Vitreous induces heme oxygenase-1 expression mediated by transforming growth factor-beta and reactive oxygen species generation in human retinal pigment epithelial cells.
OBJECTIVE
When human retinal pigment epithelial (RPE) cells come in contact with vitreous, they undergo changes in gene expression that include inflammatory and anti-oxidant responses. The effects of vitreous on expression of heme oxygenase-1 (HO-1), metallothionein (MT) -1a and -2a, and c-fos were investigated. Activator protein-1 (AP-1) binding sites are located in the promoter region of HO-1 and MT genes and the effects of vitreous on c-fos activity were investigated.


METHODS
Low passage cultures of human RPE cells were grown in the presence or absence of vitreous or transforming growth factor-beta (TGF-beta). The expression of HO-1 and MTs was measured by real time PCR and, in the case of HO-1, by immunoblotting and immunofluorescence microscopy. Specific inhibitors were used to investigate possible signaling pathways. The effect of vitreous on activation of AP-1 transcription factor was determined by immunoblotting, electrophoretic mobility shift assays, or immunofluorescence microscopy.


RESULTS
Incubation of RPE cells with vitreous resulted in increased expression of HO-1, MT-1a and MT-2a. TGF-beta caused an increase in HO-1 expression, although not to the extent mediated by vitreous, but had little effect on MT expression. Addition of inhibitors of TGF-beta signaling (SB431542 or TGF-beta-neutralizing antibodies) decreased the vitreous induction of HO-1. Several reactive oxygen species (ROS) quenchers inhibited the TGF-beta-induced or vitreous-induced elevation of HO-1 mRNA but had no effect on vitreous-mediated induction of MT expression. Inhibitors of the mitogen-activated protein kinase (p38MAPK; SB203580) and Jun N-terminal kinase (JNK; SP600125) pathways inhibited vitreous-induction of HO-1. C-fos, a component of AP-1 transcription factor complexes, exhibited increased expression and activation in the presence of vitreous.


CONCLUSIONS
TGF-beta, a known component of vitreous, can account for some but not all of the regulation of the anti-oxidant, anti-inflammatory HO-1 gene in human RPE cells, but it does not participate in the vitreous-mediated upregulation of MTs. Both vitreous and TGF-beta signals increased HO-1 expression via ROS but the latter were not involved in vitreous-mediated MT expression. Increased p38, JNK, and c-fos activation may be implicated in vitreous modulation of HO-1.
Hartung
Ren
R
Department of Pathology and Microbiology, University of South Carolina School of Medicine, Columbia, SC 29208, USA. rhartung@med.sc.edu <rhartung@med.sc.edu>
Parapuram
Sunil K
SK
Ganti
Ramapriya
R
Hunt
D Margaret
DM
Chalam
Kakarla V
KV
Hunt
Richard C
RC
D030201:Activin Receptors, Type I / Q000037:antagonists & inhibitors; D001549:Benzamides / Q000494:pharmacology; D001692:Biological Transport / Q000502:physiology; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D051547:Heme Oxygenase-1 / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D000077293:Receptor, Transforming Growth Factor-beta Type I; D018125:Receptors, Transforming Growth Factor beta / Q000037:antagonists & inhibitors; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000502:physiology*; D014822:Vitreous Body / Q000166:cytology / Q000502:physiology*
C459179:4-(5-benzo(1,3)dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)benzamide; D001549:Benzamides; D004149:Dioxoles; D020033:Protein Isoforms; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D018125:Receptors, Transforming Growth Factor beta; D018808:Transcription Factor AP-1; D016212:Transforming Growth Factor beta; D008668:Metallothionein; D051547:Heme Oxygenase-1; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases; D030201:Activin Receptors, Type I; D000077293:Receptor, Transforming Growth Factor-beta Type I