Liver cell specific TGF-beta sensitivity and outcome

Introduction:

Transforming growth factor (TGF)-beta influences a plethora of cellular processes such as matrix remodeling, proliferation and differentiation, and is a key player in the progression of chronic liver diseases. TGF-beta activates hepatic stellate cells (HSC)-a profibrogenic liver cell type-and triggers senescence, apoptosis of hepatocytes. We aimed to analyze cell type-specific genomic alterations (1) upon stimulating HSCs and hepatocytes with TGF-beta in vitro and (2) upon inducing active TGF-beta1 levels in healthy and diseased mouse liver.

Methods:

Murine hepatocytes AML-12 and human HSC LX-2 were treated with TGF-beta1 or its type I receptor (ALK5) inhibitor (LY2157299) for 2 and 24h followed by microarray analysis. The differentially expressed genes in each cell type were used for functional annotation analysis. In vivo, we induced TGF-beta1 signaling in C57BL/6 mice using TGF-beta1 adeno-associated virus. Validation of TGF-beta1 signaling activation or inhibition was performed by qPCR, immunoblotting and IHC of p-SMAD2/3.

Results:

TGF-beta1 and LY2157299 had most profound impact on AML-12, leading to differential regulation of > 1,200 genes compared to ˜400 in LX-2. In both cell types, we identified ˜80 differentially regulated genes upon the induction or inhibition of TGF-beta1 signaling, including known TGF-beta1 target genes, such as Smad7, Skil, Fn1, and novel targets, e.g. Edn1, Sox4, and Il11. Pathway enrichment analysis showed that metabolic processes such as amino acid and fatty acid pathways were most profoundly affected by TGF-beta1 signaling modulation in AML12, whereas extracellular matrix interaction, focal adhesion and the TGF-beta pathway were most influenced in LX-2. AAV-TGF-beta1 triggers Smad phosphorylation and target gene expression in vivo. Further, in vivo stimulated hepatocytes show a distinct TGF-β gene expression pattern compared to in vitro cultured cells.

Conclusion:

Our analyses reveal that liver cell types show an overlapping but partially distinct response to TGF-beta1 stimulation. Further analyses will enable a novel understanding of the role of TGF-beta1 in different disease contexts and clarify the genetic programs that determine cell type specific TGF-beta1 phenotypes/fates in healthy and diseased liver.