Regulation of VEGF mRNA expression and protein secretion by TGF-β2 in human retinal pigment epithelial cells

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Abstract

VEGF secretion by the human retinal pigment epithelium (hRPE) plays an important role in retinal and choroidal neovascularization. In this study, transforming growth factor-β2 (TGF-β2)-induced vascular endothelial growth factor (VEGF) gene expression was investigated in hRPE cells. Treatment of hRPE cells with TGF-β2 for 24 and 48 h as compared to 8 h resulted in markedly increased VEGF secretion by fivefold and nine-fold, respectively. Induced VEGF mRNA peaked within 3 h of stimulation and remained above the basal at 36 h. Stimulation of VEGF expression by TGF-β2 was blocked by cycloheximide, suggesting that de novo protein synthesis is required. Induced VEGF production was strongly inhibited by anti-inflammatory agents, dexamethasone and cyclosporin A. Despite of the weak stimulation of VEGF expression by TNF-α or bFGF alone, co-administration of either of these two cytokines synergized the effect of TGF-β2 on VEGF mRNA expression and protein production. Quantitative RT-PCR revealed that the synergy was predominantly at the level of VEGF transcription. Moreover, TGF-β2-induced RPE VEGF secretion was significantly reduced by inhibitors of mitogen-activated protein (MAP) kinase (MEK) (U0126), p38 (SB202190), c-Jun NH2-terminal kinase (JNK), Sp600125, protein tyrosine kinase (PTK) (Genistein), and phosphatidylinositol 3-kinase (PI3K) (Ly294002). Induced VEGF expression was completely abrogated by inhibitors of protein kinase C (PKC) (Ro318220), nuclear factor-κB (NF-κB) [caffeic acid phenethyl ester (CAPE)], and reactive oxygen species (ROS) [N-acetyl-cysteine (Nac) and diphenyleneiodonium (DPI)]. These results suggest that MEK, p38, JNK, PI3K, and NF-κB as well as multiple essential signaling intermediates, including PKC, PTK and ROS, are involved in hRPE VEGF up regulation by TGF-β2.

Introduction

Human retinal pigment epithelium (hRPE) cells are strategically interposed between the neurosensory retina and the choroid, and play a major role in retinal and choroidal neovascularization. A number of angiogenic factors, prominent among which is vascular endothelial growth factor (VEGF), are involved in the initiation and development of choroidal and retinal neovascularization. VEGF protein predominantly exists as a homodimer of four alternative spice variants VEGF121, VEGF165, VEGF189, and VEGF206 of a single gene (Keck et al., 1989). VEGF121 and VEGF165 are soluble proteins, whereas VEGF189 and VEGF206 are bound to heparin-containing proteoglycans on cell surfaces or in basement membranes (Houck et al., 1992). VEGF is an endothelial cell-specific mitogen, promoting vascular permeability (Keck et al., 1989). A clear temporal and spatial relationship has been found between VEGF and ocular neovascularization (Miller et al., 1994). VEGF stimulates neovascularization in corneal micropocket assays (Connolly et al., 1989) and in chicken chorioallantoic membranes (Wilting et al., 1992). It has been known that the intraocular VEGF levels are elevated in humans with proliferative diabetic retinopathy (PDR) (Adamis et al., 1994). Significantly increased VEGF immunoreactivity has been reported in retinal vascular endothelium, blood vessel walls, choriocapillaris endothelium, choroidal neovascular endothelium, and migrating human RPE cells in diabetic subjects (Lutty et al., 1996). In addition, the VEGF levels detected in vitreous from patients with active PDR have also been shown to be 15–30 times higher than that of patients with nonproliferative or quiescent diabetic retinopathy or nondiabetic control (Aiello et al., 1994).

An important function of hRPE cells, like that of endothelial cells and Müller cells, is to secrete VEGF (Adamis et al., 1993). Expression of VEGF has been known to be induced by various stimuli, including hypoxia (Aiello et al., 1995), mechanical stress, advanced glycation end products (AGE), vasopressor hormones such as angiotensin II and vasopressin, and cytokines such as interleukine-1 (IL-1), transforming growth factor-beta (TGF-β), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) (Chiarelli et al., 2000a).

TGF-β is a multifunctional regulator mediating cell proliferation, differentiation, apoptotic death and angiogenesis. However, the molecular mechanism of TGF-β-mediated angiogenesis remains poorly understood. TGF-β is mitogenic in some circumstances, but anti-mitogenic in others (Sporn and Roberts, 1992). Recent studies have demonstrated that TGF-β1, TGF-β2 and TGF-β3 induce VEGF expression in several cell types including hRPE cells (Nagineni et al., 2003). Despite of the fact that TGF-β alone is able to induce VEGF expression, little has been known whether TGF-β may work in concert with other angiogenic cytokines such as PDGF, tumor necrosis factor-alpha (TNF-α) and bFGF in VEGF gene expression (Tolentino and Adamis, 1998). In addition, most previous reports have focused on TGF-β1, but not on TGF-β2 and TGF-β3. In contrast to TGF-β3 which is functionally similar to TGF-β1, TGF-β2 appears distinct (Merwin et al., 1991a, Merwin et al., 1991b). TGF-β2 is the predominant form of TGF-β in ocular tissues (Pfeffer et al., 1994). Depending on the cell types and experimental conditions (Merwin et al., 1991b), the cellular responses to TGF-β2 differ from that to TGF-β1 in proliferation, migration and in vitro angiogenesis. In this study we investigated the effects of TGF-β2 alone and in combination with TNF-α or bFGF on hRPE VEGF mRNA expression and protein secretion, and examined the signaling pathways involved in VEGF expression in hRPE cells.

Section snippets

Materials

Recombinant human IL-1β, TNF-α, bFGF, PDGF, and TGF-β2 were purchased from R&D System (Minneapolis, MN). U0126 was obtained from Promega (Madison, WI), SB202190, Sp600125, Ly294002, Genistein, Ro318220, and AG490 from Calbiochem (San Diego, CA), CAPE (caffeic acid phenethyl ester) from BIOMOL (Plymouth Meeting, PA), Nac (N-acetyl-cysteine) and DPI (diphenyleneiodonium chloride) from Sigma–Aldrich Company (St. Louis, MO). Antibodies against actin, phosphorylated p38, and protein kinase C (PKC)

TGF-β2 induces hRPE VEGF mRNA expression and protein production

To examine stimulated VEGF secretion, hRPE cells were challenged by 10 ng/ml of TGF-β2. The growth medium was harvested at the time points ranging from 0 to 48 h after stimulation. As shown in Fig. 1A, TGF-β2 induced time-dependent, statistically significant increases in VEGF secretion. The induced VEGF expression appeared at 8 h and increased substantially at 24 and 48 h after the stimulation. When compared to 8 h, the VEGF levels at 24 and 48 h after stimulation were further increased by fivefold

Discussion

In this study, we showed that TGF-β2 strongly induced VEGF mRNA expression and protein secretion in hRPE cells, while growth factors bFGF and TNF-α stimulated VEGF very weakly. The latter results were consistent with previous observations in hRPE cells (Nagineni et al., 2003). However, our data also demonstrated that these two angiogenic factors synergized TGF-β2-induced VEGF mRNA expression and protein secretion. Synergies between TGF-β plus TNF-α and TGF-β plus bFGF have been reported for

Acknowledgements

This study was supported by NIH Grants EY-09441, EY007003, and Research to Prevent Research to Prevent Blindness, Senior Scientific Investigator Award. The authors wish to thank Margarete G. Hamlett for excellent technical assistance.

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