Induction of amyloid beta accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells.
OBJECTIVE
To investigate the intracellular mechanisms that induce amyloid beta (Abeta) accumulation and angiogenesis in the human retinal pigment epithelial cell line ARPE19.


METHODS
The authors used two endoplasmic reticulum (ER) stress-inducing reagents, thapsigargin (TG), which inhibits the sarcoplasmic/endoplasmic calcium (Ca)2+-ATPase, and tunicamycin (TM), which inhibits N-linked glycosylation. The expression pattern of Abeta-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of Abeta metabolism-related factors and angiogenic factors were evaluated by real-time RT-PCR and Western blot (VEGF). Expression of caspase-4 was examined by real-time RT-PCR and Western blot to evaluate the effect of the ER stressor. Intracellular Ca elevation by TG was evaluated by Ca2+ imaging experiments. Dimethyl sulfoxide and staurosporine were used as a nonreagent control and as an apoptosis-inducing reagent through mitochondria not ER, respectively.


RESULTS
TG-treated ARPE19 cells increased the mRNA expression of Abeta production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for Abeta. TG-treated ARPE19 cells produced increases in VEGF, TNF-alpha, TACE mRNA, and VEGF protein expressions and a decrease in PEDF mRNA expression. TG-treated ARPE19 cells induced the expression of active more than TM-treated casepase-4. The intracellular Ca concentration was elevated in only TG-treated ARPE19 cells.


CONCLUSIONS
TG-treated ARPE19 cells showed both Abeta accumulation-inducible and angiogenic factor mRNA expression patterns. This study suggests the possibility that ER stress through ER calcium disruption may induce the expression not only of Abeta deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.
Department of Anatomy and Neuroscience, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan.
Yoshihisa
Y
Koyama
Shinsuke
S
Matsuzaki
Fumi
F
Gomi
Kohei
K
Yamada
Taiichi
T
Katayama
Kohji
K
Sato
Tatsuro
T
Kumada
Atsuo
A
Fukuda
Satoshi
S
Matsuda
Yasuo
Y
Tano
Masaya
M
Tohyama
D051722:ADAM Proteins / Q000235:genetics; D000072198:ADAM17 Protein; D016229:Amyloid beta-Peptides / Q000096:biosynthesis* / Q000235:genetics; D016564:Amyloid beta-Protein Precursor; D042501:Angiogenic Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D053471:Caspases, Initiator / Q000235:genetics; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005136:Eye Proteins / Q000235:genetics; D006801:Humans; D009414:Nerve Growth Factors / Q000235:genetics; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015843:Serpins / Q000235:genetics; D019284:Thapsigargin / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D014415:Tunicamycin / Q000494:pharmacology*; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D000099174:Pigment Epithelium-Derived Factor
D051722:ADAM Proteins; D000072198:ADAM17 Protein; D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D042501:Angiogenic Proteins; D002118:Calcium; D053471:Caspases, Initiator; D004791:Enzyme Inhibitors; D005136:Eye Proteins; D009414:Nerve Growth Factors; D020033:Protein Isoforms; D012333:RNA, Messenger; D015843:Serpins; D019284:Thapsigargin; D014409:Tumor Necrosis Factor-alpha; D014415:Tunicamycin; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C467484:VEGFA protein, human; C093516:CASP4 protein, human; C000606462:ADAM17 protein, human