An estrogen receptor repressor induces cataract formation in transgenic mice

Abstract

Despite the high prevalence of age-related cataracts, there are currently no known therapies to delay or prevent their occurrence. Studies in humans and rodent models suggest that estrogen may provide protection against age-related cataracts. The discovery of ocular estrogen receptors (ERs) indicates that estrogen protection may result from direct interactions with its receptors in the eye, instead an indirect consequence from effects on another tissue. Studies in our transgenic mouse model validate the concept that estrogen is beneficial for the eye. These mice express ERΔ3, a dominant-negative form of ERα that inhibits ERα function. In the ERΔ3 transgenic mice, cortical cataracts spontaneously form in ERΔ3 females after puberty and progress with age. The cataracts initiate in the equatorial region of the lens where the epithelial cells differentiate into elongating fiber cells. Cataract formation can be prevented if the females are ovariectomized before, but not after, sexual maturity. Both male and female ERΔ3 mice develop cataracts after neonatal treatment with the potent estrogen diethylstilbestrol (DES). The incidence of spontaneous and DES-induced cataracts in ERΔ3 mice is 100%, yet these cataracts are absent from the wild-type mice. These data suggest that repression of estrogen action induces cortical cataract formation because estrogen is required to activate the ERΔ3 repressor. Evidence of DES-induced cataracts in the ERΔ3 males as well as the females suggests that estrogen is important in lens physiology in both sexes. The ERΔ3 mice provide a powerful model for assessing the role of estrogen in maintaining the transparency of the lens.

Currently there is no known therapy that can delay or prevent age-related cataracts that plague the ever-growing numbers of aging men and women. The incidence of age-related cataracts is higher in women (1), and their onset coincides with estrogen deficiency that occurs after menopause. Until recently, the eye had not been considered to be an estrogen target tissue; however, recent studies have begun to correlate estrogen status with risk of cataracts (2–4). In addition, the estrogen receptor (ER) has been detected in ocular tissues (5, 6,).‖ Estrogens acting through the ER modulate transcription of estrogen-responsive genes (7). The ER is a ligand-dependent transcription factor belonging to the nuclear receptor superfamily (8). Currently, two subtypes of the ER are known to exist, ERα and ERβ. The classic ER, which has been associated for decades with female reproductive responses, is now designated as ERα. The recently discovered second gene codes for ERβ (9). Expression of the ERα protein has been detected in the rat and bovine (5) and human (6) retina and in the rat lens‖; ERβ mRNA has been detected in the rat lens (10). The presence of these receptors indicates that the eye can respond directly to estrogens or antiestrogens, such as tamoxifen.

Studies in women suggest that estrogens may protect against the development of senile cataracts. The three types of age-related cataracts: nuclear sclerosis, cortical cataract, and posterior subcapsular opacity, occur in the central, peripheral, and posterior regions of the lens, respectively. The Beaver Dam eye study indicated a modest protective effect of lifetime estrogen exposure on cataract risk (4). In this study, a later onset of menopause was associated with a decreased risk of cortical cataracts; younger age at menarche (onset of menstruation) was associated with a protective effect regarding nuclear sclerosis; and hormone replacement therapy was associated with a lower prevalence of nuclear cataracts. In the Blue Mountains eye study, current use of hormone replacement therapy in women >65 years of age was associated with a lower prevalence of cortical cataracts (2). In addition, an increased prevalence of cortical, nuclear, and posterior subcapsular cataracts with later ages at menarche was detected (2), suggesting that a shorter lifetime exposure to estrogen may influence all types of age-related cataracts. In a study comparing lens transmittance in women taking hormone replacement therapy (measuring nuclear sclerosis), estrogen also was found to be protective (3). These data strongly suggest that estrogen may protect the lens against opacities. Furthermore, cataracts occur in women taking the antiestrogen tamoxifen to treat or prevent breast cancer (11, 12), indicating that inhibiting estrogen action can result in the loss of lens transparency.

Two rodent model systems also demonstrate that estrogen may protect the lens from developing cataracts. In an in vivo model, using ovariectomized rats treated with methylnitrosourea, estrogen treatment diminished the incidence of cortical cataracts induced by the mutagen (10). In addition, the potency of the estrogens tested influenced the efficacy of inhibiting lens opacities, with the weaker estrogen, estrone, being less effective than 17β-estradiol. In the other model, using cataracts induced by transforming growth factor-β in cultured rat lenses, subcapsular cataracts were reduced when 17β-estradiol was administered to the rat before lens culture or directly to the culture medium before transforming growth factor-β treatment (13, ∥). These data provide further evidence that estrogens can protect the lens from developing cataracts.

In this study, a transgenic mouse model is examined that expresses ERΔ3, a natural variant of ERα resulting from the in-frame deletion of exon 3 by alternative splicing. The deletion of exon 3 (Δ3) of the human ERα was originally detected in the T47D breast cancer cell line (14). Deletion of exon 3 from the ERα results in a receptor protein that is lacking only a portion of the DNA-binding domain, mainly the second zinc finger. Other functional domains for nuclear localization, transactivational domains AF-1 and AF-2, ligand-dependent dimerization, and ligand binding remain intact. In transfected HeLa cells, the human ERΔ3 variant alone did not have the ability to activate transcription of an estrogen-responsive reporter construct (14). However, when ERΔ3 is cotransfected with ERα, dominant-negative activity of the ERΔ3 receptor is evident by its ability to inhibit ERα activation of the same estrogen-responsive reporter. Transfecting a 1:10 ratio of ERα to ERΔ3 vectors was required to inhibit ≈80% of the wild-type (wt) receptor activity (14). The actual ratio of ERα to ERΔ3 required in an individual cell to suppress ERα activity has not been determined.

Transgenic mice expressing the mouse ERΔ3 cDNA were generated to examine the function of the ERΔ3 dominant-negative receptor in vivo. Expression of the ERΔ3 cDNA was directed by the rat osteocalcin promoter, truncated to ≈200 bp of 5′-flanking sequences, in conjunction with a viral murine enhancer (Harvey murine sarcoma virus long terminal repeat). In the ERΔ3 construct, truncation of the osteocalcin promoter resulted in the loss of tissue-specific expression that is usually limited primarily to bone. Therefore, expression of ERΔ3 transgene is detected in most tissues in both lines of ERΔ3 mice (V.L.D., R.R. Newbold, J.F.C., E.H. Goulding, W. Jefferson, E.M. Eddy, B.C. Bullock, and K.S.K., unpublished data). The only spontaneous phenotype evident in the ERΔ3 mice was cataract formation in the females of one line, line F. The effects of the ERΔ3 repressor on cataract development in the transgenic mice are described below.

Materials and Methods

Animals.

All procedures involving the mice were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals with a protocol approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee. All mice were housed with 12 h:12 h light:dark cycles in a temperature-controlled room with NIH 31 chow and water provided ad libitum. The ERΔ3 mice were generated on the FVB/N background strain as will be described elsewhere (V.L.D., R.R. Newbold, J.F.C., E.H. Goulding, W. Jefferson, E.M. Eddy, B.C. Bullock, and K.S.K., unpublished data). The ERΔ3 and wt FVB/N littermates were produced in the ERΔ3 colony in the National Institute of Environmental Health Sciences animal facilities. At 3 wk of age, mice were weaned, genotyped by using genomic DNA isolated from tail biopsies (15), and analyzed by PCR as described (16). Official designations for lines D and F are FVB/n-TgN(mERΔ3os)04Eme and FVB/N-TgN(mERΔ3os)06Eme, respectively.

Treatments.

For the diethylstilbestrol (DES) treatments and ovariectomized mice, wt FVB/N female mice (obtained from the National Cancer Institute Animal Program, Bethesda, MD) were bred with hemizygous ERΔ3 males. The progeny were treated with daily injections of DES (Sigma) dissolved in corn oil at the dose of 2 μg per pup per day on days 1–5 after birth. Controls were left untreated. Bilateral ovariectomies (OVXs) were performed on untreated females at either 4 wk or 2 mo of age.

Histological Analysis.

The eyes from DES-treated, control (intact), and ovariectomized ERΔ3 (transgenic) and FVB/N (wt) mice were enucleated and fixed in neutral buffered 10% formalin solution for a minimum of 10 days. Eye size was measured by using a calibrated micrometer on a Zeiss Op Mi-1 stereo dissecting microscope. Eyes were dehydrated through a graded alcohol series and embedded in glycol methacrylate. Sections (5 μm) were cut through the pupillary–optic nerve axis and stained with hematoxylin and eosin (H&E).

RNase-Protection Assay.

RNA was prepared by using a guanidine isothiocyanate/CsCl gradient procedure from whole eyes from sexually mature females, both ERΔ3 (lines D and F) and wt mice. The RNase-protection assay was performed as described (16) by using a probe generated from the 3′ end of the mouse ERα cDNA and rabbit β-globin sequences (from the vector used to generate the transgenic mice). This probe discriminates between the transcripts for the endogenous ERα and ERΔ3 transgene, protecting fragments of 368 nt and 422 nt, respectively. The cyclophilin probe, 103 nt, was used as a control probe to normalize the RNA levels between samples.

Results

Detection of Cataracts in ERΔ3 Females.

Two lines of ERΔ3 mice, lines D and F, express the ERΔ3 transgene in a variety of tissues, including the liver, lung, kidney, uterus, mammary gland, spleen, heart, brain, and bone (V.L.D., R.R. Newbold, J.F.C., E.H. Goulding, W. Jefferson, E.M. Eddy, B.C. Bullock, and K.S.K., unpublished data). In line F, 100% of hemizygous females developed lens opacities that were visible by gross inspection ≈3 mo of age. As the females age, the opacities became more apparent (Fig. 1 Upper). Histological analysis of eyes from line F females demonstrated that cataract formation was initiated in the equatorial region of the lens and progressed to the cortex and nucleus with age, whereas the lenses in aging wt females (FVB/N mice) remained normal (Fig. 2). In addition, histopathology demonstrated that cortical cataracts appeared by age 2 mo in line F females, before clouding of the lens could be detected by gross ocular examination. In the early stages of cataract formation, degenerative changes in elongating fiber cells in the equatorial region of the lens are evident (Fig. 3). With age, the cataractous changes become extensive, involving nuclear as well cortical regions of the lens (Fig. 2). The lenses of the line F ERΔ3 females are characterized by cortical vacuoles, globulin formation, and liquefaction and fragmentation in the equatorial, anterior, and posterior subcapsular cortex, in contrast to the normal pattern of even, closely apposed fibers in the wt females (Fig. 2). Cataracts were not apparent by gross examination or histological analysis in males in line F or either sex in line D at any age.

Figure 1.

Visible lens opacities in ERΔ3 mice. ERΔ3 line F females at ages 7 mo (Upper Left) and 15 mo (Upper Right). DES-treated males age 11 mo, wt (Lower Left) and line D ERΔ3 (Lower Right). Cataracts are visible by gross inspection in the ERΔ3 transgenic mice only.

Figure 2.

Histological evidence of cortical cataracts in ERΔ3 females. Sections of eyes from wt, FVB/N female at 13 mo of age (WT female) and ERΔ3, line F females at 1 mo, 3.5 mo, and 9 mo of age (ERΔ3 female) are shown at ×25 (Upper) and ×200 (Lower). The Lower panels magnify the bow region of the lens. Retinal degeneration is evident in both the wt and ERΔ3 mice because the ERΔ3 mice were generated on the FVB/N background strain, which is homozygous for rd, the retinal degeneration gene (22). (The sections were H&E stained.)

Figure 3.

Early changes in the equatorial region of the lens of ERΔ3 females. Histology sections of the eyes from a wt (Upper) and an ERΔ3 line F (Lower) female at 2 mo of age, before detecting visible cataract by gross ocular examination. The lens of the ERΔ3 female shows decreases in normal lens cortical fiber nuclei (open arrow) and increases in pyknotic nuclei (small arrows) compared with the wt female lens, indicating loss of lens cortical fibers in the transgenic mice. (The sections were H&E stained and are at a magnification of ×400.)

Effects of Estrogen on Cataract Development.

Endogenous estrogens.

The presence of cataracts only in transgenic female mice expressing an ERα variant suggested that estrogen may play a role in the formation of the cataracts observed in the ERΔ3 mice. To investigate whether estrogen was required for cataract formation, line F ERΔ3 and wt females were ovariectomized to produce estrogen-deficient mice. In sexually mature, line F ERΔ3 females, which had produced estrogens for ≈1 mo before ovariectomy (post-OVX, Fig. 4), cataracts did form, as expected. However, if the ovaries were removed from females before sexual maturity, when physiological production of estrogen is minimal (4 wk of age), the lenses of line F ERΔ3 females remained clear even 7 mo after surgery (pre-OVX, Fig. 4). Therefore, the 1 mo of exposure to ovarian hormones with post-OVX females was critical for inducing cataracts. Removing the source of estrogen production before sexual maturity in the pre-OVX group protected lens transparency. For the wt females, normal lenses were evident irrespective of hormonal status—i.e., intact and pre-OVX and post-OVX groups (data not shown).

Figure 4.

The effects of endogenous estrogen on cataract development in 8-mo-old line F female mice. These depict the lenses of ERΔ3 line F females that had their ovaries removed to induce estrogen deprivation at ≈4 wk (pre-OVX) or 2 mo of age (post-OVX) or retained their ovaries (intact). Cataracts are evident only in the intact and post-OVX ERΔ3 females. [The sections were H&E stained and are ×60 (Upper) and ×240 (Lower).]

Exogenous estrogens.

Because endogenous estrogens were required to induce cataracts in the ERΔ3 females, a potent synthetic estrogen, DES, was administered to neonatal ERΔ3 (lines D and F) and wt littermates (daily from 1 to 5 days of age) to determine whether estrogen treatment before sexual maturity could also cause cataract formation. All of the ERΔ3 males and females, both lines D and F, developed cataracts, indicating that DES, like endogenous estrogens, activated the inhibitory activity of the ERΔ3 repressor. The presence of cataracts in both lines of ERΔ3 mice is important because it verifies that cataract induction is a result of expression and activation of the ERΔ3 inhibitor vs. because of disruption of another gene at the site of transgene insertion. Gross examination of the eyes of DES-treated ERΔ3 mice demonstrated that lens opacities were often localized to the interior of the eye (Fig. 1 Lower Right). Cataracts were clearly evident in preweanlings when the eyes first opened at ≈2 wk of age (the eyes were not examined before this age). Histological analysis demonstrated severe nuclear cataracts in both ERΔ3 males and females that often result in hypermature cataracts with lens rupture (Fig. 5, 8-mo-old mice; Fig. 6, 2-wk-old mice). No lens abnormalities were evident in wt males or females treated with DES (Fig. 5).

Figure 5.

The effects of neonatal estrogen treatment on ERΔ3 mice. The mice in all of the panels were 8 mo of age. The lenses of mice neonatally treated with DES, 1–5 days of age, for wt (FVB/N) female (wt female), ERΔ3 line F female (ERΔ3 female), and ERΔ3 line F male (ERΔ3 male). Cataracts with lens rupture are evident in both ERΔ3 males and females. [The sections were H&E stained and are ×62.5 (Upper) and ×250 (Lower).]

Figure 6.

Cataracts in 2-wk-old ERΔ3 females after neonatal DES treatment. The lenses of ERΔ3 female mice (line F) that were untreated (Left) or treated daily from day 1 to 5 after birth (Right) are shown. Cataracts are evident by 2 wk of age only in the female treated with DES. Loss of photoreceptors because of retinal degeneration in the FVB/N strain has not yet occurred at this age. [The sections were H&E stained and are ×50 (Upper) and ×200 (Lower).]

In addition to cataract induction, neonatal DES treatment resulted in microphthalmia in both sexes of the ERΔ3 mice. Eye size in DES-treated ERΔ3 males and females was typically ≈80% of that measured for the treated wt mice (Fig. 7). The eyes otherwise appeared normal, except for the cataractous lens (see Figs. 1, 5, and 6). No difference in eye size was observed between untreated ERΔ3 and wt mice (data not shown).

Figure 7.

The effect of neonatal DES treatment on eye size. Eye size of DES-treated mice (treated with 2 μg per mouse s.c. on days 1–5 after birth) is shown as the average diameter of both eyes (after fixation), from both anterior-to-posterior and medial lateral measurements, using a calibrated micrometer. The mice were 12–14 mo old. Values given are mean ± SE. ERΔ3 lines F and D and wt littermates were all DES treated. wt males, n = 10; wt females, n =6; ERΔ3 line F males, n = 5 and females, n = 5; and ERΔ3 line D males, n = 4 and females, n = 10. Significance was assessed by using the unpaired Student's t test (α = 0.05). P < 0.001 for ERΔ3 line F and D males and females compared to wt.

Detection of Ocular ERΔ3 Transcript.

The ERΔ3 transcript was detected by RNase-protection assay in RNA isolated from eyes of both line D and F females (Fig. 8). ERα was also detectable in the eyes of both the ERΔ3 and wt females (Fig. 8). The levels of ERΔ3 are in excess of ERα levels, with the transgene constituting 87% and 92% of the total ERα transcripts (ERα + ERΔ3 mRNAs) in lines F and D, respectively.

Figure 8.

Levels of ERΔ3 transgene and endogenous ERα transcripts in the eyes of ERΔ3 females determined by the RNase-protection assay. Total RNA was prepared from whole eyes from wild-type (WT), ERΔ3 line F (F), and ERΔ3 line D (D) females and uterus from ERΔ3, line D female (Ut). The T arrow represents the ERΔ3 transgene message, the E arrow designates the endogenous ERα transcripts, and the cyc arrow marks the cyclophilin-protected band, used for normalization between samples. The lane labeled p is the probe only with no RNA added, and m designates the molecular weight markers. The ratio of messages for ERΔ3:ERα is 11:1 for the line D female and 6:1 for the line F female.

Discussion

Cortical cataracts develop spontaneously in ERΔ3 line F transgenic females and progress with age. Estrogen was required to induce cataracts in the ERΔ3 mice. Estrogen dependency was demonstrated by (i) the absence of cataracts in females that had their ovaries removed before, but not after, reaching sexual maturity (Fig. 4) and (ii) the development of cataracts in both males and females after neonatal treatment with the potent estrogen DES (Figs. 5 and 6). (These results are summarized in Table 1.) The RNase-protection assay data demonstrate that both the native ERα and the ERΔ3 transgene are expressed in ocular tissues (Fig. 8), indicating that the effect of estrogen on cataract development can be modulated directly in the eye.

Table 1.

Cataract incidence in ERΔ3 and wt mice

Sex	Mice	Cataract incidence, %
		Endogenous estrogens, intact	No endogenous estrogens, pre-OVX	Exogenous estrogen, DES-treated
Females	ERΔ3 line F	100	0	100
	ERΔ3 line D	0	0	100
	wt	0	0	0
Males	ERΔ3 line F	0	NA	100
	ERΔ3 line D	0	NA	100
	wt	0	NA	0

NA, not applicable. 

In the ERΔ3 transgenic mouse model, the postulated mechanism of inducing cortical cataracts is by interfering with estrogen signaling, based on the dominant-negative activity of this receptor. This is consistent with the effects of another estrogen inhibitor because cataracts are a known side effect in women treated with tamoxifen (11, 12). The reason for the estrogen dependence for cataract formation can be logically linked to the prerequisite for estrogen binding to the receptor to induce dimerization of ERΔ3 with itself, ERα, or ERβ by means of the ligand-dependent dimerization domain. The other dimerization domain, which is ligand-independent and the weaker dimerization domain for the ER (17), is lost with the exon 3 deletion. Thereby, in mice expressing the ERΔ3 receptor, estrogen would function as an inhibitor by activating the dominant-negative ERΔ3. Therefore, our data concur with other studies demonstrating that estrogen protects the lens from developing cataracts and that loss of estrogen or inhibition of its actions may be instrumental in increasing the risk of developing age-related cataracts.

In ERΔ3 mice, because DES induced estrogen-dependent cataracts in both lines of ERΔ3 mice and both lines would have random, unique sites of insertion of the transgene, cataract development must be due to ERΔ3 expression and not to disruption of another gene at the site of insertion. In addition, excessive accumulation of transgene product within the lens has been shown to disrupt the lens fiber formation simply because of the levels expressed and not related to the function of the expressed protein (18). However, because cataracts occur in the ERΔ3 mice only when sufficient levels of estrogen are present, the loss of lens transparency could not be due simply to the overexpression of the ERΔ3 protein. Therefore, these data indicate that functional ERΔ3 repressor causes cataract development in both lines of ERΔ3 mice.

Although both lines of ERΔ3 mice express the ERΔ3 repressor in the eye, only line F females develop cataracts spontaneously. The fact that a potent estrogen, DES, can induce cataracts in the line D mice suggests that insufficient levels of estrogen may be responsible for the lack of spontaneous cataracts in this line. Thereby, line F females may produce higher levels of endogenous estrogens because of differential expression in another tissue. Alternatively, the differential levels of ERΔ3 expression in specific ocular tissues in line D may prevent cataract development unless endogenous estrogen levels are supplemented. Further examinations of these lines are necessary to determine whether either or both of these factors result in the lack of spontaneous cataracts in line D females.

The single layer of lens epithelial cells is essential for maintaining the metabolic homeostasis and physiological transparency of the entire lens (19). Conditions that perturb epithelial cell physiology and differentiation can impact cell survival and result in lens opacification. In ERΔ3 mice, in the presence of estrogen, the cortical cataracts always initiate in the equatorial region of the lens, where the differentiation of the epithelial cells into the denucleated, elongating fiber cells occurs. The initiation of cataracts in this critical region suggests that estrogen may be a strategic regulatory factor in the differentiation process. The ability of the ERΔ3 repressor to induce cataracts in the male mice indicates that estrogen is important to the lens physiology in both sexes.

Induction of nuclear cataracts by neonatal DES treatment indicates that DES activation of ERΔ3 disrupts embryonic lens cell differentiation. Importantly, these effects are grossly different from the well-known effects induced by glucocorticoid administration (20). Corticosteroid cataracts are mainly delimited to the posterior subcapsular region of the lens, although ultrastructural changes have been noted in lens epithelia from patients undergoing corticosteroid therapy (21). Thus, the lenticular changes seen in the present study are different from those induced by other steroid hormones and appear to involve early development in the epithelium and differentiating fiber cells. The presentation of microophthalmia after neonatal DES treatment suggests that the ERΔ3 repressor also may alter other relevant factors in eye development.

The ERΔ3 mice were generated on the FVB/N background strain, which is homozygous for rd, the retinal degeneration gene (22). Therefore, retinal degeneration is inherent to the strain and not related to ERΔ3 expression. The effects of ERΔ3 expression on cataract development in a background strain without the −rd/−rd genotype require further examination. However, because cataracts developed in neonatal mice before retinal degeneration, retinal degeneration is not expected to contribute to ERΔ3-induced cataract development.

Estrogen influences diverse tissues, including the cardiovascular system, bone, and brain, in addition to having its effects on reproduction. Many age-related diseases that occur in menopausal women are linked to estrogen deficiency and benefit from estrogen therapy. The studies in women and rodent models, including the ERΔ3 model, support the concept that estrogen also may help preserve the transparency of the lens. Future investigations with this model should help uncover the role of estrogen in the lens pathophysiology and its potential as a therapy to delay or prevent age-related cataracts.

Abbreviations

ER

estrogen receptor

ERΔ3

ERα variant with exon 3 deletion

DES

diethylstilbestrol

OVX

ovariectomy

wt

wild type

H&E

hematoxylin/eosin