of these divisions elongate into new fiber cells and are continually added to the fiber mass at the lens equator [1,2]. In rat, as in other mammals, lens fiber cells synthesize three major classes of proteins, the α-, β-, and γ-crystallins. α-Crystallin is already present in lens epithelial cells but β- and γ-crystallin are present only in fiber cells and their presence is used as a biochemical marker for fiber differentiation [1,3]. Differentiation of fiber cells is also characterized by a sequence of morphologic changes which include cell elongation, organelle loss, and fiber denucleation [3,4]. These changes serve as morphological markers for fiber cell differentiation.

Lens growth, development, and differentiation is not an autonomous process but is directed by growth factors present in the vitreous and aqueous humors [5]. This was first shown by the classic lens reversal experiments of Coulombre and Coulombre in 1963 [6] from which it became clear that a factor in the posterior portion of the eye could trigger chicken embryo lens epithelial cells to differentiate into lens fiber cells in vivo. In mammals, primary lens cell differentiation is controlled by a member of the BMP family [7]. Signalling through TGF-β receptors is required for terminal differentiation of lens fiber cells, presumably with a member of the BMP growth factors as ligand, as TGF-β itself causes cataractous changes in vivo and in vitro [8,9]. A member of the FGF family directs secondary lens cell differentiation. Fibroblast growth factors FGF-1 and FGF-2 are sufficient to induce both the biochemical and morphologic events of secondary lens differentiation in vitro [10-12]. Studies with transgenic mice demonstrated that FGF-1 also stimulated fiber differentiation in vivo [13]. However, the natural FGF ligand stimulating secondary lens fiber differentiation in mammals remains unknown [14,15].

FGF is the only factor known to induce fiber cell differentiation of rat lens explants in vitro. Other growth factors, such as PDGF-AA and insulin/IGF-I, enhance the differentiating effect of FGF [16-18]. Insulin and IGF-I can also maintain fiber cell differentiation once it is initiated by FGF-2 [19,20]. Some hormones, like glucocorticoids and estrogen, have also been implied in lens metabolism. Prolonged use of glucocorticoids results in the formation of posterior subcapsular cataract [21-23]. On the other hand, female hormones have been suggested to have a role in protecting lens against cataracts, since the incidence of cataract in women is higher after menopause [24]. Several studies have begun to correlate estrogen levels with risk of cataract [25] and studies by Hales and associates show that estrogen confers protection against cataract induced by TGF-β in rat [26].

Crystallin gene expression is also controlled by growth factors. We have previously shown that upon withdrawal of FGF-2 from in vitro differentiating explants, crystallin gene transcription stops [27]. This growth factor effect appears to be mediated via the Maf Response Element, at least in the case of the γD-crystallin gene [28]. Furthermore, we have shown a differential effect of insulin and IGF-I on the rate of accumulation of the various crystallins in differentiating explants in vitro [29].

In the present study we tested a number of growth factors and hormones, including the factors tested by Potts et al. [30] in a study of the differentiation of chicken lens epithelial cells, for their ability to affect the synthesis of crystallins in rat lens epithelial or fiber cells. Two of the tested factors, dexamethasone and PDGF-AA showed an effect on relative crystallin levels in epithelial cells. Treatment with dexamethasone increased αB-crystallin levels, while treatment with PDGF-AA caused an unexpected increase in β- and γ-crystallin. This increase in β- and γ-crystallin was not accompanied by the typical morphological changes expected for fiber cell differentiation, indicating a discrepancy between the biochemical and the morphological markers for differentiation of lens fiber cells.

Methods

Rat lens epithelial explants

Explants were prepared from newborn (0-3 day old) rats and cultured in M199 medium containing 0.1% BSA and antibiotics (penicillin and streptomycin) as described [16]. The maintenance and treatment of animals were in full compliance with animal care guidelines comparable to those published by the Institute for Laboratory Animal Research (Guide for the Care and Use of Laboratory Animals). The University of Nijmegen's Animal Care Committee approved laboratory animal care protocols.

Dexamethasone (5x10^-7 M), estrogen (10^-9 M), PDGF-AA (30 ng/ml), EGF (5 ng/ml), NGF (10 ng/ml), M-CSF (40 ng/ml), BMP-2 (20 ng/ml), BMP-4 (20 ng/ml), and FGF-2 (50 ng/ml) were added to the medium as indicated. Medium was not refreshed during the culture period. Explants were harvested after 8 and 13 days and processed for SDS-PAGE and western blotting as described [19,20].

Dot blots

For each sample two or three explants were pooled. An extract of these explants was made by adding 100 μl buffer (50 mM Tris-HCl pH 8.0, 100 mM NaCl, 5 mM MgCl₂, 0.5% NP40) and placing the samples in a sonicating bath for 10 min. Samples then were centrifuged at 13,000 rpm for 15 min. The soluble fraction of the extract was used for dot blot analysis. Equal aliquots of each sample (1 μl) were spotted on a nitrocellulose filter (Hybond C-extra; Amersham Biosciences Europe GmbH, Roosendaal, the Netherlands) and stained as previously described [19,20] using antibodies raised against αA-, αB-, βA3/1-, βB2-, β-High-, γC-, or γS-crystallin. Note that the γC-crystallin antibody is likely to cross react with γA-, γB-, γD-, γE-, and γF-crystallin. This antibody is therefore denoted as γA/F-crystallin.

The stained blots were scanned with a BioRad GS-670 imaging densitometer at 42 μm resolution and analysed with Molecular Analyst software to quantitate the results. In all blots a standard range of water-soluble protein extracted from newborn rat lens fiber cells was included as a crystallin staining control and explant crystallin content was related to this standard.

Morphology

Explants were fixed in Carnoys solution (6:1 ethanol/acetic acid), embedded in paraffin wax, and serial sections cut at 5 μm. One out of every five sections was mounted and stained with hematoxylin and eosin. Sections were mounted in a solution of 10% Mowiol (Hoechst, Frankfurt, Germany) with 1.2% diazabicyclooctane. Sections were examined using a light microscope (Zeiss Axiovert 135 TV; Zeiss, Oberkochen, Germany).

Immunofluorescence

For detection of α-, β-, and γ-crystallin in sections, immunostaining was performed as previously described [1]. αA-, βB2-, or γS-crystallin antibodies were diluted 1:100 into a HeLa cell extract and incubated for 1 h at room temperature to reduce non-specific staining. Sections were incubated overnight with the pre-adsorbed antibodies at room temperature, washed, and stained with the secondary antibody (anti-rabbit FITC diluted 1:20; DAKO A/S, Glostrup, Denmark) for 3 h at room temperature. After washing, sections were mounted in a solution of 10% Mowiol (Hoechst) with 1.2% diazabicyclooctane. Sections were examined using a fluorescence microscope (Zeiss Axiovert 135 TV; Zeiss) with a 40x oil-immersion, 1.3 NA fluor objective lens, attached to a Coolsnap fx monochrome digital camera (Roper Scientific, Tucson, AZ).

Results

Effect of hormones and growth factors on the accumulation of crystallins

A number of factors that have been shown to affect the proliferation or differentiation of lens epithelial cells were tested for their effect on the spectrum of crystallin synthesis in explanted rat lens epithelial cells in the absence of FGF-2 (non-differentiating conditions, denoted as epithelial cells) and in explanted rat lens epithelial cells in the presence of FGF-2 (differentiating conditions, denoted as fiber cells). In addition, the factors were tested for the ability to maintain differentiation once initiated by a 24 h preincubation with FGF-2. None of the factors tested was able to do so reproducibly. Estrogen, EGF, NGF, M-CSF, BMP-2, and BMP-4 did not have any reproducible effect on crystallin accumulation in either epithelial or fiber cells. In the presence of dexamethasone and PDGF-AA a clear change in the crystallin spectrum of epithelial cells was seen and the effect of these two factors was examined further.

Effects of dexamethasone

When explanted rat lens epithelial cells were cultured with dexamethasone for up to 13 days the level of αB-crystallin increased (Figure 1, lanes marked D) as compared to explants incubated in the absence of growth factors (Figure 1, lanes marked 0). No significant increase in αB-crystallin was seen in the presence of a combination of FGF-2 and dexamethasone as compared with FGF-2 treated explants (Figure 1, lanes marked D+F and F). No increase in αA-, β-, or γ-crystallin was found (dot blots not shown) in explants incubated with dexamethasone alone or with dexamethasone and FGF-2 as compared to FGF-2 treated explants.

Effects of PDGF-AA

PDGF-AA has been previously reported to enhance fiber cell differentiation induced by FGF-2 as judged by morphology and the accumulation of β-crystallin as detected by immunolabelling [17]. In agreement with these data we found that, except for αA-crystallin, crystallin levels were higher in explants cultured for 8 days in the presence of both FGF-2 and PDGF-AA than with FGF-2 alone (Figure 2 and Figure 3, compare lanes marked F with those marked P+F). However, after 13 days of culture under differentiating conditions, there was no significant difference between the crystallin levels with or without PDGF-AA.

In the presence of PDGF-AA alone, explanted epithelial cells presented a higher level of crystallins compared with the negative control (Figure 2A, compare lanes marked P with those marked 0). To analyze this accumulation further, western blots of duplicate gels were made and stained for αA-, βB2-, β-, and γA/F-crystallin (Figure 2B-D). The quantitation of these signals showed an increase in α-, β-, and γ-crystallin levels of samples treated with PDGF-AA relative to the negative controls in lens epithelial cells. More extensive dot blot analyses (dot blots not shown), using available crystallin antisera, confirmed and extended this data. As shown in Figure 3, in the presence of PDGF-AA, αA-, βA3/1, and γA/F-crystallin reached the same levels as in FGF-2 treated explants. In contrast, the increase in βB2- and γS-crystallin was less, while the level of αB-crystallin did not change significantly in the presence of PDGF-AA. It is noteworthy that, compared to the effect of FGF-2, PDGF-AA enhanced the accumulation of βA3/1-crystallin relative to βB2-crystallin and of γA/F-crystallin relative to γS-crystallin.

Morphology of explants cultured in PDGF-AA and immunolocalization of crystallins

The finding of β- and γ-crystallin synthesis in PDGF-AA treated explants was unexpected as it has been previously reported that PDGF-AA does not induce either morphological or biochemical markers of fiber cell differentiation in explanted rat lens epithelial cells [17]. We therefore examined the morphology of explants cultured with PDGF-AA and located β- and γ-crystallin expressing cells by immunofluorescence. In these experiments we used explants cultured for 13 days for dot blot analyses (see Figure 3) showed little accumulation of γ-crystallin after 8 days of culture. As shown in Figure 4, lens explants cultured in the presence of PDGF-AA did not show the typical multilayered morphology of explants cultured with FGF-2 (compare Figure 4E with Figure 4I and Figure 4M). Compared to control explants, incubated without growth factors (Figure 4A), cells of PDGF-AA treated explants were larger and the neat cobble stone morphology of the control explants was lost. Combining PDGF-AA and BMP-4 did not lead to morphological differentiation (data not shown).

Most of the cells in control (Figure 4B) as well PDGF-AA treated explants (Figure 4F) strongly fluoresced for α-crystallin. In the PDGF-AA treated explants, some scattered cells, or occasionally a small group of cells, showed strong immunoreactivity for β- and γ-crystallin (Figure 4G and Figure 4H, respectively). No expression of β- or γ-crystallin was found in the control explants (Figure 4C,D). Note that the pictures shown were taken from the central part of the epithelium to exclude the β- or γ-crystallin containing cells that had already been triggered for differentiation in vivo as has been previously found for cells at the periphery of explants [31]. As a positive control, explants were cultured in the presence of FGF-2 (Figure 4I-L) and PDGF-AA (Figure 4M-P). Histological sections of these explants showed a larger number of layers compared with the differentiated explants in the presence of FGF-2 alone (Figure 4M compared to Figure 4I). Immunostaining showed the expression of α-, β-, and γ-crystallins in explants cultured with FGF-2 or FGF-2 and PDGF-AA (Figure 4J,K,L and Figure 4N,O,P).

Discussion

Of the hormones and growth factors tested in the experiments reported here, only dexamethasone and PDGF-AA showed an effect on crystallin expression in either lens epithelial or fiber cells. The lack of effect of BMP can be explained by the fact that this factor only induces the differentiation of primary fiber cells [7], while the in vitro differentiated fiber cells used here are secondary fiber cells.

Regulatory elements that are possible targets of growth factor signaling are a common feature of crystallin promoters. The β- and γ-crystallin promoters contain Maf Response Elements, which are the targets of the Maf transcription factors [32]. Maf transcription factors can heterodimerize with members of the AP-1 family, well known transmitters of growth factor signaling [33,34]. Both the αA- and αB-crystallin promoters contain AP-1 sites [35]. The αB-crystallin promoter also has a glucocorticoid response element (GRE) as it is activated by dexamethasone in NIH3T3 cells and in human satellite cells [36,37]. Similarly, we found that dexamethasone enhanced the expression of αB-crystallin in lens epithelial cells. Unexpectedly, it did not increase the level of αB-crystallin in fiber cells. Insulin also did not enhance αB-crystallin expression in fiber cells although it did so in epithelial cells [29]. Possibly, the rate of transcription of the αB-crystallin gene is already at its maximum level in fiber cells and cannot be further enhanced. Dexamethasone also did not increase the expression of β- or γ-crystallin genes in either epithelial or fiber cells, even though at the sequence level putative GREs are present in the promoters of these genes (unpublished data). Since the expression of at least some of these genes is increased in the presence of insulin (and FGF-2) [29] and is thus not maximal during culture with FGF-2 alone, the lack of effect of dexamethasone on transcription of these genes suggests that the sequence similarity to GRE is not biologically significant. Alternatively, the glucocorticoid receptor may be absent from fiber cells. It has been suggested that lens cells lack a glucocorticoid receptor [38], but more recent results [39,40] show that at least the lens epithelial cells do have this receptor.

In the presence of PDGF-AA some epithelial-like cells expressed β- and γ-crystallin. This finding cannot be merely explained by the presence of growth factor responsive elements in these genes. We have previously shown that expression of the γ-crystallin genes correlates with demethylation of the proximal promoter regions of these genes and that this demethylation occurs in early fiber cell differentiation [40-43]. Hence, a first differentiation step needs to occur before the promoter region of the γ-crystallin genes can be used. This suggestion is supported by the effect of insulin or IGF-I on γ-crystallin gene expression. Insulin/IGF-I activates expression of these genes in lens fiber cells but not in lens epithelial cells. By western blotting very little γ-crystallin is detected in insulin or IGF-I treated explants and immunolocalization showed that the few cells that contain β- or γ-crystallin are likely to be cells that migrated from the periphery of the explant [19]. In contrast, in PDGF-AA treated explanted rat lens epithelial cells, β- and γ-crystallin was readily detected by western blotting (Figure 2C,D) and immunolocalization showed the β- and/or γ-crystallin containing cells to be part of the epithelial layer (Figure 4G,E). Judging merely on the basis of biochemical markers of differentiation (β- and γ-crystallin accumulation) our results thus suggest that PDGF-AA is able to trigger the differentiating response in lens epithelial cells, at least in some single cells in the central part of the explants. Reneker and Overbeek [44] also found that lens epithelial cells of a transgenic mouse overexpressing PDGF-AA showed some characteristics of fiber differentiation. The anterior epithelial cells were elongated and expressed fiber-cell specific β-crystallin. However, others [17] did not find any characteristics of fiber differentiation nor β-crystallin expression in 10 day old rat lens epithelial explanted cells cultured for eight days in the presence of PDGF-AA. The discordance between those results and the ones presented here might be due to the shorter culture period used in the studies reported [17]. Furthermore, we used younger rats in our study (0 to 3 day old rats) and it has been shown that with increasing age of the lens explant donor the rate of differentiation of the explants decreases [31,45,46].

The optical properties of the lens are determined by the gradient of crystallins in the lens. This gradient is set by the development and differentiation dependent regulation of crystallin gene expression, with the additional complexity as shown here and elsewhere, that ocular growth factors influence the rate of crystallin accumulation. Due to its mode of growth (addition of layers to the outside) the mature lens retains any changes in the crystallin spectrum due to ocular growth factor changes. In the prevalence hypothesis for age-related cataract, α-crystallin chaperones the unfolding β- and γ-crystallins and cataract would ensue when the α-crystallin chaperone capacity is exhausted (reviewed in [47]). From the study of rodent model systems it is likely that (differences in) the spectrum of ocular growth factors is one of the parameters that determines the ratio of α-crystallin to β- and γ-crystallin and thus the time of onset of age-related cataract in man.

Acknowledgements

The authors would like to acknowledge the assistance of Jan Derksen and Elisabeth Pierson in the microscopy studies. This work was supported by the European Union research grant BMH4-CT98-3895.

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