IOVS BJO
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2003;44:1339-1347.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-0878
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, P.
Right arrow Articles by Jaffe, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yang, P.
Right arrow Articles by Jaffe, G. J.

Effect of Mutant I{kappa}B on Cytokine-Induced Activation of NF-{kappa}B in Cultured Human RPE Cells

Ping Yang,1 Brian S. McKay,1,2 Janice B. Allen,3 Wendy L. Roberts,1 and Glenn J. Jaffe1

1From the Departments of Ophthalmology and 2Cell Biology, Duke University Medical Center, Durham, North Carolina; and the 3Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The nuclear transcription factor (NF)-{kappa}B is a central regulator of multiple inflammatory cytokines. The current study was conducted to determine whether infection of human retinal pigment epithelial (RPE) cells by adenovirus carrying a mutant inhibitory (I)-{kappa}B (I{kappa}B) transgene inhibits cytokine-induced activity of NF-{kappa}B and expression of NF-{kappa}B-dependent cytokines by preventing degradation of I{kappa}B. The persistence of recombinant protein expression and function after the viral infection was also examined.

METHODS. Cultured human RPE cells were infected with adenovirus encoding either ß-galactosidase (LacZ) or mutant I{kappa}B and were treated with interleukin (IL)-1ß or tumor necrosis factor (TNF)-{alpha}. I{kappa}B protein expression was determined by Western blot. NF-{kappa}B nuclear translocation was evaluated by immunofluorescence, and functional NF-{kappa}B activation was determined by luciferase reporter assay. NF-{kappa}B-dependent cytokine gene expression was determined by reverse transcription-polymerase chain reaction. IL-1ß-induced monocyte chemoattractant protein (MCP)-1 protein secretion was measured by enzyme-linked immunosorbent assay.

RESULTS. Stimulation of RPE cells with IL-1ß or TNF-{alpha} caused rapid degradation of the endogenous, but not mutant, I{kappa}B protein. Expression of the mutant I{kappa}B isoform inhibited cytokine-stimulated NF-{kappa}B nuclear translocation, NF-{kappa}B transcriptional activity, NF-{kappa}B-dependent gene expression, and secretion of MCP-1. Significant levels of mutant I{kappa}B protein were expressed for at least 7 weeks after infection.

CONCLUSIONS. Infection of human RPE by an adenoviral vector carrying a mutant I{kappa}B transgene blocks NF-{kappa}B activation and expression of multiple NF-{kappa}B-dependent cytokine genes over an extended period. This technique will be useful to determine the role of NF-{kappa}B in experimental proliferative vitreoretinopathy (PVR), and may offer a novel approach to treatment of PVR with a gene therapy approach.

There is accumulating evidence that cytokines are involved in the pathogenesis of proliferative vitreoretinopathy (PVR). We and others have shown that increased levels of cytokines are found in the vitreous cavity of eyes with PVR.1 2 3 A variety of cytokines have been identified on epiretinal membranes removed surgically from patients with PVR4 5 6 and on epiretinal membranes produced experimentally.7 Furthermore, an animal model of PVR can be created by coinjecting recombinant cytokines and fibronectin.1 8 We and others have shown that human retinal pigment epithelial (RPE) cells produce the cytokines interleukin (IL)-1{alpha} and -1ß, monocyte chemoattractant protein (MCP)-1, IL-8, macrophage colony-stimulating factor (M-CSF), melanoma growth-stimulating activity (MGSA)/gro, and activin A and that the levels of these peptides are modulated by cytokines known to be increased in the vitreous of patients with PVR.9 10 11 12 13 14

The nuclear transcription factor (NF)-{kappa}B is widely expressed and is a pivotal regulator of many different genes. NF-{kappa}B is retained in an inactive form in the cytoplasm through association with a inhibitory (I)-{kappa}B (I{kappa}B) protein.15 After cellular stimulation, I{kappa}B is phosphorylated at serines 32 and 36 by I{kappa}B kinase (IKK),16 17 ubiquitinated, and degraded by the 26S proteasome complex.18 Removal of I{kappa}B protein from the NF-{kappa}B-I{kappa}B complex enables NF-{kappa}B to translocate to the nucleus where it controls the transcription of many cytokines and adhesion molecules.19 20 Many of these NF-{kappa}B-responsive gene products are involved in inflammation and wound healing.19 We have shown that TNF-{alpha} and IL-1ß cause degradation of I{kappa}B, NF-{kappa}B nuclear translocation, and increased NF-{kappa}B DNA binding activity in human RPE cells.20 NF-{kappa}B activation in human RPE cells broadly upregulates expression of a wide variety of cytokine genes, an effect that is blocked by Z-Leu-Leu-Leu-H (MG132), a 26S proteasome inhibitor.20 However, the proteasome complex degrades proteins by both NF-{kappa}B-dependent and -independent mechanisms.21 22 Thus, in our previous experiments, MG132 may have affected other signaling pathways in addition to NF-{kappa}B. The effect of specific RPE cell NF-{kappa}B blockade has yet to be determined. Furthermore, there is intense interest in developing specific NF-{kappa}B inhibitors to treat human diseases.23 24 We sought to determine whether NF-{kappa}B activation and function could be specifically blocked in cultured RPE, by using a gene therapy approach. We used an adenoviral vector that encodes a mutant form of I{kappa}B with substitutions by alanine at serine 32 and 36 to abolish cytokine-stimulated I{kappa}B protein phosphorylation and degradation. We determined whether infection of human RPE cells with this mutant I{kappa}B construct decreases cytokine-induced NF-{kappa}B activity and therefore inhibits expression of NF-{kappa}B-dependent cytokines.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture and Infection
Human donor eyes were obtained from the North Carolina Organ Donor and Eye Bank. RPE cells were harvested from eyes as previously described.25 Cells were grown in Eagle’s minimal essential medium (MEM; Gibco-BRL, Grand Island, NY) with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) at 37°C in a humidified environment containing 5% CO2. Cells (1 x 105) were seeded in six-well plates (Costar; Corning Inc., Corning, NY) and refed 24 hours later. After a 24-hour incubation with fresh medium, cells were infected with adenovirus encoding either ß-galactosidase (LacZ) or mutant I{kappa}B26 (University of North Carolina at Chapel Hill Gene Delivery Core) in medium (OPTI-MEM; Gibco-BRL) containing 1% FBS at a multiplicity of infection (MOI) of 1 or 10 and then were treated for various times with IL-1ß (5 U/mL; BD Bioscience Labware, Bedford, MA) or TNF-{alpha} (1.1 x 103 U/mL; R&D Systems, Inc., Minneapolis, MN) in MEM containing 1% FBS 24 hours after infection. We chose IL-1ß and TNF-{alpha} concentrations that we have shown previously to stimulate cytokine gene expression and cause I{kappa}B degradation in RPE cells.9 10 20 27 Short-term assays were performed at an MOI of 10, to maximize our ability to detect functional effects of mutant I{kappa}B on NF-{kappa}B activation. Experiments to determine long-term mutant I{kappa}B expression were conducted at an MOI of 1 to minimize nonspecific effects of mutant I{kappa}B overexpression. An MOI of 1 was also used to enhance our ability to detect differences between IL-1ß and TNF-{alpha} effects on I{kappa}B degradation.

Detection of LacZ Expression
To determine the infection efficiency, control experiments were conducted with an adenovirus encoding LacZ. Forty-eight hours after infection with this virus, the expression of the LacZ transgene was examined by staining the cells with 5-bromo-4-chloro-3 to 3indolyl-ß-D-galactopyranoside (X-gal) solution. Blue staining of the cells was detected by phase-contrast microscopy. The percentage of infected RPE cells at MOI of 1 was 50%, and the percentage at MOI of 10 was 100% (not shown).

Cell Extracts and Western Blot
Cells were stimulated with IL-1ß (5 U/mL) or TNF-{alpha} (1.1 x 103 U/mL) for 30 minutes. After medium was removed, cells were washed twice with cold Hanks’ balanced salt solution and lysed with RIPA buffer (50 mM Tris-HCl at pH 8.0, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS). Lysates were transferred to 1.5-mL tubes (Eppendorf, Freemont, CA) and cleared by centrifugation. Total protein in the supernatants was measured by Bradford assay (Bio-Rad, Richmond, CA) with bovine serum albumin (BSA) used to generate the curve, according to the manufacturer’s instructions. Protein (20 µg) was electrophoresed on a 12.5% SDS-polyacrylamide gel overlaid with a 3.6% polyacrylamide stacking gel. The proteins were transferred to nitrocellulose membrane (Bio-Rad) using a mini transblot apparatus (Bio-Rad), according to the manufacturer’s directions. Transfers were performed overnight at room temperature (RT). Nonspecific binding sites were blocked by immersing the membrane in 10% fat-free milk powder (SACO Foods, Inc., Middleton, WI) for 30 minutes at RT. The blocking step was repeated, and then membranes were washed three times (20 minutes per wash) in Tris-buffered saline (TBST). The membrane was incubated overnight with rabbit polyclonal antibody directed against I{kappa}B (1:2000 in TBST; Santa Cruz Biotechnology, Santa Cruz, CA) at 4°C. The blots were then washed three times (20 minutes per wash) in TBST and incubated with anti-rabbit IgG conjugated with horseradish peroxidase (1:5000 in TBST; Jackson ImmunoResearch Laboratories, Inc, West Grove, PA) at 4°C for 60 minutes. Immunoreactive bands were visualized using an enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Immunofluorescent Detection of RelA
Cells (1.3 x 104) were seeded in an eight-well chamber slides (Nalge Nunc International, Napierville, IL). One day after infection, cells were pretreated with medium alone or medium containing MG-132 (20 µM; Biomol Research Laboratories Inc., Plymouth Meeting, PA) for 60 minutes and then stimulated with IL-1ß (5 U/mL) or TNF-{alpha} (1.1 x 103 U/mL) for 40 minutes. Cells were fixed with 100% methanol for 30 minutes at RT, blocked with 10% nonimmune goat serum (NGS, Jackson ImmunoResearch Laboratories, Inc.) for 30 minutes, and incubated for 30 minutes with rabbit polyclonal antibody directed against the RelA (p65) NF-{kappa}B subunit (1:200 in 10% NGS; Rockland, Gilbertsville, PA). Cells were washed with phosphate-buffered saline (PBS) and then incubated with rhodamine isothiocyanate-conjugated goat anti-rabbit IgG antibody (1:400 in 10% NGS; Jackson ImmunoResearch Laboratories, Inc.) for 30 minutes. Cells were washed with PBS and incubated with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma, St. Louis, MO) in PBS for 5 minutes. NF-{kappa}B fluorescent staining was determined with a fluorescence light microscope. The percentage of cells with NF-{kappa}B nuclear translocation was determined by a masked observer.

Transfection and NF-{kappa}B-Driven Luciferase Reporter Assay
Transfection was performed with transfection reagent (lipoTAXI; Stratagene, La Jolla, CA) according to the manufacturer’s recommendations. Briefly, cells in triplicate wells were washed twice with 3 mL of serum-free, antibiotic-free MEM (MEM-SA) and incubated in MEM-SA at 37°C for 15 minutes. Plasmid DNA (10 µg) encoding firefly luciferase downstream from the NF-{kappa}B promoter (Stratagene) and 2 µg of DNA encoding a constitutively expressed renilla luciferase (Promega, Madison, WI) were mixed with the transfection reagent (lipoTAXI; Stratagene) and then added to cell cultures in MEM-SA medium. After a 4-hour incubation, 3 mL of 10% FBS-MEM was added to each well. Cells were refed 24 hours after transfection and maintained in fresh 10% FBS-MEM for another 24 hours before infection. Cells were stimulated with IL-1ß (5 U/mL) for 4 hours. An NF-{kappa}B luciferase reporter assay was performed with a kit (Dual Luciferase Reporter Assay System; Promega), according to the manufacturer’s instructions. Firefly luciferase activity and renilla luciferase activity in the supernatants were measured with a luminometer (LB 9501; EG&G Berghold, Bundoora, Australia). The ratio of firefly luciferase activity to renilla luciferase activity was calculated.

RNA Extraction and Amplification by Reverse Transcription-Polymerase Chain Reaction
Cells were stimulated with IL-1ß (5 U/mL) or TNF-{alpha} (1.1 x 103 U/mL) for 4 hours. At the end of the incubation period, cells were rinsed once with cold PBS, and total RNA was isolated with extraction reagent (TRIzol; Gibco-BRL), according to the manufacturer’s instructions. RNA purity was estimated by measuring optical density at 260 nm (OD260)/OD280 and RNA quantity was determined from OD260.

Total RNA was transcribed to cDNA as previously described.10 For PCR, 1 µL cDNA mixture was added to a 50-µL PCR reaction mixture consisting of 5 µL of 10x PCR buffer, 2.5 pmol dNTP, 5 pmol paired primers, 1.25 U Taq polymerase (Promega), and ultrapure water. The PCR primers used are listed in Table 1 . The reaction mixture was amplified in a PCR thermal cycler (Perkin-Elmer, Wellesley, MA) as follows: denaturation at 94°C for 1 minute, primer annealing at 55°C for 2 minutes, and extension at 72°C for 3 minutes for all primers except those for MCP-1. For reaction mixtures with MCP-1 primers, cycling parameters were as follows: denaturation at 92°C for 1 minute, primer annealing at 60°C for 1 minute, and extension at 72°C for 1 minute. PCR reaction mixtures without the addition of cDNA template were used as negative controls. Aliquots (10 µL) were removed from the reaction mixture at different cycles to ensure that PCR products were evaluated during the exponential phase of the amplification process. PCR products were run on 2% agarose (Ultrapure; Gibco-BRL) gel with TBE (Tris 0.17 M, boric acid 0.17 M, and EDTA 4 mM [pH 8.0]) running buffer. Gels were stained with ethidium bromide and photographed (type 55 film; Polaroid, Cambridge, MA). The expected bands were identified based on size. The primers were designed to span an intron to differentiate mRNA from genomic DNA.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Oligonucleotides Used in RT-PCR

 
MCP-1 Protein Measurement
IL-1ß-induced MCP-1 protein secretion in RPE cells was measured by enzyme-linked immunosorbent assay (ELISA). Cells in triplicate wells were stimulated with IL-1ß (5 U/mL) for 24 hours. Collected media were clarified by centrifugation and then stored at -70°C. The cells were rinsed with PBS and lysed with 0.1 N NaOH. Lysates were transferred to tubes and cleared by centrifugation. The protein concentration of the cell lysates was determined by Lowry protein assay28 using BSA as a standard. Secreted MCP-1 in the media supernatants was measured (Cytoscreen Immunoassay Kit; BioSource, Camarillo, CA) according to the manufacturer’s instructions. Secreted MCP-1 protein was standardized by expressing the data relative to cellular protein in each lysate.

Statistical Analysis
Data are expressed as the mean ± SD. Paired data from triplicate samples in Western blot analysis, luciferase reporter assay, and MCP-1 ELISA were analyzed by Student’s t-test. A {chi}2 test was used to compare the proportion of cells with NF-{kappa}B nuclear translocation with the proportion of cells with cytoplasmic NF-{kappa}B among the different experimental groups. P < 0.05 was considered to be statistically significant. The luciferase reporter assay, a functional assay of NF-{kappa}B activation was repeated three times with RPE cells from three different donors. More than 10 separate Western blot assays were performed, to show that overexpression of mutant I{kappa}B was resistant to IL-1ß-induced degradation of I{kappa}B. The experiment to demonstrate long-term expression of mutant I{kappa}B at various time points up to 7 weeks was performed once. Based on similar results in each of these cell lines in the luciferase assay, a single representative cell line was selected for ELISA, immunofluorescent assay, and RT-PCR.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Overexpression of Mutant I{kappa}B Protein by Adenovirus Vectors
The expression of I{kappa}B proteins in RPE cells was detected by Western blot. After treatment with IL-1ß (5 U/mL), endogenous I{kappa}B protein was rapidly degraded within 30 minutes (Fig. 1A) . Infection of cells with adenovirus that encoded either LacZ or mutant I{kappa}B did not change endogenous I{kappa}B degradation after stimulation with IL-1ß (Fig. 1A) . In contrast, mutant I{kappa}B was not degraded after treatment with IL-1ß. As a control for cytoplasmic phosphorylation and degradation we chose ß-catenin, a protein that is phosphorylated and degraded by the proteasome in response to an NF-{kappa}B-independent signal-transduction pathway. In contrast to I{kappa}B, ß-catenin was not degraded in response to IL-1ß stimulation in LacZ-expressing cells and in cells infected to express mutant I{kappa}B (Fig. 1B) . Accordingly, ß-catenin also serves as an excellent protein to standardize protein loading.



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 1. Western blot showing expression of the I{kappa}B transgene. RPE cells in triplicate wells were infected with adenovirus containing LacZ (control virus, MOI = 10) or mutant I{kappa}B (MOI = 10). One day after infection, cells were treated with medium alone or IL-1ß (5 U/mL) for 30 minutes, and cytoplasmic proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane for Western blot analysis. (A) Western blot probed with antibody to I{kappa}B. Bands at 45 kDa correspond to mutant I{kappa}B transgene and bands at 37 kDa to endogenous I{kappa}B. The relative quantity of endogenous and mutant I{kappa}B proteins, determined by densitometry, are shown separately below each lane. (B) Blot in (A) stripped and reprobed with antibody to ß-catenin, a control for gel loading. The relative quantity of ß-catenin protein is shown below each lane.

 
I{kappa}B protein levels in both in LacZ-expressing cells and in cells infected to express mutant I{kappa}B were quantified by densitometry band analysis. As shown in Table 2 , significant levels of mutant I{kappa}B protein were expressed in untreated and IL-1ß-treated cells infected to express mutant I{kappa}B. Endogenous I{kappa}B protein was not as efficiently degraded by IL-1ß treatment in cells infected to express mutant I{kappa}B as it was in LacZ-expressing cells. With or without IL-1ß treatment, ß-catenin protein levels were higher in cells infected to express mutant I{kappa}B than in LacZ-expressing cells.


View this table:
[in this window]
[in a new window]
  		TABLE 2. Quantitation of I{kappa}B and ß-Catenin Protein Expression after IL-1ß Stimulation

 
Persistent Expression of Mutant I{kappa}B Protein by Adenovirus Vectors
To investigate the feasibility of prolonged inhibition of I{kappa}B by adenoviral gene transduction in RPE, we determined whether mutant I{kappa}B protein expression persists after the infection. Significant levels of mutant I{kappa}B protein were expressed 1 and 3 days and 1, 2, 5, and 7 weeks after infection, and the mutant I{kappa}B remained resistant to IL-1ß-stimulated degradation. Endogenous I{kappa}B degradation was variable at different time points, despite constant overexpression of mutant I{kappa}B. The data shown in Figure 2A illustrate persistent transgene expression 7 weeks after infection. After treatment with IL-1ß, endogenous I{kappa}B was not efficiently degraded in cells infected to express mutant I{kappa}B 7 weeks after infection. ß-Catenin protein was not degraded by stimulation with IL-1ß in noninfected cells or in cells infected to express mutant I{kappa}B (Fig. 2B , Table 3 ).



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 2. Long-term maintenance of transgene expression. Control, noninfected RPE cells and cells infected with mutant I{kappa}B (MOI = 1) were grown in culture for 3 weeks after infection, trypsinized, replated at a dilution of 1:4, and then grown for an additional 4 weeks. Cells in triplicate wells were exposed to medium alone or to IL-1ß (5 U/mL) for 30 minutes and lysed, and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. (A) Blot probed with antibody to I{kappa}B. Bands at 45 kDa correspond to mutant I{kappa}B transgene and bands at 37 kDa to endogenous I{kappa}B. The relative quantities of endogenous and mutant I{kappa}B protein, determined by densitometry, are shown separately below each lane. (B) Blot in (A) stripped and reprobed with antibody to ß-catenin. The relative quantity of ß-catenin protein is shown below each lane.

 

View this table:
[in this window]
[in a new window]
  		TABLE 3. Long-Term Maintenance of Transgene Expression and Activation

 
Comparison of IL-1ß and TNF-{alpha}-Induced Degradation of Mutant I{kappa}B Protein
Next, we determined whether IL-1ß and TNF-{alpha} have similar effects on degradation of endogenous and mutant I{kappa}B. Mutant I{kappa}B was not degraded in response to treatment with either IL-1ß or TNF-{alpha} (Fig. 3C) . In contrast, both IL-1ß and TNF-{alpha} caused degradation of endogenous I{kappa}B in noninfected cells, in LacZ-expressing cells, and in cells infected to express mutant I{kappa}B (Figs. 3A 3B 3C) . ß-Catenin protein was not degraded in response to stimulation with either IL-1ß or TNF-{alpha} in noninfected, LacZ-expressing cells or in cells infected to express mutant I{kappa}B (Figs. 3D 3E 3F) .



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 3. Effect of IL-1ß and TNF-{alpha} on degradation of I{kappa}B. RPE cells in triplicate wells were infected with adenovirus containing LacZ (control virus, MOI = 1) or mutant I{kappa}B (MOI = 1). Noninfected cells were included as an additional control. One day after infection, cells were treated with medium alone, IL-1ß (5 U/mL), or TNF-{alpha} (1.1 x 103 U/mL) for 30 minutes and lysed, and proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. (A, B) Blot probed with antibody to I{kappa}B. Bands at 37 kDa correspond to endogenous I{kappa}B. (C) Blot probed with antibody to I{kappa}B. Bands at 45 kDa correspond to mutant I{kappa}B transgene and bands at 37 kDa to endogenous I{kappa}B. The relative quantity of endogenous and mutant I{kappa}B protein are shown separately below each lane. (D–F) Blots in (A), (B), and (C), respectively, separately stripped and reprobed with antibody to ß-catenin. The relative quantity of ß-catenin protein is shown below each lane.

 
Decreased Activation of NF-{kappa}B by Overexpression of Mutant I{kappa}B
High levels of expression of mutant I{kappa}B, together with its resistance to degradation suggests a potential inhibitory effect of this agent on NF-{kappa}B activity. To test this hypothesis, we first investigated the cellular localization of the NF-{kappa}B p65 subunit by immunofluorescence. In both noninfected cells and cells infected to express LacZ, p65 was present in the cytoplasm. Similarly, both noninfected cells and cells infected to express LacZ, had strong nuclear p65 staining after 40 minutes incubation with either IL-1ß or TNF-{alpha}. However, in cells infected to express mutant I{kappa}B, the nuclear translocation of NF-{kappa}B was efficiently blocked, even after treatment with IL-1ß or TNF-{alpha}. These differences were statistically significant (P < 0.001). At the dosage used, TNF-{alpha} induced more translocation than IL-1ß in noninfected cells and LacZ-expressing cells. MG132 used as a nonselective proteasome inhibitor also inhibited IL-1ß-induced NF-{kappa}B translocation in both LacZ-expressing cells and in cells infected to express mutant I{kappa}B (Fig. 4 , Table 4 ).



View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 4. Effect of overexpression of mutant I{kappa}B on cytokine-induced NF-{kappa}B nuclear translocation in representative RPE cells. Noninfected cells and cells infected with either LacZ (control virus, MOI = 10) or mutant I{kappa}B (MOI = 10) in duplicate wells were pretreated with MG132 (20 µM/L) for 60 minutes and then exposed to medium alone, IL-1ß (5 U/mL), or TNF-{alpha} (1.1 x 103 U/mL) for 40 minutes. One day after infection, cells were harvested and immunostained to localize p65, an NF-{kappa}B subunit (A, C, E, G, I, K, M, O, Q). Nuclei were stained with DAPI (B, D, F, H, J, L, N, P, R, T) and are shown in parallel. Arrows: p65 nuclear staining in cells treated with IL-1ß or TNF-{alpha}. Arrowheads: nucleus of treated cells. Small arrows: absence of nuclear staining in treated cells infected to express mutant I{kappa}B. (S) Negative immunofluorescence control: PBS rather than primary antibody. Bar, 10 µM.

 

View this table:
[in this window]
[in a new window]
  		TABLE 4. Effect of Mutant I{kappa}B Overexpression on NF-{kappa}B Nuclear Translocation

 
As a more sensitive and quantitative indicator of NF-{kappa}B transcriptional activity, we used a luciferase reporter assay. In this experiment, active nuclear NF-{kappa}B promotes the expression of firefly luciferase, which can then be measured in cell lysates. IL-1ß strongly induced NF-{kappa}B activity in both noninfected cells and cells infected to express LacZ, as determined by measuring the luciferase activity in cell lysates. In contrast, cells infected to express the mutant I{kappa}B demonstrated little NF-{kappa}B activity after IL-1ß treatment, as judged by the luciferase reporter assay. Overall, the baseline amount of luciferase activity in cells infected to express the mutant I{kappa}B was lower than in uninfected cells and cells infected with the LacZ virus (Fig. 5) .



View larger version (10K):
[in this window]
[in a new window]
  		FIGURE 5. Inhibition of NF-{kappa}B transcriptional activity in RPE cells infected to express the mutant I{kappa}B. RPE cells in triplicate wells were cotransfected with 10 µg plasmid DNA encoding firefly luciferase driven by the NF-{kappa}B promoter and 2 µg of DNA encoding a constitutively expressed renilla luciferase. The firefly luciferase plasmid contains a luciferase reporter gene driven by a tandem repeat of NF-{kappa}B binding elements and therefore serves as an experimental reporter of NF-{kappa}B activation. Renilla luciferase serves as control reporter to normalize the activity of the experimental reporter, based on transfection efficiency and cell viability. Two days after transfection, the cells were infected with adenovirus containing LacZ (control virus, MOI = 10) or mutant I{kappa}B (MOI = 10). Noninfected cells were included as an additional control. One day after infection, cells were exposed to medium alone or IL-1ß (5 U/mL) for 4 hours. Cells were harvested, and lysates were tested for luciferase activity with a dual-luciferase assay system. Relative luminescence reflects the ratio of firefly luciferase activity (NF-{kappa}B-dependent) to renilla luciferase (control) activity to standardize for transfection efficiency. Results are expressed as the mean ± SD of results in three experiments. *P < 0.01 versus untreated cells; **P < 0.01 versus IL-1ß-treated cells in no virus group or control virus group; #P < 0.01 versus untreated cells in control virus group.

 
Blockade of NF-{kappa}B-Dependent Gene Expression by Mutant I{kappa}B Adenovirus Vector
The strong inhibition of NF-{kappa}B transcriptional activity by overexpression of mutant I{kappa}B in RPE cells indicated that cytokine-mediated NF-{kappa}B-dependent gene induction could be downregulated by mutant I{kappa}B. Therefore, we examined NF-{kappa}B-dependent cytokine gene expression by RT-PCR. IL-1ß induction of MGSA/gro-{alpha}; IL-6, -8, and -1ß; and MCP-1 was inhibited in cells infected to express mutant I{kappa}B compared with noninfected cells and cells that expressed LacZ (Fig. 6) .



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 6. Mutant I{kappa}B inhibition of IL-1ß-induced transcription of NF-{kappa}B target genes. RPE cells in triplicate wells were either noninfected or infected with adenovirus containing LacZ (control virus, MOI = 10) or mutant I{kappa}B (MOI = 10). One day after infection, cells were treated with medium alone or IL-1ß (5 U/mL) for 4 hours. RNA was extracted, reverse transcribed to cDNA, and amplified with primers specific for NF-{kappa}B-dependent transcripts. GAPDH amplification served as a control for the molecular manipulations. Amplification of MGSA/gro-{alpha} comprised 25 cycles; amplification of IL-6 and -1ß and GAPDH, 30 cycles; amplification of IL-8, 35 cycles; and amplification of MCP-1, 40 cycles. Numbers indicate expected product size.

 
We conducted additional experiments to compare the ability of mutant I{kappa}B to block cytokine expression induced by IL-1ß versus that induced by TNF-{alpha}. For this assay, we chose to test upregulated expression of IL-1ß and MGSA/gro-{alpha}, representative NF-{kappa}B-modulated cytokines, after treatment with IL-1ß and TNF-{alpha}. IL-1ß- and TNF-{alpha}-induced MGSA/gro-{alpha} and IL-1ß gene expression was efficiently inhibited in cells infected to express mutant I{kappa}B but not in noninfected cells or LacZ-expressing cells (Fig. 7) .



View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. RT-PCR showing the effect of mutant I{kappa}B expression on IL-1ß- and TNF-{alpha}-induced transcription of NF-{kappa}B target genes. RPE cells in triplicate wells were either noninfected or infected with adenovirus containing LacZ (control virus, MOI = 1) or mutant I{kappa}B (MOI = 1). One day after infection, cells were treated with medium alone, IL-1ß (5 U/mL), or TNF-{alpha} (1.1 x 103 U/mL) for 4 hours. RNA was extracted, reverse-transcribed to cDNA, and amplified using primers specific for NF-{kappa}B-dependent transcripts. GAPDH amplification serves as a control for the molecular manipulations. Amplification of MGSA/gro-{alpha} and GAPDH comprised 25 cycles, and amplification of IL-1ß, 30 cycles. Numbers indicate expected product size.

 
Inhibition of MCP-1 Protein Secretion by Mutant I{kappa}B Adenovirus Vector
Mutant I{kappa}B overexpression blocked both IL-1ß- and TNF-{alpha}-induced cytokine gene expression. Next, we determined whether overexpression of mutant I{kappa}B could block the secretion of a cytokine that is upregulated and secreted in response to IL-1ß. We chose to examine a representative NF-{kappa}B-dependent cytokine protein, MCP-1. Noninfected cells, LacZ-expressing cells, and cells infected to express mutant I{kappa}B were stimulated with IL-1ß (5 U/mL) for 24 hours. The media were collected and tested by ELISA. MCP-1 protein was not detectable in the media under basal conditions, but secretion of MCP-1 was stimulated by IL-1ß in noninfected RPE cells and in RPE cells infected to express LacZ. In contrast, IL-1ß-induced secretion of MCP-1 was blocked in cells infected to express mutant I{kappa}B (Table 5) .


View this table:
[in this window]
[in a new window]
  		TABLE 5. Effect of Mutant I{kappa}B Overexpression on MCP-1 Protein Secretion

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In this study, a mutant I{kappa}B protein was overexpressed in human RPE cells by viral transduction and was resistant to cytokine-stimulated degradation. Moreover, mutant I{kappa}B protein expression was maintained for at least 7 weeks in culture. Finally, expression of a mutant I{kappa}B protein: (1) blocked cytokine-stimulated NF-{kappa}B nuclear translocation, (2) blocked IL-1ß-induced NF-{kappa}B transcriptional activity, (3) inhibited gene expression of multiple NF-{kappa}B-dependent cytokines in RPE cells, and (4) blocked IL-1ß-stimulated MCP-1 secretion.

NF-{kappa}B is maintained in an inactive form in the cytoplasm by its physical association with I{kappa}B protein, and many of the signals known to activate NF-{kappa}B result in phosphorylation and subsequent degradation of I{kappa}B.15 29 30 31 32 Serine residues 32 and 36 in I{kappa}B were found to be phosphorylated in response to cytokine stimulation.19 33 In this study, we explored the effect in cultured human RPE cells of a mutant I{kappa}B protein in which serines 32 and 36 were changed to alanine.26 The mutant protein was not degraded after stimulation with cytokines, which suggests that serine phosphorylation at residues 32 and 36 of I{kappa}B is a step in the normal degradation pathway of I{kappa}B in RPE cells, as has been shown in other cell types.26 Further, our results indicate that in RPE cells, I{kappa}B degradation is a normal step in the pathway of I{kappa}B action, because the endogenous protein was rapidly degraded after RPE cells were treated with IL-1ß or TNF-{alpha}. That endogenous, but not mutant, I{kappa}B was degraded indicates that the presence of the mutant transgene did not nonspecifically affect the endogenous signal-transduction pathways activated by IL-1ß and TNF-{alpha}.

ß-Catenin is a multifunctional protein involved in adhesion and transducing the Wnt signal. The Wnt signaling pathway functions reiteratively during animal development to control cellular fate.34 Free cytoplasmic ß-catenin protein was not degraded after stimulation with IL-1ß or as a consequence of adenoviral infection alone. These data suggest that the observed degradation of I{kappa}B in response to IL-1ß was specific for the NF-{kappa}B signaling pathway and not simply a nonspecific effect of IL-1ß on viral infection of NF-{kappa}B-independent signaling pathways.

NF-{kappa}B transcriptional activity, as measured by a luciferase assay, and NF-{kappa}B-dependent cytokine gene expression were inhibited by mutant I{kappa}B. These data are also consistent with immunofluorescence results that showed blockade of NF-{kappa}B nuclear translocation by mutant I{kappa}B. The exact mechanism by which mutant I{kappa}B inhibits NF-{kappa}B-dependent cytokine expression is not clear. However, because mutant I{kappa}B protein was overexpressed in this system, it is likely that the normal degradation of endogenous I{kappa}B was insufficient to allow NF-{kappa}B nuclear translocation and subsequent transcriptional activity. We hypothesize that the observed inhibition of NF-{kappa}B transcriptional activity by mutant I{kappa}B protein reflects rapid binding of free NF-{kappa}B, released by degradation of endogenous I{kappa}B, to exogenous nonphosphorylated mutant I{kappa}B protein, which is not subject to proteolytic degradation. The consumption of the free NF-{kappa}B in the cytoplasm was sufficient to effectively inhibit NF-{kappa}B transcriptional activity.

Nuclear NF-{kappa}B regulates a variety of inflammatory genes, which include IL-1, -6, and -8; MGSA/gro-{alpha}; and MCP-1. To test the functional significance of NF-{kappa}B blockade, we examined the expression of genes likely to be of significance in PVR. IL-8 is significantly increased in vitreous samples of patients with PVR.2 35 IL-1 and -6 have a broad spectrum of activity in inflammation and wound healing; IL-1, and -6 are elevated in vitreous from eyes with PVR.3 35 MCP-1 is a chemokine that promotes monocyte chemotaxis in areas of injury,36 and MCP-1 levels are significantly increased in the vitreous of eyes with PVR.2 MGSA/gro-{alpha} is a pleiotropic modulator of cell proliferation and inflammation and may contribute to the intraocular wound-healing response that characterizes PVR.27 In the current study, mutant I{kappa}B overexpression inhibited the expression of each of these genes. Further, there was significantly decreased secretion of MCP-1 protein in response to IL-1ß.

To investigate long-term expression of mutant I{kappa}B, we tested the persistence of transgene expression after initial infection. We found that RPE cells continue to express significant mutant transgene levels for at least 7 weeks after initial infection. Levels were not altered by IL-1ß stimulation. These data suggest the feasibility of achieving NF-{kappa}B blockade by mutant I{kappa}B over a period of several weeks. Persistent transgene expression is ideal for in vivo experiments in which prolonged I{kappa}B inhibition in injected cells is desired.

In conclusion, we have shown that expression of mutant I{kappa}B protein in RPE cells acts as a dominant negative regulator of NF-{kappa}B transcriptional activity. Synthesis of key proinflammatory cytokines in response to IL-1ß and TNF-{alpha}, the downstream effect of NF-{kappa}B activation, was blocked by mutant I{kappa}B expression. Modulation of this key transcription factor pathway could have an important impact on retinal proliferative diseases. Studies to determine the effectiveness of this approach to inhibit PVR in vivo are currently under way in our laboratory.


   Acknowledgements
 
The authors thank Dara Khalatbari for help with NF-{kappa}B translocation grading.


   Footnotes
 
Supported by National Eye Institute Grants R01-EY9106 (GJJ) and R01-EY11364 (JBA) and Core Grant 930EY05722, and a Research to Prevent Blindness Career Development Award (BSM).

Submitted for publication August 27, 2002; accepted September 19, 2002.

Disclosure: P. Yang, None; B.S. McKay, None; J.B. Allen, None; W.L. Roberts, None; G.J. Jaffe, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Glenn J. Jaffe, Department of Ophthalmology, Duke University Eye Center, Durham, NC 27710; jaffe001{at}mc.duke.edu.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Connor, TB, Jr, Roberts, AB, Sporn, MB, et al (1989) Correlation of fibrosis and transforming growth factor-beta type 2 levels in the eye J Clin Invest 83,1661-1666
  2. Elner, SG, Elner, VM, Jaffe, GJ, et al (1995) Cytokines in proliferative diabetic retinopathy and proliferative vitreoretinopathy Curr Eye Res 14,1045-1053[Medline][Order article via Infotrieve]
  3. Limb, GA, Little, BC, Meager, A, et al (1991) Cytokines in proliferative vitreoretinopathy Eye 5,686-693
  4. Malecaze, F, Mathis, A, Arne, JL, et al (1991) Localization of acidic fibroblast growth factor in proliferative vitreoretinopathy membranes Curr Eye Res 10,719-729[Medline][Order article via Infotrieve]
  5. Limb, GA, Alam, A, Earley, O, et al (1994) Distribution of cytokine proteins within epiretinal membranes in proliferative vitreoretinopathy Curr Eye Res 13,791-798[Medline][Order article via Infotrieve]
  6. Limb, GA, Earley, O, Jones, SE, et al (1994) Expression of mRNA coding for TNF alpha, IL-1 beta and IL-6 by cells infiltrating retinal membranes Graefes Arch Clin Exp Ophthalmol 232,646-651[Medline][Order article via Infotrieve]
  7. Planck, SR, Andresevic, J, Chen, JC, et al (1992) Expression of growth factor mRNA in rabbit PVR model systems Curr Eye Res 11,1031-1039[Medline][Order article via Infotrieve]
  8. Yeo, JH, Sadeghi, J, Campochiaro, PA, Green, WR, Glaser, BM. (1986) Intravitreous fibronectin and platelet-derived growth factor: new model for traction retinal detachment Arch Ophthalmol 104,417-421[Abstract]
  9. Jaffe, GJ, Van Le, L, Valea, F, et al (1992) Expression of interleukin-1 alpha, interleukin-1 beta, and an interleukin-1 receptor antagonist in human retinal pigment epithelial cells Exp Eye Res 55,325-335[CrossRef][Medline][Order article via Infotrieve]
  10. Jaffe, GJ, Peters, WP, Roberts, W, et al (1992) Modulation of macrophage colony stimulating factor in cultured human retinal pigment epithelial cells Exp Eye Res 54,595-603[CrossRef][Medline][Order article via Infotrieve]
  11. Jaffe, GJ, Harrison, CE, Lui, GM, et al (1994) Activin expression by cultured human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 35,2924-2931[Abstract/Free Full Text]
  12. Shattuck, RL, Wood, LD, Jaffe, GJ, Richmond, A. (1994) MGSA/GRO transcription is differentially regulated in normal retinal pigment epithelial and melanoma cells Mol Cell Biol 14,791-802[Abstract/Free Full Text]
  13. Bian, ZM, Elner, SG, Strieter, RM, et al (1996) Glycated serum albumin induces chemokine gene expression in human retinal pigment epithelial cells J Leukoc Biol 60,405-414[Abstract]
  14. Yoshida, A, Elner, SG, Bian, ZM, Elner, VM. (2001) Induction of interleukin-8 in human retinal pigment epithelial cells after denuding injury Br J Ophthalmol 85,872-876[Abstract/Free Full Text]
  15. Haskill, S, Beg, AA, Tompkins, SM, et al (1991) Characterization of an immediate-early gene induced in adherent monocytes that encodes I kappa B-like activity Cell 65,1281-1289[CrossRef][Medline][Order article via Infotrieve]
  16. Regnier, CH, Song, HY, Gao, X, et al (1997) Identification and characterization of an IkappaB kinase Cell 90,373-383[CrossRef][Medline][Order article via Infotrieve]
  17. Didonato, JA, Hayakawa, M, Rothwarf, DM, Zandi, E, Karin, M. (1997) A cytokine-responsive IkappaB kinase that activates the transcription factor NF-kappaB Nature 388,548-554[CrossRef][Medline][Order article via Infotrieve]
  18. Beg, AA, Finco, TS, Nantermet, PV, Baldwin, AS, Jr (1993) Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation Mol Cell Biol 13,3301-3310[Abstract/Free Full Text]
  19. Baldwin, AS, Jr (1996) The NF-kappa B and I kappa B proteins: new discoveries and insights Annu Rev Immunol 14,649-683[CrossRef][Medline][Order article via Infotrieve]
  20. Wang, XC, Jobin, C, Allen, JB, Roberts, WL, Jaffe, GJ. (1999) Suppression of NF-kappaB-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor Invest Ophthalmol Vis Sci 40,477-486[Abstract/Free Full Text]
  21. Glickman, MH, Ciechanover, A. (2002) The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction Physiol Rev 82,373-428[Abstract/Free Full Text]
  22. Palombella, VJ, Rando, OJ, Goldberg, AL, Maniatis, T. (1994) The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B Cell 78,773-785[CrossRef][Medline][Order article via Infotrieve]
  23. Garber, K. (2002) Cancer research: taking garbage in, tossing cancer out? Science 295,612-613[Abstract/Free Full Text]
  24. Karin, M, Cao, Y, Greten, FR, Li, ZW. (2002) NF-kappaB in cancer: from innocent bystander to major culprit Nat Rev Cancer 2,301-310[CrossRef][Medline][Order article via Infotrieve]
  25. Jaffe, GJ, Earnest, K, Fulcher, S, Lui, GM, Houston, LL. (1990) Antitransferrin receptor immunotoxin inhibits proliferating human retinal pigment epithelial cells Arch Ophthalmol 108,1163-1168[Abstract]
  26. Jobin, C, Panja, A, Hellerbrand, C, et al (1998) Inhibition of proinflammatory molecule production by adenovirus-mediated expression of a nuclear factor kappaB super-repressor in human intestinal epithelial cells J Immunol 160,410-418[Abstract/Free Full Text]
  27. Jaffe, GJ, Richmond, A, Van Le, L, et al (1993) Expression of three forms of melanoma growth stimulating activity (MGSA)/gro in human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 34,2776-2785[Abstract/Free Full Text]
  28. Lowry, O, Rosenbrough, N, Farr, A, Randall, R. (1951) Protein measurement with the folin phenol reagent J Biol Chem 193,265-275[Free Full Text]
  29. Karin, M. (1999) How NF-kappaB is activated: the role of the IkappaB kinase (IKK) complex Oncogene 18,6867-6874[CrossRef][Medline][Order article via Infotrieve]
  30. Brockman, JA, Scherer, DC, Mckinsey, TA, et al (1995) Coupling of a signal response domain in I kappa B alpha to multiple pathways for NF-kappa B activation Mol Cell Biol 15,2809-2818[Abstract]
  31. Brown, K, Gerstberger, S, Carlson, L, Franzoso, G, Siebenlist, U. (1995) Control of I kappa B-alpha proteolysis by site-specific, signal-induced phosphorylation Science 267,1485-1488[Abstract/Free Full Text]
  32. Traenckner, EB, Pahl, HL, Henkel, T, et al (1995) Phosphorylation of human I kappa B-alpha on serines 32 and 36 controls I kappa B-alpha proteolysis and NF-kappa B activation in response to diverse stimuli EMBO J 14,2876-2883[Medline][Order article via Infotrieve]
  33. Verma, IM, Stevenson, JK, Schwarz, EM, Van Antwerp, D, Miyamoto, S. (1995) Rel/NF-kappa B/I kappa B family: intimate tales of association and dissociation Genes Dev 9,2723-2735[Free Full Text]
  34. Waltzer, L, Bienz, M. (1999) The control of beta-catenin and TCF during embryonic development and cancer Cancer Metastasis Rev 18,231-246[CrossRef][Medline][Order article via Infotrieve]
  35. El-Ghrably, IA, Dua, HS, Orr, GM, Fischer, D, Tighe, PJ. (2001) Intravitreal invading cells contribute to vitreal cytokine milieu in proliferative vitreoretinopathy Br J Ophthalmol 85,461-470[Abstract/Free Full Text]
  36. Haelens, A, Wuyts, A, Proost, P, et al (1996) Leukocyte migration and activation by murine chemokines Immunobiology 195,499-521[Medline][Order article via Infotrieve]



This article has been cited by other articles:


Home page
 IOVSHome page
N. Zhang, J. J. Peairs, P. Yang, J. Tyrrell, J. Roberts, R. Kole, and G. J. Jaffe
The Importance of Bcl-xL in the Survival of Human RPE Cells
Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3846 - 3853.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
D. J. Hall, M. E. Bates, L. Guar, M. Cronan, N. Korpi, and P. J. Bertics
The Role of p38 MAPK in Rhinovirus-Induced Monocyte Chemoattractant Protein-1 Production by Monocytic-Lineage Cells
J. Immunol., June 15, 2005; 174(12): 8056 - 8063.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. Yang, J. L. Wiser, J. J. Peairs, J. N. Ebright, Z. J. Zavodni, C. Bowes Rickman, and G. J. Jaffe
Human RPE Expression of Cell Survival Factors
Invest. Ophthalmol. Vis. Sci., May 1, 2005; 46(5): 1755 - 1764.
[Abstract] [Full Text] [PDF]

Home page
 IOVSHome page
P. Yang, B. S. McKay, J. B. Allen, and G. J. Jaffe
Effect of NF-{kappa}B Inhibition on TNF-{alpha}-Induced Apoptosis in Human RPE Cells
Invest. Ophthalmol. Vis. Sci., July 1, 2004; 45(7): 2438 - 2446.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yang, P.
Right arrow Articles by Jaffe, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation