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.
12–14 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,
17–19 including several different IGFBPs,
20–25 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 human
29,30 and murine
31 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).
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.
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.
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.
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.
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).
Expression of IGFBP Family Proteins, IGF-1, and -2 in Conditioned Medium of Various Conditions and in AM Extracts
Restoration of Corneal Phenotype by IGFBP2 in TGFβ1-Induced Corneal Myofibroblasts
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 study
33 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 fibrogenesis
44 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 previously
45 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.
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