Oligomeric TTR V30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B

Moreira, Luciana V.; Beirão, João M.; Beirão, Idalina; Costa, Paulo P.

http://hdl.handle.net/10400.18/3294

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2015 Moreira L - Oligomeric TTRV30M aggregates compromise cell viability, erythropoietin gene expression and promoter activity in the human hepatoma cell line Hep3B.pdf		393.65 KB	Adobe PDF	Download
2015 Moreira L - Oligomeric TTRV30M aggregates - supplementary material.pdf		42.5 KB	Adobe PDF	Download

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Abstract(s)

Familial amyloidotic polyneuropathy, ATTRV30M (p. TTRV50M) amyloidosis, is a neurodegenerative disease characterized by systemic extracellular amyloid deposition of a mutant transthyretin, TTR V30M. Anemia, with low erythropoietin (EPO) levels and spared kidney function, affects about 25% of symptomatic patients, suggesting a blockage of EPO-producing cells. Early non-fibrillar TTR aggregates are highly cytotoxic, inducing oxidative stress, the expression of apoptosis-related molecules and secretion of pro-inflammatory cytokines, factors capable of inhibiting EPO production. Low EPO levels in these patients are not related to renal amyloid deposition or the presence of circulating TTR V30M. However, the role of early non-fibrillar TTR aggregates remains unexplored. We used the EPO producing Hep3B human hepatoma cell line to study the effect of TTR oligomeric aggregates on EPO expression. Hep3B cells were incubated with soluble and oligomeric TTR V30M, and cell proliferation as well as caspase 3/7 activation was evaluated. Relative quantification of EPO mRNA transcripts was performed by real-time PCR. Significant reductions in cell viability (13 ± 7.3%) and activation of caspases 3/7 were seen after 24 h in the presence of oligomeric TTR V30M. Also, EPO expression was significantly reduced (50 ± 2.8%), in normoxic conditions. A reporter assay was constructed with a PCR fragment of the EPO promoter linked to the luciferase gene to evaluate the role of transcription factors targeting the promoter. A significant reduction of EPO promoter activity (53 ± 6.5%) was observed in transfected cells exposed to TTR oligomers. Our results show that oligomeric TTR V30M reduces EPO expression, at least in part through inhibition of promoter activity.

Keywords

Hereditary Amyloidosis ATTR Amyloidosis TTR V30M Erythropoietin Anemia Molecular Biology Molecular Medicine EPO expression EPO promoter Familial Amyloid Polyneuropathy Transthyretin Doenças Genéticas Determinantes da Saúde e da Doença