Mutual regulation between IGF‐1R and IGFBP‐3 in human corneal epithelial cells (original) (raw)
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The IGF/Insulin-IGFBP Axis in Corneal Development, Wound Healing, and Disease
Frontiers in Endocrinology
The insulin-like growth factor (IGF) family plays key roles in growth and development. In the cornea, IGF family members have been implicated in proliferation, differentiation, and migration, critical events that maintain a smooth refracting surface that is essential for vision. The IGF family is composed of multiple ligands, receptors, and ligand binding proteins. Expression of IGF type 1 receptor (IGF-1R), IGF type 2 receptor (IGF-2R), and insulin receptor (INSR) in the cornea has been well characterized, including the presence of the IGF-1R and INSR hybrid (Hybrid-R) in the corneal epithelium. Recent data also indicates that each of these receptors display unique intracellular localization. Thus, in addition to canonical ligand binding at the plasma membrane and the initiation of downstream signaling cascades, IGF-1R, INSR, and Hybrid-R also function to regulate mitochondrial stability and nuclear gene expression. IGF-1 and IGF-2, two of three principal ligands, are polypeptide growth factors that function in all cellular layers of the cornea. Unlike IGF-1 and IGF-2, the hormone insulin plays a unique role in the cornea, different from many other tissues in the body. In the corneal epithelium, insulin is not required for glucose uptake, due to constitutive activation of the glucose transporter, GLUT1. However, insulin is needed for the regulation of metabolism, circadian rhythm, autophagy, proliferation, and migration after wounding. There is conflicting evidence regarding expression of the six IGF-binding proteins (IGFBPs), which function primarily to sequester IGF ligands. Within the cornea, IGFBP-2 and IGFBP-3 have identified roles in tissue homeostasis. While IGFBP-3 regulates growth control and intracellular receptor localization in the corneal epithelium, both IGFBP-2 and IGFBP-3 function in corneal fibroblast differentiation and myofibroblast proliferation, key events in stromal wound healing. IGFBP-2 has also been linked to cellular overgrowth in pterygium. There is a clear role for IGF family members in regulating tissue homeostasis in the cornea. This review summarizes what is known regarding the function of IGF and related proteins in corneal development, during wound healing, and in the pathophysiology of disease. Finally, we highlight key areas of research that are in need of future study.
IGFBP-3 Mediates Metabolic Homeostasis During Hyperosmolar Stress in the Corneal Epithelium
Investigative Ophthalmology & Visual Science, 2021
The insulin-like growth factor binding protein-3 (IGFBP-3) is a multifunctional secretory protein with well-known roles in cell growth and survival. Data in our laboratory suggest that IGFBP-3 may be functioning as a stress response protein in the corneal epithelium. The purpose of this study is to determine the role of IGFBP-3 in mediating the corneal epithelial cell stress response to hyperosmolarity, a well-known pathophysiological event in the development of dry eye disease. METHODS. Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used in this study. Cells were cultured in serum-free media with (growth) or without (basal) supplements. Hyperosmolarity was achieved by increasing salt concentrations to 450 and 500 mOsM. Metabolic and mitochondrial changes were assessed using Seahorse metabolic flux analysis and assays for mitochondrial calcium, polarization and mtDNA. Levels of IGFBP-3 and inflammatory mediators were quantified using ELISA. Cytotoxicity was evaluated using a lactate dehydrogenase assay. In select experiments, cells were cotreated with 500 ng/mL recombinant human (rh)IGFBP-3. RESULTS. Hyperosmolar stress altered metabolic activity, shifting cells towards a respiratory phenotype. Hyperosmolar stress further altered mitochondrial calcium levels, depolarized mitochondria, decreased levels of ATP, mtDNA, and expression of IGFBP-3. In contrast, hyperosmolar stress increased production of the proinflammatory cytokines IL-6 and IL-8. Supplementation with rhIGFBP-3 abrogated metabolic and mitochondrial changes with only marginal effects on IL-8. CONCLUSIONS. These findings indicate that IGFBP-3 is a critical protein involved in hyperosmolar stress responses in the corneal epithelium. These data further support a new role for IGFBP-3 in the control of cellular metabolism.
Scientific Reports
Insulin and insulin-like growth factor-1 (IGF-1) are present in human tears and likely play an important role in mediating ocular surface homeostasis. We previously characterized the IGF-1/insulin hybrid receptor (Hybrid-R) in corneal epithelial cells and found that it was activated by IGF-1 and not insulin; and reported the novel finding that it localized to the corneal epithelial cell nucleus. Since the corneal epithelium is an insulin insensitive tissue and does not require insulin for glucose uptake, this study investigated the function of insulin in corneal epithelial cells. We show that stress induced by growth factor deprivation triggers transcriptional upregulation and de novo nuclear accumulation of Hybrid-R through the homodimeric insulin receptor (INSR). This occurs independent of PI3K/Akt signaling. Nuclear accumulation of Hybrid-R was associated with partial cell cycle arrest and a corresponding reduction in mitochondrial respiration. Treatment with insulin, and not IGF-1, attenuated IGF-1R and INSR transcription and restored cell cycle and metabolic homeostasis. Together, these findings support that insulin mediates receptor homeostasis in corneal epithelial cells, favoring an IGF-1 mediated pathway. This may have important implications in diabetic corneal disease and wound healing. Insulin Receptor (INSR) and Insulin-like Growth Factor Type 1 Receptor (IGF-1R) are members of the receptor tyrosine kinase superfamily 1. They play an important role in the regulation of essential biological and molecular processes including proliferation, migration, metabolism, differentiation, and survival 2. This occurs through ligand binding of the receptor at the plasma membrane, leading to autophosphorylation and downstream activation of phosphoinositide 3-kinase (PI3K) and extracellular signal regulated kinase (ERK) pathways 3. Known extracellular ligands for INSR and IGF-1R include insulin, IGF-1, and IGF-2, all of which display different affinities for each receptor 1. Structurally, INSR and IGF-1R are transmembrane glycoproteins composed of two extracellular alpha subunits that form the ligand-binding domain and two transmembrane beta subunits that possess tyrosine kinase activity 4. Overall, the two receptors exhibit greater than 50% homology in their amino acid sequences. This ranges from 45% to 65% in the alpha subunit binding domain, rising to 84% homology within the tyrosine kinase domain. The structural similarity between INSR and IGF-1R make possible the formation of insulin and IGF-1 hybrid receptors (Hybrid-R) 5-7. It is unknown what drives formation of Hybrid-R. Some hypothesize that formation of Hybrid-R is driven by the ratio between IGF-1R and INSR 8. Others speculate that Hybrid-R is regulated developmentally 9. In addition to formation of Hybrid-R, the functional significance of Hybrid-R remains controversial. An increase in Hybrid-R expression has been reported in skeletal muscle and adipose tissue in diabetes 10-12. Hybrid-R has also been shown to bind IGF-1 with a greater affinity than insulin 13,14. Thus, increased expression of Hybrid-R in diabetic tissue may alter insulin sensitivity 7,10-12. A reduction in insulin sensitivity represents a key hallmark of diabetes. In the diabetic cornea, epithelial erosions, persistent epithelial defects, corneal neuropathy and ulceration can result in painful and often permanent loss of vision 15-18. While the corneal epithelium has been previously reported to be an insulin-insensitive tissue, meaning that it does not require insulin for glucose uptake, studies have shown that supraphysiological levels of insulin applied topically to the eye promotes corneal wound healing
In Vivo, 2011
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An ocular view of the IGF–IGFBP system
Growth Hormone & IGF Research, 2013
IGFs and their binding proteins have been shown to exhibit both protective and deleterious effects in ocular disease. Recent studies have characterized the expression patterns of different IGFBPs in retinal layers and within the vitreous. IGFBP-3 has roles in vascular protection stimulating proliferation, migration, and differentiation of vascular progenitor cells to sites of injury. IGFBP-3 increases pericyte ensheathment and shows anti-inflammatory effects by reducing microglia activation in diabetes. IGFBP-5 has recently been linked to mediating fibrosis in proliferative vitreoretinopathy but also reduces neovascularization. Thus, the regulatory balance between IGF and IGFBPs can have profound impact on target tissues. This review discusses recent findings of IGF and IGFBP expression in the eye with relevance to different retinopathies.
2003
PURPOSE. Growth hormone (GH), insulin-like growth factor (IGF), and somatostatin (SST) modulate each other's actions. SST analogues have been successfully used to treat proliferative diabetic retinopathy (PDR) that is unresponsive to laser therapy and to retard the progression of severe nonproliferative retinopathy to PDR. In this study, the endogenous expression of IGF-binding protein (IGFBP)-3 was examined in human retinal endothelial cells (HRECs), the direct effects of IGFBP-3 on HRECs were evaluated, and the possible involvement of IGFBP-3 in mediating the growth inhibitory effects of SST receptor (SSTR) agonists in HRECs was assessed. METHODS. The cellular localization of IGFBP-3 was examined with anti-IGFBP-3 and fluorescein-conjugated goat anti-rabbit IgG. HRECs were exposed to varying concentrations of human recombinant IGFBP-3, and growth inhibition was evaluated by thiazolyl blue (MTT) conversion. Apoptosis was examined using fluorochrome-annexin V staining. Conditioned media (CM) from SSTR2 agonist (L779976)-treated or SSTR3 agonist (L796778)-treated HRECs were analyzed by ELISA for changes in expression of IGFBP-3. RESULTS. HREC immunostaining showed cell surface and cytoplasmic IGFBP-3. Exogenous IGFBP-3 induced a dose-dependent inhibition of HREC proliferation and staining with fluorochrome-annexin V showed numerous apoptotic HRECs. HRECs exposed to the SSTR2 or SSTR3 agonists expressed IGFBP-3 in a concentration-dependent manner. CONCLUSIONS. Cultured HRECs expressed endogenous IGFBP-3. Exogenous administration of IGFBP-3-induced growth inhibition and apoptosis, supporting a regulatory role for IGFBP-3 in endothelial cells. SSTR agonists mediate their growth-inhibitory effect, in part, by increasing expression of IGFBP-3. (Invest Ophthalmol Vis Sci. 2003;44:365-369)
AJP: Cell Physiology, 2004
Hypoxia-induced physiological stress plays a central role in various neovascular diseases of the eye. Increased expression of hypoxia-inducible factor 1α (HIF-1α) and subsequent formation of HIF-1 dimers active at the vascular endothelial growth factor (VEGF) promoter lead to expression of this potent angiogenic factor in the retina, including retinal pigment epithelial (RPE) cells. We previously demonstrated that insulin-like growth factor I (IGF-I) stimulates VEGF and IGF binding protein (IGFBP)-3 secretion in RPE cells. In this study we examined IGF-I-induced HIF-1α expression, VEGF and IGFBP-3 secretion, and the autocrine actions of VEGF and IGFBP-3 on these processes in the spontaneously transformed RPE cell line ARPE-19. Cells were treated with CoCl2, IGF-I, recombinant human (rh)IGFBP-3, and rhVEGF. Immunoblot analysis revealed IGF-I-induced upregulation of total HIF-1α protein, whereas luciferase reporter assays of HIF-1 transcriptional activity demonstrated accumulation of ...
Investigative Ophthalmology & Visual Science
PURPOSE. Insulin-like growth factors (IGFs) and either substance P (SP) or an SP-derived peptide (FGLM-amide) synergistically facilitate corneal epithelial wound healing in vitro and in vivo. The mechanism of this synergism and the clinical potential of these agents were further investigated by determination of the relevant functional domain of IGFs. METHODS. The effects of IGF-derived peptides on corneal epithelial cell migration were evaluated with the rabbit cornea in an organ culture system. Corneal epithelial wound closure in vivo was also evaluated in rabbits after epithelial debridement with n-heptanol. RESULTS. In the presence of FGLM-amide, peptides corresponding to the C domain of IGF-1 or -2 significantly promoted corneal epithelial migration in vitro to an extent similar to that apparent with the full-length molecules. In contrast, peptides corresponding to the D domain of these growth factors had no such effect. Mutation of serine-34 in the C domain of IGF-1 to alanine abolished the synergistic effect with FGLM-amide on corneal epithelial migration. The C peptide of proinsulin did not affect corneal epithelial migration in the absence or presence of FGLM-amide. The administration of eye drops containing both the C-domain peptide of IGF-1 and FGLM-amide significantly promoted corneal epithelial wound closure in vivo. CONCLUSIONS. The C domain of IGF-1 or -2, for which no biological function has previously been identified, is essential for the synergistic effect of these growth factors with SP on corneal epithelial migration. (Invest Ophthalmol Vis Sci.
IGFBPs modulate IGF-I- and high glucose-controlled growth of human retinal endothelial cells
Journal of Endocrinology, 2001
Insulin-like growth factor binding proteins (IGFBPs) are important local factors in the development of proliferative diabetic retinopathy. We investigated the effects of IGF-I and increased glucose concentrations on the release of IGFBPs and the growth of human retinal endothelial cells (HRECs). HRECs secrete IGFBPs-2 to -5. IGF-I stimulated thymidine incorporation and modified the pattern of IGFBPs, decreasing the inhibitory IGFBP-4 through down-regulation of its mRNA, and increasing IGFBP-5 which, per se, was able to modulate HREC growth, exerting post-transcriptional control. Studies using an antibody (alpha IR3) against the IGF-I receptor, and compounds with low affinity for IGFBPs, such as insulin and des(1-3)IGF-I, showed that an interaction between IGF-I and IGFBP-5 was necessary to detach this IGFBP from its binding sites. The dose of IGF-I that significantly decreased the IGFBP-4/IGFBP-5 ratio was the same that stimulated HREC growth. Chronic exposure to high concentrations...
Investigative Opthalmology & Visual Science, 2004
To examine insulin-like growth factor (IGF)-1 stimulation of expression of hypoxia inducible factor (HIF)-1␣ and secretion of vascular endothelial growth factor (VEGF) and IGF binding protein (IGFBP)-3 in human retinal pigment epithelial (RPE) cell line D407. METHODS. D407 cells cultured in dishes or Transwell inserts were treated with cobalt chloride or varying doses of IGF-1. Whole cell lysates were assayed by immunoblot for HIF-1␣ expression, whereas conditioned medium was TCA precipitated and assayed by immunoblot for VEGF and ligand blot for IGFBP-3. Cells grown on coverslips were similarly treated and probed with antibodies to HIF-1␣, VEGF, and IGFBP-3, and visualized by epifluorescence microscopy. Cells grown on Transwell inserts were probed with antibodies to the Na ϩ /K ϩ-ATPase ␣-1 subunit and either the alpha or beta subunits of the IGF-1 receptor and visualized in Z-section using confocal microscopy. RESULTS. Immunoblot analysis of whole cell lysates from IGF-1-treated D407 cells revealed the upregulation of HIF-1␣ protein. Epifluorescence microscopy demonstrated a positive correlation between HIF-1␣ expression and nuclear localization, VEGF and IGFBP-3 synthesis and export, and IGF-1 action. Western and ligand blot analyses of RPE cell-conditioned medium indicated that IGF-1 induced increases in VEGF and IG-FBP-3 secretion. Cells grown on Transwell inserts exhibited constitutive apical secretion of VEGF and IGFBP-3, which increased on apical or basolateral treatment with IGF-1. Confocal analysis of Transwell-cultured D407 cells confirmed the apical localization of the Na ϩ /K ϩ-ATPase ␣-1 subunit, characteristic of polarized RPE, with IGF-1 receptor ␣ and  subunits exhibiting a nonpolarized distribution. CONCLUSIONS. IGF-1 stimulates increased HIF-1␣ expression as well as VEGF and IGFBP-3 secretion in D407 cells. Similar to their in vivo counterparts, D407 cells maintain reversed epi-thelial polarity. Apical secretion of VEGF and IGFBP-3 increases in response to either apical or basolateral IGF-1 stimulation consistent with the nonpolarized distribution of IGF-1 receptors.