ABSTRACT FINAL ID: 1700
TITLE: Ganglioside GD3 acetylation contributes to hepatic stellate cell activation
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Hepatic stellate cells (HSC) activation to myofibroblast like phenotype is a crucial step in liver fibrogenesis. HSC activation cause extracellular matrix remodeling reflected in the accumulation of specific types of collagen. Active HSC are refractory to apoptosis. Ganglioside GD3 is a glycosphingolipid synthesized from GM3 by GD3 synthase that induces apoptosis by a dual mechanism involving mitochondrial targeting, eliciting mitochondrial membrane permeability transition, and inactivation of NF-κB-dependent survival program. Recent findings have shown that GD3 acetylation by O-acetyl disialoganglioside synthase (OAcGD3S), which acetylates GD3 to 9- O-acetyl GD3, antagonizes the proapoptotic actions of ganglioside GD3. It has been reported that human cirrhotic liver exhibit increased expression of OAcGD3S. Therefore, the aim of the study was to investigate the status of GD3 acetylation during HSC activation, the susceptibility of HSC to GD3-mediated cell death and the role of OAcGD3S in HSC biology and activation.
Eight weeks old male C57BL/6J mice were fed a 60% high fat diet enriched in 0.5% with cholesterol (HFHC) from 1 week to three months. HSC from control and HFHC-fed mice were analyzed for a-SMA, Col1A1 TGFb, PDGF, GD3 levels and OAcGD3S expression. HSC apoptosis was examined by TUNEL and caspase 3 activity. The impact of OAcGD3S silencing was examined in HSC activation. GD3 immnuhistochemistry analyses and OAcGD3S expression was monitored in human biopsies from subjects with chronic liver disease.
Freshly isolated HSC from HFHC-fed mice exhibited increased expression of GD3 synthase and OAcGD3S compared to HSC prepared from mice fed chow diet. In vitro HSC activation (1-10 days) further enhanced the expression of both enzymes, but the levels were still higher in HSC isolated from HFHC-fed mice. Quiescent HSC were markedly sensitive to GD3-induced TUNEL and caspase 3 activity, and this susceptibility decreased in active HSC, correlating with the time-dependent appearance of 9-O-acetyl GD3. This event paralleled the activation of HSC with increased expression of α-SMA, Col1A1, TGFβ and PDGF. Similar effects were observed in human LX2 cells, in which increased 9-O-acetyl GD3 levels correlating with enhanced profibrotic phenotype. OAcGD3S silencing in HSC increased ganglioside GD3 levels and ameliorated HSC transdifferentiation decreasing α-SMA, Col1A1, TGFβ and PDGF levels. Finally, liver samples from patients with fibrosis exhibited increased OAcGD3S and acetylated GD3.
Targeting of OAcGD3S could be a new therapeutic target for the treatment of liver fibrosis by regulating the apoptotic potential of ganglioside GD3.