Zeisberg M, Yang C, Martino M, Duncan MB, Rieder F, Tanjore H, et al. Fibroblasts derive from hepatocytes in liver fibrosis via epithelial to mesenchymal transition. J Biol Chem 2007;282:23337–23347. (Reprinted with permission.)
Activated fibroblasts are key contributors to the fibrotic extracellular matrix accumulation during liver fibrosis. The origin of such fibroblasts is still debated, although several studies point to stellate cells as the principal source. The role of adult hepatocytes as contributors to the accumulation of fibroblasts in the fibrotic liver is yet undetermined. Here, we provide evidence that the pro-fibrotic growth factor, TGF- 1, induces adult mouse hepatocytes to undergo phenotypic and functional changes typical of epithelial to mesenchymal transition (EMT). We perform lineage-tracing experiments using AlbCre.R26RstoplacZ double transgenic mice to demonstrate that hepatocytes that undergo EMT contribute substantially to the population of FSP1-positive fibroblasts in CCl4-induced liver fibrosis. Furthermore, we demonstrate that bone morphogenic protein-7 (BMP7), a member of the TGF superfamily, which is known to antagonize TGF signaling, significantly inhibits progression of liver fibrosis in these mice. BMP7 treatment abolishes EMT-derived fibroblasts, suggesting that the therapeutic effect of BMP7 was at least partially due to the inhibition of EMT. These results provide direct evidence for the functional involvement of adult hepatocytes in the accumulation of activated fibroblasts in the fibrotic liver. Furthermore, our findings suggest that EMT is a promising therapeutic target for the attenuation of liver fibrosis.
The extracellular matrix (ECM) composition in the space of Disse has a fundamental impact on liver function. When quiescent, the 3 cell types in the space of Disse, hepatocytes, hepatic stellate cells (HSCs) and endothelial cells, contribute to the ECM. HSCs primarily express collagen (CN) types III and IV and laminin, endothelial cells express CN-IV, and hepatocytes express fibronectin. In chronic injury, the levels of fibrillar collagen types I, III, and IV, but predominantly CN-I, increase and disrupt normal wound healing. As fibrosis progresses, abnormal ECM accumulates in the periportal and perisinusoidal spaces and compromises hepatocyte function. Activated hepatic myofibroblasts produce most of these fibrillar collagens, so the HSC is central to the current paradigm of understanding hepatic fibrosis.1
Stimulators of HSC activation and proliferation include transforming growth factor (TGF) β1, platelet-derived growth factor (PDGF), connective tissue growth factor, and fibroblast growth factor, whereas interleukin 10 and interferon γ are antifibrogenic. TGFβ1 is the dominant HSC-activating factor.1
Activated hepatic myofibroblasts are produced by activated HSC transdifferentiation. However, the heterogeneity of these fibroblasts suggests that they have additional origins.2 Proteins expressed by most of the activated HSCs in humans include glial fibrillary acidic protein, fibroblast activation protein, α-smooth muscle actin (α-SMA), neural cell adhesion molecule (NCAM), intercellular adhesion molecule-1 (ICAM-1), CN-I, synaptophysin, PDGF receptor, nerve growth factor, neurotropin 3, reelin, matrix metalloproteinases (MMPs) and their inhibitors, and tyrosine kinase receptors including TRK3. Alpha-SMA is most commonly used as a marker of activated HSCs and myofibroblasts. However, none of these markers specifically defines the entire population of activated HSCs and myofibroblasts. DNA microarray studies have revealed more than 800 genes differentially expressed between quiescent and activated HSCs, so this area is not completely understood. Zeisberg et al. have recently reported that fibroblast specific protein 1 (FSP1), also called S100A4, is a hepatic myofibroblast marker in experimental fibrosis.3 Interestingly, they observed FSP1 in only 10% of α-SMA–positive cells, but up to 60% of hepatic fibroblasts were FSP1-positive. However, the exact intrahepatic localization of these apparently distinct subpopulations was not identified.
The HSC paradigm of liver fibrosis was preceded by the view that hepatocytes, which form 80% of the liver mass, were the major ECM source. Interestingly, the thinking in kidney biology has remained focused on a predominant role of epithelial cells in tissue fibrosis. In kidney and lung biology, the epithelial cell is thought to transform into an ECM-producing myofibroblast.
The process of cellular plasticity in which epithelial cells become fibroblasts is called epithelial–mesenchymal transition (EMT). EMT is essential for organ morphogenesis in embryonic development and has been studied in pathological conditions, primarily tumor progression and kidney fibrosis. EMT involves gradual loss of epithelial phenotypic features including cell-cell adhesion, tight junctions, E-cadherin, zona occludins 1 (ZO-1) and cytokeratins, and acquisition of a fibroblastic phenotype including increased motility and expression of N-cadherin, vimentin, fibronectin, CN-I, Snail, Slug, Twist, MMP2, and MMP9 (Table 1).4
|Epithelial cell markers||Myofibroblast markers||Increased in myofibroblasts|
It is firmly established that EMT can be induced by TGFβ1 in kidney and mammary epithelial cells. The TGFβ1 molecule complexes with activin-like kinase receptor 5 (ALK5), which phosphorylates Smad2 and Smad3. These Smads, β-catenin, Snail, Slug, Twist, and T cell factor (TCF) translocate into the nucleus. The relevance of EMT to liver disease pathogenesis has been uncertain. However, a recent paper in HEPATOLOGY showed EMT in biliary epithelial cells (BECs) in a primary biliary cirrhosis transplant recipient by observing BECs expressing S100A4/FSP1, vimentin, and Smad2/3.5 Zeisberg and colleagues have now clearly demonstrated EMT in experimental fibrosis and in primary hepatocyte cultures.3 The clearest evidence of EMT was obtained using a genetic marker of cell lineage to irreversibly tag hepatocytes. The genetic tool was a double transgenic mouse (AlbCre.R26RstoplacZ double transgenic) expressing both Cre-recombinase under albumin promoter control and a lacZ gene that can be made functional by exposure to the Cre enzyme. The R26RstoplacZ mouse has a LoxP-flanked transcriptional stop cassette in a lacZ transgene. Cre-recombinase cuts at LoxP sites, so Cre-expressing cells irreversibly remove the stop cassette and thereby express LacZ. Having an albumin promoter for Cre confers hepatocyte specificity upon LacZ expression such that in the untreated double transgenic mice, only hepatocytes express LacZ.
In the double transgenic mice, most hepatocytes were LacZ-positive but, remarkably, following chronic CCl4 treatment, up to 45% of the FSP1-positive cells were positive for LacZ, indicating that about half those fibroblasts were hepatocyte-derived. FSP1/S100A4 is a fibroblast-specific cytoplasmic calcium-binding protein expressed by EMT-derived kidney and pulmonary fibroblasts.6 Furthermore, FSP1 is a driver of EMT: Kidney tubular epithelial cells undergo EMT following FSP1 overexpression.7 FSP1 expression is up-regulated when CArG box-binding factor-A (CBF-A) binds to a promoter of FSP1 transcription. Up-regulating CBF-A expression is sufficient to induce EMT in the kidney epithelial cell line MCT.8 In vitro, most hepatocytes underwent EMT in response to TGFβ1, gradually losing epithelial morphology and albumin expression and gaining motility and fibroblastic morphology. Both in vitro and in vivo, an intermediate albumin-positive, FSP1-positive cell stage was observed (Fig. 1).3
The other challenge that EMT brings to the HSC paradigm is in the mechanisms by which fibrosis resolves. The resolution phase following several weeks of CCl4 liver injury in rodents is thought to involve HSC apoptosis.9 However, EMT has an antiapoptotic outcome so it would be interesting to discover how EMT-derived fibroblasts dissipate and whether this involves reversing EMT by mesenchymal–epithelial transition (MET), as occurs in the kidney.10
A further intriguing result in the Zeisberg paper relates to a role for bone morphogenic protein 7 (BMP7) in liver fibrosis. BMP7, also called osteogenic protein 1, is a 35-kDa protein of the TGFβ1 superfamily that can be up-regulated in cirrhotic human liver.11 BMP7 is an antagonist of TGFβ1, binding to ALK3 and signaling via Smad1, Smad5, and Smad8. Exogenous BMP7 inhibits renal fibrosis.10 Recently, BMP7 has been shown to suppress CN-I and α-SMA expression in the LX-2 stellate cell line and suppress thioacetimide-induced experimental liver fibrosis.12 Zeisberg and colleagues showed that BMP7 treatment also suppresses fibrosis in the CCl4 model but this seemed to be via inhibition of EMT. These results imply an important role for BMP7 at multiple levels in the fibrotic process.
Recent knowledge discussed above suggests the universality of cell plasticity and mutes the concept of terminal differentiation. Potential therapies arising from this work include exploiting BMP7, endoglin,13 and the CBF-A signal. Although the HSC liver fibrosis paradigm has a firm basis, the roles of other cell types including the hepatocyte is being revisited. Emerging from this endeavor are indications that fibrosis is of greater cellular and molecular complexity than previously thought.