Liver cell type specific discrimination of TGF-beta signaling and outcome in vitro and in vivo

Introduction:

Transforming growth factor (TGF)-β plays an important role in the progression of chronic liver diseases by influencing a plethora of cellular processes such as matrix remodeling, proliferation and differentiation. TGF-β1 can activate hepatic stellate cells therewith promoting liver fibrosis, and also possesses cytostatic and apoptotic effects towards hepatocytes. We now aimed to analyze liver cell type-specific gene expression patterns upon stimulating HSCs and hepatocytes with TGF-β1 in vitro as well as in liver tissue after expression of active TGF-β1.

Methods:

Murine hepatocytes (AML-12) and human HSC (LX-2) were treated with TGF-β1 or its type I receptor (ALK5) inhibitor (LY2157299) for 24h followed by microarray analysis. The differentially expressed genes in each cell type were used for functional annotation analysis. In vivo, TGF-β1 signaling was induced in C57BL/6 mice via TGF-β1 adeno-associated virus injection and analyzed the effects within the entire liver and in several liver cell types upon cell isolation. Validation of TGF-β1 signaling activity was performed by qPCR, immunoblotting and IHC of p-SMAD2/3. Liver damage parameterswere tested from serum.

Results:

TGF-β1 and LY2157299 impacts more 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-β1 signaling, including known TGF-β1 target genes, such as Smad7, Skil, Fn1, and novel targets, e.g. Edn1, Sox4, and Il11. Pathway enrichment analysis also showed distinct responses in AML12 and LX-2 after TGF-β1 signaling modulation. Plasma of AAV-TGF-β1 infected mice displayed significantly elevated AST levels as compared to controls, whereas ALT and ALP were unchanged. In tissue, we identified AAV-TGF-b1 induced Smad phosphorylation and signaling, but no differences in expression of TGF-b target genes like Smad7, Pai1, and IL11 on RNA level were detected. For a more specific analysis, cells were isolated and we found that TGF-β1 is overexpressed in hepatocytes and Kupffer cells. Signaling is in majority triggered in hepatocytes and LSEC after AAV-TGF-β1 injection, as shown by increased phospho-Smad3 levels. However, besides in hepatocytes, Smad7 was also induced in Kupffer cells on RNA level, whereas this bona fide target gene was not induced in LSEC at this time point of analysis.

Conclusion:

TGF-β1 affects liver cell types differently, shows overlapping but partially distinct responses to stimulation in vitro. The ongoing in depth analyses will enable a novel understanding of the role and mechanisms of TGF-β1 in different disease contexts and clarify the genetic programs that determine cell type specific TGF-β1 phenotypes/fates in healthy and diseased liver.