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.

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.

Age-Related Changes of 14-3-3 Isoforms in Midbrain of A53T-SNCA Overexpressing Mice.

BACKGROUND: Parkinson's disease (PD) is characterized by loss of midbrain dopaminergic neurons, which are affected by cytoplasmic inclusions, named Lewy pathology. The main component is alpha-synuclein (SNCA), a protein modulating SNARE-complex dependent neurotransmission. SNCA mutations trigger dominantly inherited PD variants and sporadic cases of PD via aggregation and transmission. SNCA and isoforms of the 14-3-3 family show sequence homology, protein interaction and joint aggregation, so 14-3-3 s may be key molecules of pathogenesis. OBJECTIVE: We aimed to identify the relevant isoforms in midbrain and to distinguish for the first time the changes that occur very early versus those that progress with pathology. METHODS: We assessed expression of the 14-3-3 family with quantitative RT-PCR and immunoblots of differential solubility fractions in mice with A53T-SNCA overexpression longitudinally at different ages. RESULTS: Transcript levels showed reductions at age 3 months with increases at later ages for the beta, eta and zeta isoforms. Protein levels at age 3 months exhibited a concordant reduction only for beta, while increased insolubility was observed for epsilon and zeta. At age 18 months only the reduction of 14-3-3 beta protein remained significant. Thus, the toxic gain-of-function of alpha-synuclein leads to early transitory alterations of several 14-3-3 isoforms. When the levels of soluble 14-3-3 proteins become apparently normal during later life, increasing amounts of beta, eta and zeta mRNA are produced, possibly to compensate for protein insolubility and aggregation in a SNCA/14-3-3 complex. CONCLUSIONS: These data may contribute to identify key molecular events that reflect Parkinson's disease risk and progression.

Complexin-1 and Foxp1 Expression Changes Are Novel Brain Effects of Alpha-Synuclein Pathology.

As the second most frequent neurodegenerative disorder of the aging population, Parkinson's disease (PD) is characterized by progressive deficits in spontaneous movement, atrophy of dopaminergic midbrain neurons and aggregation of the protein alpha-synuclein (SNCA). To elucidate molecular events before irreversible cell death, we studied synucleinopathy-induced expression changes in mouse brain and identified 49 midbrain/brainstem-specific transcriptional dysregulations. In particular complexin-1 (Cplx1), Rabl2a and 14-3-3epsilon (Ywhae) downregulation, as well as upregulation of the midbrain-specific factor forkhead box P1 (Foxp1) and of Rabgef1, were interesting as early mRNA level effects of alpha-synuclein triggered pathology. The protein levels of complexin-1 were elevated in midbrain/brainstem tissue of mice with A53T-SNCA overexpression and of mice with SNCA-knockout. The response of CPLX1 and Foxp1 levels to SNCA deficiency supports the notion that these factors are regulated by altered physiological function of alpha-synuclein. Thus, their analysis might be useful in PD stages before the advent of Lewy pathology. Because both alpha-synuclein and complexin-1 modulate vesicle release, our findings support presynaptic dysfunction as an early event in PD pathology.