We report recessive mutations in the gene for the latent transforming growth factor-beta binding protein 4 (LTBP4) in four unrelated patients with a human syndrome disrupting pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development. All patients had severe respiratory distress, with cystic and atelectatic changes in the lungs complicated by tracheomalacia and diaphragmatic hernia. Three of the four patients died of respiratory failure. Cardiovascular lesions were mild, limited to pulmonary artery stenosis and patent foramen ovale. Gastrointestinal malformations included diverticulosis, enlargement, tortuosity, and stenosis at various levels of the intestinal tract. The urinary tract was affected by diverticulosis and hydronephrosis. Joint laxity and low muscle tone contributed to musculoskeletal problems compounded by postnatal growth delay. Craniofacial features included microretrognathia, flat midface, receding forehead, and wide fontanelles. All patients had cutis laxa. Four of the five identified LTBP4 mutations led to premature termination of translation and destabilization of the LTBP4 mRNA. Impaired synthesis and lack of deposition of LTBP4 into the extracellular matrix (ECM) caused increased transforming growth factor-beta (TGF-beta) activity in cultured fibroblasts and defective elastic fiber assembly in all tissues affected by the disease. These molecular defects were associated with blocked alveolarization and airway collapse in the lung. Our results show that coupling of TGF-beta signaling and ECM assembly is essential for proper development and is achieved in multiple human organ systems by multifunctional proteins such as LTBP4.

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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.