Inhibition by Female Sex Hormones of Collagen Gel Contraction Mediated by Retinal Pigment Epithelial Cells

Abstract

Purpose.: Collagen contraction mediated by retinal pigment epithelial (RPE) cells contributes to the pathogenesis of proliferative vitreoretinopathy (PVR). We examined the effects of sex hormones on this process.

Methods.: Mouse RPE cells were cultured in a type I collagen gel and exposed to 17β-estradiol, progesterone, or dehydro-epiandrosterone. Collagen contraction induced by transforming growth factor–β2 (TGF-β2) was determined by measurement of gel diameter. Expression of α–smooth muscle actin (α-SMA), as well as phosphorylation of Smad2 and myosin light chain (MLC), was examined by immunoblot analysis. Matrix metalloproteinase (MMP) release was evaluated by gelatin zymography. Fibronectin and interleukin-6 secretion was measured with immunoassays.

Results.: The female sex hormones 17β-estradiol and progesterone inhibited TGF-β2–induced collagen contraction mediated by RPE cells, whereas the male sex hormone dehydro-epiandrosterone had no such effect. The TGF-β2–induced release of MMP-2 and MMP-9 from RPE cells was also inhibited by 17β-estradiol and progesterone, and the MMP inhibitor GM6001 attenuated TGF-β2–induced collagen contraction. Expression of the mesenchymal markers α-SMA and fibronectin, interleukin-6 release, and Smad2 and MLC phosphorylation induced by TGF-β2 were all inhibited by 17β-estradiol and progesterone. Immunohistochemical analysis also detected nuclear immunoreactivity for estrogen and progesterone receptors in proliferative fibrocellular membranes of PVR patients.

Conclusions.: Female sex hormones inhibited TGF-β2–induced collagen contraction mediated by RPE cells. This action appeared to be mediated through inhibition both of MMP, α-SMA, and fibronectin expression as well as of Smad2 and MLC phosphorylation. Female sex hormones might thus prove effective for the treatment of PVR.

Introduction

Proliferative vitreoretinopathy (PVR) is a major complication of rhegmatogenous retinal detachment and is characterized by the formation of proliferative fibrocellular membranes on the retinal surface as well as in the vitreous and subretinal space. Contraction of the retina mediated by these proliferative fibrocellular membranes can result in tractional retinal detachment and consequent failure of retinal detachment surgery and a marked reduction in visual acuity. The pathogenesis of PVR is also affected by local inflammation. ¹ Retinal pigment epithelial (RPE) cells contribute to the formation of proliferative fibrocellular membranes in response to stimulation by various growth factors and cytokines—such as platelet-derived growth factor, transforming growth factor–β (TGF-β), and hepatocyte growth factor—that pass through the blood–retinal barrier after retinal detachment. ^2–4 The RPE cells undergo transdifferentiation to become fibroblast-like cells and produce extracellular matrix (ECM), thereby giving rise to the proliferative fibrocellular tissue on or adjacent to the detached retina. These cells are characterized by the expression of α–smooth muscle actin (α-SMA), fibronectin, and vimentin, all of which are markers of the epithelial–mesenchymal transition (EMT), ⁵ and they also produce matrix metalloproteinases (MMPs) and connective tissue growth factor (CTGF). ⁶ Transforming growth factor–β plays a key role in induction of the EMT, which is mediated predominantly by the Smad signaling pathway. The TGF-β–induced phosphorylation of Smad2 and Smad3 triggers their translocation to the nucleus and the consequent transcriptional activation of genes associated with the EMT. ⁷ The contraction of cells that have undergone the EMT is mediated in part by the actomyosin system, which is activated as a result of myosin light chain (MLC) phosphorylation. ⁸  

The major sex hormones are estrogens and progesterone in females and androgens in males, and the effects of these steroid hormones are mediated mostly through their interaction with specific nuclear receptors. Although their levels differ, both male and female sex hormones are present in both sexes, where they contribute to various biological processes including development and metabolism in large part through the regulation of gene expression. ^9–11 The physiology of ocular tissue is maintained by sex hormones. ^12,13 Specific receptors for these hormones are thus expressed in the eye, ^14,15 and the synthesis of 17β-estradiol has been detected in the rat retina. ¹⁶ Furthermore, progesterone, testosterone, and 17β-estradiol have all been shown to prevent the swelling of retinal glial cells. ¹⁷ We have also previously shown that female sex hormones inhibit collagen degradation mediated by corneal fibroblasts. ^18,19  

Contraction of a collagen gel mediated by various cell types in culture has been studied as a model of cell-mediated wound contraction. ²⁰ Cells cultured in collagen gels adopt a morphology similar to that apparent in vivo. ^19,21,22 We have now examined the effects of sex hormones (17β-estradiol, progesterone, and dehydro-epiandrosterone [DHEA]) on collagen gel contraction mediated by mouse RPE cells exposed to TGF-β2 as a model for the contraction of proliferative fibrocellular membranes in PVR. We also determined the effects of sex hormones on the expression of α-SMA, fibronectin, and MMPs in these cells, and we examined the expression of the estrogen receptor (ER) and progesterone receptor (PR) in proliferative fibrocellular membranes of patients with PVR. 

Methods

Materials

A mixture of Dulbecco's modified Eagle's medium and Nutrient Mixture F-12 (DMEM/F-12) as well as minimum essential medium (MEM), fetal bovine serum (FBS), and trypsin-EDTA were obtained from Invitrogen-Gibco (Rockville, MD, USA). Cell culture flasks (100-mm diameter) and 24-well culture plates were from Corning (Corning, NY, USA). Native porcine type I collagen (acid solubilized) and reconstitution buffer were obtained from Nitta Gelatin (Osaka, Japan); bovine serum albumin (BSA) was from Nacalai Tesque (Kyoto, Japan); and a protease inhibitor cocktail, 17β-estradiol, progesterone, and DHEA, were from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human TGF-β2 and an enzyme-linked immunosorbent assay (ELISA) kit for mouse interleukin-6 (IL-6) were obtained from R&D Systems (Minneapolis, MN, USA), and a chemiluminescence immunoassay (CLIA) kit for fibronectin was from USCN Life Science (Wuhan, China). GM6001 was from Merck Millipore (Darmstadt, Germany). Mouse monoclonal antibodies to α-SMA were obtained from Sigma-Aldrich, and those to total or phosphorylated forms of Smad2 or MLC were from Cell Signaling (Beverly, MA, USA). Rabbit polyclonal antibodies to ER-α were from Abcam (Bristol, UK), and those to PR were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rhodamine-phalloidin and Cyto59 were obtained from Invitrogen. Horseradish peroxidase–conjugated goat secondary antibodies and ECL Plus detection reagents were from Amersham Biosciences (Little Chalfont, UK). Alexa Fluor 488–conjugated secondary antibodies were from Invitrogen. Tissue-Tek OCT compound was obtained from Sakura Finechemical (Tokyo, Japan), silanized glass slides were from Dako (Glostrup, Denmark), and immunohistochemical staining kits for ER and PR were from Nichirei (Tokyo, Japan). 

Isolation and Culture of Mouse RPE Cells

Retinal pigment epithelial cells were isolated from adult male mice as described previously. ^23,24 The cells were maintained under a humidified atmosphere of 5% CO₂ at 37°C in culture dishes containing DMEM/F-12 supplemented with 10% FBS. All animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 

Collagen Gel Contraction Assay

Collagen gels were prepared as described previously. ^18,21,22 In brief, 24-well culture plates were coated with 1% BSA (1 mL per well) for 1 hour at 37°C. Retinal pigment epithelial cells were harvested by exposure to trypsin-EDTA, washed twice with serum-free MEM, and resuspended in serum-free MEM. Type I collagen (3 mg/mL), 10× MEM, reconstitution buffer, RPE cell suspension (1.1 × 10⁷ cells/mL in MEM), and de-ionized water were mixed on ice in a volume ratio of 7:1:1:0.2:1.8 (final concentration of type I collagen, 1.9 mg/mL; final cell density, 2 × 10⁵/mL). A portion (0.5 mL) of the mixture was added to each BSA-coated well of the culture plates and was allowed to solidify by incubation at 37°C under 5% CO₂ for 1 hour. The collagen gels were freed from the sides of the wells with the use of a microspatula, and serum-free MEM (0.5 mL) containing the test agents was then added on top of each gel. The diameter of the gels was measured daily with a ruler, and the extent of gel contraction was calculated by subtracting the diameter at each time point from the initial diameter. 

Immunoblot Analysis

Cells incubated in collagen gels were lysed in a solution containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 5 mM NaF, 1% Nonidet P-40 (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM Na₃VO₄, and 1% protease inhibitor cocktail. The cell lysates were subjected to SDS-polyacrylamide gel electrophoresis on a 10% gel, and the separated proteins were transferred electrophoretically to a nitrocellulose membrane. Nonspecific sites of the membrane were blocked before incubation with primary antibodies. Immune complexes were detected with the use of horseradish peroxidase–conjugated secondary antibodies and enhanced chemiluminescence reagents. The intensity of immunoreactive bands was measured with the use of NIH ImageJ software (version 1.46r; Bethesda, MD, USA). 

Gelatin Zymography

Gelatin zymography was performed as described previously. ^18,25 In brief, culture supernatants (8 μL) from collagen gel incubations were mixed with 4 μL nonreducing SDS sample buffer (125 mM Tris-HCl [pH 6.8], 20% glycerol, 2% SDS, 0.002% bromophenol blue), and 5 μL of the resulting mixture was subjected to SDS-polyacrylamide gel electrophoresis in the dark at 4°C on a 10% gel containing 0.1% gelatin. The gel was then washed with 2.5% Triton X-100 (Sigma-Aldrich) for 1 hour before incubation for 18 hours at 37°C in a reaction mixture containing 50 mM Tris-HCl (pH 7.5), 5 mM CaCl₂, and 1% Triton X-100. The gel was finally stained with Coomassie brilliant blue. 

Assays for IL-6 and Fibronectin Production

Assay of IL-6 was performed as described previously. ²⁶ Culture medium from collagen gel incubations was centrifuged at 120g for 5 minutes, and the resultant supernatant was frozen at −80°C for subsequent assay of IL-6 and fibronectin with ELISA and CLIA kits, respectively. 

Fluorescence Microscopy

The cells were fixed for 15 minutes at room temperature with 3.7% formaldehyde in phosphate-buffered saline (PBS), washed with PBS, air-dried, and permeabilized for 5 minutes with 1% Triton X-100 in PBS. After blocking of nonspecific sites with 1% BSA in PBS, the cells were incubated for 1 hour at room temperature with antibodies to α-SMA (1:100 dilution in PBS containing 1% BSA). The specimens were then incubated for 1 hour with Alexa Fluor 488–conjugated donkey antibodies to mouse IgG (1:1000 dilution in PBS containing 1% BSA) and Cyto-59 (1:1000 dilution in PBS containing 1% BSA) to stain nuclei. The cells were examined with a laser-scanning confocal microscope (Axiovert200M; Carl Zeiss, Tokyo, Japan). 

Human Tissue Preparation and Immunohistochemical Analysis

This aspect of the study was approved by the Institutional Review Board of Yamaguchi University Hospital and adhered to the tenets of the Declaration of Helsinki. Specimens of proliferative fibrocellular membranes from five individuals with PVR (four men and one woman; mean age ± SD, 57.4 ± 16.7 years; age range, 41–81 years) were obtained at the time of vitrectomy. All specimens were transferred to 4% paraformaldehyde immediately after collection. They were subsequently immersed in Tissue-Tek OCT compound and immediately frozen with liquid nitrogen. Frozen sections (thickness, 5 μm) were cut with a Microm HM505E cryostat (Leica, Wetzlar, Germany), transferred to silanized glass slides, and subjected to immunohistochemical staining for ER and PR with kits. In brief, the sections were incubated with mouse monoclonal antibodies to ER-α (1D5) or to PR (A9621A), or with mouse immunoglobulin G as a negative control, and they were then washed three times with phosphate-buffered saline before incubation with horseradish peroxidase–conjugated goat polyclonal antibodies to mouse immunoglobulin G. The sections were then washed again before detection of immune complexes by exposure to 3,3′-diaminobenzidine and counterstaining with hematoxylin. The stained sections were observed with an inverted microscope (AxiovertS100; Carl Zeiss, Jena, Germany). 

Statistical Analysis

Data are presented as means ± SEM. All experiments were performed in triplicate and repeated at least three times. Statistical analysis was performed with Dunnett's multiple-comparison test. A P value of <0.05 was considered statistically significant. 

Results

Effects of Sex Hormones on Collagen Gel Contraction Mediated by RPE Cells

We first examined the effects of sex hormones on collagen contraction mediated by mouse RPE cells. The cells were cultured in a three-dimensional collagen gel in the absence or presence of TGF-β2 and various concentrations of progesterone, 17β-estradiol, or DHEA for up to 48 hours. Transforming growth factor–β2 increased the extent of collagen contraction mediated by RPE cells. This effect of TGF-β2 was inhibited by the female sex hormones progesterone (Fig. 1) and 17β-estradiol (Fig. 2) in a concentration- and time-dependent manner, whereas it remained unchanged in the presence of the male sex hormone DHEA (Fig. 3). The inhibitory effects of progesterone and 17β-estradiol on TGF-β2–induced collagen contraction at 48 hours were significant at concentrations of ≥5 μM, whereas those exerted by the hormones at a concentration of 10 μM were significant at 24 and 48 hours. 

Figure 1

 

Concentration- and time-dependent inhibition by progesterone of RPE cell–mediated collagen contraction induced by TGF-β2. Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and either in the presence of the indicated concentrations of progesterone for 48 hours (A) or in the absence or presence of 10 μM progesterone for the indicated times (B). The extent of gel contraction was then determined. Data are means ± SD of triplicates from experiments that were repeated a total of three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured with TGF-β2 alone.

Figure 2

 

Concentration- and time-dependent inhibition by17β-estradiol of RPE cell–mediated collagen contraction induced by TGF-β2. Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and either in the presence of the indicated concentrations of 17β-estradiol for 48 hours (A) or in the absence or presence of 10 μM 17β-estradiol for the indicated times (B). The extent of gel contraction was then determined. Data are means ± SD of triplicates from experiments that were repeated a total of three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured with TGF-β2 alone.

Figure 3

 

Lack of effect of DHEA on TGF-β2–induced collagen contraction mediated by RPE cells. Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and either in the presence of the indicated concentrations of DHEA for 48 hours (A) or in the absence or presence of 10 μM DHEA for the indicated times (B). The extent of gel contraction was then determined. Data are means ± SD of triplicates from experiments that were repeated a total of three times with similar results. *P < 0.05 (Dunnett's test) versus the corresponding value for cells cultured without addition.

Effects of 17β-Estradiol and Progesterone on MMP Expression in RPE Cells

We next examined the effects of 17β-estradiol and progesterone on MMP abundance and activity in culture supernatants of RPE cells with the use of gelatin zymography. The cells were incubated in collagen gels with TGF-β2 (1 ng/mL) in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 48 hours, after which the culture supernatants were collected for analysis. Transforming growth factor–β2 increased the production of both MMP-2 and MMP-9 by RPE cells, and these effects were inhibited by 17β-estradiol and progesterone (Fig. 4A). Moreover, the MMP inhibitor GM6001 attenuated the effect of TGF-β2 on RPE cell–mediated collagen contraction in a concentration-dependent manner (Fig. 4B). We also investigated the effects of 17β-estradiol and progesterone on Smad signaling induced by TGF-β2 in RPE cells. Immunoblot analysis revealed that both sex hormones inhibited the phosphorylation of Smad2 induced by TGF-β2 (Fig. 5). 

Figure 4

 

Inhibition of TGF-β2–induced MMP expression in RPE cells by 17β-estradiol and progesterone. (A) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 48 hours, after which the culture supernatants were subjected to gelatin zymography. Bands corresponding to MMP-9 and MMP-2 are indicated. Similar results were obtained in three separate experiments. (B) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the presence of the indicated concentrations of GM6001 for 48 hours. The extent of gel contraction was then determined. Data are means ± SD of triplicates from an experiment that was repeated a total of three times with similar results. *P < 0.05 (Dunnett's test) versus the value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the value for cells cultured with TGF-β2 alone.

Figure 5

 

Inhibitory effects of 17β-estradiol and progesterone on TGF-β2–induced Smad2 phosphorylation in RPE cells. (A) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 24 hours. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated forms of Smad2. (B) Quantitation of immunoblot signals for cells treated as in (A). Data are expressed relative to the value for control cells and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test) versus the value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the value for cells cultured with TGF-β2 alone.

Effects of 17β-Estradiol and Progesterone on the Expression of α-SMA and Fibronectin in RPE Cells

We examined the effects of 17β-estradiol and progesterone on the expression of α-SMA and fibronectin associated with the EMT in RPE cells cultured in collagen gels. Immunoblot analysis revealed that TGF-β2 increased the abundance of α-SMA in these cells and that this effect was inhibited by both 17β-estradiol and progesterone (Figs. 6A, 6B). Immunofluorescence analysis also showed that both 17β-estradiol and progesterone inhibited TGF-β2–induced α-SMA expression in RPE cells (Fig. 6C). Similarly, assay of culture supernatants revealed that TGF-β2 induced the release of fibronectin by RPE cells and that this effect was also inhibited by 17β-estradiol and by progesterone (Fig. 7). 

Figure 6

 

Inhibitory effects of 17β-estradiol and progesterone on TGF-β2–induced α-SMA expression in RPE cells. (A) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 48 hours. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to α-SMA and to β-actin (internal control). (B) Quantitation of immunoblot signals for cells treated as in (A). Data are expressed relative to the value for control cells and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test) versus the value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the value for cells incubated with TGF-β2 alone. (C) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 48 hours. They were then stained with antibodies to α-SMA and Alexa Fluor 488–conjugated secondary antibodies (green). Nuclei were stained blue with Cyto59. Scale bar: 50 μm. Data are representative of three independent experiments.

Figure 7

 

Inhibitory effects of 17β-estradiol and progesterone on TGF-β2–induced fibronectin production by RPE cells. Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 48 hours, after which the culture supernatants were assayed for fibronectin. Data are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test) versus the value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the value for cells cultured with TGF-β2 alone.

Effects of 17β-Estradiol and Progesterone on MLC Phosphorylation and IL-6 Release in RPE Cells

Immunoblot analysis revealed that TGF-β2 induced the phosphorylation of MLC in RPE cells cultured in collagen gels and that this effect was inhibited by 17β-estradiol and progesterone (Fig. 8). Furthermore, TGF-β2 stimulated the release of the pro-inflammatory cytokine IL-6 from these cells, and again this effect was inhibited by both 17β-estradiol and progesterone (Fig. 9). 

Figure 8

 

Inhibitory effects of 17β-estradiol and progesterone on TGF-β2–induced MLC phosphorylation in RPE cells. (A) Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 24 hours. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to total or phosphorylated forms of MLC. (B) Quantitation of immunoblot signals for cells treated as in (A). Data are expressed relative to the value for control cells and are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test) versus the value for cells cultured without addition. †P < 0.05 (Dunnett's test) versus the value for cells incubated with TGF-β2 alone.

Figure 9

 

Inhibitory effects of 17β-estradiol and progesterone on TGF-β2–induced IL-6 production by RPE cells. Cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) and in the absence or presence of 17β-estradiol (50 μM) or progesterone (50 μM) for 24 hours, after which the culture supernatants were assayed for IL-6. Data are means ± SEM from three independent experiments. *P < 0.05 (Dunnett's test) versus the value for cells incubated without addition. †P < 0.05 (Dunnett's test) versus the value for cells cultured with TGF-β2 alone.

Expression of ER and PR in RPE Cells and PVR Lesions

The effects of estrogens and progesterone are mediated in large part by their nuclear receptors. Immunoblot analysis revealed the presence of both ER-α and PR in cultured RPE cells, but the amounts of these receptors were not affected by TGF-β2 (Figs. 10A, 10B). We also examined the expression of ER and PR in proliferative fibrocellular membrane specimens obtained from individuals with PVR. Immunohistochemical analysis revealed nuclear immunoreactivity for ER-α and PR in cells of these lesions from all five PVR patients examined (Fig. 10C). 

Figure 10

 

Expression of ER and PR in cultured mouse RPE cells and in proliferative fibrocellular membranes of PVR patients. (A) Retinal pigment epithelial cells were cultured in collagen gels with or without TGF-β2 (1 ng/mL) for 48 hours. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to ER-α, to PR, and to β-actin (internal control). (B) Quantitation of immunoblot signals for cells treated as in (A). Data are expressed relative to the corresponding value for control cells and are means ± SEM from three independent experiments. (C) Representative immunohistochemical staining of ER-α and PR in proliferative fibrocellular membranes of PVR patients. The control represents staining with nonspecific immunoglobulin G instead of specific primary antibodies. Arrows indicate specific nuclear immunoreactivity. Scale bar: 20 μm.

Discussion

We showed here that 17β-estradiol and progesterone each inhibited in a concentration- and time-dependent manner the TGF-β2–induced contraction of a collagen gel mediated by mouse RPE cells, whereas DHEA had no such effect. The two female sex hormones also inhibited the expression of MMP-2 and MMP-9 induced by TGF-β2 in these cells, and an MMP inhibitor attenuated TGF-β2–induced cell-mediated collagen contraction. Furthermore, both 17β-estradiol and progesterone inhibited the expression of α-SMA and fibronectin as well as the phosphorylation of Smad2 and MLC induced by TGF-β2 in RPE cells. These results thus suggest that female sex hormones inhibit TGF-β2–induced collagen contraction through downregulation of MMP expression as well as attenuation of the EMT and actomyosin activation in RPE cells. Moreover, these effects of female sex hormones may be mediated, at least in part, through inhibition of Smad2 signaling elicited by TGF-β2. 

Proliferative vitreoretinopathy is characterized by the formation of proliferative fibrocellular membranes at the surface of the retina as well as in the vitreous and subretinal space, and the contraction of these structures promotes retinal detachment and can lead to loss of vision. The vitreous fluid of individuals with PVR has been found to contain various cytokines and growth factors including TGF-β and IL-6, ^27,28 with the former contributing to induction of the EMT in RPE cells and contraction of the proliferative fibrocellular membranes and the latter promoting local inflammation. Estrogen has been found to suppress cytokine production in murine macrophages. ²⁹ Moreover, female sex hormones attenuate inflammation in airway diseases, ^30,31 and estrogen also ameliorates hepatic fibrosis. ^32,33 We now showed that 17β-estradiol and progesterone each inhibited the TGF-β2–induced contraction of RPE cells as well as suppressed the production of IL-6 by these cells. Moreover, we detected both ER and PR in the proliferative fibrocellular membranes of individuals with PVR. These results thus suggest that female sex hormones might suppress the formation or contraction of proliferative fibrocellular membranes as well as attenuate the local retinal inflammation associated with PVR. 

Female sex hormones have been shown to influence the immune response in various tissues, with estrogens in particular contributing to cellular immunity. ^34,35 We found that 17β-estradiol and progesterone each at a relatively high concentration inhibited the TGF-β2–induced production of IL-6 by cultured mouse RPE cells, with the effect of 17β-estradiol appearing greater than that of progesterone. It is thus possible that 17β-estradiol might prove more effective than progesterone for immunosuppression in the eye. 

The expression of α-SMA correlates with contractile force in various cell types. ³⁶ Phosphorylation of MLC also activates the actomyosin system and thereby promotes tissue contraction. ^37,38 Moreover, reorganization of the ECM can influence cellular contractility ²² and plays an important role in many pathophysiological processes. ³⁹ We now showed that TGF-β2–induced α-SMA expression and MLC phosphorylation in RPE cells are inhibited by both 17β-estradiol and progesterone. Moreover, both TGF-β2–induced fibronectin production and expression of MMPs, which mediate remodeling of the ECM and contribute to collagen gel contraction, ⁴⁰ were also attenuated by these female sex hormones in RPE cells. These various effects of female sex hormones may thus give rise to inhibition of the contraction of proliferative fibrocellular membranes stimulated by TGF-β in PVR. 

The ECM plays an important role in tissue remodeling including the formation and contraction of fibrotic membranes. Collagen types I and III accumulate at the synovial membrane during joint remodeling. ⁴¹ Extracellular fibronectin also contributes to the formation of collagen fibrillar networks and modulates contraction of fibrotic membranes. ⁴² We have previously shown that fibronectin modulates TGF-β1–induced collagen contraction mediated by tenon fibroblasts. ²¹ Fibrous tissue of the retina has also been found to contain collagen fibrils. ⁴³ We now showed that TGF-β2–induced collagen contraction mediated by RPE cells and the production of fibronectin by these cells are inhibited by female sex hormones. Matrix metalloproteinases are responsible for the degradation of collagen and other ECM proteins associated with various physiological and pathologic conditions. ⁴⁴ Matrix metalloproteinases also contribute to wound healing in the eye and to retinal disease, ⁴⁵ with MMP-2 and MMP-9 having been found to play an important role in the pathogenesis of diabetic retinopathy. ^46,47 We found that the expression of MMP-2 and MMP-9 induced by TGF-β2 in RPE cells was inhibited by female sex hormones. Our results thus suggest that female sex hormones might attenuate ECM production and degradation associated with PVR. 

The EMT is responsible for epithelial cell transdifferentiation during tissue fibrosis ⁴⁸ and is characterized by upregulation of mesenchymal marker proteins such as α-SMA, vimentin, fibronectin, and N-cadherin as well as by phenotypic changes that are associated with the secretion of MMPs, ⁴⁹ increased expression of CTGF, ⁵⁰ and cell migration. ⁵¹ Transforming growth factor–β is a potent profibrotic factor and primary inducer of the EMT in various tissues, ^51–53 with EMT induction by this growth factor being mediated predominantly by the Smad signaling pathway. ⁵⁴ We now showed that TGF-β2 induces the expression of α-SMA and fibronectin as well as upregulates MMP-2 and MMP-9 in RPE cells, and that these effects are accompanied by Smad2 phosphorylation. Furthermore, these actions of TGF-β2 were inhibited by17β-estradiol and progesterone. The binding of female sex hormones to their specific receptors regulates gene expression. ^55,56 In addition to serving as nuclear transcription factors, however, ER and PR modulate cell signaling pathways. ^57–59 Female sex hormones might thus attenuate induction of the EMT by TGF-β2 in RPE cells by regulating the expression of their target genes or by modulating TGF-β2 signal transduction. We detected the expression of both ER and PR in cultured mouse RPE cells. Both receptors are also regulated by posttranslational modification such as ubiquitination, phosphorylation, and acetylation, ^60–62 adding an additional level of complexity to regulation of the actions of female sex hormones. 

Estrogens and progesterone bind to their specific receptors composed of ER-α or ER-β and of PR-A or PR-B, respectively, and thereby induce their biological responses. ⁶³ Expression of ER-α has previously been detected in the retina and retinal pigment epithelium of young females but not in those of men or postmenopausal women. ¹⁴ Androgen receptor, ER-α, ER-β, or PR mRNAs have also been detected in the retina and retinal pigment epithelial cells of rats, rabbits, or humans. ¹⁵ We now showed that cultured RPE cells derived from adult male mice express both ER-α and PR. The expression levels of ER and PR affect the clinical and biological responses of breast cancer to specific endocrine therapy ⁶⁴ as well as determine cancer growth and invasion. ⁶⁵ We found that ER-α and PR were also expressed in proliferative fibrocellular membranes of PVR patients without regard to age or sex. 

In summary, we showed that the female sex hormones 17β-estradiol and progesterone each inhibited TGF-β2–induced collagen contraction mediated by RPE cells. These hormones also inhibited the expression of α-SMA and phosphorylation of MLC as well as the upregulation of MMP-2 and MMP-9 and the production of fibronectin and IL-6 in TGF-β2–stimulated RPE cells, with these effects likely resulting at least in part from attenuation of TGF-β2–induced Smad2 signaling. We further demonstrated the expression of ER and PR in mouse RPE cells as well as in the proliferative lesions of PVR patients. Although it is important that estradiol or progesterone levels in plasma or tissues be maintained at appropriate levels to achieve therapeutic benefit without the induction of side effects, ^66,67 our results suggest that female sex hormones warrant further investigation as potential drugs for the treatment of PVR and other proliferative retinal diseases. 

Acknowledgments

The authors thank Yukari Mizuno and Shizuka Murata for technical assistance. 

Supported by Takeda Science Foundation. 

Disclosure: K. Kimura, None; T. Orita, None; Y. Fujitsu, None; Y. Liu, None; M. Wakuta, None; N. Morishige, None; K. Suzuki, None; K.-H. Sonoda, None