The Rho GTPase RAC1 in Osteoblasts Controls Their Function.

The regulation of the differentiation of the bone-forming cells, the osteoblasts, is complex. Many signaling pathways converge on the master regulator of osteoblast differentiation Runx2. The role of molecules that integrate several signaling pathways such as the Rho GTPases need to be better understood. We, therefore, asked at which stage Rac1, one of the Rho GTPase, is needed for osteoblast differentiation and whether it is involved in two pathways, the anabolic response to parathyroid hormone and the stimulatory effect of fibronectin isoforms on integrins. Genetic deletion of Rac1 in preosteoblasts using the osterix promoter diminished osteoblast differentiation in vitro. This effect was however similar to the presence of the promoter by itself. We, therefore, applied a Rac1 inhibitor and confirmed a decrease in differentiation. In vivo, Rac1 deletion using the osterix promoter decreased bone mineral density as well as histomorphometric measures of osteoblast function. In contrast, deleting Rac1 in differentiating osteoblasts using the collagen α1(I) promoter had no effects. We then evaluated whether intermittent parathyroid hormone (PTH) was able to affect bone mineral density in the absence of Rac1 in preosteoblasts. The increase in bone mineral density was similar in control animals and in mice in which Rac1 was deleted using the osterix promoter. Furthermore, stimulation of integrin by integrin isoforms was able to enhance osteoblast differentiation, despite the deletion of Rac1. In summary, Rac1 in preosteoblasts is required for normal osteoblast function and bone density, but it is neither needed for PTH-mediated anabolic effects nor for integrin-mediated enhancement of differentiation.

EDA-Fibronectin Originating from Osteoblasts Inhibits the Immune Response against Cancer.

Osteoblasts lining the inner surface of bone support hematopoietic stem cell differentiation by virtue of proximity to the bone marrow. The osteoblasts also modify their own differentiation by producing various isoforms of fibronectin (FN). Despite evidence for immune regulation by osteoblasts, there is limited knowledge of how osteoblasts modulate cells of the immune system. Here, we show that extra domain A (EDA)-FN produced by osteoblasts increases arginase production in myeloid-derived cells, and we identify α5ß1 as the mediating receptor. In different mouse models of cancer, osteoblasts or EDA-FN was found to up-regulate arginase-1 expression in myeloid-derived cells, resulting in increased cancer growth. This harmful effect can be reduced by interfering with the integrin α5ß1 receptor or inhibiting arginase. Conversely, in tissue injury, the expression of arginase-1 is normally beneficial as it dampens the immune response to allow wound healing. We show that EDA-FN protects against excessive fibrotic tissue formation in a liver fibrosis model. Our results establish an immune regulatory function for EDA-FN originating from the osteoblasts and identify new avenues for enhancing the immune reaction against cancer.

Fibronectins containing extradomain A or B enhance osteoblast differentiation via distinct integrins.

Fibronectin is a multidomain protein secreted by various cell types. It forms a network of fibers within the extracellular matrix and impacts intracellular processes by binding to various molecules, primarily integrin receptors on the cells. Both the presence of several isoforms and the ability of the various domains and isoforms to bind to a variety of integrins result in a wide range of effects. In vivo findings suggest that fibronectin isoforms produced by the osteoblasts enhance their differentiation. Here we report that the isoform characterized by the presence of extradomain A activates α4ß1 integrin and augments osteoblast differentiation. In addition, the isoform containing extradomain B enhances the binding of fibronectin through the RGD sequence to ß3-containing integrin, resulting in increased mineralization by and differentiation of osteoblasts. Our study thus reveals novel functions for two fibronectin isoforms and the mediating receptors in osteoblast differentiation.

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-ß 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.

Mutations in LTBP4 cause a syndrome of impaired pulmonary, gastrointestinal, genitourinary, musculoskeletal, and dermal development.

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.

Regulation of TGF-ß storage and activation in the human idiopathic pulmonary fibrosis lung.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of unknown cause. The pathogenesis of the disease is characterized by fibroblast accumulation and excessive transforming growth factor-ß (TGF-ß) activation. Although TGF-ß activation is a complex process involving various protein interactions, little is known of the specific routes of TGF-ß storage and activation in human lung. Here, we have systematically analyzed the expression of specific proteins involved in extracellular matrix targeting and activation of TGF-ß. Latent TGF-ß-binding protein (LTBP)-1 was found to be significantly upregulated in IPF patient lungs. LTBP-1 expression was especially high in the fibroblastic foci, in which P-Smad2 immunoreactivity, indicative of TGF-ß signaling activity, was less prominent. In cultured primary lung fibroblasts and epithelial cells, short-interfering-RNA-mediated downregulation of LTBP-1 resulted in either increased or decreased TGF-ß signaling activity, respectively, suggesting that LTBP-1-mediated TGF-ß activation is dependent on the cellular context in the lung. Furthermore, LTBP-1 was shown to colocalize with fibronectin, fibrillin-1 and fibrillin-2 proteins in the IPF lung. Fibrillin-2, a developmental gene expressed only in blood vessels in normal adult lung, was found specifically upregulated in IPF fibroblastic foci. The TGF-ß-activating integrin ß8 subunit was expressed at low levels in both control and IPF lungs. Alterations in extracellular matrix composition, such as high levels of the TGF-ß storage protein LTBP-1 and the re-appearance of fibrillin-2, probably modulate TGF-ß availability and activation in different pulmonary compartments in the fibrotic lung.

An O-Glycosylation of Fibronectin Mediates Hepatic Osteodystrophy Through α4ß1 Integrin.

Patients with cholestatic liver disease experience increased fracture risk. Higher circulating levels of a fibronectin isoform called oncofetal fibronectin (oFN) were detected in a subset of such patients. Administering this isoform to mice suppresses osteoblast differentiation and diminishes bone mineral density in vivo, suggesting it is responsible for bone loss in cholestatic liver disease. The aim of this study was to define the mechanism by which oFN affects osteoblast function and evaluate possible modifiers in experimental hepatic osteodystrophy. The fibronectin isoform oFN is characterized by the presence of various glycosylations. In line with this, adding oFN that underwent enzymatic O-deglycosylation to osteoblasts normalized nodule formation in vitro. Of three possible O-glycosylation sites in oFN, only a mutation at AA 33 of the variable region or binding of this glycosylated site with an antibody normalized osteoblast differentiation. Because the responsible site is located in the variable region of fibronectin, which binds to α4ß1 or α4ß7 integrins, these integrins were evaluated. We show that integrin α4ß1 mediates the inhibitory effect of oFN both in vitro as well as in vivo. In a hepatic osteodystrophy mouse model, we demonstrate that liver fibrosis is associated with increased circulating oFN and diminished BMD. In addition, trabecular bone loss induced by oFN injection or fibrosis induction could be prevented by either administering an antibody that binds to α4 integrin (PS/2) or the CS1 peptide, which contains a binding site for α4ß1 integrin. In summary, oFN inhibits osteoblast activity. This is because of an O-glycosylation in the variable region that results in decreased integrin-mediated signaling. This deleterious effect can be thwarted by binding α4ß1 integrin. Thus, we have characterized the defect and the receptor mediating bone loss in patients with hepatic osteodystrophy and evaluated possible therapeutic interventions in a murine model. © 2016 American Society for Bone and Mineral Research.

Identification and characterization of a unique role for EDB fibronectin in phagocytosis.

UNLABELLED: Plasma fibronectin is a circulating protein that facilitates phagocytosis by connecting bacteria to immune cells. A fibronectin isoform, which includes a sequence of 90 AA called extra-domain B (EDB), is synthesized de novo at the messenger RNA (mRNA) level in immune cells, but the reason for its expression remains elusive. We detected an 80-fold increase in EDB-containing fibronectin in the cerebrospinal fluid of patients with bacterial meningitis that was most pronounced in staphylococcal infections. A role for this isoform in phagocytosis was further suggested by enhanced EDB fibronectin release after internalization of Staphylococcus aureus in vitro. Using transgenic mouse models, we established that immune cell production of fibronectin contributes to phagocytosis, more so than circulating plasma fibronectin, and that accentuated release of EDB-containing fibronectin by immune cells improved phagocytosis. In line with this, administration of EDB fibronectin enhanced in vitro phagocytosis to a larger extent than plasma fibronectin. This enhancement was mediated by αvß3 integrin as shown using inhibitors or cells from ß3 integrin knockout mice. Thus, we identified both a novel function for EDB fibronectin in augmenting phagocytosis over circulating plasma fibronectin, as well as the mediating receptor. Our data also establish for the first time, a direct role for ß3 integrin in bacterial phagocytosis in mammals. KEY MESSAGES: • Fibronectin containing an extra domain called EDB is released in bacterial meningitis. • EDB-containing fibronectin enhances phagocytosis more than plasma fibronectin. • The enhancement is mediated by activation of αvß3 integrin in the presence of EDB.

Circulating fibronectin controls tumor growth.

Fibronectin is ubiquitously expressed in the extracellular matrix, and experimental evidence has shown that it modulates blood vessel formation. The relative contribution of local and circulating fibronectin to blood vessel formation in vivo remains unknown despite evidence for unexpected roles of circulating fibronectin in various diseases. Using transgenic mouse models, we established that circulating fibronectin facilitates the growth of bone metastases by enhancing blood vessel formation and maturation. This effect is more relevant than that of fibronectin produced by endothelial cells and pericytes, which only exert a small additive effect on vessel maturation. Circulating fibronectin enhances its local production in tumors through a positive feedback loop and increases the amount of vascular endothelial growth factor (VEGF) retained in the matrix. Both fibronectin and VEGF then cooperate to stimulate blood vessel formation. Fibronectin content in the tumor correlates with the number of blood vessels and tumor growth in the mouse models. Consistent with these results, examination of three separate arrays from patients with breast and prostate cancers revealed that a high staining intensity for fibronectin in tumors is associated with increased mortality. These results establish that circulating fibronectin modulates blood vessel formation and tumor growth by modifying the amount of and the response to VEGF. Furthermore, determination of the fibronectin content can serve as a prognostic biomarker for breast and prostate cancers and possibly other cancers.

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

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

Mechanisms of emphysema in autosomal dominant cutis laxa.

Heterozygous elastin gene mutations cause autosomal dominant cutis laxa associated with emphysema and aortic aneurysms. To investigate the molecular mechanisms leading to cutis laxa in vivo, we generated transgenic mice by pronuclear injection of minigenes encoding normal human tropoelastin (WT) or tropoelastin with a cutis laxa mutation (CL). Three independent founder lines of CL mice showed emphysematous pulmonary airspace enlargement. No consistent dermatological or cardiovascular pathologies were observed. One CL and one WT line were selected for detailed studies. Both mutant and control transgenic animals showed elastin deposition into pulmonary elastic fibers, indicated by increased desmosine levels in the lung and by colocalization of transgenic and endogenous elastin by immunostaining. CL mice showed increased static lung compliance and decreased stiffness of lung tissue. In addition, markers of transforming growth factor-ß (TGFß) signaling and the unfolded protein response (UPR) were elevated together with increased apoptosis in the lungs of CL animals. We conclude that the synthesis of mutant elastin in CL activates multiple downstream disease pathways by triggering a UPR, altered mechanical signaling, increased release of TGFß and apoptosis. We propose that the combined effects of these processes lead to the development of an emphysematous pulmonary phenotype in CL.