Bile salt-induced pro-fibrogenic proliferation of hepatic stellate cells is partially mediated by PI3K p110a signalling

Question Chronic cholestatic liver diseases frequently progress to liver fibrosis. To date, therapeutic options remain scarce. The hydrophobic bile salt Chenodeoxycholic acid (CDCA), accumulating in cholestasis, has been previously shown to stimulate proliferation of hepatic stellate cells (HSCs) and we recently characterised the pro-fibrogenic effects of such signalling (Cells 2020;9(2):281). Here, we test the role of Phosphatidyl-inositol-3-kinase dependent signalling on bile salt induced activation of HSCs, since this signalling cascade had been implicated in other causes of HSC activation.

Methods HSCs were derived from female FVB/N mice. Expression of catalytic PI3K subunits and activation of this signalling cascade were confirmed by Western Blotting. HSCs were incubated with CDCA (100 µM) in presence or absence of the PI3K isoform p110a specific inhibitor Alpelisib (up to 5 µM) for up to 14 days. DNA quantification was performed as a surrogate of cell number. In vitro collagen deposition was quantified photometrically. The human hepatic stellate cell line LX-2 was activated by TGF-b (10 ng/ml) in presence or absence of Alpelisib (0.5 – 25 µM). PI3K p110a protein expression in LX-2 cells was suppressed by siRNA, followed by 24 h incubation with TGF-b. LX-2 cell activation was evaluated by aSMA quantification.

Results PI3K p110a was found to be expressed in both human and murine HSCs as well as in LX-2 cells. An increase in PKB phosphorylation upon stimulation with CDCA indicated engagement of this signalling cascade. Following incubation with the PI3K inhibitor specific for p110a, Alpelisib, PKB phosphorylation and CDCA-induced increase in DNA amount were diminished. Furthermore, CDCA-induced collagen deposition decreased. Activation of LX-2 cells by TGF-b was reduced by Alpelisib, too. PI3K p110a protein expression was reduced after treatment with siRNA. Lack of p110a prevented TGF-b-induced activation of LX-2 cells.

Conclusion Our results indicate that the pro-fibrogenic properties of CDCA in murine HSCs may partially be mediated by PI3K p110a. In line with this observation, pro-fibrotic signalling in human LX-2 cells seems to depend on this signalling cascade, too. These results warrant further investigation of this signalling pathway in in vivo models of cholestasis and liver fibrosis.

Publikationsverlauf

Artikel online veröffentlicht:
04. Januar 2021