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The Role of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2) in the Regulation of Corneal Fibroblast Differentiation

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The Role of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2) in the Regulation of Corneal Fibroblast Differentiation Cornea The Role of Insulin-Like Growth Factor Binding Protein 2 (IGFBP2) in the Regulation of Corneal Fibroblast Differentiation Soo Hyun Park, Kyoung Woo Kim, and Jae Chan Kim Department of Ophthalmology, College of Medicine, Chung-Ang University Hospital, Seoul, Korea Correspondence: Jae Chan Kim, De- PURPOSE. Previously, we reported that keratocyte-conditioned medium (KCM) facilitates the partment of Ophthalmology, Chung- differentiation of human mesenchymal stem cells (hMSCs) into corneal keratocyte–like cells. Ang University Hospital, 224-1, This study is designed to investigate the roles of insulin-like growth factor binding protein 2 Heukseok-dong, Dongjak-gu, Seoul (IGFBP2) for the regulation of corneal fibroblast differentiation as a newly unveiled 156-755, Korea; component of KCM. [email protected]. Submitted: February 4, 2015 METHODS. Immunodot blot analysis was performed to identify the factors that are highly Accepted: October 5, 2015 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 Citation: Park SH, Kim KW, Kim JC. fibroblasts (HCFs) by analyzing expression patterns of alpha-smooth muscle actin (a-SMA) and The role of insulin-like growth factor binding protein 2 (IGFBP2) in the keratocyte markers including keratocan, lumican and aldehyde dehydrogenase 1 family regulation of corneal fibroblast differ- member A1 (ALDH1A1). Furthermore, to specify the role of IGFBP2, the expression of a-SMA entiation. Invest Ophthalmol Vis Sci. and keratocyte markers was determined in transforming growth factor b 1 (TGFb1)-induced 2015;56:7293–7302. DOI:10.1167/ corneal myofibroblast and in HCFs after knockdown of IGFBP2. iovs.15-16616 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 a-SMA expression in hMSCs and HCFs. Insulin-like growth factor binding protein 2 inhibited TGFb1-induced upregulation of a-SMA and increased expressions of keratocan and ALDH1A1 in HCFs. Furthermore, the knockdown of IGFBP2 increased a-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. Keywords: insulin-like growth factor binding protein 2, alpha-smooth muscle actin, keratocytes, corneal fibroblasts nsulin-like growth factor (IGF)-1 and -2 are polypeptides that factor binding proteins 1, 2, 3, and 5 have been reported to I exhibit mitogenic, metabolic, and differentiative effects on a bind to the cell surface or ECM.3,7,8 The binding affinity of IGFs variety of cell types. Because of the relative abundance of both to IGFBPs decreases when IGFBPs are bound to the cell surface IGF-1 and IGF-2 during development, the IGFs are thought to or ECM.5,9 play an especially important role in the proliferation and Recently, the complex actions of the IGFBPs in skeletal differentiation of embryonic tissues.1,2 The insulin-like growth muscle have become more apparent, with IGFBP2 implicated factor binding proteins (IGFBP1–6) are a family of circulating in skeletal muscle cell proliferation and differentiation.10,11 proteins that were initially defined by their capacity to Additionally, IGFBPs are thought to have an inhibitory effect on differentially modulate (positively or negatively) the actions of both IGF-1 and -2.12–14 Insulin-like growth factor binding IGF ligands. Insulin-like growth factor binding proteins are protein 2 binds to IGF-1 or -2 with high affinity and can present in serum and in a variety of biological fluids, including manipulate their binding to the IGF-1 receptor. This activity is amniotic, follicular, cerebrospinal, and seminal fluid, as well as modulated by the interaction of the binding protein with milk.3 Insulin-like growth factor binding proteins have also proteases.15 Furthermore, IGFBP2 is expressed in fetal tissues been identified in the extracellular environment and inside that are highly proliferative, and its expression significantly cells, and play distinct physiological roles in growth and decreases after birth.16 development. Insulin-like growth factor binding proteins may Several components of an IGF autocrine–paracrine sys- be differentially targeted to different tissues depending on both tem,17–19 including several different IGFBPs,20–25 have been their primary structure and their posttranslational modifica- identified in ocular tissues. Some studies have reported that tions.4 It has been postulated that a number of IGFBPs can IGFBPs in the vitreous humor exhibit an expression pattern interact with the extracellular matrix (ECM) or cell surface via different to those in serum. This suggests the possibility of local glycoproteins, collagens, and integrins.5,6 Insulin-like growth synthesis of IGFBPs in the eye rather than uptake from the Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc. iovs.arvojournals.org j ISSN: 1552-5783 7293 Downloaded from iovs.arvojournals.org on 09/28/2021 Regulatory Role of Fibroblast Lineage of IGFBP2 IOVS j November 2015 j Vol. 56 j No. 12 j 7294 systemic circulation.26,27 The unique expression of IGFBP2 in mL on 2.5 cm 3 2.5 cm denuded AM Cells were cultured on the the eye suggests that it could be involved in the regulation of AM for 15 days in a medium containing DMEM/F12 supple- ocular growth and differentiation as well as in homeostasis in mented with 10% fetal bovine serum (FBS), and the medium the mature eye.28 However, there is little research focused on was replaced every 2 to 3 days. intraocular IGFBP, and its role remains unclear. The amniotic membrane (AM) is the innermost layer of the Collection of KCM fetal membrane. Studies have demonstrated that human29,30 and murine31 keratocyte, as judged by their characteristic Conditioned medium was obtained by culture of human dendritic morphology as well as expression of corneal stroma- corneal keratocytes on an AM as described above. Confluent specific keratocan, can maintain their phenotype without stromal matrix cells growing on an AM were washed with PBS differentiation into alpha-smooth muscle actin (a-SMA)–ex- before adding 13 mL of DMEM/F12 supplemented with 10% pressing myofibroblasts. This can occur when the keratocytes FBS. The medium was then harvested after 2 days and are cultured on the AM stromal surface even when TGF-b is centrifuged. The supernatant was collected by filtration added in a serum-containing medium.31 Additionally, AM through a 0.22-lm filter and used as KCM, which was directly stromal extract not only helps maintain the fibroblast transferred onto MSC cultures. phenotype of AM stromal cells (AMSC; isolated mesenchymal cells from human AM stromal matrix) in vitro, but can also Primary Culture of HCFs reverse differentiated myofibroblasts back to fibroblasts.32 Moreover, we previously reported that keratocyte-conditioned For fibroblast isolation, corneal stromal tissue was cut into 6 to medium (KCM) has the capacity to facilitate the differentiation 8 pieces and placed in 6-well plates. After 10 minutes of of MSCs into corneal keratocyte–like cells.33 However, the adhesion, each explant was covered with DMEM/F12 supple- factors controlling the differentiation of the keratocyte and mented with 10% FBS and 100 units/mL penicillin/streptomy- fibroblast lineages remain unclear. cin (WelGENE), and then placed in a humidified incubator We hypothesized that factors present in KCM or AM extract (378C, 5%, CO2). The medium was changed every 4 to 5 days. might play an important role in differentiation and mainte- nance of keratocyte characteristics. In this study, IGFBP2 Culture of hMSCs showed distinct expression in both KCM and AM extract compared with other IGFBP family proteins, IGF-1 and -2; Bone marrow–derived MSCs (BM-MSCs, BM3.B10), which were therefore, we investigated the involvement of IGFBP2 in the obtained from human fetal spinal vertebrae at 12 to 15 weeks regulation of the differentiation of human corneal fibroblasts of gestation with an amphotropic, replication-incompetent (HCFs). 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). MATERIALS AND METHODS Bone marrow–derived MSCs were cultured in alpha-minimum essential medium (a-MEM; Invitrogen-Gibco) supplemented Study Design with L-glutamine, deoxyribonucleosides, ribonucleosides, 10% FBS, and 1% penicillin/streptomycin. Our study was designed as follows: 1. Composition analysis of conditioned medium (from In Vitro Drug Treatment keratocytes grown on AM; from corneal fibroblasts Cultured HCFs were treated with IGFBP2 (R&D Systems, Inc., grown on plastic dishes; from AM) and AM extract; Minneapolis, MN, USA) at a concentration of 100 to 500 ng/mL 2. Analysis of effects of IGFBP2 on keratocyte differentia- for various durations ranging from 24 to 72 hours. TGFb1 tion; investigation of specific markers in human (ProSpec-Tany Technogene Ltd., Rehovot, Israel) was used for mesenchymal stem cells (hMSCs) and HCFs induced by chemically-induced fibrosis of HCFs. treatment with IGFBP2 or cultured in KCM; 3. Investigation of IGFBP2-mediated inhibition of TGFb1- induced corneal myofibroblast transformation by mea- Quantitative RT-PCR (qRT-PCR) suring alteration of a-SMA expression; and RNA isolation was performed using RNAiso plus (Takara Bio, 4. Investigation of the change of corneal phenotype after Inc., Otsu, Japan) according to the manufacturer’s instructions. knockdown of IGFBP2 in HCFs. For semiquantitative RT-PCR, total RNA was reverse transcribed into complementary DNA (cDNA synthesis kit; Takara Bio, Primary Culture of Human Keratocytes on AM Inc.). Equal amounts of samples were used for PCR amplifica- tion of cDNA with primers specific for human a-SMA or the Human donor corneal tissue was obtained and stored in IGFBP family.
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