αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Keijiro Ishikawa; Parameswaran G Sreekumar; Christine Spee; Hossein Nazari; Danhong Zhu; Ram Kannan; David R Hinton

doi:10.1016/j.ajpath.2015.11.014

. 2016 Apr;186(4):859–873. doi: 10.1016/j.ajpath.2015.11.014

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Keijiro Ishikawa ^∗,^†, Parameswaran G Sreekumar ^∗, Christine Spee ^†, Hossein Nazari ^†, Danhong Zhu ^‡, Ram Kannan ^∗, David R Hinton ^†,^‡,^∗

PMCID: PMC4822331 PMID: 26878210

Abstract

Subretinal fibrosis is an end stage of neovascular age-related macular degeneration, characterized by fibrous membrane formation after choroidal neovascularization. An initial step of the pathogenesis is an epithelial-mesenchymal transition (EMT) of retinal pigment epithelium cells. αB-crystallin plays multiple roles in age-related macular degeneration, including cytoprotection and angiogenesis. However, the role of αB-crystallin in subretinal EMT and fibrosis is unknown. Herein, we showed attenuation of subretinal fibrosis after regression of laser-induced choroidal neovascularization and a decrease in mesenchymal retinal pigment epithelium cells in αB-crystallin knockout mice compared with wild-type mice. αB-crystallin was prominently expressed in subretinal fibrotic lesions in mice. In vitro, overexpression of αB-crystallin induced EMT, whereas suppression of αB-crystallin induced a mesenchymal-epithelial transition. Transforming growth factor-β2–induced EMT was further enhanced by overexpression of αB-crystallin but was inhibited by suppression of αB-crystallin. Silencing of αB-crystallin inhibited multiple fibrotic processes, including cell proliferation, migration, and fibronectin production. Bone morphogenetic protein 4 up-regulated αB-crystallin, and its EMT induction was inhibited by knockdown of αB-crystallin. Furthermore, inhibition of αB-crystallin enhanced monotetraubiquitination of SMAD4, which can impair its nuclear localization. Overexpression of αB-crystallin enhanced nuclear translocation and accumulation of SMAD4 and SMAD5. Thus, αB-crystallin is an important regulator of EMT, acting as a molecular chaperone for SMAD4 and as its potential therapeutic target for preventing subretinal fibrosis development in neovascular age-related macular degeneration.

Age-related macular degeneration (AMD) is a leading cause of blindness because of progressive degeneration of the macular region of the retina that is responsible for visual acuity and color vision. The natural history of AMD is a progression from its early stage to the two forms of late stage of AMD: geographic atrophy and neovascular AMD (nAMD).¹

The visual prognosis for nAMD is poor; the condition progresses rapidly with the development of choroidal neovascularization (CNV) and subsequent subretinal fibrosis. Although the commonly used treatment with anti-vascular endothelial growth factor (VEGF) drugs improves visual acuity in nAMD patients, the subretinal scarring (fibrosis) that may develop in approximately half of all anti–VEGF-treated eyes within 2 years has been identified as a cause of unsuccessful outcomes.² Thus, therapeutic strategies for the inhibition of subretinal fibrosis are currently an active area of investigation. Among the critical growth factors involved in subretinal fibrosis, platelet-derived growth factor (PDGF) is a potential therapeutic target. Currently, several clinical trials for the treatment for nAMD have been evaluating the efficacy of dual VEGF/PDGF inhibitors, such as the following: E10030 (Ophthotech, New York, NY), an anti-PDGF pegylated aptamer as an adjunct to anti-VEGF therapy; sorafenib, an inhibitor of VEGF receptor, PDGF receptor, and Raf kinases; and pazopanib, an inhibitor of VEGF receptor, PDGF receptor, and c-kit.³ In addition, it has been shown that nucleotide-binding oligomerization domain-, leucine-rich repeat domain-, and pyrin domain–containing 3 inflammasome activation is implicated in the pathogenesis of nAMD.⁴ The recent implication of inflammasome activation in the pathogenesis of hepatic fibrosis⁵ suggests that the inflammasome should be further evaluated for its potential role in subretinal fibrosis. Important inflammasome effector cytokines, IL-1β and IL-18, can be potential therapeutic targets for the treatment of subretinal fibrosis in nAMD. In support of this contention, inhibition of IL-1β has been shown to inhibit the development of experimental CNV in mice.⁶

Subretinal fibrosis results from an excessive wound healing response, characterized by fibrous membrane formation after CNV. In fibrous membranes, the main cellular components are myofibroblasts, the cells immunoreactive for α-smooth muscle actin (α-SMA). Previous histological studies imply that the source of myofibroblasts can be both bone marrow–derived cells and retinal pigment epithelium (RPE) cells.7, 8 After injury to RPE, the cells undergo epithelial-mesenchymal transition (EMT), which enables transdifferentiation, resulting in the conversion of epithelial cells to myofibroblasts. CNV induction can result in the recruitment of more inflammatory cells and fibroblasts, which can be a direct or indirect source of additional myofibroblasts. Myofibroblasts play important roles in the development of subretinal fibrosis, such as proliferation, migration, and extracellular matrix remodeling.⁸ Although previous studies have indicated the involvement of several growth factors and cytokines in EMT,⁸ the precise molecular mechanism and the critical regulators of this process remain to be determined.

The soluble cytoplasmic protein αB-crystallin is a prominent member of the small heat shock protein family. The small heat shock proteins exert diverse biological activities in both normal and stressed cells. They can act as molecular chaperones by binding misfolded proteins to prevent their denaturation and aggregation.⁹ αB-crystallin can bind to and stabilize cytoskeleton proteins, such as desmin and actin, and help to maintain cytoskeletal integrity.¹⁰ The role of αB-crystallin in EMT in liver and lung fibrosis has been recently reported.11, 12

Our previous work has suggested an important role for αB-crystallin in both the early and late stages of AMD. The early stage of AMD is characterized by the accumulation of drusen between the RPE and Bruch's membrane, accompanied by RPE cell death and synaptic dysfunction.¹³ Geographic atrophy is caused by extensive atrophy and loss of the RPE and the overlying photoreceptors that rely on the RPE for trophic support.¹ αB-crystallin can be seen in RPE, associated with drusen and identified as one of the components of drusen.14, 15, 16 Our laboratory has shown that RPE cells lacking αB-crystallin are more susceptible to oxidative and endoplasmic reticulum stress compared with normal RPE.17, 18, 19, 20 Furthermore, RPE-overexpressing αB-crystallin shows resistance to apoptosis.²¹ These findings suggest that αB-crystallin plays a cytoprotective role against multiple stress stimuli that can cause RPE cell death, resulting in drusen formation and geographic atrophy. In addition, we previously demonstrated that αB-crystallin plays a regulatory role by functioning as a chaperone for VEGF in ocular angiogenesis and may play a part in CNV formation in nAMD.²² However, the involvement of αB-crystallin in subretinal fibrosis in nAMD has not been studied.

Herein, we examined the pathogenesis of subretinal fibrosis in αB-crystallin^−/− and wild-type (WT) mice; we further investigated the role of αB-crystallin in EMT and fibrotic process in cultured RPE cells.

Materials and Methods

Study Approval

All procedures used in these studies were conducted in accordance with applicable regulatory guidelines at the University of Southern California (Los Angeles, CA), principles of human research subject protection in the Declaration of Helsinki, and principles of animal research in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Human fetal eyes (16 to 18 weeks of gestation) were obtained from Advanced Bioscience Resources Inc. (Alameda, CA) and Novogenix Laboratories LLC (Los Angeles, CA), and written informed consent was obtained from all donors.

RPE Cell Culture

Human RPE cells were isolated from human fetal eyes (gestational age, 16 to 18 weeks) obtained from Advanced Bioscience Resources Inc. and Novogenix Laboratories LLC. Primary cultures of RPE cells were established, as described previously.²³ The experiments used cultured RPE cells at two to four passages with normal morphology.

Mice

αB-crystallin^−/− mice on the 129 S6/SvEvTac background were generated at the National Eye Institute (Bethesda, MD).²⁴ The 129 S6/SvEvTac WT mice were purchased from Taconic Farms (Germantown, NY). Male mice, aged between 6 and 8 weeks, were used in all studies to eliminate sex-related bias in size of laser-induced CNV lesions.²⁵

Laser-Induced CNV

CNVs were generated as described previously.²² Briefly, laser photocoagulation (532 nm, 150 mW, 50 milliseconds, 75 μm) was applied to each fundus using a coverslip as a contact lens on day 0. We made four laser spots per eye for immunohistochemistry and 20 laser spots for extracting protein for enzyme-linked immunosorbent assay (ELISA) analysis. The production of a subretinal bubble at the time of laser application indicated a rupture of Bruch's membrane. We excluded animals that developed burns with bleeding.

CNV and Choroidal Fibrosis Volume Measurement

The volumes of the CNV and choroidal fibrous tissue were measured in choroidal flat mounts on days 7, 21, and 35 after laser photocoagulation. Mouse eye cups were fixed in 4% paraformaldehyde and then permeabilized in 1% Triton X-100 (ICN Biomedicals, Irvine, CA) for 2 hours. After removal of the anterior segment and neural retina, fluorescein-labeled isolectin-B4 (Vector Laboratories, Burlingame, CA) and collagen type I antibody (Rockland Immunochemicals Inc., Limerick, PA) were added to the mouse eye cup and incubated at 4°C overnight to detect CNV and choroidal fibrous tissue, respectively. The secondary antibody against collagen type I was goat anti-rabbit IgG secondary antibody, Alexa Fluor 647 conjugate (Life Technologies, Grand Island, NY). Samples were coverslipped with Vectashield medium (Vector Laboratories) and examined by use of the Perkin Elmer spinning disk confocal microscope (Perkin Elmer, Waltham, MA). Fluorescence volume measurements were recorded by generating image stacks of optical slices within lesions, as previously described.²⁶

ELISA

Total protein was isolated from the sonicated mouse RPE-choroid complexes using tissue protein extraction reagent with protease inhibitor (Thermo Scientific, Waltham, MA). Each experiment, consisting of two pooled RPE-choroid complexes, was performed in triplicate (biological replicates). The concentrations of αB-crystallin and bone morphogenetic protein 4 (BMP4) in the mouse RPE-choroid complexes were measured with ImmunoSet αB-Crystallin ELISA kit (ENZO Life Sciences, Farmingdale, NY) and BMP4 BioAssay ELISA Kit (US Biological, Salem, MA), respectively, according to the manufacturer's instructions.

Transfection

RPE cells were cultured for 24 hours in 6- or 12-well plates or 4-well chamber slides to a confluence of 70% to 90%. siRNA targeting αB-crystallin or control siRNA (Qiagen, Valencia, CA) (sequences included in Table 1) were mixed (10 nmol/L at final concentration) with RNA transfection reagent (Lipofectamine RNAiMAX; Life Technologies). To overexpress αB-crystallin, pCMV6-XL5 plasmid encoding human αB-crystallin (OriGene Technologies, Rockville, MD) or empty vector was mixed with Lipofectamine LTX (Life Technologies) or X-tremeGENE DNA (Roche Applied Science, Indianapolis, IN), following the manufacturer's protocol. Twenty-four hours after transfection, the composite transfection mixture was removed and replaced with Dulbecco's modified Eagle's medium containing 3% fetal bovine serum or serum-free medium for 24 hours, followed with recombinant transforming growth factor (TGF)-β2 (Sigma-Aldrich, St. Louis, MO) or BMP4 (R&D Systems, Minneapolis, MN) treatment before each assay.

Table 1.

siRNA Sequences

αB-crystallin	S r(5′-CCAGGGAGUUCCACAGGAA-3′) dTdT
AS r(5′-UUCCUGUGGAACUCCCUGG-3′) dTdT
Scrambled control	S r(5′-UUCUCCGAACGUGUCACGU-3′) dTdT
AS r(5′-ACGUGACACGUUCGGAGAA-3′) dTdT

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AS, antisense; S, sense.

Western Blot Analysis

Western blot analysis was performed as described previously.¹⁷ After a 48-hour incubation in medium with or without recombinant TGF-β2 or BMP4, total cell lysates were extracted with lysis buffer. In separate experiments, empty vector or αB-crystallin transfected cells were treated with 10 ng TGF-β2 for 2 hours and nuclear and cytosolic fractions were separated. The fractionation of the cells into cytosolic and nuclear fractions was performed using a nuclear/cytosol fractionation kit (BioVision, Milpitas, CA). The extracted cell lysates were subjected to Tris-HCl polyacrylamide gels, and the blots were incubated with antibodies (Abs) (Table 2) and visualized using an enhanced chemiluminescence (Amersham Pharmacia Biotech, Cleveland, OH) detection system. Glyceraldehyde-3-phosphate dehydrogenase or histone H3 was used as the loading control.

Table 2.

List of Antibodies Used in This Study

Target molecule	Antibody type	Application	Source
Human αB-crystallin	Mouse monoclonal	WB, IHC	ENZO Life Sciences
Mouse αB-crystallin	Rabbit polyclonal	IHC	ENZO Life Sciences
Pan-cytokeratin	Mouse monoclonal	IHC	Santa Cruz Biotechnology (Dallas, TX)
α-SMA	Mouse monoclonal	WB, IHC	Sigma-Aldrich (St. Louis, MO)
E-cadherin (24E10)	Rabbit monoclonal	WB, IHC	Cell Signaling
SNAIL	Rabbit polyclonal	WB	Abgent (San Diego, CA)
SLUG	Rabbit polyclonal	WB	Santa Cruz Biotechnology
SMAD4	Rabbit polyclonal	IHC	Santa Cruz Biotechnology
SMAD5	Rabbit polyclonal	WB	Cell Signaling
SMAD2/3	Rabbit polyclonal	WB	Cell Signaling
β-Catenin	Rabbit monoclonal	WB	Cell Signaling
NF-κB p65	Rabbit monoclonal	WB	Cell Signaling
Notch2	Rabbit monoclonal	WB	Cell Signaling
Fibronectin	Mouse monoclonal	IHC	R&D Systems
Collagen type I	Rabbit polyclonal	WB, IHC	Rockland Immunochemicals
Ubiquitin	Rabbit polyclonal	WB	Thermo Scientific
Histone H3	Rabbit monoclonal	WB	Cell Signaling
GAPDH	Mouse monoclonal	WB	EMD Millipore (Billerica, MA)
β-Actin	Mouse monoclonal	WB	Santa Cruz Biotechnology

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α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IHC, immunohistochemistry; WB, Western blotting.

RNA Isolation and Real-Time Quantitative RT-PCR

Total RNA was isolated from cells using the RNeasy kit (Qiagen), according to the manufacturer's instructions. Real-time quantitative RT-PCR was performed, as described previously.²² To eliminate contamination of genomic or plasmid DNA, we pretreated total RNA with DNase (Life Technologies) before proceeding to reverse transcription. The primer sequences are shown in Table 3. Gene expression levels were normalized relative to glyceraldehyde-3-phosphate dehydrogenase mRNA and reported as fold-change over controls.

Table 3.

Primer Sequences Used for Real-Time RT-PCR

Target molecule	Forward primer sequence	Reverse primer sequence
αB-crystallin	5′-TCCCCAGAGGAACTCAAAGTTAAG-3′	5′-GGCGCTCTTCATGTTTCCA-3′
α-SMA	5′-TCTGTAAGGCCGGCTTTGC-3′	5′-TGTCCCATTCCCACCATCA-3′
E-cadherin	5′-ATTTTTCCCTCGACACCCGAT-3′	5′-TCCCAGGCGTAGACCAAGA-3′
SNAIL	5′-TCGGAAGCCTAACTACAGCGA-3′	5′-AGATGAGCATTGGCAGCGAG-3′
SLUG	5′-CGAACTGGACACACATACAGTG-3′	5′-CTGAGGATCTCTGGTTGTGGT-3′
GAPDH	5′-ACAGTCGCCGCATCTTCTT-3′	5′-ACGACCAAATCCGTTGACTC-3′

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α-SMA, α-smooth muscle actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Immunohistochemistry and Immunofluorescence Staining

RPE cells were cultured in 4-well multichamber slides (Thermo Scientific) in serum-free medium for 12 hours and then stimulated with recombinant TGF-β2 for up to 2 days. The cells were rinsed in phosphate-buffered saline (PBS) for 3 minutes and fixed and permeabilized with ice-cold methanol for 20 minutes. After a 30-minute blocking step with 10% goat serum, the cells were incubated with primary Abs (Table 2) for 24 hours at 4°C and then incubated with secondary Abs. Cryostat sections (6 μm thick) of snap-frozen mouse eyes were obtained from four WT mice and four αB-crystallin^−/− mice. For immunohistochemistry, the sections were treated with 0.3% hydrogen peroxide for 10 minutes to block endogenous peroxide. The sections were then blocked in 10% normal goat serum for 30 minutes and incubated with Abs (Table 2) overnight at 4°C. Slides were washed with PBS and incubated with biotinylated IgG (Vector Laboratories) for 1 hour. Immunoperoxidase was detected with aminoethylcarbazole as the chromogen, using an aminoethylcarbazole kit (Life Technologies), according to the manufacturer's protocol. Immunofluorescence staining for the sections was performed as described previously.²² Pan-cytokeratin-positive cells or cells positive for α-SMA or E-cadherin were calculated as the number of the immunoreactive cells per mm² of CNV area at day 35 after laser, according to the method previously reported.⁷ The average percentage of the cells immunoreactive for α-SMA or E-cadherin in pan-cytokeratin-positive cells was calculated from four eyes. The specimens were mounted in mounting medium, including DAPI (Vector Laboratories), and examined with a Perkin Elmer spinning disk confocal microscope.

Immunofluorescence Localization of SMAD4 and SMAD5

RPE cells transfected with empty vector or vector with αB-crystallin grown on 4-well chamber slides were serum starved overnight and then treated with 10 ng/mL TGF-β2 for 2 hours in serum-free medium. After treatment, cells were washed, fixed in 4% paraformaldehyde, blocked in 5% goat serum, and incubated with primary antibody for anti-rabbit SMAD4 or anti-rabbit SMAD5 (1:100 dilution; Cell Signaling Technology, Danvers, MA) overnight at 4°C. Cells were washed in PBS, incubated with fluorescein isothiocyanate–conjugated anti-rabbit secondary antibody, mounted with DAPI-containing medium (Vector Laboratories), and viewed with a spinning disk confocal microscope.

Isolation of Ubiquitin Conjugates

Monotetraubiquitin- and polyubiquitin-conjugated protein was isolated from cultured RPE using the Pierce Ubiquitin Enrichment kit (Thermo Scientific). Briefly, 150 μL of cell lysates (1 mg/mL) was applied to 150 μL of sample dilution buffer and 20 μL of ubiquitin affinity resin. The mixtures were incubated at 4°C overnight and then centrifuged using a spin column. A resin in this spin column binds polyubiquitin containing four or more ubiquitin subunits. Thus, the flow-through is harvested as monotetraubiquitin-conjugated protein, whereas the polyubiquitinated proteins were recovered in the elution buffer. Laemmli sample buffer (Bio-Rad Laboratories Inc., Irvine, CA) was added to the column and heated at 95°C for 5 minutes.

Cell Proliferation Assay

Proliferation in RPE cells was measured using 5-bromo-2′-deoxyuridine ELISA (Roche Applied Science), according to the manufacturer's instructions, with a 2-hour 5-bromo-2′-deoxyuridine incubation.

Cell Migration Assay

A cell migration assay was performed using an Oris 96-well cell migration assay kit (Platypus Technologies, Madison, WI), according to the manufacturer's instructions, as described previously.²⁷ Briefly, 5 × 10⁴ cells were seeded in each well and transfected with or without siRNAs. After 1 hour of pretreatment with Dulbecco's modified Eagle's medium with 3% fetal bovine serum containing recombinant TGF-β2 with 5 μmol/L aphidicolin (Sigma-Aldrich) to inhibit cell division, the stoppers were removed to allow cells to migrate into the detection zone. The cells were incubated for 48 hours after initiating migration and stained with PBS containing calcein AM (Life Technologies) for 1 hour. The area of cell migration was determined using Photoshop software version CS3 (Adobe Systems, San Jose, CA).

Statistical Analysis

All results are expressed as means ± SEM. The statistical significance of differences between groups was analyzed using the Tukey's (honest significant difference) test or the two-tailed t-test. In comparison with the control group, Dunnett's t-test was applied. Differences were considered significant at P < 0.05. Statistical analyses were performed using JMP version 7.0.1 (SAS Institute, Cary, NC).

Results

Attenuation of Subretinal Fibrosis in αB-Crystallin^−/− Mice

To evaluate a time-dependent alteration of CNV and fibrosis development after laser treatment, we labeled RPE/choroid flat mounts with Abs against isolectin B4 and collagen type I to represent CNV and fibrosis, respectively. CNV reaches a maximum on day 7; thereafter, it begins to regress, disappearing almost completely within 35 days after laser. Fibrosis continues to increase for up to 35 days after laser (Figure 1A). As shown previously,²² CNV volume at day 7 after laser was reduced in αB-crystallin^−/− mice compared with the WT mice. At days 21 and 35 after laser, no significant difference was seen in CNV volume between WT and αB-crystallin^−/− mice. Fibrosis volume was significantly (P < 0.01) reduced in αB-crystallin^−/− mice compared with the WT at days 7, 21, and 35 after laser (Figure 1, A and B). In WT mice, prominent subretinal fibrosis was seen at 35 days after laser with extensive deposition of collagen type I in the lesions; however, fibrosis in αB-crystallin^−/− mice was markedly reduced (Figure 1C). These findings demonstrated significant attenuation of subretinal fibrosis that occurs subsequent to CNV in αB-crystallin^−/− mice.

αB-crystallin (αB) knockout (KO; αB^−/−) mice have less subretinal fibrosis development after laser injury. Wild-type (WT) and αB^−/− mice had laser photocoagulation (four lesions per eye) on day 0. A: Representative images of choroidal neovascularization (CNV; stained by isolectin B4) and fibrosis (stained by collagen type I) at days 7, 21, and 35 after laser in WT and αB^−/− mice. B: The mean volume of the CNV and fibrosis at days 7, 21, and 35 after laser. C: Histological section through a CNV membrane at day 35 after laser in WT and αB^−/− mice. Fibrous tissue can be seen (**dotted circles**) in the subretinal space. ^∗∗P < 0.01. n = 8 per group (B). Scale bars: 100 μm (A); 50 μm (C). INL, inner nuclear layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium.

Impact of αB-Crystallin on EMT in Subretinal Fibrosis of Mouse Model

BMP4, a member of the TGF-β superfamily, is expressed in subretinal fibrosis lesions in AMD patients²⁸ and up-regulates αB-crystallin in microvascular endothelial cells.²⁹ To examine the expression changes of αB-crystallin and BMP4 in the development of subretinal fibrosis in mice, we measured αB-crystallin and BMP4 in the RPE-choroid complexes after laser injury of WT mice.³⁰ The concentration of αB-crystallin and BMP4 gradually increased, peaking at day 21 after laser (Figure 2A). Prominent expression of αB-crystallin can be seen in the lesion at day 21 after laser, whereas expression of αB-crystallin was weak in control eyes (Figure 2B).

αB-crystallin (αB) is up-regulated and has an impact on epithelial-mesenchymal transition markers in retinal pigment epithelium (RPE) cells in subretinal fibrous tissue. A: Levels of αB and bone morphogenetic protein (BMP)-4 in protein extracted from the RPE-choroidal complexes after 20 laser lesions. B: Histological section of the choroidal neovascularization (CNV) membrane at day 21 after laser. Immunoreactivity to αB can be seen in the subretinal space (**arrows**). C and D: Triple immunofluorescence staining for α-smooth muscle actin (α-SMA; mesenchymal marker), E-cadherin (epithelial marker), and pan-cytokeratin (a marker for RPE cells) in eyes without laser treatment (C) and eyes with subretinal fibrosis (D) of wild-type (WT) and αB knockout (KO; αB^−/−) mice. Nuclei are counterstained with DAPI (blue). E: Bar graph shows average number of cells immunoreactive for both α-SMA and pan-cytokeratin and average number of the cells solely immunoreactive for pan-cytokeratin in the subretinal lesion. The proportions of cells immunoreactive for α-SMA in the cells immunoreactive for pan-cytokeratin are calculated in the eyes of WT and αB^−/− mice at day 35 after laser. F: Bar graph shows average number of the cells immunoreactive for both E-cadherin and pan-cytokeratin and average number of cells solely immunoreactive for pan-cytokeratin is in the subretinal lesion. The proportions of cells immunoreactive for E-cadherin in the cells immunoreactive for pan-cytokeratin are calculated in the eyes of WT and αB^−/− mice at day 35 after laser. ^∗P < 0.05 versus control (Ctrl) without laser treatment. n = 3 per group (A); n = 4 per group (E and F). Scale bars: 50 μm (B); 100 μm (C and D).

To investigate the difference in the cellular expression of EMT markers in the subretinal fibrosis lesion between WT and αB-crystallin^−/− mice, we stained the sections with Abs against α-SMA (mesenchymal marker), E-cadherin (epithelial marker), and pan-cytokeratin (a marker for RPE cells). As a control, we evaluated expression of EMT-related markers in the RPE layer in mice without laser treatment. No significant difference was seen between WT and αB-crystallin^−/− mice in the expression of EMT-related markers in RPE. In both WT and αB-crystallin^−/− mice, RPE were positive for pan-cytokeratin and E-cadherin, but were negative for α-SMA (Figure 2C). In the subretinal fibrosis lesions, RPE cells in WT mice expressed high levels of α-SMA and low E-cadherin in the lesion at day 35 after laser. In contrast, RPE cells in αB-crystallin^−/− mice expressed less α-SMA and more E-cadherin compared with WT mice (Figure 2D). Average numbers of RPE cells immunoreactive for α-SMA were lower in αB-crystallin^−/− mice compared with WT mice. The proportion of the RPE cells immunoreactive for α-SMA in the total RPE population was significantly reduced in αB-crystallin^−/− mice compared with WT mice (Figure 2E). Average numbers of RPE cells immunoreactive for E-cadherin were higher in αB-crystallin^−/− mice compared with WT mice. The proportion of the RPE cells immunoreactive for E-cadherin in the total RPE population was significantly increased in αB-crystallin^−/− mice compared with WT mice (Figure 2F). Taken together, these findings indicate that in the absence of αB-crystallin, RPE in CNV lesions show a prominent inhibition of the EMT phenotype.

αB-Crystallin Induces EMT through Regulation of SNAIL and SLUG Expression

To test whether modulation of αB-crystallin expression can change EMT in RPE cells, we examined the expression changes of E-cadherin, α-SMA, SNAIL, and SLUG in RPE cells transfected with αB-crystallin siRNA and αB-crystallin–encoding vector. SNAIL and SLUG are transcription factors that can repress E-cadherin while stimulating α-SMA³¹; SNAIL is known to be expressed in CNV membranes from patients with nAMD and to induce EMT in RPE cells in vitro.32, 33 TGF-β2 is the predominant TGF-β isoform in the posterior segment of the eye and a crucial EMT inducer for RPE cells.³⁴ TGF-β2 treatment at 10 ng/mL for 48 hours significantly reduced E-cadherin and increased α-SMA, SNAIL, and SLUG but did not alter αB-crystallin at the protein or mRNA levels (Figure 3, A and B, and Supplemental Figure S1). Suppression of αB-crystallin by siRNA significantly increased E-cadherin and decreased α-SMA, SNAIL, and SLUG, whereas overexpression of αB-crystallin by DNA vector decreased E-cadherin and increased α-SMA, SNAIL, and SLUG in protein and mRNA levels (Figure 3, A and B, and Supplemental Figure S1). The TGF-β2–induced decrease of E-cadherin and the increase of α-SMA, SNAIL, and SLUG could be inhibited by suppression of αB-crystallin and enhanced by overexpression of αB-crystallin (Figure 3, A and B, and Supplemental Figure S1). These results suggest that suppression of αB-crystallin induced a mesenchymal-epithelial transition (increased E-cadherin and decreased α-SMA), whereas overexpression of αB-crystallin induced EMT (decreased E-cadherin and increased α-SMA). TGF-β2–induced EMT was inhibited by suppression of αB-crystallin.

αB-crystallin (αB) induces epithelial-mesenchymal transition through regulation of SNAIL and SLUG expression. After transfection with control (Ctrl) siRNA, αB-crystallin siRNA, empty (Ctrl) vector, and αB-encoding (αB⁺) vector, retinal pigment epithelium (RPE) cells were stimulated with 10 ng/mL transforming growth factor (TGF)-β2 for 48 hours. A: Western blot analysis of E-cadherin, α-smooth muscle actin (α-SMA), SNAIL, SLUG, αB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the cell lysates of RPE cells. Quantification shown in Supplemental Figure S1. B: mRNA expression of E-cadherin, α-SMA, SNAIL, SLUG, and αB is shown as relative fold to control siRNA normalized to GAPDH. C: Triple immunofluorescence staining for α-SMA and E-cadherin in RPE cells. Nuclei are stained blue. Data are presented as means ± SEM (B). n = 4 per group (B). ^∗P < 0.05, ^∗∗P < 0.01. Scale bar = 10 μm (C).

Immunofluorescence staining validated the effect of αB-crystallin modulation on the expression changes of E-cadherin and α-SMA, with or without TGF-β2 treatment (Figure 3C). These results suggest that αB-crystallin plays a significant role in EMT of RPE cells.

BMP4 Up-Regulates αB-Crystallin, Resulting in EMT

Because BMP4 was induced in the CNV lesion after laser treatment in mice and up-regulates αB-crystallin in microvascular endothelial cells,²⁹ we investigated whether BMP4 up-regulates αB-crystallin in RPE cells. Expression levels of αB-crystallin protein and mRNA were strikingly up-regulated at 48 hours after BMP4 stimulation at 25, 50, and 100 ng/mL in a dose-dependent manner (Figure 4, A and B, and Supplemental Figure S2A). BMP4 treatment (50 ng/mL) significantly reduced E-cadherin and increased α-SMA, SNAIL, and SLUG in protein and mRNA levels; under these experimental conditions, αB-crystallin protein levels increased 4.5-fold. Next, we explored the potential role of αB-crystallin in BMP4-inducible EMT. We inhibited αB-crystallin expression by siRNA in RPE cells treated with BMP4. In αB-crystallin siRNA-transfected cells, BMP4-induced expression changes of E-cadherin, α-SMA, SNAIL, and SLUG were inhibited at the protein and mRNA levels (Figure 4, C and D, and Supplemental Figure S2B). These findings indicate that BMP4 up-regulates αB-crystallin, resulting in the induction of EMT in RPE cells.

Bone morphogenetic protein (BMP)-4 induces epithelial-mesenchymal transition through up-regulation of αB-crystallin (αB). A: Western blot analysis of αB and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in retinal pigment epithelium (RPE) cells stimulated with the indicated concentrations of BMP4 for 48 hours. Quantification shown in Supplemental Figure S2A. B: Expression of αB mRNA shown as relative fold to control (Ctrl) normalized to GAPDH. C: After transfection with control siRNA and αB siRNA, RPE cells were stimulated by 50 ng/mL BMP4 for 48 hours. Western blot analysis of E-cadherin, α-smooth muscle actin (α-SMA), SNAIL, SLUG, αB, and GAPDH in the cell lysates of RPE cells. Quantification shown in Supplemental Figure S2B. D: mRNA expression of E-cadherin, α-SMA, SNAIL, SLUG, and αB shown as relative fold to control normalized to GAPDH. Data are presented as means ± SEM (B and D). n = 4 per group (B and D). ^∗P < 0.05, ^∗∗P < 0.01.

Suppression of αB-Crystallin Inhibits Nuclear Translocation of SMAD4 Mediated by Its Monotetraubiquitination

αB-crystallin is a molecular chaperone that facilitates translocation of proteins by binding and stabilizing the target protein.¹⁴ EMT can occur through nuclear translocation of proteins, such as β-catenin, NF-κB, Notch, R-SMADs, and SMAD4, which regulate SNAIL gene expression.³⁵ We hypothesized that the effect of αB-crystallin modulation on EMT can be mediated by a change in nuclear translocation of these proteins. Nuclear and cytosolic fractions from RPE cells were used to investigate nuclear translocation of β-catenin, NF-κB, Notch, R-SMADs, and SMAD4. Suppression of αB-crystallin by siRNA did not change levels of β-catenin, NF-κB, Notch2, or SMAD2/3 in nuclear and cytosolic fractions (Supplemental Figure S3). In contrast, the SMAD4 level was decreased in the nuclear fraction and increased in the cytosolic fraction by αB-crystallin suppression. SMAD4 was increased in the nuclear fraction and decreased in the cytosolic fraction at 24 hours after treatment by TGF-β2 at 10 ng/mL. Suppression of αB-crystallin inhibited the TGF-β2–induced expression change of SMAD4 in the cytosolic and nuclear fractions (Figure 5A).

αB-crystallin (αB) silencing impairs nuclear translocation of SMAD4 mediated by its monotetraubiquitination. Retinal pigment epithelium (RPE) cells were transfected with control (Ctrl) siRNA and αB siRNA. A: Western blot (WB) analysis of SMAD4, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control for cytosolic protein), histone H3 (loading control for nuclear protein), and αB in nuclear and cytoplasmic fractions extracted from RPE cells with or without 10 ng/mL transforming growth factor (TGF)-β2 stimulation for 24 hours. B: Triple immunofluorescence staining for SMAD4 and αB in RPE cells, with or without 10 ng/mL TGF-β2 stimulation for 24 hours. Nuclei are counterstained with DAPI (blue). C: Western blot analysis of ubiquitin and β-actin in monotetraubiquitinated and polyubiquitinated protein isolated from RPE cells. D: Monotetraubiquitinated SMAD4 and polyubiquitinated SMAD4 detected by Western blot of ubiquitinated proteins; densitometric quantitation of blots from three independent experiments is shown. ^∗P < 0.05. Scale bar = 10 μm (B).

Next, we performed immunofluorescence staining to confirm the Western blot findings. Treatment with αB-crystallin siRNA markedly suppressed αB-crystallin expression in the cytosol. In RPE cells treated with control siRNA, SMAD4 is present in both cytosol and nucleus, and TGF-β2 treatment (10 ng/mL for 24 hours) induced accumulation of SMAD4 in the nucleus. SMAD4 expression was not seen in the nucleus after suppression of αB-crystallin by siRNA with or without TGF-β2 treatment (Figure 5B). These findings clearly demonstrated that suppression of αB-crystallin inhibits SMAD4 nuclear translocation in RPE cells.

Monoubiquitin is a regulator of the location and activity of diverse cellular proteins.³⁶ Recently, a regulatory role of αB-crystallin in SMAD4 monoubiquitination was reported.¹² We first isolated monotetraubiquitinated or polyubiquitinated protein from RPE cells treated with control or αB-crystallin siRNA. Western blot analysis of ubiquitin in the whole gels shows a similar pattern of monotetraubiquitinated proteins in RPE cells transfected with αB-crystallin siRNA compared with control (Figure 5C) and showed equal loading of β-actin (Figure 5C). The level of monotetraubiquitinated SMAD4 was increased in the αB-crystallin siRNA-treated cells (Figure 5D). Analysis of polyubiquitinated proteins showed they are similarly represented in αB-crystallin siRNA-treated samples and in controls (Figure 5C) with equal level of polyubiquitinated SMAD4 (Figure 5D). These results suggest that the inhibitory effect of αB-crystallin suppression on SMAD4 nuclear translocation could be mediated by monoubiquitination.

Overexpression of αB-Crystallin Modulates Nuclear Translocation of SMAD4 and SMAD5

On the basis of our finding that knockdown of αB-crystallin prevented nuclear translocation of SMAD4, we hypothesized that an increased nuclear translocation and accumulation of SMAD4 occurs in αB-crystallin–overexpressed RPE cells. To test this hypothesis, we transiently overexpressed αB-crystallin in RPE cells. The efficacy of overexpression was verified using a green fluorescent protein–encoding plasmid and immunoblot analysis of total cell extracts probed for αB-crystallin (Figure 6A). A significant >4.5-fold increase in αB-crystallin protein expression was obtained in αB-crystallin–overexpressed RPE cells versus empty vector control cells (Figure 6A). Immunofluorescence studies revealed a prominent increase in nuclear accumulation of SMAD4 in αB-crystallin–overexpressed cells (Figure 6B). Immunoblot analysis for SMAD4 in nuclear fractions of the cells showed a significant (P < 0.01) increase in the nuclear levels of SMAD4 (Figure 6C) in αB-crystallin–overexpressed cells when compared with empty vector control cells. TGF-β2 treatment also significantly up-regulated SMAD4 accumulation in the nuclear fraction of empty vector control cells; however, no further accumulation of SMAD4 was observed in αB-crystallin–overexpressed cells (Figure 6C).

αB-crystallin (αB) overexpression increases nuclear translocation of SMAD4 in retinal pigment epithelium (RPE) cells. RPE cells were transfected with αB-encoding vector or empty vector, followed by treatment with 10 ng/mL transforming growth factor (TGF)-β2 for 2 hours. A: RPE cells transfected with green fluorescent protein (GFP)–encoding plasmid using X-tremeGENE HP DNA transfection reagent showing transfection efficiency. Immunoblot analysis of αB in vector-only and αB transfected RPE cells; densitometric analysis of the blots from three independent experiments is shown. B: Immunofluorescence staining of SMAD4 in RPE cells transfected with empty vector or with αB-encoded vector. αB overexpression enhances nuclear expression of SMAD4. C: Immunoblot analysis of the nuclear fraction of SMAD4 after cell fractionation of RPE cells transfected with αB or empty vector; densitometric analysis of the blots from three independent experiments is shown. ^∗∗P < 0.01. Scale bar = 25 μm (A and B). GAPDH, glyceraldehye-3-phosphate dehydrogenase.

Because SMAD4 is the common mediator or co-SMAD that displays continuous shuttling between the nucleus and the cytoplasm,37, 38 we determined whether its accumulation in the nucleus after αB-crystallin overexpression was associated with nuclear accumulation of receptor-regulated R-SMADs. We first evaluated SMAD2/3 because these are activated in the canonical TGF-β/SMAD signaling pathway. To determine whether αB-crystallin overexpression can alter SMAD2/3 translocation and accumulation, we extracted nuclear and cytosolic fractions from RPE cells transfected with αB-crystallin–encoding vector. Overexpression of αB-crystallin did not alter the accumulation of SMAD2/3 in the nuclei (Supplemental Figure S3). We next evaluated R-SMAD5 because recent studies showed that SMAD5 activation may occur as a noncanonical TGF-β signaling pathway in macrophages.³⁹ Interestingly, confocal immunofluorescence studies and immunoblot studies of nuclear fractions revealed increased SMAD5 accumulation in the nuclei of RPE after treatment with TGF-β2 (Figure 7). Just as with SMAD4, analysis of αB-crystallin–overexpressing cells showed increased SMAD5 accumulation in the nuclei when compared with vector-only control cells (Figure 7).

αB-crystallin (αB) induces nuclear translocation of SMAD5. Retinal pigment epithelium (RPE) cells were transfected with αB-encoding vector or empty vector, followed by treatment with 10 ng/mL transforming growth factor (TGF)-β2 for 2 hours. A: Immunofluorescence staining showing increased nuclear translocation of SMAD5 in αB-overexpressed RPE cells versus vector-only control cells. TGF-β2 treatment enhances nuclear translocation of SMAD5 in both vector-only and αB-overexpressed cells. B: Immunoblot analysis of SMAD5 in the nuclear extracts from the vector and αB-overexpressed cells with and without TGF-β2 treatment; densitometric analysis of the blots from three independent experiments is shown. ^∗P < 0.05. Scale bar = 25 μm (A).

Suppression of αB-Crystallin Inhibits Cell Proliferation, Migration, and Fibronectin Synthesis

EMT of RPE cells is an initial step in subretinal fibrosis that is followed by cell proliferation, migration, and ECM remodeling.8, 40 Fibronectin and collagen I are the most prominent ECM components in human subretinal fibrotic lesions.⁴¹ Fibronectin provides a provisional matrix for cell migration of RPE cells.40, 42 Procollagen I synthesis leads to collagen type I deposition during tissue fibrosis.⁴³ Because αB-crystallin induced EMT, we next tested the effect of αB-crystallin inhibition on cell proliferation, migration, and fibronectin and procollagen I synthesis in RPE cells. Inhibition of αB-crystallin expression by siRNA significantly inhibited cell proliferation, as determined by decreased 5-bromo-2′-deoxyuridine incorporation at 24 hours after transfection (Figure 8A). Immunofluorescence staining showed prominently increased fibronectin expression at 12 hours after treatment with TGF-β2. RPE cells treated by αB-crystallin siRNA showed less expression of fibronectin stimulated with or without TGF-β2 (Figure 8B). Procollagen I expression and TGF-β2–induced expression of procollagen I were decreased after silencing αB-crystallin in RPE cells (Figure 8C). In the cell migration assay, TGF-β2 stimulation significantly promoted cell migration. Inhibition of αB-crystallin expression by siRNA significantly suppressed the cell migration stimulated with or without TGF-β2 (Figure 8D).

Inhibition of αB-crystallin (αB) expression prevents cell proliferation, migration, and fibronectin synthesis. Retinal pigment epithelium (RPE) cells were transfected with control (Ctrl) siRNA and αB siRNA. A: 5-Bromo-2′-deoxyuridine (BrdU) incorporations were measured to assay proliferation. B: Immunofluorescence staining of fibronectin in RPE cells with or without 10 ng/mL transforming growth factor (TGF)-β2 stimulation for 12 hours. C: Western blot analysis of procollagen I, αB, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the cell lysates of RPE cells. D: For the migration assay, RPE cells were incubated after transfection for 48 hours with or without 10 ng/mL TGF-β2. The areas of the cells (stained by calcein AM) that migrated into the detection zone (**white dotted circle**) were measured. Data are presented as means ± SEM (A and D). n = 4 per group (A and D). ^∗∗P < 0.01. Scale bars: 10 μm (B); 1 mm (D).

Discussion

This study demonstrates, for the first time, the functional role of αB-crystallin in EMT of RPE and its association with subretinal fibrosis. We further show that inhibition of αB-crystallin down-regulates nuclear translocation of SMAD4 in RPE cells undergoing EMT through an increase in monotetraubiquitination that can impair nuclear localization of SMAD4. We also demonstrate that overexpression of αB-crystallin results in increased nuclear accumulation of co-SMAD4 and R-SMAD5. Our studies thus identify αB-crystallin as a molecular chaperone for SMAD4, and SMAD5 as a component of a noncanonical TGF-β2 signaling pathway in RPE that could bind with SMAD4, allowing their nuclear accumulation.

In the natural history of nAMD, CNV progresses to an end-stage fibrous plaque/disciform scar.44, 45 Similarly, in WT mice, after laser-induced CNV reaches a maximum at day 7 after laser and starts to regress, fibrous tissue increases for up to 35 days. By contrast, in αB-crystallin knockout mice, fibrous tissue does not increase after day 21 after laser, whereas CNV regresses. In addition, the αB-crystallin expression level reaches a peak at day 21, which can imply the facilitating role of αB-crystallin in subretinal fibrosis development. Decreased mesenchymal RPE cells in the subretinal fibrous tissue in αB-crystallin knockout mice is supported by the in vitro data showing that inhibition of αB-crystallin expression repressed EMT of RPE cells. Because EMT is an essential step of fibrotic processes, such as cell proliferation, migration, and fibronectin production,⁴⁰ the effect of αB-crystallin inhibition on the process shown in vitro can cause less fibrous tissue formation in vivo.

Two recent studies demonstrated a role for αB-crystallin in the EMT process in the pathogenesis of hepatocellular carcinoma and pulmonary fibrosis.11, 12 Huang et al¹¹ showed that αB-crystallin protects 14-3-3ζ from its degradation by the proteasome leading to the activation of extracellular signal–regulated kinase signaling pathway, which can induce EMT of hepatocellular carcinoma cells. Bellaye et al12, 46 found the overexpression of αB-crystallin in human and rodent fibrotic lung tissue. The authors elucidated the causal role of αB-crystallin in EMT by demonstrating that overexpression of αB-crystallin disrupted monoubiquitination of SMAD4 by interacting with transcriptional intermediary factor 1γ, E3–ubiquitin ligase and thus limiting its nuclear export.12, 46 Our study in RPE showed that knockdown of αB-crystallin increased the monotetraubiquitination of SMAD4 by threefold and decreased its nuclear localization in both TGF-β2 treated and untreated cells. Interestingly, in RPE, EMT is induced by both TGF-β2, the major TGF-β isoform in the RPE, and BMP4. BMP4 may be mediating its effects through regulation of αB-crystallin expression. BMP4 induction of EMT was associated with a 4.5-fold increase in αB-crystallin, and knocking down αB-crystallin in the BMP4-treated cells reversed the induction of EMT. Previous histological findings demonstrated the expression of αB-crystallin and BMP4 in subretinal disciform scarring of human nAMD,15, 28 indicating that BMP4-inducible up-regulation of αB-crystallin may play a role in the pathogenesis of subretinal fibrosis.

Overexpression of αB-crystallin led to a prominent increase in SMAD4 translocation/accumulation. In canonical TGF-β signaling, nuclear translocation of R-SMAD2/3 facilitates binding to Smad binding elements via masking the SMAD4 nuclear export signal.37, 38 However, we did not find evidence that nuclear SMAD2/3 accumulation was altered after αB-crystallin knockdown or overexpression. Instead, we considered the possibility that SMAD4 stabilization in the nucleus involved other R-SMADs. Herein, we identified an alternate TGF-β2 pathway in which treatment of RPE with TGF-β2 increased the nuclear translocation/accumulation of SMAD5. Although SMAD5 is an integral component of the BMP signaling pathway, the noncanonical TGF-β1–mediated activation of SMAD5 has been demonstrated in epithelial cells⁴⁷ and recently in B-cell lymphomas.⁴⁸ Similarly, TGF-β1, and not BMP, proteins activated SMAD1/5 signaling in macrophages.³⁹ Recently, TGF-β1 regulation of posterior lateral line formation in zebrafish was shown to be mediated by SMAD5.⁴⁹ Skewing of canonical Wnt signaling by TGF-β toward the ALK1/SMAD1/5/8 pathway was reported to cause chondrocyte hypertrophy.⁵⁰ Our new data in RPE cells reveal that αB-crystallin overexpression increases nuclear SMAD5 accumulation. In support, overexpression of αA-crystallin, another member of the α-crystallin family, was associated with increased SMAD3/5 expression in a pancreatic cell line.⁵¹ Thus, SMAD4 and SMAD4/SMAD5 complex play an important role in αB-crystallin–mediated EMT in RPE cells.

In the physiological condition, RPE maintains the epithelial phenotype of highly polarized cells located between the neural retina and the choroid.²³ Physiological RPE cells are mitotically quiescent, with cell-cell contact inhibition mediated by the homotypic adhesion of cadherins on adjacent cells.⁵² In the late phase of AMD, RPE dissociation can occur because of RPE and retinal detachment subsequent to CNV formation.¹ Once these contacts are disrupted and exposed by profibrotic growth factors (ie, TGF-β and PDGF), RPE cells undergo EMT. This enables RPE to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity and elevated resistance to apoptosis, as well as increased production of ECM components and proangiogenic factors, including VEGF.8, 53, 54 Therefore, the novel property of αB-crystallin as an EMT inducer of RPE might account for the known functional roles of αB-crystallin in AMD, such as cytoprotection and VEGF production in response to stress stimuli.21, 22

The physiological RPE monolayer with cell-cell contact showed no difference in the expression of EMT markers between WT and αB-crystallin knockout mice. RPE cells with cell-cell contact are known to preferentially express E-cadherin.⁵⁵ The high expression of E-cadherin can prevent cells from undergoing EMT by limiting SNAIL gene transcription. Thus, when the epithelial phenotype is maintained with adherent junctions, the cells will not be affected by the action of stimuli triggering EMT.³¹ These findings might elucidate the mechanism of the absence of αB-crystallin silencing effect on EMT markers in the physiological RPE cells. Our results indicate that αB-crystallin inhibition can affect only RPE cells that have lost cadherin-mediated adhesions, but it does not influence the cellular phenotypes of the normal RPE monolayer.

Although anti-VEGF therapy for nAMD is effective as an early treatment intervention to prevent consequential fibrotic progression, its direct suppressive effect on fibrosis is yet to be proved.² Notably, some studies reported that anti-VEGF therapy is associated with development of fibrosis in nAMD and proliferative diabetic retinopathy.56, 57, 58 The present study demonstrated that local αB-crystallin inhibition could suppress development of subretinal fibrosis through EMT repression. Recently, a small-molecule inhibitor that blocks the interaction between αB-crystallin and VEGF-165 was identified⁵⁹; this provides a proof of concept that perhaps an inhibitor of αB-crystallin and SMAD4 binding could be identified. Therefore, αB-crystallin would be an attractive therapeutic target for the treatment of nAMD with an advantage in controlling both CNV and subretinal fibrosis.

Acknowledgment

We thank Ernesto Barron for technical assistance.

Footnotes

Supported by NIH grants EY03040 and EY01545, the Arnold and Mabel Beckman Foundation, the Japanese Society for the Promotion of Science, and a Japan Society for the Promotion of Science Postdoctoral Fellowships for Research Abroad fellowship (K.I.).

R.K. and D.R.H. contributed equally to this work as senior authors.

Disclosures: None declared.

Current address of K.I., Department of Ophthalmology, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan.

Supplemental material for this article can be found at http://dx.doi.org/10.1016/j.ajpath.2015.11.014.

Supplemental Data

Supplemental Figure S1

αB-crystallin induces epithelial-mesenchymal transition through regulation of SNAILand SLUG expression. Quantification of data shown in Figure 3A. After transfection with control (Ctrl) siRNA, αB-crystallin siRNA (αB siRNA), empty vector, and αB-crystallin–encoding vector (αB vector), retinal pigment epithelium (RPE) cells were stimulated with 10 ng/mL transforming growth factor (TGF)-β2 for 48 hours. Western blot analysis was performed to examine the expression of E-cadherin, α-smooth muscle actin (α-SMA), SNAIL, SLUG, αB-crystallin (αB), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the cell lysates of RPE cells. Protein expression quantified by densitometry is shown as relative fold to control siRNA normalized to GAPDH. Data are presented as means ± SEM. n = 3 per group. ^∗P < 0.05, ^∗∗P < 0.01.

mmc1.pdf^{(934.6KB, pdf)}

Supplemental Figure S2

Bone morphogenetic protein (BMP)-4 induces epithelial-mesenchymal transition through up-regulation of αB-crystallin. Quantification of data shown in Figure 4. A: Western blot analysis of αB-crystallin (αB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in retinal pigment epithelium (RPE) cells stimulated with the indicated concentrations of BMP4 for 48 hours. Protein expression quantified by densitometry is shown as relative fold to control (Ctrl) normalized to GAPDH. B: After transfection with control siRNA and αB siRNA, RPE cells were stimulated by 50 ng/mL BMP4 for 48 hours. Western blot analysis was performed to examine the expression of E-cadherin, α-smooth muscle actin (α-SMA), SNAIL, SLUG, αB, and GAPDH in the cell lysates of RPE cells. Protein expression quantified by densitometry is shown as relative fold to control siRNA normalized to GAPDH. Data are presented as means ± SEM (A and B). n = 3 per group (A and B). ^∗P < 0.05, ^∗∗P < 0.01.

mmc2.pdf^{(510.3KB, pdf)}

Supplemental Figure S3

A: αB-crystallin silencing does not alter nuclear translocation of β-catenin, NF-κB, Notch2, and SMAD2/3. Retinal pigment epithelium (RPE) cells were transfected with control siRNA and αB-crystallin siRNA. Western blot analysis of β-catenin, NF-κB, Notch2, SMAD2/3, glyceraldehyde-3-phosphate dehydrogenase (GAPDH; loading control for cytosolic protein), histone H3 (loading control for nuclear protein), and αB-crystallin in nuclear and cytoplasmic fractions extracted from the RPE cells. B: αB-crystallin overexpression does not alter nuclear translocation of SMAD2/3. After transfection with empty (Ctrl) vector, and αB-crystallin–encoding (αB⁺) vector, RPE cells were stimulated by 10 ng/mL transforming growth factor (TGF)-β2 for 24 hours. Western blot analysis of SMAD2/3, GAPDH, and histone H3 in nuclear and cytoplasmic fractions extracted from the RPE cells.

mmc3.pdf^{(485.1KB, pdf)}

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PERMALINK

αB-Crystallin Regulates Subretinal Fibrosis by Modulation of Epithelial-Mesenchymal Transition

Keijiro Ishikawa

Parameswaran G Sreekumar

Christine Spee

Hossein Nazari

Danhong Zhu

Ram Kannan

David R Hinton

Abstract

Materials and Methods

Study Approval

RPE Cell Culture

Mice

Laser-Induced CNV

CNV and Choroidal Fibrosis Volume Measurement

ELISA

Transfection

Table 1.

Western Blot Analysis

Table 2.

RNA Isolation and Real-Time Quantitative RT-PCR

Table 3.

Immunohistochemistry and Immunofluorescence Staining

Immunofluorescence Localization of SMAD4 and SMAD5

Isolation of Ubiquitin Conjugates

Cell Proliferation Assay

Cell Migration Assay

Statistical Analysis

Results

Attenuation of Subretinal Fibrosis in αB-Crystallin−/− Mice

Figure 1.

Impact of αB-Crystallin on EMT in Subretinal Fibrosis of Mouse Model

Figure 2.

αB-Crystallin Induces EMT through Regulation of SNAIL and SLUG Expression

Figure 3.

BMP4 Up-Regulates αB-Crystallin, Resulting in EMT

Figure 4.

Suppression of αB-Crystallin Inhibits Nuclear Translocation of SMAD4 Mediated by Its Monotetraubiquitination

Figure 5.

Overexpression of αB-Crystallin Modulates Nuclear Translocation of SMAD4 and SMAD5

Figure 6.

Figure 7.

Suppression of αB-Crystallin Inhibits Cell Proliferation, Migration, and Fibronectin Synthesis

Figure 8.

Discussion

Acknowledgment

Footnotes

Supplemental Data

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Attenuation of Subretinal Fibrosis in αB-Crystallin^−/− Mice