November 2015
Volume 56, Issue 12
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Cornea  |   November 2015
The Role of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2) in the Regulation of Corneal Fibroblast Differentiation
Author Affiliations & Notes
  • Soo Hyun Park
    Department of Ophthalmology College of Medicine, Chung-Ang University Hospital, Seoul, Korea
  • Kyoung Woo Kim
    Department of Ophthalmology College of Medicine, Chung-Ang University Hospital, Seoul, Korea
  • Jae Chan Kim
    Department of Ophthalmology College of Medicine, Chung-Ang University Hospital, Seoul, Korea
  • Correspondence: Jae Chan Kim, Department of Ophthalmology, Chung-Ang University Hospital, 224-1, Heukseok-dong, Dongjak-gu, Seoul 156-755, Korea; jck50ey@daum.net
Investigative Ophthalmology & Visual Science November 2015, Vol.56, 7293-7302. doi:10.1167/iovs.15-16616
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      Soo Hyun Park, Kyoung Woo Kim, Jae Chan Kim; The Role of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2) in the Regulation of Corneal Fibroblast Differentiation. Invest. Ophthalmol. Vis. Sci. 2015;56(12):7293-7302. doi: 10.1167/iovs.15-16616.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Previously, we reported that keratocyte-conditioned medium (KCM) facilitates the differentiation of human mesenchymal stem cells (hMSCs) into corneal keratocyte–like cells. This study is designed to investigate the roles of insulin-like growth factor binding protein 2 (IGFBP2) for the regulation of corneal fibroblast differentiation as a newly unveiled component of KCM.

Methods: Immunodot blot analysis was performed to identify the factors that are highly secreted, especially in KCM. Then, we investigated whether IGFBP2 differentiates hMSCs into keratocyte-like cells and whether maintains the phenotypes of keratocyte in human corneal fibroblasts (HCFs) by analyzing expression patterns of alpha-smooth muscle actin (α-SMA) and keratocyte markers including keratocan, lumican and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). Furthermore, to specify the role of IGFBP2, the expression of α-SMA and keratocyte markers was determined in transforming growth factor β 1 (TGFβ1)-induced corneal myofibroblast and in HCFs after knockdown of IGFBP2.

Results: The most prominent factor in both KCM and amniotic membrane extract was IGFBP2. Insulin-like growth factor binding protein 2 increased the expression of IGFBP2, keratocan, and ALDH1A1, and decreased α-SMA expression in hMSCs and HCFs. Insulin-like growth factor binding protein 2 inhibited TGFβ1-induced upregulation of α-SMA and increased expressions of keratocan and ALDH1A1 in HCFs. Furthermore, the knockdown of IGFBP2 increased α-SMA expression and decreased ALDH1A1 level in HCFs.

Conclusions: Insulin-like growth factor binding protein 2 is strongly associated with restoration of keratocyte phenotype in HCFs. Our results show an important novel role of IGFBP2 in regulation of corneal fibroblast differentiation and suggest that IGFBP2 can be a therapeutic candidate for corneal antifibrotic strategy.

Insulin-like growth factor (IGF)-1 and -2 are polypeptides that exhibit mitogenic, metabolic, and differentiative effects on a variety of cell types. Because of the relative abundance of both IGF-1 and IGF-2 during development, the IGFs are thought to play an especially important role in the proliferation and differentiation of embryonic tissues.1,2 The insulin-like growth factor binding proteins (IGFBP1–6) are a family of circulating proteins that were initially defined by their capacity to differentially modulate (positively or negatively) the actions of IGF ligands. Insulin-like growth factor binding proteins are present in serum and in a variety of biological fluids, including amniotic, follicular, cerebrospinal, and seminal fluid, as well as milk.3 Insulin-like growth factor binding proteins have also been identified in the extracellular environment and inside cells, and play distinct physiological roles in growth and development. Insulin-like growth factor binding proteins may be differentially targeted to different tissues depending on both their primary structure and their posttranslational modifications.4 It has been postulated that a number of IGFBPs can interact with the extracellular matrix (ECM) or cell surface via glycoproteins, collagens, and integrins.5,6 Insulin-like growth factor binding proteins 1, 2, 3, and 5 have been reported to bind to the cell surface or ECM.3,7,8 The binding affinity of IGFs to IGFBPs decreases when IGFBPs are bound to the cell surface or ECM.5,9 
Recently, the complex actions of the IGFBPs in skeletal muscle have become more apparent, with IGFBP2 implicated in skeletal muscle cell proliferation and differentiation.10,11 Additionally, IGFBPs are thought to have an inhibitory effect on both IGF-1 and -2.1214 Insulin-like growth factor binding protein 2 binds to IGF-1 or -2 with high affinity and can manipulate their binding to the IGF-1 receptor. This activity is modulated by the interaction of the binding protein with proteases.15 Furthermore, IGFBP2 is expressed in fetal tissues that are highly proliferative, and its expression significantly decreases after birth.16 
Several components of an IGF autocrine–paracrine system,1719 including several different IGFBPs,2025 have been identified in ocular tissues. Some studies have reported that IGFBPs in the vitreous humor exhibit an expression pattern different to those in serum. This suggests the possibility of local synthesis of IGFBPs in the eye rather than uptake from the systemic circulation.26,27 The unique expression of IGFBP2 in the eye suggests that it could be involved in the regulation of ocular growth and differentiation as well as in homeostasis in the mature eye.28 However, there is little research focused on intraocular IGFBP, and its role remains unclear. 
The amniotic membrane (AM) is the innermost layer of the fetal membrane. Studies have demonstrated that human29,30 and murine31 keratocyte, as judged by their characteristic dendritic morphology as well as expression of corneal stroma-specific keratocan, can maintain their phenotype without differentiation into alpha-smooth muscle actin (α-SMA)–expressing myofibroblasts. This can occur when the keratocytes are cultured on the AM stromal surface even when TGF-β is added in a serum-containing medium.31 Additionally, AM stromal extract not only helps maintain the fibroblast phenotype of AM stromal cells (AMSC; isolated mesenchymal cells from human AM stromal matrix) in vitro, but can also reverse differentiated myofibroblasts back to fibroblasts.32 Moreover, we previously reported that keratocyte-conditioned medium (KCM) has the capacity to facilitate the differentiation of MSCs into corneal keratocyte–like cells.33 However, the factors controlling the differentiation of the keratocyte and fibroblast lineages remain unclear. 
We hypothesized that factors present in KCM or AM extract might play an important role in differentiation and maintenance of keratocyte characteristics. In this study, IGFBP2 showed distinct expression in both KCM and AM extract compared with other IGFBP family proteins, IGF-1 and -2; therefore, we investigated the involvement of IGFBP2 in the regulation of the differentiation of human corneal fibroblasts (HCFs). 
Materials and Methods
Study Design
Our study was designed as follows: 
  1.  
    Composition analysis of conditioned medium (from keratocytes grown on AM; from corneal fibroblasts grown on plastic dishes; from AM) and AM extract;
  2.  
    Analysis of effects of IGFBP2 on keratocyte differentiation; investigation of specific markers in human mesenchymal stem cells (hMSCs) and HCFs induced by treatment with IGFBP2 or cultured in KCM;
  3.  
    Investigation of IGFBP2-mediated inhibition of TGFβ1-induced corneal myofibroblast transformation by measuring alteration of α-SMA expression; and
  4.  
    Investigation of the change of corneal phenotype after knockdown of IGFBP2 in HCFs.
Primary Culture of Human Keratocytes on AM
Human donor corneal tissue was obtained and stored in Optisol-GC (Bausch & Lomb, Rochester, NY, USA) for less than 3 days. Human keratocytes were isolated from the corneal stroma by sequential collagenase digestion as described previously.33 
Human AM preserved in Dulbecco's modified Eagle's medium (DMEM; WelGENE, Daegu, South Korea) and pure glycerol (1:1) at −80°C was thawed and incubated in a solution of versene and trypsin-EDTA (1:1; Invitrogen-Gibco, Carlsbad, CA, USA) for 30 minutes at 37°C, and the amniotic epithelium was removed from the AM using a scraper. Epithelium-free AM was placed on a 3 × 3-cm piece of stainless mesh with the stromal matrix facing upward. The suspended keratocytes extracted from the corneal tissues were seeded at 1 × 105 cells/mL on 2.5 cm × 2.5 cm denuded AM Cells were cultured on the AM for 15 days in a medium containing DMEM/F12 supplemented with 10% fetal bovine serum (FBS), and the medium was replaced every 2 to 3 days. 
Collection of KCM
Conditioned medium was obtained by culture of human corneal keratocytes on an AM as described above. Confluent stromal matrix cells growing on an AM were washed with PBS before adding 13 mL of DMEM/F12 supplemented with 10% FBS. The medium was then harvested after 2 days and centrifuged. The supernatant was collected by filtration through a 0.22-μm filter and used as KCM, which was directly transferred onto MSC cultures. 
Primary Culture of HCFs
For fibroblast isolation, corneal stromal tissue was cut into 6 to 8 pieces and placed in 6-well plates. After 10 minutes of adhesion, each explant was covered with DMEM/F12 supplemented with 10% FBS and 100 units/mL penicillin/streptomycin (WelGENE), and then placed in a humidified incubator (37°C, 5%, CO2). The medium was changed every 4 to 5 days. 
Culture of hMSCs
Bone marrow–derived MSCs (BM-MSCs, BM3.B10), which were obtained from human fetal spinal vertebrae at 12 to 15 weeks of gestation with an amphotropic, replication-incompetent retroviral vector containing v-myc as previously reported,34 were provided by Seung U. Kim (Professor Emeritus of Neurology, University of British Columbia, Vancouver, Canada). Bone marrow–derived MSCs were cultured in alpha-minimum essential medium (α-MEM; Invitrogen-Gibco) supplemented with L-glutamine, deoxyribonucleosides, ribonucleosides, 10% FBS, and 1% penicillin/streptomycin. 
In Vitro Drug Treatment
Cultured HCFs were treated with IGFBP2 (R&D Systems, Inc., Minneapolis, MN, USA) at a concentration of 100 to 500 ng/mL for various durations ranging from 24 to 72 hours. TGFβ1 (ProSpec-Tany Technogene Ltd., Rehovot, Israel) was used for chemically-induced fibrosis of HCFs. 
Quantitative RT-PCR (qRT-PCR)
RNA isolation was performed using RNAiso plus (Takara Bio, Inc., Otsu, Japan) according to the manufacturer's instructions. For semiquantitative RT-PCR, total RNA was reverse transcribed into complementary DNA (cDNA synthesis kit; Takara Bio, Inc.). Equal amounts of samples were used for PCR amplification of cDNA with primers specific for human α-SMA or the IGFBP family. Real-time quantitative RT-PCR (qRT-PCR) was performed using SYBR Premix ExTaq (Takara Bio, Inc.). SybrGreen fluorescence of the amplified cDNA products was quantified using the CFX96 Real-Time PCR Detection System (BioRad, Hercules, CA, USA) and an appropriate standard curve from autonomous qPCR assay reactions. Relative gene quantities were obtained using the comparative cycle threshold (Ct) method after normalization to a reference gene (glyceraldehyde-3-phosphate dehydrogenase [GAPDH]). The results of the qRT-PCR analysis are presented as the average amount of each gene expressed relative to average GAPDH expression. The specific primers that were used for α-SMA, IGFBP1, IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGF-1, IGF-2, and GAPDH are shown in the Table
Table
 
Sequences of PCR Primers
Table
 
Sequences of PCR Primers
Western Blot Analysis
Western blot analysis of cultured HCFs was performed as previously described.10 Primary mouse monoclonal antibodies against human keratocan, lumican (1:1000; MD Biosciences, Inc., St. Paul, MN, USA), ALDH1A1 (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), and a α-SMA (1:1000; Merck Millipore, Darmstadt, Germany) were diluted in tris-buffered saline (TBS), applied to the membrane, and incubated overnight at 4°C. Secondary antibodies were then diluted in TBS (1:2000), applied to the membrane, and incubated for 1 hour at room temperature. The protein signal after the application of secondary antibody was visualized using an enhanced chemiluminescence (ECL) Western blotting detection kit (Pierce Biotechnology, Inc., Rockfold, IL, USA), and β-actin (Sigma-Aldrich Corp., St. Louis, MO, USA) was used as a loading control. Image analysis of the immunobands was performed using ImageJ software ver. 1.46 (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Immunocytochemistry
Human corneal fibroblasts were cultured on coverslips. The coverslips were briefly washed with PBS, fixed in 4% paraformaldehyde for 15 minutes at room temperature (RT) and then washed three times, 5 minutes each time, with PBS. The fixed cells were permeabilized by incubation with 0.2% Triton X-100 for 15 minutes at RT and then rinsed three times with PBS. To prevent nonspecific binding, the slides were incubated with a blocking agent (2% bovine serum albumin in PBS) for 30 minutes at RT. The slides were then incubated with anti–α-SMA (1:50; Merck Millipore) antibodies. After three rinses in PBS for 5 minutes each time, the slides were incubated with secondary antibodies conjugated with fluorescein isothiocyanate (FITC; 1:100, Bethyl Laboratories, Montgomery, TX, USA) for 1 hour in the dark. After washing with PBS (5 minutes each time, three times for each slide), coverslips were mounted using Fluoroshield with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich Corp.) to visualize nuclei and combat fading of the immunolabeling. 
Immunodot Blot Analysis
The levels of growth factors in the conditioned medium were evaluated using a custom cytokine antibody array kit (RayBiotech, Inc., Norcross, GA, USA). Cytokine array membranes were analyzed according to manufacturer's protocols. The membranes were detected using an ECL Plus detection system included with the kit and signals were directly digitized using ChemiDoc XRS (BioRad). Blot density measurements were obtained with a Personal Molecular Imager FX (BioRad) supported by imaging analysis software (Quantity One, Imaging Research, Inc., Ontario, Canada). 
RNA Interference
Small interfering RNAs (siRNAs) targeting the human mRNA sequences of IGFBP2 were purchased from Dharmacon (SMARTpool: siGENOME IGFBP2 siRNA). A siGENOME Nontargeting siRNA pools (Dharmacon Products, Lafayette, CO, USA) was used as the negative control. Human corneal fibroblasts with 70% to 80% confluence were transfected with IGFBP2 or control siRNA by using lipofectamine (RNA iMAX; Invitrogen-Gibco) following manufacturer's instructions. The efficacy of knockdown of IGFBP2 was assessed by qRT-PCR and Western blotting. 
Statistical Analysis
Data are presented as the mean ± SD. Analysis of variance (ANOVA) followed by Bonferroni's post hoc analysis and two-tailed Student's t-test were used for statistical analysis of multiple groups and pairwise comparison, respectively. All experiments in this study were repeated in three or more separate trials. SPSS software version 20.0 (SPSS, Chicago, IL, USA) was used for all statistical analyses and P less than 0.05 indicated statistical significance. 
Results
Expression of IGFBP Family Proteins, IGF-1, and -2 in Conditioned Medium of Various Conditions and in AM Extracts
We intended to identify the factors, which are expressed significantly higher in both of KCM and AM extracts compared with corneal fibroblast conditioned medium (CFCM) from CFs which were grown on plastic dishes. Moreover, AM conditioned medium (AMCM) without keratocytes was analyzed to exclude the AM-related effects in KCM, which is conditioned medium from keratocytes grown on AM. Thus, the immunodot blot assay was performed to detect growth factors in conditioned medium derived from keratocytes, corneal fibroblasts, AM and in AM extracts (Figs. 1A, 1B). 
Figure 1
 
Secreted level of IGFBP family proteins IGF-I and -II in diverse conditioned media and amniotic membrane (AM) extract. (A) Immunodot blot analysis of conditioned media derived from keratocytes, corneal fibroblasts and AM, and of AM extracts. (B) Custom human growth factor antibody array map used in immunodot blot analysis. (C) The level of IGFBP1, 2, 4, 6, and IGF-II significantly differed according to four groups. The IGFBP2 level revealed higher in both keratocyte conditioned medium (KCM) and AM extract than in corneal fibroblast conditioned medium (CFCM), while IGFBP1, 4, and 6 were highly expressed in KCM or AM extract only, when compared with CFCM. ***P < 0.001 (ANOVA among four groups; KCM, CFCM, AMCM, and AM extract); ###P < 0.001, #P < 0.05 (Bonferroni's post hoc, KCM, or AM extract versus CFCM).
Figure 1
 
Secreted level of IGFBP family proteins IGF-I and -II in diverse conditioned media and amniotic membrane (AM) extract. (A) Immunodot blot analysis of conditioned media derived from keratocytes, corneal fibroblasts and AM, and of AM extracts. (B) Custom human growth factor antibody array map used in immunodot blot analysis. (C) The level of IGFBP1, 2, 4, 6, and IGF-II significantly differed according to four groups. The IGFBP2 level revealed higher in both keratocyte conditioned medium (KCM) and AM extract than in corneal fibroblast conditioned medium (CFCM), while IGFBP1, 4, and 6 were highly expressed in KCM or AM extract only, when compared with CFCM. ***P < 0.001 (ANOVA among four groups; KCM, CFCM, AMCM, and AM extract); ###P < 0.001, #P < 0.05 (Bonferroni's post hoc, KCM, or AM extract versus CFCM).
Among IGFBP family proteins, IGF-1 and -2, only IGFBP2 showed significantly higher expression in both KCM (P < 0.001) and AM extracts (P < 0.001) compared with in CFCM. Moreover, the difference of IGFBP2 level between in KCM and in conditioned medium from AM was significant (P < 0.001) indicating that prominent existence of IGFBP2 in KCM may not be derived from AM Although IGFBP1 revealed high expression in AM extracts (P < 0.001, in KCM versus in CFCM), IGFBP1 level between in KCM and CFCM showed no significant difference (P = 1.000). Likewise, IGFBP4 and IGFBP6 levels were higher in KCM than in CFCM (P = 0.015 and P < 0.001, respectively), but no significant difference was noted between in CFCM and in AM extract (P = 1.000 and P = 0.059, respectively). Insulin-like growth factor 2 expression was most prominent in CFCM and levels of IGFBP and IGF-2 showed no difference according to mediums (Fig. 1C). 
Acquisition of Corneal Phenotype by IGFBP2 in HMSCS
Human mesenchymal stem cells were seeded on a culture dish, and then cultured with KCM. When MSCs were grown in KCM for two passages, mRNA expression of IGFBP2 was significantly increased (Fig. 2A), but other IGFBP family proteins, IGF-1 and -2 were not changed by real-time PCR analysis (data not shown). Furthermore, in order to clarify the effects of IGFBP2 in hMSC cultured in KCM, MSCs were treated with IGFBP2 protein and we analyzed the expression pattern of markers of MSC and keratocyte. After treating the hMSCs with various concentrations (100–500 ng/mL) of the IGFBP2 for 24 hours, mRNA level of α-SMA was decreased (Fig. 2B). A similar effect was observed when 100 ng/mL of IGFBP2 was added for various durations (24–72 hours; Fig. 2C). 
Figure 2
 
Upregulation of IGFBP2 and inhibition of α-SMA in human mesenchymal stem cells (hMSCs). (A) The relative expression of IGFBP2 mRNA in MSCs statistically increased in the presence of KCM. After MSCs were treated with IGFBP2, (B) α-SMA was downregulated at various concentrations (at 24 hours) and (C) at various durations ranging from 24 to 72 hours. There was no significant difference of α-SMA mRNA expression according to the concentration (B) or treating time (C) of IGFBP2. (D) Insulin-like growth factor-BP2 downregulated protein expression of α-SMA and upregulated the expression of IGFBP2 and corneal markers including keratocan and ALDH1A1 with statistical significance. Especially, IGFBP 500 ng/mL showed prominent alteration of expressions of α-SMA, IGFBP2, keratocan, and ALDH1A1 compared with lower concentrations of IGFBP2. **P < 0.01, *P < 0.05, versus control. ##P < 0.01, #P < 0.05.
Figure 2
 
Upregulation of IGFBP2 and inhibition of α-SMA in human mesenchymal stem cells (hMSCs). (A) The relative expression of IGFBP2 mRNA in MSCs statistically increased in the presence of KCM. After MSCs were treated with IGFBP2, (B) α-SMA was downregulated at various concentrations (at 24 hours) and (C) at various durations ranging from 24 to 72 hours. There was no significant difference of α-SMA mRNA expression according to the concentration (B) or treating time (C) of IGFBP2. (D) Insulin-like growth factor-BP2 downregulated protein expression of α-SMA and upregulated the expression of IGFBP2 and corneal markers including keratocan and ALDH1A1 with statistical significance. Especially, IGFBP 500 ng/mL showed prominent alteration of expressions of α-SMA, IGFBP2, keratocan, and ALDH1A1 compared with lower concentrations of IGFBP2. **P < 0.01, *P < 0.05, versus control. ##P < 0.01, #P < 0.05.
We confirmed the downregulation of α-SMA, an MSC marker, by IGFBP2 in hMSCs using Western blot analysis. Additionally, expression of keratocyte markers, including keratocan and ALDH1A1, was strongly increased in hMSCs by IGFBP2, especially at the concentration of 500 ng/mL compared with lower concentrations of IGFBP2. However, lumican expression was not clearly influenced by IGFBP2 (Fig. 2D). 
Maintenance of Corneal Phenotype by IGFBP2 in HCFs
Stromal cells grown in AM are good at maintaining their cell shape and marker expression.29 However, when cultured in plastic dishes, corneal stromal cells rapidly lose their dendritic morphology and acquire a fibroblastic shape.35,36 Therefore, the former is called a keratocyte, while the latter is referred to as a corneal fibroblast. 
Keratocytes strongly expressed keratocan, lumican, ALDH1A1, and IGFBP2, but α-SMA expression was negligible. However, in corneal fibroblasts, α-SMA expression gradually increased and expression of IGFBP2, keratocan, and ALDH1A1 decreased over the culture passages (Fig. 3A). In addition, the expression of α-SMA was inhibited, and keratocan and ALDH1A1 were upregulated by IGFBP2 in HCFs, which was similar to the effect of KCM itself (Figs. 3B, 3C). Moreover, treatment with IGFBP2 or culture in KCM increased the expression of intracellular IGFBP2 in HCFs. The expression of keratocan and ALDH1A1 were increased depending on the increased expression of IGFBP2. However, there was no significant change in lumican expression. Human corneal fibroblasts without IGFBP2 or KCM did not alter the expression level of all of α-SMA, IGFBP2, keratocan, ALDH1A1, and lumican until 72 hours (data not shown). 
Figure 3
 
Expression of marker proteins in cultured HCFs according to with or without IGFBP2 or KCM. (A) As the passage of culture progresses, IGFBP2, keratocan, and ALDH1A1 were downregulated, while α-SMA was upregulated in HCFs. (B) However, when incubated with IGFBP2 or KCM, α-SMA expression decreased steadily and the expression of IGFBP2, keratocan, and ALDH1A1 increased. (C) The protein expression pattern by Western blot analysis (A, B) was confirmed statistically based on density measurements. Change of lumican expression by IGFBP2 was not statistically significant. **P < 0.01, *P < 0.05; #, versus keratocytes; ¥, versus control (HCF [P8]).
Figure 3
 
Expression of marker proteins in cultured HCFs according to with or without IGFBP2 or KCM. (A) As the passage of culture progresses, IGFBP2, keratocan, and ALDH1A1 were downregulated, while α-SMA was upregulated in HCFs. (B) However, when incubated with IGFBP2 or KCM, α-SMA expression decreased steadily and the expression of IGFBP2, keratocan, and ALDH1A1 increased. (C) The protein expression pattern by Western blot analysis (A, B) was confirmed statistically based on density measurements. Change of lumican expression by IGFBP2 was not statistically significant. **P < 0.01, *P < 0.05; #, versus keratocytes; ¥, versus control (HCF [P8]).
Restoration of Corneal Phenotype by IGFBP2 in TGFβ1-Induced Corneal Myofibroblasts
Transforming growth factor–β1 is known to play a central role in fibroblast activation and fibroblast-to-myofibroblast differentiation, and induces the expression of α-SMA, a marker of myofibroblasts.37 Therefore, we treated HCFs with various concentrations of TGFβ1 (5–20 ng/mL) for various durations (24–72 hours) to prepare the corneal myofibroblasts. The expression of α-SMA in HCFs increased dependent upon treatment duration, but there was no definite change of expression pattern of α-SMA according to the concentration of TGFβ1 used (Fig. 4A). 
Figure 4
 
The change in protein expression of myofibroblast and keratocyte markers by IGFBP2. (A) Human corneal fibroblasts HCFs were treated with TGFβ1 under various concentrations and times. Transforming growth factor–β1 induced expression of α-SMA in HCFs, the myofibroblast transdifferentiation marker. (B) After HCFs were cotreated with TGFβ1 and IGFBP2, relative mRNA expression of TGFβ1-induced α-SMA was downregulated and keratocan was upregulated by treatment with IGFBP2 for 24 hours (α-SMA) and for 24 to 48 hours (keratocan), respectively, although there was no change when treated for 72 hours. (C) Protein expression of α-SMA, keratocan, and ALDH1A1 by Western blot analysis after treated with TGFβ1 and IGFBP2 revealed a pattern similar to the results of qRT-PCR. (D) Immunofluorescence staining of α-SMA expression in HCFs with TGFβ1 and IGFBP2 according to treating time (24–72 hours). Expressed α-SMA–positive stress fibers were decreased by IGFBP2 cotreatment for 24 and 48 hours. The proposed mechanism explaining such a phenomenon is also illustrated. **P < 0.01, *P < 0.05.
Figure 4
 
The change in protein expression of myofibroblast and keratocyte markers by IGFBP2. (A) Human corneal fibroblasts HCFs were treated with TGFβ1 under various concentrations and times. Transforming growth factor–β1 induced expression of α-SMA in HCFs, the myofibroblast transdifferentiation marker. (B) After HCFs were cotreated with TGFβ1 and IGFBP2, relative mRNA expression of TGFβ1-induced α-SMA was downregulated and keratocan was upregulated by treatment with IGFBP2 for 24 hours (α-SMA) and for 24 to 48 hours (keratocan), respectively, although there was no change when treated for 72 hours. (C) Protein expression of α-SMA, keratocan, and ALDH1A1 by Western blot analysis after treated with TGFβ1 and IGFBP2 revealed a pattern similar to the results of qRT-PCR. (D) Immunofluorescence staining of α-SMA expression in HCFs with TGFβ1 and IGFBP2 according to treating time (24–72 hours). Expressed α-SMA–positive stress fibers were decreased by IGFBP2 cotreatment for 24 and 48 hours. The proposed mechanism explaining such a phenomenon is also illustrated. **P < 0.01, *P < 0.05.
The α-SMA expression induced by TGFβ1 was decreased by IGFBP2, although IGFBP2 could not reverse the α-SMA expression in HCFs when treating time of TGFβ1 was extended to 72 hours. Moreover, IGFBP2 upregulated the expression of ALDH1A1 and keratocan in TGFβ1-treated HCFs, but IGFBP2 could not restore the corneal phenotype of HCFs when they had been treated with TGFβ1 for 72 hours (Figs. 4B, 4C). Expression of lumican was not affected by IGFBP2 (data not shown). 
We confirmed this phenomenon again using immunocytochemical staining. The formation of prominent stress fibers by TGFβ1 in HCFs was visualized by α-SMA staining. Nearly all cells obtained a myofibroblast-like shape and showed strong expression of α-SMA depending on the duration of TGFβ1 treatment. Alpha-SMA–expressing stress fibers, which were more prominent in TGFβ1-treated HCFs than control HCFs, were attenuated by IGFBP2 for 24 to 24 hours. There was no change of expression pattern of α-SMA in HCFs after 72 hours (Fig. 4D). 
Attenuation of Corneal Phenotypes by Knockdown of IGFBP2 in HCFs
To more specifically elucidate the role of IGFBP2 in HCFs, we performed knockdown of IGFBP2 expression in HCFs using IGFBP2 siRNA. Insulin-like growth factor binding protein 2 siRNA–transfected HCFs revealed decreased of IGFBP2 expression in mRNA and protein level to approximately 0.5-fold and 0.3-fold, respectively, while nontargeting (negative control) siRNA-transfected cells and vehicle alone (mock) showed no difference. Knockdown of IGFBP2 in HCFs cells increased α-SMA expression and inhibited the expression of ALDH1A1 significantly (Figs. 5A, 5B). 
Figure 5
 
Aggravated myofibroblast transformation and attenuated expression of keratocyte phenotypes induced by IGFBP2 siRNA in HCFs. (A) Knockdown of IGFBP2 increased α-SMA mRNA level and decreased ALDH1A1 mRNA level. (B) Western blot analysis of α-SMA, ALDH1A1, and keratocan revealed that knockdown of IGFBP2 protein in HCFs attenuated ALDH1A1 level and uninhibited α-SMA expression. There was no significant change in keratocan expression before and after knockdown of IGFBP2 similar to in mRNA level. N/C, negative control. **P < 0.01, *P < 0.05.
Figure 5
 
Aggravated myofibroblast transformation and attenuated expression of keratocyte phenotypes induced by IGFBP2 siRNA in HCFs. (A) Knockdown of IGFBP2 increased α-SMA mRNA level and decreased ALDH1A1 mRNA level. (B) Western blot analysis of α-SMA, ALDH1A1, and keratocan revealed that knockdown of IGFBP2 protein in HCFs attenuated ALDH1A1 level and uninhibited α-SMA expression. There was no significant change in keratocan expression before and after knockdown of IGFBP2 similar to in mRNA level. N/C, negative control. **P < 0.01, *P < 0.05.
Discussion
Corneal stromal cells, keratocytes, secrete cornea-specific ECM components, including keratocan sulfate, lumican, and keratocan.38 They play an important role in the maintenance of corneal transparency.39,40 In conditions of disrupted tissue homeostasis, such as after injury, during wound healing, or chronic inflammation, keratocytes tend to differentiate into fibroblasts and myofibroblasts and deposit a less-organized collagen-fibrillar construct in a pattern with similarities to corneal scar tissue due to a lack of cornea-specific ECM components.38 If this differentiation process can be alleviated, the regulation of corneal scar formation may be possible. In this study, we analyzed the components of the KCM and AM extract that are likely involved in the homeostatic maintenance of corneal stroma, and IGFBP2 was identified as a key candidate for such a mechanism. 
In the present study, IGFBP2 was strongly identified in both KCM and AM extract among IGFBP family proteins, IGF-1 and -2 when compared with in CFCM and AMCM. It is interesting that IGFBP2 is abundant in AM itself as appears by the immune-expression of IGFBP2 in AM extract, while there is lack of IGFBP2 in AMCM although IGFBP2 is highly expressed in KCM from keratocytes cultured on AM. We thought that IGFBP2 in KCM was derived not from AM but from keratocytes and inherited IGFBP2 in AM might interact with or force keratocyte to excrete IGFBP2. 
We confirmed that the hMSCs would differentiate into keratocyte-like cells after treatment with IGFBP2. This is similar to the effect of KCM treatment as reported in our previous study33 in which the differentiation-inducing effect of a single treatment of IGFBP2 was confirmed to be equally powerful as KCM. Thereafter, we investigated whether IGFBP2 has the effect of maintaining the characteristics of the keratocytes in in vitro culture, similar to its effects in the induction of differentiation in MSCs. In addition, treating HCFs with IGFBP2 reduced their expression of α-SMA and increased the expression of keratocyte markers. The HCFs reacquired the properties of keratocytes after IGFBP2 treatment. This effect was reproduced in the same manner as KCM treatment. Furthermore, knockdown of IGFBP2 deprived HCFs of corneal phenotype (ALDH1A1) and promoted the acquisition of myofibroblast feature. These serial results suggest that IGFBP2 may normally serve to maintain the corneal phenotype against to be myofibroblast transformation. 
A central feature of activated stromal cells is the acquisition of smooth muscle features, most notably neoformation of contractile stress fibers and expression of α-SMA; hence, the name myofibroblast. The transient acquisition of this phenotype is beneficial for normal tissue repair processes when myofibroblast remodeling activities restore and preserve tissue integrity. However, persistence of myofibroblast transformation results in tissue stiffening and deformation. In fibrosis, stiff scar tissue alters normal organ function; the mechanical and chemical conditions generated by myofibroblasts promote disease progression.41 Moreover, fibrosis of the cornea can lead to corneal opacification and subsequent loss of vision.42,43 Therefore, the maintenance of keratocyte characteristics can be linked to the maintenance of corneal clarity and homeostasis. In this context, our results show antifibrotic potential that the keratocyte-fibroblast lineage that can be controlled by IGFBP2. Although IGFBP2 has been previously shown to be involved in abrogation of proliferation and of biosynthesis of collagen, fibulin and fibronectin in liver myofibroblasts against liver fibrogenesis44 with a similar mechanism to in cornea as seen in our study, there have been no studies regarding this kind of effect of IGFBP2 in cornea. 
Inhibition of α-SMA and corneal markers by IGFBP2 was affected by the status of TGFβ1-induced myofibroblast transformation. When HCFs were treated by TGFβ1 for 72 hours, there was no significant influence of IGFBP2 on the reverse of expression of α-SMA, keratocan, and ALDH1A1 unlike at 24 and 48 hours (Fig. 4C). We suggest that the expression of all those markers is probably variable depending on the exposure time to TGFβ1, which can affect the severity of myofibroblast transformation. Furthermore, it is speculated that IGFBP2 may regulate the myofibroblast transition as a possible antifibrotic strategy, particularly in the early stages of cell transformation. 
In analyses of expression alterations of α-SMA by IGFBP2 treatment in hMSCs, there was discordance of expression between mRNA and protein levels (Figs. 2B, 2D). Unlike mRNA levels, protein levels of α-SMA, IGFBP2, and corneal markers including keratocan and ALDH1A1 revealed their gradual change of expressions toward the increase of IGFBP concentrations. As a possible mechanism, we suggest that there may be possible unknown impact of microRNAs on translation or process of posttranscriptional regulation as proposed previously45 and think that this would be an interesting issue of future study. 
In conclusion, we demonstrated the novel effect of IGFBP2; regulation of the differentiation of corneal fibroblasts. Although further studies are necessary to determine the specific mechanism of IGFBP2, our results suggest the IGFBP2 may be considered to be a novel antifibrotic strategy in corneal diseases. 
Acknowledgments
Supported by grants from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (2015R1A2A2A01004643; Daejeon, South Korea), and by a grant from the Korea Healthcare technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (HI12C1376; Cheongju, Chungcheongbuk-do, South Korea). 
Disclosure: S.H. Park, None; K.W. Kim, None; J.C. Kim, None 
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Figure 1
 
Secreted level of IGFBP family proteins IGF-I and -II in diverse conditioned media and amniotic membrane (AM) extract. (A) Immunodot blot analysis of conditioned media derived from keratocytes, corneal fibroblasts and AM, and of AM extracts. (B) Custom human growth factor antibody array map used in immunodot blot analysis. (C) The level of IGFBP1, 2, 4, 6, and IGF-II significantly differed according to four groups. The IGFBP2 level revealed higher in both keratocyte conditioned medium (KCM) and AM extract than in corneal fibroblast conditioned medium (CFCM), while IGFBP1, 4, and 6 were highly expressed in KCM or AM extract only, when compared with CFCM. ***P < 0.001 (ANOVA among four groups; KCM, CFCM, AMCM, and AM extract); ###P < 0.001, #P < 0.05 (Bonferroni's post hoc, KCM, or AM extract versus CFCM).
Figure 1
 
Secreted level of IGFBP family proteins IGF-I and -II in diverse conditioned media and amniotic membrane (AM) extract. (A) Immunodot blot analysis of conditioned media derived from keratocytes, corneal fibroblasts and AM, and of AM extracts. (B) Custom human growth factor antibody array map used in immunodot blot analysis. (C) The level of IGFBP1, 2, 4, 6, and IGF-II significantly differed according to four groups. The IGFBP2 level revealed higher in both keratocyte conditioned medium (KCM) and AM extract than in corneal fibroblast conditioned medium (CFCM), while IGFBP1, 4, and 6 were highly expressed in KCM or AM extract only, when compared with CFCM. ***P < 0.001 (ANOVA among four groups; KCM, CFCM, AMCM, and AM extract); ###P < 0.001, #P < 0.05 (Bonferroni's post hoc, KCM, or AM extract versus CFCM).
Figure 2
 
Upregulation of IGFBP2 and inhibition of α-SMA in human mesenchymal stem cells (hMSCs). (A) The relative expression of IGFBP2 mRNA in MSCs statistically increased in the presence of KCM. After MSCs were treated with IGFBP2, (B) α-SMA was downregulated at various concentrations (at 24 hours) and (C) at various durations ranging from 24 to 72 hours. There was no significant difference of α-SMA mRNA expression according to the concentration (B) or treating time (C) of IGFBP2. (D) Insulin-like growth factor-BP2 downregulated protein expression of α-SMA and upregulated the expression of IGFBP2 and corneal markers including keratocan and ALDH1A1 with statistical significance. Especially, IGFBP 500 ng/mL showed prominent alteration of expressions of α-SMA, IGFBP2, keratocan, and ALDH1A1 compared with lower concentrations of IGFBP2. **P < 0.01, *P < 0.05, versus control. ##P < 0.01, #P < 0.05.
Figure 2
 
Upregulation of IGFBP2 and inhibition of α-SMA in human mesenchymal stem cells (hMSCs). (A) The relative expression of IGFBP2 mRNA in MSCs statistically increased in the presence of KCM. After MSCs were treated with IGFBP2, (B) α-SMA was downregulated at various concentrations (at 24 hours) and (C) at various durations ranging from 24 to 72 hours. There was no significant difference of α-SMA mRNA expression according to the concentration (B) or treating time (C) of IGFBP2. (D) Insulin-like growth factor-BP2 downregulated protein expression of α-SMA and upregulated the expression of IGFBP2 and corneal markers including keratocan and ALDH1A1 with statistical significance. Especially, IGFBP 500 ng/mL showed prominent alteration of expressions of α-SMA, IGFBP2, keratocan, and ALDH1A1 compared with lower concentrations of IGFBP2. **P < 0.01, *P < 0.05, versus control. ##P < 0.01, #P < 0.05.
Figure 3
 
Expression of marker proteins in cultured HCFs according to with or without IGFBP2 or KCM. (A) As the passage of culture progresses, IGFBP2, keratocan, and ALDH1A1 were downregulated, while α-SMA was upregulated in HCFs. (B) However, when incubated with IGFBP2 or KCM, α-SMA expression decreased steadily and the expression of IGFBP2, keratocan, and ALDH1A1 increased. (C) The protein expression pattern by Western blot analysis (A, B) was confirmed statistically based on density measurements. Change of lumican expression by IGFBP2 was not statistically significant. **P < 0.01, *P < 0.05; #, versus keratocytes; ¥, versus control (HCF [P8]).
Figure 3
 
Expression of marker proteins in cultured HCFs according to with or without IGFBP2 or KCM. (A) As the passage of culture progresses, IGFBP2, keratocan, and ALDH1A1 were downregulated, while α-SMA was upregulated in HCFs. (B) However, when incubated with IGFBP2 or KCM, α-SMA expression decreased steadily and the expression of IGFBP2, keratocan, and ALDH1A1 increased. (C) The protein expression pattern by Western blot analysis (A, B) was confirmed statistically based on density measurements. Change of lumican expression by IGFBP2 was not statistically significant. **P < 0.01, *P < 0.05; #, versus keratocytes; ¥, versus control (HCF [P8]).
Figure 4
 
The change in protein expression of myofibroblast and keratocyte markers by IGFBP2. (A) Human corneal fibroblasts HCFs were treated with TGFβ1 under various concentrations and times. Transforming growth factor–β1 induced expression of α-SMA in HCFs, the myofibroblast transdifferentiation marker. (B) After HCFs were cotreated with TGFβ1 and IGFBP2, relative mRNA expression of TGFβ1-induced α-SMA was downregulated and keratocan was upregulated by treatment with IGFBP2 for 24 hours (α-SMA) and for 24 to 48 hours (keratocan), respectively, although there was no change when treated for 72 hours. (C) Protein expression of α-SMA, keratocan, and ALDH1A1 by Western blot analysis after treated with TGFβ1 and IGFBP2 revealed a pattern similar to the results of qRT-PCR. (D) Immunofluorescence staining of α-SMA expression in HCFs with TGFβ1 and IGFBP2 according to treating time (24–72 hours). Expressed α-SMA–positive stress fibers were decreased by IGFBP2 cotreatment for 24 and 48 hours. The proposed mechanism explaining such a phenomenon is also illustrated. **P < 0.01, *P < 0.05.
Figure 4
 
The change in protein expression of myofibroblast and keratocyte markers by IGFBP2. (A) Human corneal fibroblasts HCFs were treated with TGFβ1 under various concentrations and times. Transforming growth factor–β1 induced expression of α-SMA in HCFs, the myofibroblast transdifferentiation marker. (B) After HCFs were cotreated with TGFβ1 and IGFBP2, relative mRNA expression of TGFβ1-induced α-SMA was downregulated and keratocan was upregulated by treatment with IGFBP2 for 24 hours (α-SMA) and for 24 to 48 hours (keratocan), respectively, although there was no change when treated for 72 hours. (C) Protein expression of α-SMA, keratocan, and ALDH1A1 by Western blot analysis after treated with TGFβ1 and IGFBP2 revealed a pattern similar to the results of qRT-PCR. (D) Immunofluorescence staining of α-SMA expression in HCFs with TGFβ1 and IGFBP2 according to treating time (24–72 hours). Expressed α-SMA–positive stress fibers were decreased by IGFBP2 cotreatment for 24 and 48 hours. The proposed mechanism explaining such a phenomenon is also illustrated. **P < 0.01, *P < 0.05.
Figure 5
 
Aggravated myofibroblast transformation and attenuated expression of keratocyte phenotypes induced by IGFBP2 siRNA in HCFs. (A) Knockdown of IGFBP2 increased α-SMA mRNA level and decreased ALDH1A1 mRNA level. (B) Western blot analysis of α-SMA, ALDH1A1, and keratocan revealed that knockdown of IGFBP2 protein in HCFs attenuated ALDH1A1 level and uninhibited α-SMA expression. There was no significant change in keratocan expression before and after knockdown of IGFBP2 similar to in mRNA level. N/C, negative control. **P < 0.01, *P < 0.05.
Figure 5
 
Aggravated myofibroblast transformation and attenuated expression of keratocyte phenotypes induced by IGFBP2 siRNA in HCFs. (A) Knockdown of IGFBP2 increased α-SMA mRNA level and decreased ALDH1A1 mRNA level. (B) Western blot analysis of α-SMA, ALDH1A1, and keratocan revealed that knockdown of IGFBP2 protein in HCFs attenuated ALDH1A1 level and uninhibited α-SMA expression. There was no significant change in keratocan expression before and after knockdown of IGFBP2 similar to in mRNA level. N/C, negative control. **P < 0.01, *P < 0.05.
Table
 
Sequences of PCR Primers
Table
 
Sequences of PCR Primers
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