Human RPE expression of cell survival factors.
OBJECTIVE
To determine basal and tumor necrosis factor (TNF)-alpha-regulated expression of retinal pigment epithelial (RPE) cell survival factors and whether regulation is dependent on nuclear transcription factor (NF)-kappaB.


METHODS
Cultured human RPE cells were infected with adenovirus encoding either mutant inhibitory (I)-kappaB or beta-galactosidase and treated with TNF-alpha for various times. Freshly prepared RPE/choroid and RPE samples were isolated from human donor eyes. Real-time reverse transcription-polymerase chain reaction, Western blot, and immunocytochemistry were used to determine survival factor gene expression, cellular protein levels, and localization, respectively.


RESULTS
Multiple survival factor genes, including cellular inhibitor of apoptosis protein (c-IAP1), c-IAP2, TNF receptor-associated factor-1 (TRAF-1), TRAF-2, B-cell leukemia/lymphoma-2 (Bcl-2), Bcl-x, A1, and cellular Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme-like inhibitory protein (c-FLIP), were expressed in basal conditions in both cultured RPE cells and RPE cells in situ, whereas survivin was expressed only by cultured cells. TNF-alpha upregulated expression of TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1. TRAF-1, c-FLIP, and to a lesser extent c-IAP2 protein levels were increased by TNF-alpha in a time-dependent manner, whereas c-IAP1, survivin, Bcl-x(L), and TRAF-2 protein levels were not influenced by TNF-alpha treatment at any time point tested. In contrast, Bcl-2 and A1 proteins were not detected under basal conditions or after TNF-alpha treatment. Overexpression of mutant IkappaB blocked TNF-alpha-induced TRAF-1, TRAF-2, c-IAP1, c-IAP2, c-FLIP, and A1 gene expression and downregulated TRAF-1 protein levels. TRAF-1 and Bcl-x(L) proteins were localized diffusely in RPE cytoplasm.


CONCLUSIONS
Multiple RPE cell survival factors are expressed by human RPE cells. TNF-alpha regulates expression of some of these factors in an NF-kappaB-dependent manner, whereas others are not influenced by NF-kappaB. RPE cell survival factors may protect RPE cells from apoptosis normally and in diseases such as age-related macular degeneration (AMD) and proliferative vitreoretinopathy (PVR).
Yang
Ping
P
Departments of Ophthalmology, Duke University Medical Center, Durham, NC 27710, USA.
Wiser
Jessica L
JL
Peairs
James J
JJ
Ebright
Jessica N
JN
Zavodni
Zachary J
ZJ
Bowes Rickman
Catherine
C
Jaffe
Glenn J
GJ
D000256:Adenoviridae / Q000235:genetics; D000368:Aged; D017209:Apoptosis; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002470:Cell Survival / Q000502:physiology*; D002478:Cells, Cultured; D015870:Gene Expression; D018014:Gene Transfer Techniques; D006801:Humans; D007150:Immunohistochemistry; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism; D008875:Middle Aged; D016328:NF-kappa B / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000378:metabolism*; D011506:Proteins / Q000235:genetics* / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D047990:TNF Receptor-Associated Factor 1 / Q000235:genetics* / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases; D051020:bcl-X Protein
C494811:BCL2L1 protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D016328:NF-kappa B; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D047990:TNF Receptor-Associated Factor 1; D047992:TNF Receptor-Associated Factor 2; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; C494833:BIRC2 protein, human; D044767:Ubiquitin-Protein Ligases