N. Kawelke

Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function

The bone matrix is composed mostly of collagen, but the initial and continuous presence of fibronectin was found to be crucial for collagen matrix integrity in vitro. It has been assumed that osteoblasts produce the fibronectin required for bone matrix formation. Using transgenic mice, we conditionally deleted fibronectin in the osteoblasts and in the liver using the cre‐loxP system. We also used mice with mutated fibronectin and conditionally deleted β1‐integrin in osteoblasts to identify the receptor involved in fibronectin effects on osteoblasts. Conditional deletion of fibronectin in the differentiating osteoblasts [using the 2.3 kb collagen‐α1(I) promoter] failed to show a decrease in fibronectin amount in the bone matrix despite evidence of successful deletion. Using these mice we established that osteoblast‐derived fibronectin solely affects osteoblast function. This effect was not mediated by integrins that bind to the RGD motif. Conditional deletion of fibronectin in the liver showed a marked decrease in fibronectin content in the matrix associated with decreased mineral‐to‐matrix ratio and changed biomechanical properties but had no effect on osteoblasts or osteoclasts. In conclusion, osteoblast fibronectin affects osteoblasts function. This does not seem to be mediated by the RGD motif on fibronectin. In contrast, liver‐derived fibronectin affects bone matrix properties without affecting osteoblast or osteoclast function. A novel role for liver‐derived circulating fibronectin thus was defined and delineated from that of locally produced fibronectin.

Fibronectin protects from excessive liver fibrosis by modulating the availability of and responsiveness of stellate cells to active TGF-β.

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-β, and increased TGF-β-mediated signaling. In vitro experiments revealed that collagen-type-I production by fibronectin-deficient hepatic stellate cells stimulated with TGF-β was more pronounced, and was associated with augmented Smad3-mediated signaling. Interfering with TGF-β 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-β in fibronectin-deficient stellate cells and normalized collagen-type-I production in response to TGF-β stimulation. Thus, excessive stellate cell activation and production of collagen results from increased active TGF-β and TGF-β signaling in the absence of fibronectin. In conclusion, our data indicate that fibronectin controls the availability of active TGF-β 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-β-mediated response.

Inhibition of fibronectin deposition improves experimental liver fibrosis

BACKGROUND & AIMS Common pathogenic steps in liver fibrosis are inflammation and accumulation of extracellular matrix proteins including collagen, which lead to disruption of tissue microarchitecture and liver dysfunction. Adequate fibronectin fibril formation is required for collagen matrix deposition in several cell types in vitro. We therefore hypothesized that preventing fibronectin fibril assembly will result in decreased collagen matrix accumulation, and hence diminish liver injury associated with fibrosis. METHODS In vitro studies on hepatic stellate cells and in vivo studies in mice were performed. RESULTS In vitro studies on hepatic stellate cells confirmed that a fibronectin assembly inhibitor, pUR4 diminishes the amount of both fibronectin and collagen, accumulating in the extracellular matrix, without affecting their production. Induction of fibrosis using CCl4 or DMN was therefore combined with pUR4-treatment. pUR4 normalized the amount of fibrotic tissue that accumulated with injury, and improved liver function. Specifically, pUR4-treatment decreased collagen accumulation, without changing its mRNA expression. Most interestingly, we did not detect any changes in Kupffer cell numbers (F4/80+) or α-smooth muscle actin expressing hepatic stellate cell numbers. Further, there was no impact on TGF-β or TNF-α. Thus, in line with the in vitro findings, decreased fibrosis is due to inhibition of matrix accumulation and not a direct effect on these cells. CONCLUSIONS In summary, a peptide that blocks fibronectin deposition results in decreased collagen accumulation and improved liver function during liver fibrogenesis. Thus, fibronectin matrix modulation offers a therapeutic benefit in preclinical models of liver fibrosis.

Isoform of fibronectin mediates bone loss in patients with primary biliary cirrhosis by suppressing bone formation.

Osteoporosis is a major cause of morbidity and decreased quality of life in patients with chronic cholestatic liver disease. It is established that this osteoporosis results from decreased bone formation, but the mechanisms for the interaction between liver and bone remain elusive. The aim of this study was to test the hypothesis that an increase in the production of cellular fibronectins during liver disease may result in decreased osteoblast‐mediated mineralization and thus explain the decrease in bone formation. We performed a prospective cross‐sectional study in patients with primary biliary cirrhosis and matched controls, followed by experiments on human and mouse osteoblasts in culture and injections in mice in vivo. In patients with primary biliary cirrhosis, the oncofetal domain of fibronectin correlated significantly with the decrease in osteocalcin, a marker of bone formation (r = −0.57, p < 0.05). In vitro, amniotic fluid fibronectin (aFN) containing mainly the oncofetal domain and EIIIA domain resulted in decreased osteoblast‐mediated mineralization in human osteoblasts (69% decrease at 100 μg/ml; p < 0.01) and mouse osteoblasts (71% decrease; p < 0.05). Removing the EIIIA domain from aFN similarly suppressed mineralization by osteoblasts (78% decrease; p < 0.05). Injection of labeled aFN in mice showed that it infiltrates the bone, and its administration over 10 days resulted in decreased trabecular BMD (17% drop; p < 0.05), mineralizing surface (30% drop; p < 0.005), and number of osteoblasts (45% drop; p < 0.05). Increased production of a fibronectin isoform containing the oncofetal domain and its release in the circulation in patients with primary biliary cirrhosis is at least partially responsible for the decrease in bone formation seen in these patients. This establishes that a molecule that has thus far been viewed as an extracellular matrix protein exerts hormone‐like actions.