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. 2018 Mar 25;233(9):6965–6974. doi: 10.1002/jcp.26486

Effect of sex steroid hormone fluctuations in the pathophysiology of male‐retinal pigment epithelial cells

Carlo Astarita 1,2, Barbara D'Angelo‐Maansson 1, Mina Massaro‐Giordano 3, Maria P Alba 1, Silvia Boffo 1, Ilaria Macchi 3,4, Antonio Giordano 1,2,✉, Marcella Macaluso 1,✉
PMCID: PMC13482521  PMID: 29336491

Abstract

Gender‐based differences may influence the occurrence of several ocular conditions suggesting the possibility that fluctuations in sex steroid homeostasis may have direct effects on the eye physiology. Here, we evaluated the effect of sex steroid hormone fluctuations in male retinal pigment epithelial cells, RPEs (ARPE‐19). To mimic hormonal fluctuations occurring during aging, we exposed ARPE‐19 to acute, prolonged or chronic estradiol, and progesterone challenges. We found that chronic estradiol treatment promotes a remarkable necrosis of RPE cells, and does not affect pRb2/p130 or PAI‐2 sub‐cellular localization. In contrast, chronic progesterone exposure induces nuclear subcellular rearrangement of pRb2/p130, co‐immunolocalization of pRb2/p130 with PAI‐2, and accumulation of cells in G2/M phase, which is accompanied by a remarkable reduction of necrosis in favour of apoptosis activation.

This study has a high clinical significance since it considers sex steroid fluctuations as inducers of milieu change in the retina able to influence pathological situations occurring with aging in non‐reproductive systems such as the eye. Exogenous administration of physiologically significant amounts of sex hormones for long periods of time is a common clinical practice for transgender patients seeking sex reassignment. In particular, our study offers the unique opportunity to unravel the effects of sex hormones, not only in determining gender differences but also in affecting the physiology of non‐reproductive systems, such as the eye, in the underserved transgender community.

Gender‐based differences may influence the occurrence of several ocular conditions suggesting the possibility that fluctuations in sex steroid homeostasis may have direct effects on the eye physiology. We evaluated the effect of sex steroid hormone fluctuations in male retinal pigment epithelial cells, RPEs (ARPE‐19). This study has a high clinical significance since it considers sex steroid fluctuations as inducers of milieu change in the retina able to influence pathological situations occurring with aging in non‐reproductive systems such as the eye.

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1. INTRODUCTION

Gender‐based differences may influence the occurrence of several ocular conditions suggesting the possibility that fluctuations in sex steroid homeostasis may have direct effects on the eye physiology (Ogueta, Schwartz, Yamashita, & Farber, 1999; Zetterberg, 2016).

Several epidemiological studies have indicated that eye disorders such as idiopathic full‐thickness macular hole, cataract, and age‐related macular degeneration (AMD) are associated with gender and age, and showed an increased incidence of eye diseases in the aging population, particularly in postmenopausal women (Klein, Klein, & Linton, 1992; Zetterberg, 2016) The prevalence of high morbidity eye diseases observed in post‐menopausal women can be explained by a sudden decline in hormone levels after the reproductive years. Furthermore, the male elderly population also endures a slow and long‐lasting drop in hormonal activity associated with a decrease in circulating sex steroids and other minor androgens (Schulster, Bernie, & Ramasamy, 2016).

AMD is a multifactorial and progressive disease of the eye characterized by progressive degeneration of the retina, resulting in permanent damage to central vision among the elderly population (Kaarniranta et al., 2014; Prasad, Schwartz, & Hubschman, 2010).

AMD pathogenesis is a very complicated process that leads to excessive accumulation of intracellular lipofuscin in lysosomes of retinal pigment epithelial cells (RPE) and formation of extracellular drusen. The accumulation of intra– and extracellular waste is mainly triggered by oxidative stress and chronic inflammation, which in turn leads to apoptosis of the RPE followed by the death of underlying photoreceptors (Bhutto & Lutty, 2012). AMD has been associated with several factors and conditions including aging, obesity, family history, diet, hypertension, arteriosclerosis, and hypercholesterolemia (Kaarniranta et al., 2014).

However, despite intensive basic and clinical research, AMD pathogenesis remains unclear probably due to the multifactorial characteristics of this disease. Of particular interest, is the controversial link between AMD and gender. Several studies have reported a prevalent incidence of AMD in post menopausal women and linked it with a great decline in circulating estrogen levels. Estrogens are known to regulate several cellular processes including inflammation (Paimela et al., 2007). In fact, due to the high level of circulating female sex hormones, pre‐menopausal women seem to be more protected from inflammatory responses, and exhibit a lower incidence of AMD compared to men. In contrast, the association between AMD and male gender is complicated by the existence of controversial studies. Some findings have indicated male‐gender as a protective factor for AMD (Srinivasan, Swaminathan, Kulothungan, Raman, & Sharma, 2017), while others have pointed out that male sex is an unreliable risk factor for AMD (Chakravarthy et al., 2010; Evans, 2001; Mitchell, Wang, Foran, & Smith, 2002).

The extracellular plasminogen activator inhibitor type‐2, PAI‐2, is an inhibitor of urokinase‐type plasminogen activator (u‐PA), and its multifunctional role has been suggested in different studies (Schroder, Major, & Suhrbier, 2011; Su, Lin, Yang, Fan, & Yang, 2016; Tang & Han, 2013). PAI‐2 upregulation has been reported in various inflammatory conditions and its aberrant expression is associated with a number of inflammatory diseases (Corsetti, 2016; Hall, Pehrson, Ekestubbe, Jemt, & Friberg, 2015). Until now, the intracellular activity of PAI‐2 is still unclear.

Previously, we have shown that Rb family proteins (Rb1/p105, pRb2/p130, and p107) interact with PAI‐2 in normal corneal and conjunctival cells (Macaluso et al., 2006) . Also, we have indicated that pRb2/p130 and PAI‐2 cooperate in modulating PAI‐2 gene expression by epigenetic mechanisms (Macaluso et al., 2006).

Here, we evaluated the effect of sex steroid hormone fluctuations in male retinal pigment epithelial cells, RPEs (ARPE‐19). To mimic hormonal fluctuations occurring during aging, we exposed ARPE‐19 to acute, prolonged or chronic estradiol, and progesterone challenges.

We found that chronic estradiol treatment promotes a remarkable necrosis of RPE cells, and does not affect pRb2/p130 or PAI‐2 sub‐cellular localization. In contrast, chronic progesterone exposure induces nuclear subcellular rearrangement of pRb2/p130, co‐immunolocalization of pRb2/p130 with PAI‐2, and accumulation of cells in G2/M phase, which is accompanied by a remarkable reduction of necrosis in favor of apoptosis activation.

This study has high clinical significance since it considers sex steroid fluctuations as inducers of milieu changes in the retina that are able to influence pathological situations occurring with aging in non‐reproductive systems such as the eye. As today, the mechanisms governing the action of sex steroid hormones in maintaining the health of the eye are still largely unknown.

Also, our investigation offers the unique opportunity to unravel the effects of sex hormones not only in determining gender differences but also in affecting the physiology of non‐reproductive systems, such as the eye, in the underserved transgender community.

2. MATERIALS AND METHODS

2.1. Cell culture

Retinal pigment epithelial cells (ARPE‐19; CRL‐2302) were purchased from (ATCC, Manassas, VA). Cells were maintained in DMEM‐F12 (ATCC) containing 10% fetal bovine serum (ATCC) at 37 °C in a humidified atmosphere of 5% CO2 to retain the proliferating condition.

2.2. Treatments

ARPE‐19 cells were incubated in DMEM‐F12 containing 10% hormone stripped FBS Twenty‐four hours after seeding, cells were treated with 1 nM (f.c.) estradiol (E2, Sigma‐Aldrich, St. Louis, MO), or 100 nM (f.c) progesterone (P4, Sigma–Aldrich) or estradiol and progesterone in combination (E2 + P4) for 48 hr (acute), 72 hr (prolonged), and 120 hr (chronic).

2.3. Immunofluorescence

ARPE‐19 cells were grown on coverslips for 48, 72, and 120 hr and treated as described above. Immunofluorescence was performed using an Immunofluorescence Application kit accordingly the manufacturer's protocol (Cell Signaling Technology, Danvers, MA). Cells were incubated with anti‐rabbit pRb2/p130 (1:100; Santa Cruz Biotechnology, Dallas, TX) and anti‐mouse PAI‐2 (1:50; Santa Cruz Biotechnology), overnight at 4 °C. The coverslips were rinsed with PBS following incubation for 1 hr at room temperature with secondary fluorochrome‐conjugated antibodies, goat anti‐rabbit IgG AlexaFluor 488 and goat anti‐mouse IgG AlexaFluor 555, respectively (Thermo Fisher Scientific, Waltham, MA). The preparations were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA). The images were captured and evaluated with an Olympus IX81 deconvolution fluorescence microscope (Olympus Microscopes, Tokyo, Japan). No staining was detected when conjugates alone were used as negative controls. Image magnification was 40×.

2.4. Western blotting

Total protein extracts were resolved using SDS–PAGE and subsequently transferred onto nitrocellulose membrane (Sigma‐Aldrich). For antibody detection, membranes were blocked in Phosphate Saline Buffer (PBS; Sigma‐Aldrich) containing 5% fat‐free milk powder (Sigma‐Aldrich) for 30 min at room temperature and then incubated with the following primary antibodies overnight: anti‐rabbit pRb2/p130 (1:800; Santa Cruz Biotechnology) or anti‐mouse PAI‐2 (1:800; Santa Cruz Biotechnology), and rabbit anti‐GADPH (Santa Cruz Biotechnology). After washing, the membranes were incubated with the appropriate peroxidase‐labelled secondary antibody (Santa Cruz Biotechnology) 1 hr at room temperature and visualized using the Super Signal Western Dura substrate (Pierce, Rockford, IL). Images were captured using the Odyssey Fc system (LI‐COR, Lincoln, NE).

2.5. Cell cycle analysis

Both treated and untreated ARPE‐19 cells (1 × 106/ml) were washed with PBS, fixed in 70% (v/v) ethanol and stored at 4 °C, according to protocols well‐established in the lab (Macaluso et al., 2012).

The cells were then centrifuged at 1,000 rpm, washed twice with PBS, and resuspended in 0.4 ml of freshly prepared PBS solution containing 50 μg/ml of propidium iodide (PI) and 0.2 mg/ml of RNase (Abcam, Cambridge, UK) (Macaluso et al., 2012). The stained samples were incubated 30 min at 37 °C and analyzed by using an Accuri C6 Flow Cytometer (Accuri, Bioscience San Jose, CA). Fluorescence signals were screened using an FL‐2 filter, with a 585/40 nm band‐pass filter. The flow cytometer was routinely operated at the Slow Flow Rate setting (14 μl sample/min), and data acquisition for a single sample typically took 5 min.

2.6. Apoptosis assay

Annexin V–fluorescein isothiocyanate (FITC) and propidium iodide (PI) were used to identify and quantify cell death (Annexin V‐FITC Apoptosis Detection Kit, Abcam, Cambridge, MA). Briefly, cells were seeded in a six‐well plate. The adherent and floating cells were combined and washed twice with cold PBS, resuspended in binding buffer and stained using Annexin V‐FITC/PI, according to the manufacturer's protocol. Samples were analyzed by FACS (Accuri C6 Flow Cytometer, Accuri Bioscience, San Jose, CA) to assess the percentage of viable cells (Annexin‐V negative and PI‐negative), apoptotic cells (Annexin‐V positive and PI‐negative), and necrotic cells (Annexin‐V positive and PI‐positive).

2.7. Statistical analysis

Statistical analysis was performed with (GraphPad Software, Inc., La Jolla, CA), and p values were obtained using one‐way analysis of variance (ANOVA) followed by post hoc Bonferroni. Data are presented as mean ± SEM with the number of independent experiments (run in triplicate). A p‐value of p ≤ 0.05 was considered significant.

3. RESULTS

3.1. Effect of acute, prolonged and chronic sex steroid hormone treatment on pRb2/p130 and PAI‐2 protein expression

Several studies have indicated that pRb2/p130 plays an important role in regulating the cell cycle progression (Henley & Dick, 2012; Weinberg, 1995). Also, there is evidence showing that pRb2/p130 is involved in different cellular mechanisms including apoptosis, senescence, and differentiation (Fiorentino, Symonds, Macaluso, & Giordano, 2009; Genovese, Trani, Caputi, & Claudio, 2006; Giacinti & Giordano, 2006; Indovina, Marcelli, Casini, Rizzo, & Giordano, 2013). Previously, we indicated that pRb2/p130 interact with PAI‐2 in cornea and conjunctival cells, and suggested that this interaction could be essential in regulating PAI‐2 gene expression, as well as, PAI‐2 protein subcellular localization (Macaluso et al., 2006).

Here, we assessed the effects of acute (48 hr), prolonged (72 hr), and chronic (120 hr) estradiol (E2), progesterone (P4), and E2 + P4 exposure on pRB2/p130 and PAI‐2 proteins expression in retinal pigment epithelial cells, ARPE‐19. Western blotting analysis was performed using total protein lysate from ARPE‐19 cells. A basal expression of pRb2/p130 and PAI‐2 was detected in untreated ARPE‐19 (CTR; Figures 1a and 1b). However, it appears that E2, P4, and E2 + P4 treatments at all time points (48, 72, and 120 hr) increase pRb2/p130 and PAI‐2 protein levels (Figures 1a and 1b).

Figure 1.

Figure 1

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Effect of sex steroid hormone treatment on pRb2/p130 and PAI‐2 protein expression. Western blotting analysis was performed to assess the effect of acute (48 hr), prolonged (72 hr), and chronic (120 hr), estradiol (E2), progesterone (P4), or estradiol + progesterone in combination (E2 + P4) on the expression of pRb2/p130 and PAI‐2 in in male retinal pigment epithelial cells, RPE, (ARPE‐19). A basal expression of pRb2/p130 and PAI‐2 was detected in untreated ARPE‐19 (CTR; a and b). However, it appears that E2, P4, and E2 + P4 treatments at all time points (48, 72, and 120 hr) increase pRb2/p130 and PAI‐2 protein levels (a and b). The expression of GADHP was assessed to normalize protein loading. The results are representative of three separate experiments

3.2. Chronic progesterone treatment induces co‐immunolocalization of pRb2/p130 with PAI‐2 in the nuclei of ARPE‐19 cells

Several studies have shown that a particular subcellular localization dictates the ability of proteins to exert a specific cellular function. In particular, in vitro investigations have reported that PAI‐2 protein is highly expressed intracellularly (Kruithof, Vassalli, Schleuning, Mattaliano, & Bachmann, 1986; Lee, Cochran, Lobov, & Ranson, 2011), suggesting that this protein has not only a classical inhibitory serpin function but also a variety of other roles such as inhibition of apoptosis, regulation of transcription, and modulation of intracellular signal transduction (Antalis et al., 1998; Dickinson, Bates, Ferrante, & Antalis, 1995; Lee et al., 2011).

In addition, our previous studies have shown that PAI‐2 interacts with pRb2/p130 and Rb1/p105, but not p107, in both the cytoplasm and nucleus of normal primary human cornea and conjunctival epithelial cells, and also indicated that PAI‐2 cooperates with pRb2/p130 in modulating PAI‐2 gene expression by chromatin remodeling (Macaluso et al., 2006).

Here we assessed the effects of acute (48 hr), prolonged (72 hr), and chronic (120 hr) estradiol (E2), progesterone (P4) and E2 + P4 exposure on pRB2/p130 and PAI‐2 sub‐cellular localization in retinal pigment epithelial cells.

The expression and distribution of pRb2/p130 and PAI‐2 were evaluated by immunofluorescence staining. Surprisingly, in untreated ARPE‐19, pRb2/p130, known for being a nuclear protein, was prevalently distributed in the cytoplasm at 48 hr, where this protein seems to be mostly localized in perinuclear aggregates (Figure 2a; CTR). pRb2/p130 perinuclear distribution was more evident at 72 hr (Figure 3a; CTR) and 120 hr (Figure 4a; CTR). On the other hand, PAI‐2 was prevalently observed in the nuclei of untreated cells at 48 hr (Figure 2e; CTR), 72 hr (Figure 3e; CTR), and 120 hr (Figure 4e; CTR). No pRb2/p130/PAI‐2‐ co‐immunolocalization was detected in untreated cells (Figures 2i, 2i, and 4i; CTR).

Figure 2.

Figure 2

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Effect of acute sex steroid hormone treatment on pRb2/p130 and PAI‐2 subcellular localization. The expression and distribution of pRb2/p130 and PAI‐2, under acute (48 hr) estradiol, progesterone (P4) or estradiol + progesterone (E2 + P4) in combination exposure, were evaluated by immunofluorescence staining. pRb2/p130 (green), and PAI‐2 (red) staining are combined in merged images (I, L, M, and N) . Images were captured under identical conditions, at 40× magnification, and processed in the same manner. (Bar 20 μm). The results are representative of three separate experiments

Figure 3.

Figure 3

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Effect of prolonged sex steroid hormone treatment on pRb2/p130 and PAI‐2 subcellular localization. The expression and distribution of pRb2/p130 and PAI‐2, under prolonged (72 hr) estradiol, progesterone (P4) or estradiol + progesterone (E2 + P4) in combination exposure, were evaluated by immunofluorescence staining. pRb2/p130 (green), and PAI‐2 (red) staining are combined in merged images (I, L, M, and N) . Images were captured under identical conditions, at 40× magnification, and processed in the same manner (Bar 20 μm). The results are representative of three separate experiments

Figure 4.

Figure 4

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Effect of chronic sex steroid hormone treatment on pRb2/p130 and PAI‐2 subcellular localization. The expression and distribution of pRb2/p130 and PAI‐2, under chronic (120 hr) estradiol, progesterone (P4) or estradiol + progesterone (E2 + P4) in combination exposure, were evaluated by immunofluorescence staining. pRb2/p130 (green), and PAI‐2 (red) staining are combined in merged images (I, L, M, and N) . Images were captured under identical conditions, at 40X magnification, and processed in the same manner (Bar 20 μm). The results are representative of three separate experiments

Similar to untreated cells, pRb2/p130‐perinuclear aggregates were also observed in ARPE‐19 exposed to acute (48 hr), prolonged (72 hr), and chronic (120 hr) E2 challenges (Figures 2b, 3b, and 4b; E2). In these cells, however, PAI‐2 exhibited both cytoplasmic and nuclear staining (Figures 2f, 3f, and 4f; E2). No pRb2/p130/PAI‐2‐ co‐immunolocalization was detected in E2 treated cells (Figures 2l, 3l, and 4l; E2).

Similar to untreated cells, ARPE‐19 exposed to acute (48 hr) and prolonged (72 hr) progesterone (P4) treatment exhibited diffuse perinuclear and cytoplasmic staining (Figures 2c and 3c; P4) of pRb2/p130. Interestingly, chronic P4 challenge induced an accumulation of pRb2/p130 in the nuclei of these cells (Figure 4c; P4). Diffuse nuclear and cytoplasmic PAI‐2 staining was detected at both 48 hr and 72 hr (Figures 2h and 3g; P4). However, chronic (120 hr) P4 exposure induced a remarkable nuclear accumulation of PAI‐2 (Figure 4g; p4), and also a nuclear co‐immunolocalization of PAI‐2 with pRb2/p130 (Figure 4m).

After acute (48 hr) and prolonged (72 hr) treatment of ARPE‐19 with E2 + P4 together, a diffuse cytoplasmic and perinuclear staining of pRb2/p130 was detected (Figures 2d and 3d; E2 + P4), as well as a cytoplasmic and nuclear PAI‐2 localization (Figures 2h and 3h; E2 + P4). On the other hand, under chronic (120 hr) E2 + P4 challenge, ARPE‐19 exhibited a similar staining pattern of untreated cells with a prevalent pRb2/p130 perinuclear aggregation (Figure 4d; E2 + P4) and a pronounced nuclear accumulation of PAI‐2 (Figure 4h; E2 + P4). No pRb2/p130/PAI‐2‐ co‐immunolocalization was detected in these cells at any time point (Figures 2n, 3n, and 4n; E2 + P4).

3.3. Effect of acute, prolonged and chronic sex steroid hormone treatment on ARPE‐19 cell cycle regulation

The cell cycle of sex steroid hormone‐treated and untreated ARPE‐19 cells was analyzed using flow cytometry. As expected, untreated, cycling cells were under proliferative conditions at all the time points analyzed: G1/G0 64.5%, S 16.9%, G2/M 21.3% (48 hr) (Figure 5; CTR); G1/G0 65.8%, S 16.2%, G2/M 18.1% (72 hr) (Figure 5; CTR); G1/G0 62.3%, S 16.6%, G2/M 20.3% (120 hr) (Figure 5; CTR). Prolonged and chronic E2 exposure induced an accumulation of cells in S and G2/M phases compared to untreated ARPE‐19 (72 hr: G1/G0 55.3%, S 20.9%, G2/M 22%; 120 hr: G1/G0 54.3%, S 19.2%, G2/M 23.0%) (Figure 5; E2). On the other hand, chronic P4 challenge induced an accumulation of cells in G2/M phase with respect to control cells (120 hr: G1/G0 58.5%, S 15.1%, G2/M 24.1%) (Figure 5; P4). Furthermore, acute and prolonged treatment of these cells with E2 + P4 together has no effect on the cell cycle of ARPE‐19 as compared to untreated cells. However, chronic E2 + P4 exposure induces an accumulation of these cells in G2/M phase (120 hr: G1/G0 57.3%, S 14.6%, G2/M 24.7%) (Figure 5; E2 + P4). These results indicated that prolonged and chronic exposure to estradiol increases the proliferation of ARPE‐19, causing cells to enter a growth phase. In contrast, progesterone acts as a homeostatic factor, opposing the estradiol effect on cell growth by decreasing the number of cells in G1/G0 and S phases.

Figure 5.

Figure 5

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Effect of sex steroid hormone treatment on ARPE‐19 cell cycle regulation. Fluorescence‐activated cell sorting (FACS) was performed to assess the effect of acute (48 hr), prolonged (72 hr) and chronic (120 hr) estradiol (E2), progesterone (P4), or estradiol + progesterone in combination (E2 + P4) on the cell cycle of ARPE‐19 cells. Fluorescence signals were screened using an FL‐2 filter, with a 585/40 nm band‐pass filter. The results are representative of three separate experiments

3.4. Chronic estradiol challenge induces cell death of ARPE‐19 via necrosis

We assessed whether E2 exposure induces cell death of ARPE‐19 cells besides triggering cell growth. Cell apoptosis and necrosis were assessed using a standard flow cytometry‐based Annexin V/PI apoptosis assay. Viable ARPE‐19 cells were both Annexin V and PI negative (48 hr: 79.5.4%; 72 hr: 76.2%; 120 hr: 70.6%;) (Figure 6a; CTR). Interestingly, ARPE‐19 cells under chronic E2 treatment showed reduced cell apoptosis (p ≤ 0.0005; E2 vs. CTR) (Figures 6a and 6b) and a significant amount of cells in necrosis (p ≤ 0.0001; E2 vs. CTR) compared to untreated cells (Figures 6a and 6C). On the other hand, neither P4 or E2 + P4 challenge affected the percentage of viable cells with respect to the control cells (Figure 6a–c). However, chronic E2 + P4 treatment induced a significant increase of apoptotic cells (p ≤ 0.0005; E2 + P4 vs. E2) and a decrease of necrotic cells (p ≤ 0.0001; E2 + P4 vs. E2) compared to E2 challenge alone (Figure 6a–c). These results indicated that chronic exposure to estradiol induces cell death via necrosis, perhaps a result of activation of inflammatory pathways. On the other hand, progesterone activates homeostatic mechanisms, such as apoptosis, to help to restore normal cellular condition.

Figure 6.

Figure 6

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Effect of sex steroid hormone treatment on ARPE‐19 cell death Cell apoptosis and necrosis were assessed using a standard flow cytometry‐based Annexin V/PI apoptosis assay. The analysis was performed was performed to assess the effect of acute (48 hr), prolonged (72 hr), and chronic (120 hr), estradiol (E2), progesterone (P4), or estradiol + progesterone in combination (E2 + P4) on cell apoptosis (a, b) and cell necrosis (a, c). In panel B, the ratio of apoptosis among different experimental groups was reported. Apoptosis ratio was early apoptosis percentage plus late apoptosis percentage. The data were presented as the means ± SE. Columns, mean of three independent experiments; bars, SE; ***p ≤ 0.0005; SE; **p ≤ 0.005. In panel C, the ratio of necrosis among different experimental groups was reported. The data were presented as the means ± SE. Columns, mean of three independent experiments; bars, SE; ****p ≤ 0.0001

4. DISCUSSION

AMD, glaucoma, and diabetic retinopathy are the principal causes of permanent blindness worldwide (Zetterberg, 2016) .

In the last two decades, several clinical and epidemiological studies have explored the effects of hormonal fluctuations, gender, and hormonal therapy use in retinal/ocular diseases, including age‐related diseases. Although these studies are clinically relevant, they still lack in providing the molecular mechanisms underlying the phenomena being observed, therefore leaving important unresolved questions (Eisner, 2015; Zetterberg & Celojevic, 2015). Recently, new studies have attempted to dissect the exogenous estrogen mechanisms in retinal disorders, but again the results are still elusive (Kaarniranta et al., 2014). Also, research focused on understanding the link between AMD and gender has led to conflicting results. A neuroprotective effect of estrogens has been linked to less incidence of AMD in pre‐menopausal women (Klein et al., 1992; Zetterberg, 2016). On the other hand, the link between male gender and AMD is more controversial and inconsistent, as some studies indicate male‐gender as a protective factor for AMD (Srinivasan et al., 2017), while others point out that male sex is an unreliable risk factor for AMD (Chakravarthy et al., 2010; Evans, 2001; Mitchell et al., 2002) . Due to the high level of circulating female sex hormones, pre‐menopausal women seem to be more protected from inflammatory responses, and exhibit a lower incidence of AMD compared to men.

While estrogens have historically been associated with women physiology, over the last two decades, several studies have also shown that these hormones play a fundamental role in men (Cooke, Nanjappa, Ko, Prins, & Hess, 2017). Moreover, in vivo experiments have revealed that estrogens are also essential for male physiology targeting not only reproductive organs but also non‐reproductive systems such as the brain, bone, adipose, cardiovascular, skeletal muscle, and immune tissues (Cooke et al., 2017).

As men age, estrogen levels begin to rise because testosterone is being converted to estrogen through aromatase actions (Vermeulen, Kaufman, Goemaere, & van Pottelberg, 2002). Since most progesterone in males is produced during testicular production of testosterone, progesterone levels in aging men drop sharply as estrogen levels rise, affecting a variety of physiological processes as well as increasing the risk of developing serious illnesses such as osteoporosis, arthritis, and prostate cancer (Vermeulen et al., 2002).

Fluctuation in circulating levels of sex steroid hormones has been proposed to influence retinal/ocular physiology. For instance, it has been reported that estrogen deficiency promotes early aging of the optic nerve (Vajaranant & Pasquale, 2012). Moreover, it has been suggested that the de novo synthesis of progesterone and testosterone, along with their metabolites, controls the sex steroid microenvironment of the retina (Cascio, Deidda, Russo, & Guarneri, 2015).

However, the molecular mechanisms governing the action of sex steroid hormones in maintaining the health of the eye remain elusive, and further investigation is needed to tackle the physiological link between estrogen, testosterone and progesterone in this system.

In our study, we explored the effect of sex steroid hormone fluctuations in affecting the physiology of human male RPE cells. Also, our goal was to define whether male gender can be considered a risk factor in developing age‐related retinal disease such as AMD.

To mimic sex steroid hormone fluctuations occurring with aging, we exposed retinal pigment epithelial cells, ARPE‐19, to acute, prolonged or chronic estradiol, and progesterone challenges. As expected, we found that exposure to estradiol increased the proliferation of RPE cells while progesterone antagonized the estradiol effect on cell growth. Interestingly, chronic exposure to estradiol induced cell death via necrosis, perhaps as a result of activation of chronic inflammatory pathways. On the other hand, progesterone activated homeostatic mechanisms, such as apoptosis, to help to restore normal cellular condition.

Previously, we have indicated that the cross‐talk between PAI‐2 and RB family proteins plays an important role in the physiology of cornea and conjunctival cells (Macaluso et al., 2006). Here, we observed that in untreated ARPE‐19 cells, pRb2/p130 was mainly localized in the cytoplasm, while PAI‐2 was prevalently accumulated in the nuclei. Interestingly, estradiol treatment did not change the subcellular localization of pRb2/p130 and PAI‐2 in these cells, suggesting that perhaps the proliferative effect of E2 could be executed by maintaining pRb2/p130 out of the nuclei. Based on our previous findings (Macaluso et al., 2006), it is reasonable to speculate that the peculiar subcellular localization of PAI‐2 and pRb2/p130, observed in RPE cells, is necessary to modulate PAI‐2 transcription and the activation of death pathways in response to aberrant proliferation. In support of this hypothesis, we observed that chronic progesterone exposure induced nuclear subcellular rearrangement of pRb2/p130, along with co‐immunolocalization of pRb2/p130 with PAI‐2 and accumulation of cells in G2/M phase, accompanied by a remarkable reduction of necrosis (see chronic E2 treatment) in favor of apoptosis activation.

This study provides novel insights on the effect of sex steroid hormone fluctuations on eye physiology occurring during aging in the male. Also, it provides essential information for clinicians involved in the prevention and treatment of eye disorders, including AMD, and for individuals undergoing transgender hormone replacement therapy.

Exogenous administration of physiologically significant amounts of sex hormones for long periods of time is a common clinical practice for transgender patients seeking sex reassignment. In particular, our study offers a unique opportunity to unravel the effects of sex hormones, not only in determining gender differences but also in affecting the physiology of non‐reproductive systems, such as the eye, in the underserved transgender community.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

ACKNOWLEDGMENTS

This work was supported by the Pennsylvania Department of Health for Sbarro Health Research Organization (SHRO), the Dry Eye Research Fund and The Douglas Charitable Trust.

Astarita C, D'Angelo‐Maansson B, Massaro‐Giordano M, et al. Effect of sex steroid hormone fluctuations in the pathophysiology of male‐retinal pigment epithelial cells. J Cell Physiol. 2018;233: 6965–6974. 10.1002/jcp.26486

Carlo Astarita and Barbara D'Angelo contributed equally to the work.

Contributor Information

Antonio Giordano, Email: giordano@temple.edu.

Marcella Macaluso, Email: macaluso@temple.edu.

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