Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins.

Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs). IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I. Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa. Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present. Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity. IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains. In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage. None of the crosslinked IGF-BPs exhibit O-linked side chains. Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation. Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable. To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr. Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity. This is the first report of IGF-BP degrading activity in a cell culture system. These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity. Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.     

Bone morphogenetic protein-4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells secrete vascular endothelial growth factor (VEGF), a cytokine known to promote angiogenesis. Results from RNase protection assays (RPAs) show that RPE from non-diabetic human donors and from adult retinal pigment epithelium-19 (ARPE-19) cells expressed significant bone morphogenetic protein-4 (BMP-4) message. In addition, ARPE-19 cells cultured in high glucose (25 mM), compared to those in physiological glucose (5.5 mM) released significantly more BMP-4 into the conditioned media (CM). However, the effect of BMP-4 on the release of VEGF by ARPE-19 cells has not been studied. Accordingly, ARPE-19 cells were treated with BMP-4 to determine VEGF secretion. BMP-4 and VEGF levels in the CM and cell lysates were measured by enzyme-linked immunosorbent assay (ELISA). Cells treated with exogenous BMP-4 had higher VEGF in the CM and this treatment effect was dose- and time-dependent, while cell lysates had low levels of VEGF. Addition of cycloheximide (CHX) or actinomycin-D (ACT) significantly reduced VEGF secretion from cells treated with BMP-4, suggesting that the BMP-4-induced secretion of VEGF requires new RNA and protein synthesis. Our results suggest that BMP-4 may play a role in the regulation of ocular angiogenesis associated with diabetic retinopathy (DR) by stimulating VEGF release from RPE cells.     

Regulation of insulin-like growth factor 1 binding protein 3 levels by epidermal growth factor and retinoic acid in cervical epithelial cells

Insulin-like growth factors (IGFs) are important regulators of epithelial cell growth. The mitogenic activity of these factors is influenced by the levels of extracellular IGF binding proteins, including insulin-like growth factor binding protein 3 (IGFBP-3). In the present report we study the effects of epidermal growth factor (EGF) and all-trans-retinoic acid (RA) on IGFBP-3 RNA and protein levels in human papillomavirus-immortalized cervical epithelial cells. Treatment of ECE16-1 cells with 3–20 ng/ml EGF causes a marked reduction in IGFBP-3 levels. In contrast, 1 μM RA increases IGFBP-3 mRNA and protein levels in the presence or absence of 20 ng/ml EGF. The response is concentration dependent with a half-maximal increase observed at 1 nM RA. RA is able to reverse the EGF suppression when added simultaneously or 3 days after initiation of EGF treatment. Conversely, when cells are treated with RA, IGFBP-3 levels increase within 24 h and subsequent addition of EGF is without effect. Thus, the RA-dependent increase in IGFBP-3 levels is dominant over the EGF suppression. The increased IGFBP-3 levels are correlated with RA suppression of proliferation. Similar RA effects on IGFBP-3 mRNA levels were observed in other cervical epithelial cell lines (i.e., ECE16-D1, ECE16-D2, and CaSki). These results suggest that RA may act to inhibit cervical cell growth by increasing IGFBP-3 levels and reducing the extracellular concentration of free insulin-like growth factor I (IGFI) and/or, alternatively, IGFBP-3 may inhibit cell growth by direct effects on the cell, independent of IGFI. © 1994 Wiley-Liss, Inc.     

Platelet-derived growth factor gene expression in cultured human retinal pigment epithelial cells.

Gene expression of platelet-derived growth factor (PDGF) and its receptors in cultured human retinal pigment epithelial (RPE) cells was studied by using semiquantitative polymerase chain reaction. The RPE cells were found to express PDGF A- and B-chain genes as well as alpha- and beta-receptor genes with dominant expression of B-chain and beta-receptor isoforms. Phorbol myristate acetate (PMA) and thrombin increased the expression of PDGF B-chain gene to 19.8 +/- 1.75 and 15.9 +/- 1.84 fold (n = 3) of the control without affecting beta-receptor gene expression. PDGF produced by the RPE cells may play an important role in the pathogenesis of some ocular proliferative diseases.     

Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells

We previously demonstrated that differentiated retinal pigment epithelial (RPE) cells express high levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), and a critical balance between VEGF and PEDF is important to prevent the development of choroidal neovascularization. We report here that VEGF secreted by RPE cells upregulates PEDF expression via VEGFR-1 in an autocrine manner. PEDF mRNA and protein expression was downregulated by neutralizing antibody against VEGF in differentiated human RPE cells. VEGFR-1 neutralization decreased PEDF mRNA and protein expression whereas anti-VEGFR-2 antibody had no effect. Addition of placenta growth factor (PlGF) restored PEDF expression in the presence of anti-VEGF antibody. These results demonstrate a regulatory interaction between angiogenesis stimulators and inhibitors to maintain homeostasis in normal human retina.     

Ceramide inhibits connective tissue growth factor expression by human retinal pigment epithelial cells

Connective tissue growth factor (CTGF) is known to be involved in retinal fibrotic disorders. We used human retinal pigment epithelial cells (HRPE), which play critical roles in retinal fibrosis, to examine the expression of CTGF and its regulation by ceramide and TGF-β. Real-time PCR analysis showed downregulation of CTGF mRNA by C₂ ceramide and upregulation by TGF-β. C₂ ceramide also inhibited constitutive and TGF-β-enhanced CTGF secretion by HRPE cells. Predominant secretion (>80% of total) of CTGF from the apical side was observed in highly polarized HRPE cells. Fumonosin, an inhibitor of ceramide synthesis, stimulated CTGF secretion while 4HPR, an activator of ceramide synthesis, downregulated CTGF secretion. Based on these results demonstrating ceramide regulation of CTGF secretion by HRPE, we suggest that ceramide may have therapeutic potential for the treatment of retinal fibrotic diseases by inhibiting CTGF production.     

Efflux protein expression in human stem cell-derived retinal pigment epithelial cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE-derived diseases, drug testing and targeted drug therapy.     

Transforming growth factor-beta induces expression of vascular endothelial growth factor in human retinal pigment epithelial cells: involvement of mitogen-activated protein kinases

Vascular endothelial growth factor (VEGF) is a major agent in choroidal and retinal neovascularization, events associated with age-related macular degeneration (AMD) and diabetic retinopathy. Retinal pigment epithelium (RPE), strategically located between retina and choroid, plays a critical role in retinal disorders. We have examined the effects of various growth factors on the expression and secretion of VEGF by human retinal pigment epithelial cell cultures (HRPE). RT-PCR analyses revealed the presence of three isoforms of mRNA corresponding to VEGF 121, 165, and 189 that were up regulated by TGF-beta1. TGF-beta1, beta2, and beta3 were the potent inducers of VEGF secretion by HRPE cells whereas bFGF, PDGF, TGF-alpha, and GM-CSF had no effects. TGF-beta receptor type II antibody significantly reversed induction of VEGF secretion by TGF-beta. In contrast activin, inhibin and BMP, members of TGF-beta super family, had no effects on VEGF expression in HRPE. VEGF mRNA levels and protein secretion induced by TGF-beta were significantly inhibited by SB203580 and U0126, inhibitors of MAP kinases, but not by staurosporine and PDTC, protein kinase C and NF-kappaB pathway inhibitors, respectively. TGF-beta also induced VEGF expression by fibroblasts derived from human choroid of eye. TGF-beta induction of VEGF secretion by RPE and choroid cells may play a significant role in choroidal neovascularization (CNV) in AMD. Since the secretion of VEGF by HRPE is regulated by MAP kinase pathways, MAP kinase inhibitors may have potential use as therapeutic agents for CNV in AMD.     

Regulation of granulocyte-macrophage colony-stimulating factor in human retinal pigment epithelial cells by IL-1beta and IFN-gamma.

GM-CSF production by RPE cells, which form part of the blood-retina barrier, is upregulated by IL-1beta and this increase can be reversed by IFN-gamma. IL-1beta up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1beta to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1beta pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1beta-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-kappaB. IFN-gamma inhibition of the GM-CSF response to IL-1beta acted via NF-kappaB, reducing the translocation of NF-kappaB to the nuclei of RPE cells treated with IL-1beta and IFN-gamma. The results show that IFN-gamma down-regulation acts either directly on NF-kappaB or its activation or by blockade of a pathway upstream of NF-kappaB. However, any such blockade does not involve PKC or intracellular calcium.     

Vasculotropin/Vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultued in vitro

Vasculotropin (VAS), also called vascular endothelial growth factor (VEGF) or vascular permeability factor, is a secreted growth factor whose target cell specificity has been reported as restricted to vascular endothelium. Its effects are mediated by at least two distinct membrane-spanning tyrosine kinase receptors, KDR and flt-1, the expression of which also seems restricted to vascular endothelium. We describe here that cultured human retinal pigment epithelial (HRPE) cells express both KDR and flt-1 receptors, bind VAS/VEGF on two high affinity sites (apparent Kd of 9 and 210 pM corresponding to 940 and 18,800 sites per cell) and proliferate or migrate upon recombinant VAS/VEGF addition. HRPE cells also express the mRNA corresponding to the 121 and 165 amino acid forms of VAS/VEGF. HRPE cells release in their own culture medium and store in their extracellular matrix self-mitogenic and chemoattractant factors indistinguishable from 121 and 165 VAS/VEGF isoforms. The autocrine role of VAS/VEGF was confirmed by the inhibition of these bioactivities by neutralizing specific anti-VAS/VEGF antibodies. © 1995 Wiley-Liss, Inc.     

Role for cyclic adenosine monophosphate in modulating insulin-like growth factor binding protein secretion by muscle cells

The modulation of insulin-like growth factor-binding protein (IGFBP) secretion is an important variable affecting muscle cell metabolism, proliferation, and differentiation. We have previously shown that secretion of IGFBP-4 and IGFBP-5 by L6 and BC₃H-1 muscle cells was stimulated by treatment with either insulin, IGF-I, or IGF-II. Herein, these cells were used to further identify mechanisms involved in controlling IGFBP secretion. Agents that elevate intracellular cAMP concentrations (dcAMP, forskolin, isoproterenol, and prostaglandin [PGE₁]) increase secretion of IGFBP-4 and IGFBP-5 from L6 cells. Similar increases in IGFBP secretion were found by treatment with either insulin, IGF-I, or dcAMP. The effects of dcAMP and either insulin or IGF-I were additive, but the effects of insulin and IGF-I were not additive. These results suggest that insulin/IGF-I and dcAMP are acting via distinct mechanisms to stimulate IGFBP secretion. Indomethacin, which blocks endogenous prostaglandin synthesis, and progesterone, which decreases intracellular cAMP levels, decreased IGFBP-4 and IGFBP-5 secretion. IGFBP-5 secretion by BC₃H-1 cells was increased by either insulin or IGF-I. Agents which elevate intracellular cAMP concentrations did not increase IGFBP-5 secretion. Additionally, these agents were not synergistic with either insulin or IGF-I. However, indomethacin and progesterone depressed IGFBP-5 secretion by BC₃H-1 cells. In summary, there appear to be at least two intracellular signaling mechanisms controlling IGFBP-4 and IGFBP-5 secretion by L6 and BC₃H-1 muscle cells. IGFBP secretion by L6 cells is stimulated by both insulin/IGF-I and cAMP-dependent pathways, whereas IGFBP-5 secretion by BC₃H-1 cells is stimulated only by the insulin/IGF pathway. IGFBP secretion by both cell lines can be decreased by agents which depress cAMP levels. Our results suggest that two divergent but synergistic pathways modulate IGFBP production and these mechanisms can potentially modulate IGF activity during muscle cell proliferation and differentiation. J. Cell. Physiol. 174:293–300, 1998. © 1998 Wiley-Liss, Inc.     

A novel human insulin-like growth factor binding protein secreted by osteoblast-like cells.

Insulin-like growth factor binding proteins (IGFBPs) modulate the cellular action of the insulin-like growth factors. Inhibition or enhancement of IGF effects by these cell-secreted binding proteins have been described. We have purified two IGFBPs (23 and 29 kDa) from media conditioned by U-2 human osteosarcoma cells using ligand-affinity chromatography and reversed phase HPLC. N-terminal amino acid analysis of the 23 kDa protein revealed a unique sequence with variable homology to IGFBPs 1-4. The 29 kDa IGFBP was found to be nearly identical to a recently reported IGFBP. Because the affinity purified U-2 IGFBPs enhanced IGF-I-stimulated osteoblast mitogenesis, we suggest that one or both of these binding proteins enhance IGF action in bone.     

Hepatocyte growth factor promotes the proliferation of human embryonic stem cell derived retinal pigment epithelial cells

Research that pertains to the molecular mechanisms involved in retinal pigment epithelial (RPE) development can significantly contribute to cell therapy studies. The effects of periocular mesenchymal cells on the expansion of RPE cells remain elusive. We have examined the possible proliferative role of hepatocyte growth factor (HGF) as a mesenchymal cell secretory factor against human embryonic stem cell derived RPE (hESC-RPE). We found that the conditioned medium of human mesenchymal stem cells from apical papilla and/or exogenous HGF promoted proliferation of the hESC-RPE cells as single cells and cell sheets, in addition to rabbit RPE sheets in vitro. Blockage of HGF signaling by HGF receptor inhibitor, PHA-665752, inhibited proliferation of hESC-RPE cells. However, differentiation of hESCs and human-induced pluripotent stem cells to a rostral fate and eye-field specification was unaffected by HGF. Our in vivo analysis showed HGF expression in periocular mesenchymal cells after optic cup formation in chicken embryos. Administration of HGF receptor inhibitor at this developmental stage in chicken embryos led to reduced eye size and disorganization of the RPE sheet. These findings suggested that HGF administration could be beneficial for obtaining higher numbers of hESC-RPE cells in human preclinical and clinical trials.     

Regulation of taurine transporter expression by NO in cultured human retinal pigment epithelial cells

Taurine is activelytransported at the retinal pigment epithelial (RPE) apical membrane inan Na⁺- and Cl-dependent manner. Diabetes mayalter the function of the taurine transporter. Because nitric oxide(NO) is a molecule implicated in the pathogenesis of diabetes, we askedwhether NO would alter the activity of the taurine transporter incultured ARPE-19 cells. The activity of the transporter was stimulatedin the presence of the NO donor 3-morpholinosydnonimine. Thestimulatory effects of 3-morpholinosydnonimine were not observed duringthe initial 16-h treatment; however, stimulation of taurine uptake waselevated dramatically above control values with 20- and 24-htreatments. Kinetic analysis revealed that the stimulation wasassociated with an increase in the maximal velocity of the transporterwith no significant change in the substrate affinity. The NO-induced increase in taurine uptake was inhibited by actinomycin D and cycloheximide. RT-PCR analysis and nuclear run-on assays provided evidence for upregulation of the transporter gene. This study providesthe first evidence of an increase in taurine transporter geneexpression in human RPE cells cultured under conditions of elevatedlevels of NO.

     

Regulation of the reduced-folate transporter by nitric oxide in cultured human retinal pigment epithelial cells

The regulation of the reduced-folate transporter (RFT) by nitric oxide (NO) was analyzed in human retinal pigment epithelial (HRPE) cells. NO inhibited specifically and reversibly the uptake of N5-methyltetrahydrofolate by a cGMP-independent mechanism. The inhibition was associated with a decrease in substrate affinity. The NO-induced inhibition was prevented by antioxidants and NO scavengers. Agents capable of modifying thiol groups in proteins inhibited RFT, indicating that the likely mechanism of NO-induced inhibition is via modification of essential thiol groups in this protein. These studies suggest that NO produced during retinal disease may affect the function of RFT in adjacent RPE cells.     

Involvement of the protein kinase pathway in melanin synthesis by chick retinal pigment epithelial cells

Protein kinases are involved in a variety of cellular functions and cell proliferation in eyes. We have explored the involvement of protein kinase C (PKC) in cell proliferation and melanin synthesis by chick retinal pigment epithelial (RPE) cells in vitro. This was achieved by incubation of confluent RPE cells with known inhibitors of protein kinase, H-7, W-7, H-8, and staurosporine. Chick RPE cells were cultured in the presence or absence of the protein kinase inhibitors for a 10-day period. Effects of the inhibitors on cell proliferation and melanin synthesis, as an indication of cell differentiation, were assessed by counting the number of surviving cells and by measuring the melanin content in the cells, respectively. H-7, W-7, and staurosporine inhibited cell proliferation and increased melanin synthesis in a concentration-dependent manner during culture; however, H-8 did not produce these cellular effects. These findings indicate that PKC and calcium/calmodulin-dependent kinase pathways are involved in the proliferation and differentiation of chick RPE cells.