Fibronectin Protects from Excessive Liver Fibrosis by Modulating the Availability of and Responsiveness of Stellate Cells to Active TGF-β

et al. (2011) Fibronectin Protects from Excessive Liver Fibrosis by Modulating the Availability of and Responsiveness of Stellate Cells to Active TGF-b. PLoS ONE 6(11): e28181. doi:10.1371/journal.pone.0028181 Fibronectin Protects from Excessive Liver Fibrosis by Modulating the Availability of and Responsiveness of Stellate Cells to Active TGF-b Nina Kawelke 0 Matthaeus Vasel 0 Carla Sens 0 Anja von Au 0 Steven Dooley 0 Inaam A. 0 Carol Feghali-Bostwick, University of Pittsburgh, United States of America 0 1 Translational Medicine, Max-Planck Institute for Biochemistry , Martinsried, Germany , 2 Institute for Immunology, University of Heidelberg , Heidelberg, Germany , 3 Department of Medicine II, University of Heidelberg at Mannheim , Mannheim , Germany Fibrotic tissue in the liver is mainly composed of collagen. Fibronectin, which is also present in fibrotic matrices, is required for collagen matrix assembly in vitro. It also modulates the amount of growth factors and their release from the matrix. We therefore examined the effects of the absence of fibronectin on the development of fibrosis in mice. Conditional deletion of fibronectin in the liver using the Mx promoter to drive cre expression resulted in increased collagen production and hence a more pronounced fibrosis in response to dimethylnitrosamine in mice. Exclusive deletion of fibronectin in hepatocytes or normalization of circulating fibronectin in Mx-cKO mice did not affect the development of fibrosis suggesting a role for fibronectin production by other liver cell types. The boosted fibrosis in fibronectin-deficient mice was associated with enhanced stellate cell activation and proliferation, elevated concentrations of active TGF-b, and increased TGF-b-mediated signaling. In vitro experiments revealed that collagen-type-I production by fibronectin-deficient hepatic stellate cells stimulated with TGF-b was more pronounced, and was associated with augmented Smad3-mediated signaling. Interfering with TGF-b signaling using SB431542 normalized collagen-type-I production in fibronectin-deficient hepatic stellate cells. Furthermore, precoating culture plates with fibronectin, but not collagen, or providing fibronectin fibrils unable to interact with RGD binding integrins via the RGD domain significantly diminished the amount of active TGF-b in fibronectin-deficient stellate cells and normalized collagentype-I production in response to TGF-b stimulation. Thus, excessive stellate cell activation and production of collagen results from increased active TGF-b and TGF-b signaling in the absence of fibronectin. In conclusion, our data indicate that fibronectin controls the availability of active TGF-b in the injured liver, which impacts the severity of the resulting fibrosis. We therefore propose a novel role for locally produced fibronectin in protecting the liver from an excessive TGF-b-mediated response. - Funding: This work was funded by the Max-Planck Society and the University of Heidelberg. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The development of liver fibrosis is one of the early steps in the pathogenesis of advanced liver disease and liver failure. Hepatic stellate cells play a key role in this process by producing extracellular matrix molecules that become incorporated in a distorted fibrogenous network hindering normal function in hepatocytes [1]. Transforming-growth-factor-b (TGF-b) has been described as a major stimulator of stellate cell activation, and hence, extracellular matrix production [2]. Fibronectin is one of the molecules produced by hepatic stellate cells [3]. It is also part of the extracellular matrix [4]. Several isoforms have been described and many functions have been attributed to fibronectin. Fibronectin is important for the assembly of a collagen matrix in vitro [5,6]. Its continuous presence also supports matrix integrity, both in vitro and in vivo [5,7]. It further regulates cell proliferation and cell cycle progression [8]. We have shown that two isoforms of fibronectin reflect the severity of liver fibrosis in patients with chronic hepatitis C raising the possibility that fibronectin itself may play a role in the pathogenesis of fibrosis [9]. In order to define the role of fibronectin in the liver on the development of liver fibrosis we undertook the following study in which fibronectin was conditionally deleted in various cell types in mice. Liver injury was induced using dimethylnitrosamine (DMN) and experiments on liver tissue from these mice were complemented with in vitro experiments in isolated stellate cells. Our results show that deletion of fibronectin leads to an increase in stellate cell activation, both at baseline and after stimulation with TGF-b. This is due to an increase in TGF-b bioavailability and results in a more pronounced fibrosis. Deletion of fibronectin in hepatic stellate cells, hepatocytes, Kupffer cells and endothelial cells Hepatic stellate cells were isolated from control mice (CT) and conditional knockout mice carrying the Mx promoter attached to cre recombinase (Mx-cKO). All mice were homozygote for the floxed fibronectin gene. The Mx promoter was activated in vivo in Mx-cKO mice and through the resulting expression of the attached cre enzyme the homozygote fibronectin floxed genes present in the cells were deleted (Figures 1A and 1B) [10]. 9762% of the isolated hepatic stellate cells stained for either desmin or glial fibrillary acidic protein (GFAP), both of which are markers used for identifying stellate cells [11] suggesting a high purity. Deletion of fibronectin was confirmed at the DNA (Figures 1A and 1B), mRNA (Figure 1C) and protein level (Figure 1D: Western blot; Figure 1E: ELISA of conditioned media) in cells and conditioned media. Thus, activation of the Mx promoter successfully deleted fibronectin in hepatic stellate cells. In addition to deleting fibronectin in stellate cells, the activated Mx promoter can efficiently delete fibronectin in hepatocytes, which are responsible for the production of circulating fibronectin. Therefore, loss of fibronectin in hepatocytes in mice that express Mx-cre and are homozygote for floxed fibronectin can be easily confirmed by measuring circulating plasma fibronectin, which in this case confirmed successful deletion (Figure 1F). Other cell types that can be affected by the activation of the Mx promoter are Kupffer and endothelial cells. The identity of isolated Kupffer cells was confirmed by staining with F4/80 and absence of staining with desmin or GFAP (data not shown). ELISA of conditioned media confirmed deletion of fibronectin to 14% of control values (Figure 1G). The identity of isolated endothelial cells was confirmed by positive staining with acetylated-LDL and absence of staining with F4/80, desmin and GFAP (Data not s (...truncated)