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.
