November 2005
Volume 46, Issue 11
Free
Biochemistry and Molecular Biology  |   November 2005
Aberrant Accumulation of Fibulin-3 in the Endoplasmic Reticulum Leads to Activation of the Unfolded Protein Response and VEGF Expression
Author Affiliations
  • C. Nathaniel Roybal
    From the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico; and the
  • Lihua Y. Marmorstein
    Department of Ophthalmology, University of Arizona, Tucson, Arizona.
  • David L. Vander Jagt
    From the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico; and the
  • Steve F. Abcouwer
    From the Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico; and the
Investigative Ophthalmology & Visual Science November 2005, Vol.46, 3973-3979. doi:10.1167/iovs.05-0070
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      C. Nathaniel Roybal, Lihua Y. Marmorstein, David L. Vander Jagt, Steve F. Abcouwer; Aberrant Accumulation of Fibulin-3 in the Endoplasmic Reticulum Leads to Activation of the Unfolded Protein Response and VEGF Expression. Invest. Ophthalmol. Vis. Sci. 2005;46(11):3973-3979. doi: 10.1167/iovs.05-0070.

      Download citation file:


      © 2015 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. The inherited early-onset macular degenerative disease known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense mutation leading to production of a mutant fibulin-3 protein (R345W). R345W is poorly secreted and accumulates in the RPE of ML/DHRD retinas. Accumulation of misfolded proteins within the endoplasmic reticulum (ER) causes activation of unfolded protein response (UPR) signaling and expression of ER stress–responsive genes, including vascular endothelial growth factor (VEGF). Therefore, we hypothesized that the expression of R345W activates the UPR, leading to VEGF expression.

methods. Adenoviral vectors were used to overexpress fibulin-3 wild-type (Wt) and R345W mutant proteins in ARPE-19 cells. Secretion and intracellular accumulation of Wt and R345W were compared by Western blot analysis and immunocytochemistry. Activation of the UPR was evaluated by measuring the expression of glucose-regulated protein 78 (GRP78 [BiP]) and editing of the X-box binding protein (XBP-1) mRNA. VEGF expression and transcriptional activation of the VEGF promoter were determined by Northern blot analysis, Western blot analysis, and use of a novel VEGF promoter-reporter construct containing 8.2 kb of the human VEGF gene.

results. R345W was poorly secreted by ARPE-19 cells and accumulated in the ER, leading to UPR activation and increased VEGF expression. Compared with Wt mutant proteins, the expression of R345W was more effective at causing UPR activation, increasing VEGF expression, and stimulating transcription from the VEGF promoter.

conclusions. These findings demonstrated that the expression of mutated fibulin-3 caused UPR activation and increased VEGF expression. Expression of mutant fibulin proteins may contribute to macular degeneration and choroidal neovascularization by causing ER stress leading to RPE dysfunction and increased VEGF expression.

Age-related macular degeneration (AMD) is a leading cause of vision loss in the Western world, 1 yet its pathogenesis remains poorly understood. An in-depth understanding of AMD has been hindered by its late-onset, complex genetics and the numerous environmental factors contributing to its development. 2 However, studies of inherited maculopathies with earlier clinical presentation and simpler Mendelian inheritance patterns have identified genetic mutations associated with these disorders and have made possible the analysis of signaling pathways that may be involved in the development of AMD. 3 4  
An early-onset autosomal dominant form of maculopathy known as malattia leventinese (ML) and Doyne honeycomb retinal dystrophy (DHRD) have been linked to a missense point mutation in fibulin-3 (also known as epithelial growth factor [EGF]–containing fibulin-like extracellular matrix protein 1 [EFEMP1]) resulting in an arginine-to-tryptophan (Arg345Trp [R345W]) alteration in the protein. 5 ML/DHRD exhibits autosomal dominance, suggesting a gain-of-function mutation. The proposed involvement of mutant fibulins in the development of macular degeneration has been strengthened by the identification of mutations in fibulin-6 and fibulin-5 in subsets of patients with AMD. 6 7 Therefore, the expression of mutant fibulins may activate a common pathway that leads to macular dysfunction and, in some cases, choroidal neovascularization (CNV). 
Fibulins are a family of six secreted extracellular matrix–associated proteins minimally defined as having a series of calcium-binding, epidermal growth factor (EGF)–like modules followed by a carboxyl-terminal fibulin-type module. 8 9 It has been proposed that mutant fibulins could promote macular degeneration by causing abnormal deposition of the extracellular matrix underlying the RPE cell layer, leading to RPE dysfunction. 10 RPE dysfunction is thought to be central to the development of AMD. The RPE provides nutritional, metabolic, and phagocytic support for the overlying photoreceptors. Late-stage AMD is characterized by geographic atrophy of the RPE and subretinal neovascularization. The neovascularization observed in AMD is thought to be caused in part by the overexpression of vascular endothelial growth factor (VEGF) by the RPE. 11 However, the mechanism by which aberrant VEGF expression by RPE cells is triggered is unknown. 
Fibulin-3 is highly expressed in the RPE. Insight into the effects of mutant fibulin-3 has been provided by the recent demonstration that overexpressed fibulin-3 R345W mutant protein is poorly secreted in a retinal pigmented epithelial cell line (RPE-J). 12 Furthermore, pronounced accumulation of R34W was observed in the retinal pigment epithelium (RPE) cell layer and not in the retinal drusen of a patient with ML. 12 Poor secretion of R345W has implications for the mechanism of macular degeneration. Lack of secretion suggests that R345W could not directly alter the characteristics of the extracellular matrix. Although reduced expression of wild-type (Wt) fibulin-3 protein may result in an extracellular matrix that is deficient in fibulin-3 protein, it is doubtful that the matrix is altered by the presence of small amounts of the mutant protein. 
Accumulation of misfolded proteins within the endoplasmic reticulum (ER) activates a signaling pathway termed the unfolded protein response (UPR). This pathway, which is conserved from yeast to mammals, leads to the activation of stress-responsive gene expression resulting in increased ER protein processing capacity and increased ability to clear and degrade proteins aggregating within the ER. 13 We recently demonstrated that activation of the UPR also induced the expression of VEGF. 14 15 Therefore, the expression of mutant fibulin-3 may cause ER stress, leading to activation of the UPR and to increased expression of VEGF, a protein important to the development of CNV. 
Herein, we hypothesize that the poorly secreted mutant fibulin-3 aggregates in the ER and activates the UPR. To investigate the activation of this response, we used adenoviral delivery of Wt and mutant fibulin-3 to investigate UPR activation and VEGF upregulation. Findings suggest that the UPR is effectively activated by expression of mutant fibulin-3, resulting in the upregulation of VEGF expression. This upregulation is caused, at least in part, by the activation of transcription from the VEGF promoter. Thus, ER stress may contribute to the retinal dysfunction observed in ML/DHRD and fibulin mutation–associated AMD, and this stress could contribute to the expression of VEGF and the development of CNV. 
Materials and Methods
Cell Culture
ARPE-19 cells (American Type Culture Collection [ATCC], Manassas, VA) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; low-glucose formulation) supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 Us/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B. For Northern blotting experiments, cells were plated in 10-cm tissue culture dishes and grown to confluence. One day before treatment, cells were rinsed with Dulbecco’s phosphate-buffered saline (DPBS) and fed with fresh media. For experiments using adenoviral vectors, subconfluent cultures of ARPE-19 cells were treated with fibulin-3 Wt vector, fibulin-3 mutant (R345W) vector, or an empty AdEasy vector (Empty). 
Fibulin-3 Adenoviral Vector Construction
Wild-type and mutant fibulin-3 cDNAs were expressed using an adenoviral vector system 16 provided by Bert Vogelstein (AdEasy; Howard Hughes Institute, Johns Hopkins University, Baltimore, MD). To clone into the adenoviral vector system, fibulin-3 Wt and R345W coding sequences from plasmid vectors (described previously 12 ) were amplified by polymerase chain reaction (PCR) using a 5′-NotI–containing (underlined) forward primer (5′- ATAAGAATGCGGCCGCTTCACAATGTTGAAAGCCCTTTTCC-3′) and a 5′-HindIII–containing (underlined) reverse primer (5′-CCCAAGCTTCTAAAATGAAAATGGCCCCAC-3′). The PCR reaction was carried out with Taq polymerase (Applied Biosystems, Foster City, CA) under the following conditions: 95°C 1 minute, 58°C 2 minutes, 72°C 1 minute, for 35 cycles followed by 7-minute incubation at 72°C. PCR products were cloned into the pAdTrack-CMV adenoviral shuttle vector encoding kanamycin resistance and containing a second expression cassette encoding green fluorescence protein (GFP). The GFP cassettes were then removed with HneI restriction enzyme, and the DNAs were ligated to create pAE-Empty, pAE-fibulin-3-Wt, and pAE-fibulin-3-R345W vectors. From these plasmids, ΔGFP Empty and fibulin-3 adenoviral vectors were created, as previously described. 15 Viral titers in the form of tissue culture 50% infective dosages (TC-ID50) were obtained (Virapur, San Diego, CA). TC-ID50 concentrations of viral stocks were 1.0 × 109 infectious units per milliliter (IU/mL) for Empty virus, 5.6 × 108 IU/mL for fibulin-3 Wt vector, and 1.8 × 109 IU/mL for fibulin-3 R345W vector. Dilutions of viral stocks used were determined by examining and normalizing the fibulin-3 mRNA levels of Wt and R345W vector–infected cells so that approximately equal levels of fibulin-3 mRNA expression would result (for example, see Fig. 3A ). 
Western Blot Analysis
ARPE-19 cells were grown to confluence in 60-cm2 dishes and were treated as described in the figure legends. After treatment, cells were washed in cold PBS and were lysed for 20 minutes on ice in lysis buffer (0.1% SDS, 0.5% Na-deoxycholate, 1% Triton X-100, 50 mM Tris HCl, pH 8.0) containing protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Protein contents of cleared lysates were determined with a BCA Protein Assay Kit (Pierce, Rockford, IL), and equal amounts of proteins (20 μg) were loaded into each lane and separated on an 8% SDS-PAGE gel. Protein bands were then transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA) and were probed with antibodies specific for VEGF, 78 kDa glucose-regulated protein (GRP78; Santa Cruz Biotechnology, Santa Cruz, CA), fibulin-3, 12 or β-actin (Sigma, St Louis, MO). Proteins were detected through enhanced chemiluminescence (ECL Chemiluminescence Kit; Amersham Biosciences, Piscataway, NJ) according to the manufacturer’s instructions. Membranes were scanned and viewed (MultiGenius Bioimaging System; Syngene, Cambridge, UK). 
Immunohistochemistry
ARPE-19 cells were plated onto culture slides (Falcon; Becton Dickinson Labware, Franklin Lakes, NJ) and infected with fibulin-3–encoding or empty adenoviral vectors 1 day before fixation. Cells were rinsed with PBS and fixed in 4% formaldehyde in PBS for 5 minutes Membranes were permeabilized with successive rinses with 70%, 80%, and 100% ethanol followed by 5-minute incubation in 100% ethanol. The ethanol was aspirated, and the slides were allowed to dry and then were blocked for 1 hour with PBS containing 1 mM Ca2+, 5% normal rabbit serum, and 1% BSA. Slides were incubated overnight at 4°C with primary antibody (mouse anti–fibulin-3 12 and rabbit anti-GRP78) in blocking solution, washed four times with PBS containing 1 mM Ca2+, incubated for 1 hour at room temperature with secondary antibody (FITC-labeled anti–mouse or Cy3-labeled anti–rabbit) in blocking solution, and washed four times with PBS containing 1 mM Ca2+. Cells were then covered with 50 μL mounting solution (Mowiol; Calbiochem, La Jolla, CA) and visualized by fluorescence microscopy. 
Northern Blot Analysis
RNA isolation and Northern blot analysis were performed as previously described 15 using random-primed probes obtained from cDNA templates corresponding to human VEGF (dbEST189750), GRP78 (HAEAC89; ATCC), and fibulin-3 (derived by PCR as described above). For each sample, hybridization to probe obtained from an 18S rRNA template was used to normalize results for mRNAs. The 18S rRNA cDNA template was RT-PCR generated from total rat kidney RNA using R18F2 sense (5′-GCTACCACATCCAAGGAAGGC-3′) and R18B1 antisense (5′-CCCGTGTTGAGTCAAATTAAGCC-3′) primers. Hybridization of probe was quantified (STORM phosphorimager and ImageQuant software; Molecular Dynamics, Sunnyvale, CA). Fold inductions were determined by dividing normalized mRNA band intensity volumes for experimental samples to that of control (empty vector–treated or time 0) samples. 
RT-PCR Analysis
For analysis of X-box binding protein 1 (XBP-1) mRNA editing by IRE-1, cDNA was synthesized from 100 ng total RNA using the RNA PCR reagent kit (GeneAmp Gold RNA PCR Reagent Kit; Applied Biosystems, Foster City, CA). PCR amplification was performed using XBP-1 primers amplifying the edited segment, as previously described. 15 The PCR products were size-fractionated by sieving agarose gel electrophoresis with gels containing 3.3% sieving agent (Synergel; Diversified Biotech, Boston, MA) according to the manufacturer’s recommendations and stained with 0.5 μg/mL ethidium bromide. PCR products were photographed under UV light using an imaging system (Genegnome; Syngene). 
Construction of a VEGF Promoter-Reporter Gene Plasmid
HindIII restriction enzyme digest was performed on the 137-kb PAC clone (GenBank accession number AL136131). After double gel purification (Qiagen, Valencia, CA), the 11.7-kb HindIII fragment was blunt ended (PCRTerminator end repair kit; Lucigen, Middleton, WI). The fragment was ligated into the pSMART-LC-Kan vector using a blunt end cloning kit (Clonesmart; Lucigen). The pSMART-LC-Kan-VEGF clone was digested with XhoI and the blunt end cutter EcoRV, excising an 8.2-kb fragment ranging from bases –6363 to +1886 relative to the VEGF transcription start site. The 8.2-kb fragment was then ligated into the XhoI- and SmaI-digested PGL-3-basic reporter vector (Promega, Madison, WI) creating the reporter vector pVEGF8.2-Luc. Sequencing performed by the UNM DNA Core Facility determined proper insert orientation and size. 
VEGF Promoter Activity Assays
ARPE-19 cells were plated at 50% confluence in 12-well plates and cotransfected with a CaCl2-DNA precipitate containing 2.5 μg pAE-Empty, pAE-fibulin-3-Wt, or pAE-fibulin-3-R345W; 2.5 μg pVEGF8.2-Luc; and 0.2 μg pRL-CMV vector (Promega) and were incubated for 4 hours. Cells were treated with 1 mL glycerol shock solution (4 × TBS, 15% glycerol, and 600 μM Na2Hpo 4) for 90 seconds and were washed twice with TBST (142 mM NaCl, 2.7 mM KCl, 25 mM Tris, 1% Tween 20). To prepare cell extracts, cells were washed with PBS and incubated at room temperature in 200 μL passive lysis buffer (Promega) for 15 minutes. Lysates were collected, and 10 μL cell extract was used to determine luciferase activity as recommended by the assay manual (Dual-luciferase; Promega). Luminescence was measured using a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA). Firefly luciferase relative light units (RLU Luc) in each sample were normalized to the Renilla luciferase relative light units (RLU Ren), and the sample mean ratio was divided by the control mean ratio to give fold inductions of VEGF promoter activity. Statistical significance of differences among triplicate samples was determined with the two-sample t-test and was shown as probability value. 
Results
Fibulin-3 R345W Accumulates in the Endoplasmic Reticulum
R345W was previously shown to be poorly secreted and to accumulate within RPE cells. 12 To further characterize this accumulation, subconfluent ARPE-19 cells were treated with increasing dilutions of Empty, fibulin-3 Wt, or fibulin-3 R345W adenoviral vectors for 24 hours. Dilutions of viral stocks used were determined by examining and normalizing the fibulin-3 mRNA levels of Wt and R345W vector-infected cells so that approximately equal levels of fibulin-3 mRNA expression would result (for example, see Fig. 3A ). Conditioned media were collected, and Western blotting was performed to measure the secreted fibulin-3 (Fig. 1A) . Equal sample loading was demonstrated by blotting for the bovine serum albumin contributed by the 10% fetal bovine serum present in the culture medium. No fibulin-3 was detected in the conditioned media from Empty vector–infected cells. Fibulin-3 Wt protein was readily secreted by cells infected with the fibulin-3 Wt vector, as demonstrated by a single band migrating at 60 kDa that was measurable in media samples from cultures treated with 1:80, 1:160, and 1:320 dilutions of fibulin-3 Wt vector stocks. These dilutions corresponded to approximately 7.0 × 106, 3.5 × 106, and 1.8 × 106 IU/mL, respectively. In contrast, R345W protein was not readily secreted. A small amount of fibulin-3 was detected in the medium from cultures treated with the highest amount of R345W viral vector treatment, 1:80 dilution, but was not detected in the media from cultures treated with 1:160 and 1:320 dilutions of vector stock. These dilutions corresponded to approximately 2.2 × 107, 1.1 × 107, and 5.6 × 106 IU/mL, respectively. Secreted fibulin-3 was not detected in media samples from cultures treated with 1:160 and 1:320 dilutions of the Empty vector. These dilutions corresponded to approximately 6.2 × 106 and 3.1 × 106 IU/mL, respectively. There was little variation in sample loading, as demonstrated by nearly identical amounts of albumin, the loading control. 
To confirm R345W accumulation in the ER, immunocytochemistry was performed on ARPE-19 cells treated with Empty (1:320 dilution, 3.1 × 106 IU/mL), fibulin-3 Wt (1:320 dilution, 1.8 × 106 IU/mL), and fibulin-3 R345W (1:640 dilution, 2.8 × 106 IU/mL) adenoviral vectors (Fig. 1B) . Immunostaining for GRP78/BiP was used as an indication of UPR activation and for ER localization. Cells were immunostained for GRP78 (red; a, b, c) and fibulin-3 (green; d, e, f). Neither GRP78 nor fibulin-3 was detected in cells treated with Empty virus (a and d, respectively). Fibulin-3 Wt viral vector-treated cells (b, e, h) exhibited perinuclear GRP78 protein staining (b) and faint staining for fibulin-3 Wt (e) in an identical perinuclear pattern (see merged image h). R345W viral vector–treated cells (c, f, i) exhibited intense, mainly perinuclear GRP78 (c) and fibulin-3 (f) staining that was completely colocalized (i). Fluorescence intensities suggested that GRP78 and fibulin-3 accumulated to markedly higher levels in R345W-expressing cells than in cells expressing exogenous fibulin-3 Wt protein. These data suggest that R345W aggregated in the ER and caused UPR activation, as indicated by dramatically increased endogenous GRP78 expression. Overexpression of fibulin-3 Wt activated the UPR and increased GRP78 expression, as would be expected because of the increased ER protein processing demand, but to a lesser extent than the mutant protein. 
Activation of GRP78 and VEGF Expression by Fibulin-3 Wt and R345W Expression
Western blot analysis was used to further analyze the effect of expressing fibulin-3 Wt and its mutant counterpart on intracellular levels of GRP78 and VEGF protein. Subconfluent ARPE-19 cells were treated with increasing amounts of fibulin-3 Wt or fibulin-3 R345W adenoviral vectors for 24 hours. Total cell lysates were prepared and immunoblotted for VEGF, GRP78, fibulin-3, and β-actin proteins (Fig. 2A 2B) . The dilutions of viral vector stocks used were 1:2560, 1:1280, 1:640, 1:320, 1:160, and 1:80 for Wt and R345W fibulin-3. These correspond to viral concentrations of approximately 2.2 × 105 to 7.0 × 106 IU/mL for the Wt vector and 7.0 × 105 to 2.2 × 107 IU/mL for the R345W vector. Endogenous fibulin-3 expression was undetectable, as evidenced by the lack of an immunoreactive band at the lowest viral dilutions. Intracellular concentrations of exogenous fibulin-3 were detectable at viral dilutions of 1:640 and increased with an increasing amount of viral vectors added to the cells. GRP78 and VEGF protein levels increased in amounts proportional to intracellular fibulin-3 protein accumulation. However, the ratio of GRP78 and VEGF proteins to fibulin-3 protein was greater in cells expressing the mutant protein. Thus, intracellular accumulation of fibulin-3 Wt and R345W proteins caused UPR activation and VEGF expression. However, the mutant protein caused these effects at lower expression levels than its Wt counterpart. 
Characterization of UPR Activation and VEGF Expression in Response to Fibulin-3 Expression
Unlike the intracellular levels of Wt and mutant fibulin-3 proteins, intracellular amounts of the corresponding mRNAs should provide a relatively accurate measure of the expression levels of these proteins. Therefore, the effects of fibulin-3 Wt and R345W expression on GRP78 and VEGF mRNA levels were examined, and these mRNA levels were compared with fibulin-3 mRNA levels. For this analysis ARPE-19 cells were treated with Empty, fibulin-3 Wt, or fibulin-3 R345W viral vectors for 4 days using 1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions of Empty, Wt, and R345W viral stocks. These correspond to viral concentrations of approximately 7.8 × 105 to 1.2 × 107 IU/mL for the Empty vector; 4.4 × 105 to 7.0 × 106 IU/mL for the Wt vector; and 1.4 × 106 to 2.2 × 107 IU/mL for the R345W vector. Northern blot analysis was performed to determine the intracellular contents of VEGF, GRP78, and fibulin-3 mRNAs (Fig. 3A) . Fibulin-3 mRNA was detectable in cells infected with the Empty vector, but the levels of fibulin-3 mRNA did not change as more viral vector was added. In fibulin-3 vector-infected cells, fibulin-3 Wt mRNA reached levels that were approximately fourfold higher than fibulin-3 R345W mRNA at corresponding viral dilutions, even though viral IU concentrations at corresponding dilutions were 3.2-fold higher for the R345W stock. VEGF mRNA levels were only slightly induced by expression of the Wt fibulin-3, whereas expression of mutant fibulin-3 dramatically increased VEGF mRNA levels. GRP78 mRNA levels were increased by the expression of Wt and mutant fibulin-3. Similarly, expression of the mutant seemed more potent at increasing GRP78 mRNA expression. 
To quantify the effects of Wt and mutant fibulin-3 expression on GRP78 and VEGF expression, fold inductions GRP78 mRNA and VEGF mRNA were plotted as a function of fibulin-3 mRNA levels, and linear regression was performed. Plotting GRP78 mRNA induction levels against Wt fibulin-3 mRNA levels (Fig. 3B)yielded a strong correlation (r = 0.98) with a slope of 2.1. Plotting GRP78 mRNA against Wt fibulin-3 mRNA (Fig. 3B)also yielded a strong correlation (r = 0.96), but with a greater slope, 7.0. This suggests that the fibulin-3 mutant was more than threefold more potent at inducing GRP78 mRNA expression than the Wt protein. When applied to VEGF mRNA inductions, this analysis again yielded strong linear correlations, with regression values of r = 0.88 and r = 0.97 for fibulin-3 Wt and R345W, respectively. When VEGF mRNA inductions were plotted against fibulin-3 Wt mRNA values, a slope of 0.057 was obtained. For VEGF mRNA inductions plotted against R345W mRNA, the slope obtained was 0.46. Therefore, expression of the mutant protein was eightfold more effective at inducing VEGF mRNA expression. Assuming that fibulin-3 mRNA levels are indicative of the corresponding protein expression rate, the data demonstrate that the mutant is a more potent inducer of UPR and VEGF expression than its Wt counterpart. 
These RNA samples were also used to further test for an indication of UPR activation in response to fibulin-3 expression. XBP-1 is a transcription factor that is activated as part of the UPR and that acts to increase the expression of genes needed for the expansion of secretory capacity. 17 XBP-1 protein is expressed only when the endonuclease activity of the ER resident transmembrane kinase IRE-1 is activated, resulting in the removal of a 26-nucleotide intron from XBP-1 mRNA and the formation of a new open reading frame that encodes the transcription factor. 18 Using PCR primers flanking the splice site, RT-PCR analysis of XBP-1 processing was performed on the RNA samples obtained for the experiment described (Fig. 3D) . Processed XBP-1 mRNA (PR) was not detectable in cells treated with the empty virus, as noted by the single PCR species obtained from unprocessed (UP) XBP-1 as template. XBP-1 mRNA splicing was evident in RNA samples from cells expressing fibulin-3 Wt and R345W and closely mirrored GRP78 mRNA expression levels observed in these cells (Fig. 3A) . This analysis demonstrated activation of the endonuclease activity of IRE-1 and thus confirmed activation of the UPR by fibulin-3 Wt and mutant protein expression. It should be noted that although the extent of XBP-1 mRNA processing is similar for fibulin-3 Wt and R345W samples, Wt fibulin-3 mRNA expression is greater than that of R345W mRNA in the corresponding samples. 
Effect of Fibulin-3 Expression on VEGF Promoter Activity
VEGF is transcriptionally upregulated in response to ER stress. 14 15 The effects of fibulin-3 expression on the transcriptional activation of the VEGF promoter were tested using a reporter gene construct containing an 8.2-kb region of the VEGF promoter (pVEGF8.2Luc). This region of the VEGF promoter extends from bases –6363 to +1886 relative to the VEGF transcription start site. Cells were cotransfected with Empty (pAE-Empty), fibulin-3 Wt (pAE-fibulin-3-Wt), or fibulin-3 R345W (pAE-fibulin-3-R345W) encoding plasmid vectors, VEGF8.2Luc, and pRL-CMV. Cell lysates were obtained at 24 hours (Fig. 4A)and 48 hours (Fig. 4B)after transfection and were analyzed for firefly and Renilla luciferase activities. After 24 hours of incubation, fibulin-3 Wt induced transcriptional activity 1.6-fold (P = 0.06), whereas mutant fibulin-3 significantly induced transcription 2.1-fold (P ≤ 0.001). The ratio of firefly luciferase to that of Renilla luciferase was significantly induced at 48 hours by fibulin-3 Wt 1.9-fold (P ≤ 0.005) and by fibulin-3 R345W 2.3-fold (P ≤ 0.005), indicating that transcription from the VEGF promoter was increased. This analysis suggests that the effect of fibulin-3 protein expression on VEGF expression is, at least partly, caused by transcriptional activation. 
Discussion
A missense mutation in the fibulin-3 gene leading to the production of R345W mutant protein causes the autosomal dominant disease ML/DHRD. 5 Fibulin-3 R345W aggregates within the RPE cell layer of patients with ML. 12 The RPE cell is a polarized, highly metabolic cell that is central to the proper functioning of the retina. Photoreceptors are isolated from the choroid by the RPE and thus rely on the RPE for filtering of nutrients from the blood. The RPE is also charged with processing waste material, primarily in the form of shed photoreceptor outer segments. Aggregation of fibulin-3 within the RPE is significant because a dysfunctional RPE cell layer is thought to be responsible for the pathophysiologic conditions associated with AMD. 
Fibulin-3 is an extracellular glycoprotein and thus is translated into the ER, where it is folded and processed before transport to the Golgi and eventual secretion. Our findings confirm that the poorly secreted fibulin-3 R345W protein accumulates within the ER of cultured RPE cells. The ER, the first organelle in the secretory pathway, is particularly susceptible to stress caused by protein misfolding, which leads to UPR activation. UPR signaling leads to transcriptional activation of an array of genes required for secreted protein processing, ER expansion, ER–Golgi trafficking, and ER-associated degradation (ERAD), which collectively relieve stress within the ER. 19 Expression of fibulin-3 R345W caused a pronounced increase in expression of the ER-resident protein-folding chaperone GRP78/BiP. The extent of GRP78 expression was proportional to the intracellular levels of fibulin-3. Activation of the UPR was further evidenced by XBP-1 mRNA processing through the action of IRE-1 endonuclease activity. The extent of XBP-1 editing matched GRP78 induction well. Although overexpression of the mutant fibulin-3 was more effective at upregulating GRP78 expression and activating XBP-1 mRNA processing, overexpression of Wt fibulin-3 also had these effects. This was not unexpected, for when additional nascent protein production is targeted to the ER, the UPR is activated in an attempt to match ER capacity to increased ER processing demand. Such an increase in demand would be caused by Wt fibulin-3 overexpression. 
ER stress could also explain the autosomal dominant inheritance pattern seen with the fibulin-3 R345W mutation. It should be noted that 163 of 164 ML patients genotyped were heterozygous for the fibulin-3 R345W mutation. 5 There is no proven explanation of how the mutation of a single fibulin-3 allele results in the early-onset macular degeneration seen in ML/DHRD patients. It has been suggested that the aggregation of the mutated protein from one allele may also cause misfolding of the protein from the unaffected allele, thus causing aberrant extracellular matrix deposition. 12 Our data suggest that the mutant fibulin-3 could confer a dominant mode of inheritance by aggregating in the ER and activating the UPR. Mutation of a single allele would thus be sufficient to cause RPE dysfunction. The structural features of fibulins may make them prone to causing ER stress. These proteins contain multiple calcium-binding domains and numerous putative intramolecular disulfide bonds. The multiple calcium-binding sites found in the EGF-like domains could lead to the sequestration of calcium by fibulin proteins accumulated within the ER. Loss of free calcium ions within the ER is a potent inducer of the UPR. 13 Furthermore, the formation of improper cysteine disulfide bonds followed by their reduction can produce a futile cycle that consumes reduced glutathione, produces reactive oxygen species, and contributes to ER stress–mediated cell death. 20  
UPR activation in the RPE of AMD patients should be further evaluated. UPR and protein misfolding have now become recognized in several other chronic diseases, including Alzheimer’s, Parkinson’s, diabetes, and obesity. 21 22 Fibulin-3 mutation is directly linked to ML/DHRD. However, it is possible that mutations in fibulins 5 and 6, which have been linked to AMD, also lead to protein misfolding and ER stress. ER stress could explain RPE atrophy observed during ML/DHRD and the end stages of macular degeneration. Progressive ER stress could lead to RPE dysfunction and ultimately to RPE cell apoptosis. Expression of fibulin-3 R345W for periods greater than 4 days does lead to progressive ARPE-19 cell death (Roybal CN, unpublished results, July 2005). 
Choroidal neovascularization often results in dramatic loss of vision in patients with exudative AMD. 23 The RPE is a major source for the increase in VEGF secretion seen in AMD patients. 24 VEGF is sufficient and necessary to cause neovascularization in AMD animal models. 25 26 The stimulus for increased VEGF production in AMD patients is still unknown. Ischemia/hypoxia is a major stimulator of VEGF in RPE cells. 27 Deposits between Bruch’s membrane and RPE cells, damage to the choriocapillaris, or thickening of Bruch’s membrane could cause hypoxia in overlying RPE cells, resulting in increased VEGF expression. 28 However, it has not been documented that Bruch’s membrane thickening or abnormalities in choroidal blood flow are sufficient to induce a hypoxia response in the RPE–choroid in AMD eyes. The present results suggest a novel hypothesis of VEGF induction caused by the accumulation of mutant fibulin proteins in the ER of RPE cells, leading to the upregulation of VEGF expression through activation of the UPR. Although studies have described CNV and subretinal hemorrhage in ML/DHRD patients, 29 30 it is generally understood that drusen formation, rather than CNV, is the typical finding associated with ML/DHRD. Therefore, future studies are needed to determine whether the accumulation of mutant fibulin-3 is sufficient for the stimulation of neovascularization. Animal studies have shown that VEGF overexpression alone is not sufficient to cause CNV in animal models. 31 To better understand the role of mutant fibulin-3 accumulation and the UPR in retinal angiogenesis, a more comprehensive study is needed of the effects on expression of angiogenic and antiangiogenic factors. 
 
Figure 1.
 
Accumulation of fibulin-3 R345W in ARPE-19 cells. (A) Western blot analysis of conditioned media to detect secreted fibulin-3 protein. ARPE-19 cells were infected with fibulin-3 Wt (1:640, 1:320, 1:160, 1:80 dilutions; lanes 1–4), R345W (1:640, 1:320, 1:160, 1:80 dilutions; lanes 5–8), or Empty (1:320 and 1:160 dilutions; lanes 9,10) adenoviral vector stocks for 24 hours (see text for viral IU concentrations). Conditioned media were then immunoblotted for fibulin-3 and albumin. (B) Immunocytochemical analysis of GRP78 (a, b, c) and fibulin-3 (d, e, f) in ARPE-19 cells infected with either Empty viral stock at 1:320 dilution (a, d, g), fibulin-3 Wt viral stock at 1:320 dilution (b, e, h), or R345W viral stock at 1:160 dilution (c, f, i) for 24 hours. Yellow represents the colocalization of GRP78 and fibulin-3 shown in merged images (g, h, i).
Figure 1.
 
Accumulation of fibulin-3 R345W in ARPE-19 cells. (A) Western blot analysis of conditioned media to detect secreted fibulin-3 protein. ARPE-19 cells were infected with fibulin-3 Wt (1:640, 1:320, 1:160, 1:80 dilutions; lanes 1–4), R345W (1:640, 1:320, 1:160, 1:80 dilutions; lanes 5–8), or Empty (1:320 and 1:160 dilutions; lanes 9,10) adenoviral vector stocks for 24 hours (see text for viral IU concentrations). Conditioned media were then immunoblotted for fibulin-3 and albumin. (B) Immunocytochemical analysis of GRP78 (a, b, c) and fibulin-3 (d, e, f) in ARPE-19 cells infected with either Empty viral stock at 1:320 dilution (a, d, g), fibulin-3 Wt viral stock at 1:320 dilution (b, e, h), or R345W viral stock at 1:160 dilution (c, f, i) for 24 hours. Yellow represents the colocalization of GRP78 and fibulin-3 shown in merged images (g, h, i).
Figure 2.
 
Effect of fibulin-3 Wt and R345W expression on GRP78 and VEGF protein expression. (A) Western blot analysis of GRP78 and VEGF levels in fibulin-3 Wt–expressing cells. ARPE-19 cells were infected with increasing amounts of fibulin-3 Wt vector (1:2560, 1:1280, 1:640. 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins. (B) Western blot analysis of GRP78 and VEGF levels in fibulin-3 R345W–expressing cells. ARPE-19 cells were infected with increasing amounts of R345W vector (1:2560, 1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were then immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins (see text for viral UI concentrations).
Figure 2.
 
Effect of fibulin-3 Wt and R345W expression on GRP78 and VEGF protein expression. (A) Western blot analysis of GRP78 and VEGF levels in fibulin-3 Wt–expressing cells. ARPE-19 cells were infected with increasing amounts of fibulin-3 Wt vector (1:2560, 1:1280, 1:640. 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins. (B) Western blot analysis of GRP78 and VEGF levels in fibulin-3 R345W–expressing cells. ARPE-19 cells were infected with increasing amounts of R345W vector (1:2560, 1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were then immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins (see text for viral UI concentrations).
Figure 3.
 
Effect of fibulin-3 expression on VEGF and GRP78 mRNA expression. (A) Northern blot analysis of ARPE-19 cells infected with various amounts of empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. Cells were infected with increasing amounts of respective adenoviral vectors (1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions), and mRNA levels were examined 4 days later. (B) GRP78 mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴). (C) VEGF mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴) and mRNA levels, respectively. (D) XBP-1 splicing by ARPE-19 cells infected with various amounts of Empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. RT-PCR analysis with primers flanking the XBP-1 splice site was performed on the RNA samples previously subjected to Northern blotting shown in panel A (the four highest viral dose samples—1:640. 1:320, 1:160, and 1:80—dilutions of viral vector stocks). Arrows highlight the PCR products obtained from unprocessed (UP) and processed (PR) XBP-1 mRNA templates (see text for viral UI concentrations).
Figure 3.
 
Effect of fibulin-3 expression on VEGF and GRP78 mRNA expression. (A) Northern blot analysis of ARPE-19 cells infected with various amounts of empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. Cells were infected with increasing amounts of respective adenoviral vectors (1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions), and mRNA levels were examined 4 days later. (B) GRP78 mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴). (C) VEGF mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴) and mRNA levels, respectively. (D) XBP-1 splicing by ARPE-19 cells infected with various amounts of Empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. RT-PCR analysis with primers flanking the XBP-1 splice site was performed on the RNA samples previously subjected to Northern blotting shown in panel A (the four highest viral dose samples—1:640. 1:320, 1:160, and 1:80—dilutions of viral vector stocks). Arrows highlight the PCR products obtained from unprocessed (UP) and processed (PR) XBP-1 mRNA templates (see text for viral UI concentrations).
Figure 4.
 
Effects of exogenous fibulin-3 expression on VEGF promoter activity. ARPE-19 cells were cotransfected with pAE-Empty, pAE-fibulin-3-Wt, or pAE-fibulin-3-R345W expression vectors, together with pVEGF8.2Luc and pRL-CMV luciferase reporter vectors. At 24 and 48 hours after transfection, cells were harvested and dual luciferase assays were performed. Firefly luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren) in each sample. Data are presented as mean ± SD (n = 3) *P ≤ 0.005. (A) 24-hour incubation. (B) 48-hour incubation.
Figure 4.
 
Effects of exogenous fibulin-3 expression on VEGF promoter activity. ARPE-19 cells were cotransfected with pAE-Empty, pAE-fibulin-3-Wt, or pAE-fibulin-3-R345W expression vectors, together with pVEGF8.2Luc and pRL-CMV luciferase reporter vectors. At 24 and 48 hours after transfection, cells were harvested and dual luciferase assays were performed. Firefly luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren) in each sample. Data are presented as mean ± SD (n = 3) *P ≤ 0.005. (A) 24-hour incubation. (B) 48-hour incubation.
The authors thank Brant Wagner for his kind help in obtaining the confocal imagery seen in Figure 1 . CNR is also indebted to the participants in the biomedical research writing class and to Dorothy Vander Jagt and Robert Glew for their valuable comments and help during the initial preparation of this manuscript. 
CongdonNG, FriedmanDS, LietmanT. Important causes of visual impairment in the world today. JAMA. 2003;290:2057–2060. [CrossRef] [PubMed]
GorinMB, BreitnerJC, De JongPT, et al. The genetics of age-related macular degeneration. Mol Vis. 1999;5:29. [PubMed]
BessantDA, AliRR, BhattacharyaSS. Molecular genetics and prospects for therapy of the inherited retinal dystrophies. Curr Opin Genet Dev. 2001;11:307–316. [CrossRef] [PubMed]
ZackDJ, DeanM, MoldayRS, et al. What can we learn about age-related macular degeneration from other retinal diseases?. Mol Vis. 1999;5:30. [PubMed]
StoneEM, LoteryAJ, MunierFL, et al. A single EFEMP1 mutation associated with both Malattia Leventinese and Doyne honeycomb retinal dystrophy. Nat Genet. 1999;22:199–202. [CrossRef] [PubMed]
SchultzDW, KleinML, HumpertAJ, et al. Analysis of the ARMD1 locus: evidence that a mutation in HEMICENTIN-1 is associated with age-related macular degeneration in a large family. Hum Mol Genet. 2003;12:3315–3323. [CrossRef] [PubMed]
StoneEM, BraunTA, RussellSR, et al. Missense variations in the fibulin 5 gene and age-related macular degeneration. N Engl J Med. 2004;351:346–353. [CrossRef] [PubMed]
ArgravesWS, GreeneLM, CooleyMA, GallagherWM. Fibulins: physiological and disease perspectives. EMBO Rep. 2003;4:1127–1131. [CrossRef] [PubMed]
ChuML, TsudaT. Fibulins in development and heritable disease. Birth Defects Res Part C Embryo Today. 2004;72:25–36. [CrossRef]
JohnsonLV, AndersonDH. Age-related macular degeneration and the extracellular matrix. N Engl J Med. 2004;351:320–322. [CrossRef] [PubMed]
LopezPF, SippyBD, LambertHM, ThachAB, HintonDR. Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes. Invest Ophthalmol Vis Sci. 1996;37:855–868. [PubMed]
MarmorsteinLY, MunierFL, ArsenijevicY, et al. Aberrant accumulation of EFEMP1 underlies drusen formation in malattia leventinese and age-related macular degeneration. Proc Natl Acad Sci USA.. 2002;99:13067–13072. [CrossRef]
HardingHP, CalfonM, UranoF, NovoaI, RonD. Transcriptional and translational control in the mammalian unfolded protein response. Annu Rev Cell Dev Biol. 2002;18:575–599. [CrossRef] [PubMed]
AbcouwerSF, MarjonPL, LoperRK, Vander JagtDL. Response of VEGF expression to amino acid deprivation and inducers of endoplasmic reticulum stress. Invest Ophthalmol Vis Sci. 2002;43:2791–2798. [PubMed]
RoybalCN, YangS, SunCW, et al. Homocysteine increases the expression of vascular endothelial growth factor by a mechanism involving endoplasmic reticulum stress and transcription factor ATF4. J Biol Chem. 2004;279:14844–14852. [CrossRef] [PubMed]
HeTC, ZhouS, da CostaLT, YuJ, KinzlerKW, VogelsteinB. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA. 1998;95:2509–2514. [CrossRef] [PubMed]
CalfonM, ZengH, UranoF, et al. IRE1 couples endoplasmic reticulum load to secretory capacity by processing the XBP-1 mRNA. Nature. 2002;415:92–96. [CrossRef] [PubMed]
YoshidaH, MatsuiT, YamamotoA, OkadaT, MoriK. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell. 2001;107:881–891. [CrossRef] [PubMed]
TraversKJ, PatilCK, WodickaL, LockhartDJ, WeissmanJS, WalterP. Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell. 2000;101:249–258. [CrossRef] [PubMed]
HaynesCM, TitusEA, CooperAA. Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death. Mol Cell. 2004;15:767–776. [CrossRef] [PubMed]
OzcanU, CaoQ, YilmazE, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science. 2004;306:457–461. [CrossRef] [PubMed]
SelkoeDJ. Cell biology of protein misfolding: the examples of Alzheimer’s and Parkinson’s diseases. Nat Cell Biol. 2004;6:1054–1061. [CrossRef] [PubMed]
CampochiaroPA, SolowayP, RyanSJ, MillerJW. The pathogenesis of choroidal neovascularization in patients with age-related macular degeneration. Mol Vis. 1999;5:34. [PubMed]
IdaH, TobeT, NambuH, MatsumuraM, UyamaM, CampochiaroPA. RPE cells modulate subretinal neovascularization, but do not cause regression in mice with sustained expression of VEGF. Invest Ophthalmol Vis Sci. 2003;44:5430–5437. [CrossRef] [PubMed]
TakahashiK, SaishinY, MoriK, et al. Topical nepafenac inhibits ocular neovascularization. Invest Ophthalmol Vis Sci. 2003;44:409–415. [CrossRef] [PubMed]
TakahashiK, SaishinY, SilvaRL, et al. Intraocular expression of endostatin reduces VEGF-induced retinal vascular permeability, neovascularization, and retinal detachment. FASEB J. 2003;17:896–898. [PubMed]
MousaSA, LorelliW, CampochiaroPA. Role of hypoxia and extracellular matrix-integrin binding in the modulation of angiogenic growth factors secretion by retinal pigmented epithelial cells. J Cell Biochem. 1999;74:135–143. [CrossRef] [PubMed]
CaoJ, McLeodS, MergesCA, LuttyGA. Choriocapillaris degeneration and related pathologic changes in human diabetic eyes. Arch Ophthalmol. 1998;116:589–597. [CrossRef] [PubMed]
DantasMA, SlakterJS, NegraoS, FonsecaRA, KagaT, YannuzziLA. Photodynamic therapy with verteporfin in mallatia leventinese. Ophthalmology. 2002;109:296–301. [CrossRef] [PubMed]
PagerCK, SarinLK, FedermanJL, et al. Malattia leventinese presenting with subretinal neovascular membrane and hemorrhage. Am J Ophthalmol. 2001;131:517–518. [CrossRef] [PubMed]
OshimaY, OshimaS, NambuH, et al. Increased expression of VEGF in retinal pigmented epithelial cells is not sufficient to cause choroidal neovascularization. J Cell Physiol. 2004;201:393–400. [CrossRef] [PubMed]
Figure 1.
 
Accumulation of fibulin-3 R345W in ARPE-19 cells. (A) Western blot analysis of conditioned media to detect secreted fibulin-3 protein. ARPE-19 cells were infected with fibulin-3 Wt (1:640, 1:320, 1:160, 1:80 dilutions; lanes 1–4), R345W (1:640, 1:320, 1:160, 1:80 dilutions; lanes 5–8), or Empty (1:320 and 1:160 dilutions; lanes 9,10) adenoviral vector stocks for 24 hours (see text for viral IU concentrations). Conditioned media were then immunoblotted for fibulin-3 and albumin. (B) Immunocytochemical analysis of GRP78 (a, b, c) and fibulin-3 (d, e, f) in ARPE-19 cells infected with either Empty viral stock at 1:320 dilution (a, d, g), fibulin-3 Wt viral stock at 1:320 dilution (b, e, h), or R345W viral stock at 1:160 dilution (c, f, i) for 24 hours. Yellow represents the colocalization of GRP78 and fibulin-3 shown in merged images (g, h, i).
Figure 1.
 
Accumulation of fibulin-3 R345W in ARPE-19 cells. (A) Western blot analysis of conditioned media to detect secreted fibulin-3 protein. ARPE-19 cells were infected with fibulin-3 Wt (1:640, 1:320, 1:160, 1:80 dilutions; lanes 1–4), R345W (1:640, 1:320, 1:160, 1:80 dilutions; lanes 5–8), or Empty (1:320 and 1:160 dilutions; lanes 9,10) adenoviral vector stocks for 24 hours (see text for viral IU concentrations). Conditioned media were then immunoblotted for fibulin-3 and albumin. (B) Immunocytochemical analysis of GRP78 (a, b, c) and fibulin-3 (d, e, f) in ARPE-19 cells infected with either Empty viral stock at 1:320 dilution (a, d, g), fibulin-3 Wt viral stock at 1:320 dilution (b, e, h), or R345W viral stock at 1:160 dilution (c, f, i) for 24 hours. Yellow represents the colocalization of GRP78 and fibulin-3 shown in merged images (g, h, i).
Figure 2.
 
Effect of fibulin-3 Wt and R345W expression on GRP78 and VEGF protein expression. (A) Western blot analysis of GRP78 and VEGF levels in fibulin-3 Wt–expressing cells. ARPE-19 cells were infected with increasing amounts of fibulin-3 Wt vector (1:2560, 1:1280, 1:640. 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins. (B) Western blot analysis of GRP78 and VEGF levels in fibulin-3 R345W–expressing cells. ARPE-19 cells were infected with increasing amounts of R345W vector (1:2560, 1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were then immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins (see text for viral UI concentrations).
Figure 2.
 
Effect of fibulin-3 Wt and R345W expression on GRP78 and VEGF protein expression. (A) Western blot analysis of GRP78 and VEGF levels in fibulin-3 Wt–expressing cells. ARPE-19 cells were infected with increasing amounts of fibulin-3 Wt vector (1:2560, 1:1280, 1:640. 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins. (B) Western blot analysis of GRP78 and VEGF levels in fibulin-3 R345W–expressing cells. ARPE-19 cells were infected with increasing amounts of R345W vector (1:2560, 1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions) for 24 hours. Total cell lysates (20 μg) were then immunoblotted with antibodies to VEGF, GRP78, fibulin-3, and β-actin proteins (see text for viral UI concentrations).
Figure 3.
 
Effect of fibulin-3 expression on VEGF and GRP78 mRNA expression. (A) Northern blot analysis of ARPE-19 cells infected with various amounts of empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. Cells were infected with increasing amounts of respective adenoviral vectors (1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions), and mRNA levels were examined 4 days later. (B) GRP78 mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴). (C) VEGF mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴) and mRNA levels, respectively. (D) XBP-1 splicing by ARPE-19 cells infected with various amounts of Empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. RT-PCR analysis with primers flanking the XBP-1 splice site was performed on the RNA samples previously subjected to Northern blotting shown in panel A (the four highest viral dose samples—1:640. 1:320, 1:160, and 1:80—dilutions of viral vector stocks). Arrows highlight the PCR products obtained from unprocessed (UP) and processed (PR) XBP-1 mRNA templates (see text for viral UI concentrations).
Figure 3.
 
Effect of fibulin-3 expression on VEGF and GRP78 mRNA expression. (A) Northern blot analysis of ARPE-19 cells infected with various amounts of empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. Cells were infected with increasing amounts of respective adenoviral vectors (1:1280, 1:640, 1:320, 1:160, and 1:80 dilutions), and mRNA levels were examined 4 days later. (B) GRP78 mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴). (C) VEGF mRNA inductions were plotted as a function of R345W (▪) mRNA levels and fibulin-3 Wt (▴) and mRNA levels, respectively. (D) XBP-1 splicing by ARPE-19 cells infected with various amounts of Empty, fibulin-3 Wt, and fibulin-3 R345W retroviral vectors. RT-PCR analysis with primers flanking the XBP-1 splice site was performed on the RNA samples previously subjected to Northern blotting shown in panel A (the four highest viral dose samples—1:640. 1:320, 1:160, and 1:80—dilutions of viral vector stocks). Arrows highlight the PCR products obtained from unprocessed (UP) and processed (PR) XBP-1 mRNA templates (see text for viral UI concentrations).
Figure 4.
 
Effects of exogenous fibulin-3 expression on VEGF promoter activity. ARPE-19 cells were cotransfected with pAE-Empty, pAE-fibulin-3-Wt, or pAE-fibulin-3-R345W expression vectors, together with pVEGF8.2Luc and pRL-CMV luciferase reporter vectors. At 24 and 48 hours after transfection, cells were harvested and dual luciferase assays were performed. Firefly luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren) in each sample. Data are presented as mean ± SD (n = 3) *P ≤ 0.005. (A) 24-hour incubation. (B) 48-hour incubation.
Figure 4.
 
Effects of exogenous fibulin-3 expression on VEGF promoter activity. ARPE-19 cells were cotransfected with pAE-Empty, pAE-fibulin-3-Wt, or pAE-fibulin-3-R345W expression vectors, together with pVEGF8.2Luc and pRL-CMV luciferase reporter vectors. At 24 and 48 hours after transfection, cells were harvested and dual luciferase assays were performed. Firefly luciferase relative light units (RLU Luc) were normalized to Renilla luciferase relative light units (RLU Ren) in each sample. Data are presented as mean ± SD (n = 3) *P ≤ 0.005. (A) 24-hour incubation. (B) 48-hour incubation.
Copyright 2005 The Association for Research in Vision and Ophthalmology, Inc.
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×