Induction of amyloid β accumulation by ER calcium disruption and resultant upregulation of angiogenic factors in ARPE19 cells

PURPOSE. To investigate the intracellular mechanisms that induce amyloid beta (A beta) 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 A beta-precursor protein (APP) splice variants was investigated by reverse transcription (RT)-PCR. Cellular expressions of both a series of A beta 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 stauro-sporine 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 A beta production-inducing APP splice variants and reduced that of neprilysin, a catabolic enzyme for A beta. 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 A beta 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 A beta deposit-promoting factors but also angiogenic factors in the retinal pigment epithelium.