Insulin-like Growth Factor Binding Protein-3 expression in the human corneal epithelium

Abstract

Insulin-like Growth Factor Binding Protein-3 (IGFBP3) is a high affinity binding protein shown to regulate cell growth, differentiation, and apoptosis in a variety of cellular systems. The primary aim of this study was to characterize IGFBP3 expression in the human corneal epithelium and in a corneal epithelial cell line and to establish a potential role for IGFBP3 mediated apoptotic signaling in corneal epithelial cells.Using a telomerase-immortalized human corneal epithelial (hTCEpi) cell line cultured in serum-free media and fresh human eye bank donor tissue, expression and localization of IGFBP3 were established in situ and in vitro by indirect immunofluorescence and western blotting. Real time PCR was used to measure IGFBP3 mRNA levels following Trichostatin-A (TSA) treatment and as a function of confluence. IGFBP3 protein levels were assessed in resting human tears and in conditioned media by western blotting; as was the ability of recombinant human IGFBP3 protein to associate with the cell surface. Apoptotic signaling was assessed in vitro using TSA and recombinant human (rh)IGFBP3. Apoptosis was measured by Viability/Cytotoxicity, Annexin V, and TUNEL assays.IGFBP3 localized to the plasma membrane of human corneal epithelial cells in situ and was upregulated in surface cells in the central cornea. IGFBP3 was secreted in conditioned media of growing cells, with a robust upregulation following confluence (P<0.001) and differentiation. IGFBP3 was undetectable in human tears. Addition of TSA to the culture media resulted in an upregulation of IGFBP3 mRNA (P<0.001) and protein. In addition, TSA treatment led to a significant increase in Annexin V positive cells at 18 and 24 hours (P<0.001) and TUNEL positive cells at 24 and 48 hours (P<0.001). The addition of rhIGFBP3 to the cell culture media appeared to induce occasional membrane blebbing, but cells failed to become positive with Annexin V or TUNEL.Taken together, these results demonstrate that cell membrane-associated IGFBP3 is produced by corneal epithelial cells and associates with the plasma membrane of superficial cells in situ and in cultured cells, but is not present in human tears. The differential localization and effect(s) on apoptosis suggest that the effects of IGFBP3 are likely tissue compartment and receptor specific and may be regulated by glycosylation.

Role of Insulin-Like Growth Factor Binding Protein-3 in the Pathogenesis of Herpes Stromal Keratitis

“…IGFBP-3 is reported to express in corneal epithelial cells. 27 Our results showed an upregulation of IGFBP-3 mRNA expression in HSV-1-infected cornea during clinical disease period. Several factors such as TGF-β1, IGF-I, retinoic acid, TNF-α, and hypoxia are known to upregulate IGFBP-3 mRNA expression.…”

“…The decrease in serum level of IGFBP-3 protein at 5 and 10 days postinfection was associated with an increased amount of IGFBP-3 protein in HSV-1-infected cornea as evident from the protein array data shown in Figure 1, suggesting a possibility of IGFBP-3 influx in inflamed cornea from the peripheral circulation. However, IGFBP-3 is also reported to express in the corneal epithelial cells, 27 and an increased expression of IGFBP-3 in inflamed cornea could also contribute to the higher level of IGFBP-3 that is detected in HSK-developing corneas. To measure the gene expression of IGFBP-3 molecule, qRT-PCR assay was carried out on uninfected and HSV-1-infected corneas.…”

“…In addition, IGFBP-3 is also known to act in IGFindependent mode and exhibits anti-proliferative and proapoptotic effects. 15,[24][25][26] IGFBP-3 is reported to present in human corneal epithelial cells 27 and in the tears of diabetic individuals. 28,29 However, the role of IGFBP-3, if any, in regulating the pathogenesis of HSK is not known.…”

Elevated IGFBP3 Levels in Diabetic Tears: A Negative Regulator of IGF-1 Signaling in the Corneal Epithelium

“…14 The IGF-1R is a glycosylated, transmembrane receptor tyrosine kinase that has vital roles in development and normal tissue maintenance. 15 Both IGFBP3 and IGF-1R have been previously identified in the corneal epithelium and in cultured corneal epithelial cells 16, 17 ; however, the functional significance of this localization in mediating homeostatic renewal is unknown. In addition to mediating IGF-1R signaling, IGFBP3 has also been shown to regulate insulin resistance 18, 19 and apoptosis 20–24 in a variety of cell types via IGF-1 independent pathways.…”

C-Terminal Cleavage of ΔNp63α Is Associated with TSA-Induced Apoptosis in Immortalized Corneal Epithelial Cells

“…We have previously shown that incubation of hTCEpi cells in TSA induces apoptotic-mediated cell death as measured by annexin V and TUNEL assays. 34 In this study, we demonstrated that treatment with TSA is associated with apoptosis through the activation of caspase. Significantly, treatment with TSA was also associated with a concurrent downregulation of endogenous ⌬Np63␣.…”