R-spondin (RSpo) proteins amplify Wnt signaling and stimulate regeneration in a variety of tissues. to repair tissue in a tissue-specific manner, tissue-targeted RSPO mimetic molecules are desired. Here, we mutated RSPO (RSPO2 F105R/F109A) to eliminate LGR binding while preserving ZNRF3/RNF43 binding and targeted the mutated RSPO to a liver specific receptor, ASGR1. The resulting bi-specific molecule (αASGR1-RSPO2-RA) enhanced Wnt signaling effectively in vitro, and its activity was limited to ASGR1 expressing cells. Systemic administration of αASGR1-RSPO2-RA in mice specifically upregulated Wnt target genes and stimulated cell proliferation in liver but not intestine (which is more responsive to non-targeted RSPO2) in healthy mice, and improved liver function in diseased mice. These results not only suggest that a tissue-specific RSPO mimetic protein can stimulate regeneration in a cell-specific manner, but also provide a blueprint of how a tissue-specific molecule might be constructed for applications in a broader context. The Wnt ("Wingless-related integration site" or "Wingless and Int-1" or "Wingless-Int") signaling pathway plays a key role in the development, homeostasis and regeneration of many essential organs and tissues 1. Timely activation, modulation, or enhancement of Wnt signaling holds potential for the treatment of various degenerative diseases and pathologies in which tissue regeneration could confer a therapeutic benefit. R-spondins 1-4 (RSPO1-4) are a family of ligands that amplify Wnt signals through a receptor complex containing the zinc and ring finger 3 (ZNRF3) and ring finger protein 43 (RNF43) proteins and the coreceptor leucine-rich repeat-containing G-protein coupled receptors 4-6 (LGR4-6) 2-4. RSPOs contain two furin (Fu) repeats, Fu1 and Fu2, which in combination are sufficient to recapitulate Wnt signaling enhancing activity 5. Fu1 primarily interacts with ZNRF3/RNF43 and Fu2 interacts with LGR4-6 5-7. ZNRF3 and RNF43 are membranebound E3 ligases that specifically target Wnt receptors (FZD1-10 and LRP5 or LRP6) for degradation 8,9. Binding of RSPOs to ZNRF3/RNF43 and LGR4-6 causes clearance or sequestration of the ternary complex, which stabilizes Wnt receptors and amplifies Wnt signaling. In addition, R-spondins might work through mechanisms that are independent of LGRs 10,11. RSPOs may be beneficial in adult tissue repair 12,13 , particularly in situations where the expression of endogenous Wnt ligands is upregulated but signaling (presumably limited by receptor stability) is insufficient to overcome tissue damage. In liver, RSPO function is important for metabolic zonation and for hepatocyte proliferation and regeneration 12,14. Therefore, RSPO may provide therapeutic benefit for various acute and chronic liver injury and diseases. One major challenge to exploring RSPO for tissue repair and regeneration is limiting RSPO effects to specific tissue of interest such as liver, as LGR4-6 and ZNRF3/RNF43 are widely expressed in various tissues. The mucosa of the gastrointestinal tract h...
ResolvinE1(RvE1), an endogenous lipid mediator derived from eicosapentaenoic acid (omega 3 fatty acids), contributes to the resolution of an allergic inflammatory response. We investigated the effects of RvE1 and Omega 3 fatty acids in airway reactivity and inflammation using an asthmatic murine model. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. RvE1 was administered intraperitoneally post‐allergen challenge while fish oil was administered via oral gavage once daily (day 1 through 13). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. RvE1attenuated airway responsiveness to methacholine (MCh; 48mg/ml) in treated mice (150±27.88% in SEN vs. 54±7.52% in SEN+RvE1, p<0.05). However, no difference was observed with either omega‐3 supplementation alone (115±19.28%in SEN+omega‐3) or additive effect of omega‐3 on RvE1 treatment (39±12.37% in SEN+RvE1+omega‐3 vs. 54±7.52% in SEN+RvE1). Differential BAL cell analysis showed that post‐treatment with RVE1 decreased eosinophils (40 ± 2.7% in SEN vs.10.2 ± 1.4% in SEN+RvE1, p<0.005). Omega‐3 treated SEN showed no difference (45 ± 3%) while SEN+RvE1+omega‐3 group had similar results as RvE1 treated mice (10.2 ± 1.4% in SEN+RvE1 vs.10.6 ± 1.4% in SEN+RvE1+omega‐3), suggesting that RvE1 only attenuated the eosinophilia. Our data suggests that omega‐3 supplementation has little effect on airway inflammation and reactivity. Further, omega‐3 fatty acid, although a precursor for resolvin formation, did not have additive effects on resolvin‐mediated decreases in airway inflammation and airway reactivity. Support or Funding Information Long Island University start‐up funds (DSP) This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Animal models of asthma have shown that limonene, a naturally occurring terpene in citrus fruits, can reduce inflammation and airway reactivity. However, the mechanism of these effects is unknown. We first performed computational and molecular docking analyses that showed limonene could bind to both A 2A and A 2B receptors. The pharmacological studies were carried out with A 2A adenosine receptor knock-out (A 2A KO) and wild-type (WT) mice using ovalbumin (OVA) to generate the asthma phenotype. We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) and NECA (nonselective adenosine analog) by administering limonene as an inhalation prior to OVA aerosol challenges in one group of allergic mice for both WT and KO. In whole-body plethysmography studies, we observed that airway responsiveness to MCh in WT SEN group was significantly lowered upon limonene treatment but no effect was observed in A 2A KO. Limonene also attenuated NECA-induced airway responsiveness in WT allergic mice with no effect being observed in A 2A KO groups. Differential BAL analysis showed that limonene reduced levels of eosinophils in allergic WT mice but not in A 2A KO. However, limonene reduced neutrophils in sensitized A 2A KO mice, suggesting that it may activate A 2B receptors as well. These data indicate that limonene-induced reduction in airway inflammation and airway reactivity occurs mainly via activation of A 2A AR but A 2B receptors may also play a supporting role.
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