ABSTRACT
BACKGROUND: Canonical Wnt signaling is involved in a variety of biological processes including stem cell renewal and differentiation, embryonic development, and tissue regeneration. Previous studies reported the stage-specific roles of the Wnt signaling in heart development. Canonical Wnt signal activation by recombinant Wnt3a in the early phase of differentiation enhances the efficiency of myocardial cell production from pluripotent stem cells. However, the hydrophobicity of Wnt proteins results in high cost to produce the recombinant proteins and presents an obstacle to their preparation and application for therapeutics, cell therapy, or molecular analysis of Wnt signaling. METHODS: To solve this problem, we generated an inexpensive molecule-responsive differentiation-inducing chimeric antigen receptor (designated as diCAR) that can activate Wnt3a signaling. The extracellular domains of low-density-lipoprotein receptor-related protein 6 (LRP6) and frizzeled-8 (FZD8) were replaced with single-chain Fv of anti-fluorescein (FL) antibody, which can respond to FL-conjugated bovine serum albumin (BSA-FL) as a cognate ligand. We then analyzed the effect of this diCAR on Wnt signal activation and cardiomyocyte differentiation of mouse embryonic stem cells in response to BSA-FL treatment. RESULTS: Embryonic stem cell lines stably expressing this paired diCAR, named Wnt3a-diCAR, showed TCF/ß-catenin-dependent transactivation by BSA-FL in a dose-dependent manner. Treatment with either Wnt3a recombinant protein or BSA-FL in the early phase of differentiation revealed similar changes of global gene expressions and resulted in efficient myocardial cell differentiation. Furthermore, BSA-FL-mediated signal activation was not affected by a Wnt3a antagonist, Dkk1, suggesting that the signal transduction via Wnt3a-diCAR is independent of endogenous LRP6 or FZD8. CONCLUSION: We anticipate that Wnt3a-diCAR enables target-specific signal activation, and could be an economical and powerful tool for stem cell-based regeneration therapy.
ABSTRACT
We evaluated effects of local administration of selective inhibitors of group IVA phospholipase A(2) (GIVA PLA(2)) and cyclooxygenase (COX) on exogenous prostaglandin (PG) F(2alpha)-induced luteal regression in pseudopregnant rats. Intra-bursal treatment with a GIVA PLA(2) inhibitor AACOCF(3) just prior to PGF(2alpha) (30microg, subcutaneously) on day 6 of pseudopregnancy (PSP6) prevented a decline in circulating progesterone and inhibited TUNEL-positive reactions of steroidogenic cell. Its treatment on PSP9 failed to inhibit functional regression, but reduced significantly apoptosis of steroidogenic cells and vascular endothelial cells, and suppressed the infiltration of macrophages. A COX-2-selective inhibitor NS398 inhibited the decline of progesterone and apoptosis of steroidogenic cells on PSP6 but not on PSP9. A COX-1 inhibitor SC560 exerted insignificant anti-luteolytic effects. Overall data suggest that luteal GIVA PLA(2) plays multiple promoting roles in PGF(2alpha)-induced luteal regression at least partly by a COX-2 activity-related mechanism in pseudopregnant rats.
