SERPINA1 levels dictate TTR expression in HepG2 cells

Question:

Transthyretin (TTR), synthesized mainly by liver, can cause amyloidosis due to deposition of wildtype TTR, mostly observed in the elderly, or of mutated TTR (ATTR). We recently employed induced pluripotent stem cell (iPSC)-derived hepatocyte-like cells (HLCs) and identified alpha-1-antitrypsin (SERPINA1) to be related to TTR amyloidosis [1]. Moreover, SerpinA1 was found to bind to TTR and could prevent TTR in vitro aggregation. Surprisingly, knockdown of TTR resulted in induction of SERPINA1, both at the mRNA and protein level. In this study we explored the impact of modulating SERPINA1 expression with respect to TTR.

Methods:

The human hepatoma cell line HepG2 was used for SERPINA1knockdown and overexpression. For SERPINA1 knockdown, HepG2 cells were seeded in 6-well plates and treated with 50 pmol Silencer® pre-designed siRNA. Scrambled siRNA was used as a control. To achieve SERPINA1 overexpression, HepG2 cells were treated with 10 – 100µM Oncostatin-M (OSM). mRNA levels of SERPINA1 and TTR were examined 24h after treatment using real-time RT-PCR. Gene expression of hepatic markers (e.g. ALB, TF) and chaperone genes (e.g. ATF6, HSPA5) were also assessed. SerpinA1 and TTR protein found in tissue culture supernatants were determined by ELISA.

Results:

siRNA treatment of cells resulted in a 2.7 ± 0.5-fold downregulation of SERPINA1 as compared to scrambled siRNA. Following SERPINA1 knockdown, TTR mRNA was upregulated by a factor of 2.2 ± 0.8. Similarly, SerpinA1 protein levels were downregulated by a factor of 4.6 ± 0.1, while TTR was induced by a factor of 3.0 ± 0.4. OSM treatment of HepG2 cells resulted in a dose-dependent up-regulation of SERPINA1 mRNA and a concomitantly downregulation of TTR. Gene expression was affected by OSM (100µM) at a fold change of 3.3 ± 1.5 and 2.0 ± 0.9 for SERPINA1 and TTR, respectively. In cell culture supernatants, SerpinA1 increased by 2.0 ± 0.7 and TTR decreased by a factor of 6.0 ± 0.1 after OSM treatment.

Conclusions:

Our data indicate that expression of SERPINA1 and TTR are tightly regulated in hepatic cells. We speculate that manipulation of SERPINA1 expression might represent a novel target of disease progression in TTR amyloidosis.

[1] C Niemietz, L Fleischhauer, V Sandfort, S Guttmann, A Zibert, H Schmidt. Hepatocyte-Like Cells Reveal Novel Role of SerpinA1 in Transthyretin Amyloidosis. J Cell Sci. 2018. doi: 10.1242/jcs.219824. [Epub ahead of print].