Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor- superfamily. Mouse embryos lacking either Cripto or Nodal have the same lethal phenotype at embryonic day 7.5. Previous studies suggest that O-fucosylation of the epidermal growth factor-like (EGF) repeat in Cripto is essential for the facilitation of Nodal signaling. Substitution of Ala for the Thr to which O-fucose is attached led to functional inactivation of both human and mouse Cripto. However, embryos null for protein O-fucosyltransferase 1, the enzyme that adds O-fucose to EGF repeats, do not exhibit a Cripto null phenotype and die at about embryonic day 9.5. This suggested that the loss of O-fucose from the EGF repeat may not have led to the inactivation of Cripto in previous studies. Here we investigate this hypothesis and show the following: 1) protein O-fucosyltransferase 1 is indeed the enzyme that adds O-fucose to Cripto; 2) Pofut1 ؊/؊ embryonic stem cells behave the same as Pofut1 ؉/؉ embryonic stem cells in a Nodal signaling assay; 3) Pofut1 ؊/؊ and Pofut1 ؉/؉ embryoid bodies are indistinguishable in their ability to differentiate into cardiomyocytes; and 4) none of 10 amino acid substitutions at Thr 72 , including Ser which acquires O-fucose, rescues the activity of mouse Cripto in Nodal signaling assays. Therefore, the Thr to which O-fucose is linked in Cripto plays a key functional role, but O-fucose at Thr 72 is not required for Cripto to function in cell-based signaling assays or in vivo. By contrast, we show that O-fucose, and not the Thr to which it is attached, is required in the ligand-binding domain of Notch1 for Notch1 signaling.Nodal, a member of the TGF- 4 superfamily, plays essential roles in the embryonic development of vertebrates, including mesoderm formation and the generation of left-right asymmetry (1). The major components of the Nodal-signaling pathway are the soluble ligand Nodal, activin membrane receptors (ActRIIB and ALK4) to which Nodal binds, Smad2 and Smad4 signal-transducing molecules, and the transcription factor Fast1 (FoxH1). In addition, Cripto, a membrane glycoprotein with a glycosylphosphatidylinositol anchor, is an essential coreceptor for Nodal and is required for Nodal signaling. Cripto contains the following two functional domains that play distinct roles in Nodal signaling: a truncated epidermal growth factor-like (EGF) repeat and the CFC domain (2). Cripto and Nodal null mouse embryos exhibit extremely similar phenotypes, both lacking embryonic mesoderm and definitive endoderm (2-5). Cripto is also a co-receptor for GDF1/Vg1, another member of the TGF- superfamily (6). In addition, mouse Gdf3 (growth-differentiation factor 3) is similar to Nodal in requiring Cripto for its signaling activity (7). More recently, other factors were found to regulate Nodal signaling via interaction with Cripto, including activin (8), Lefty (9, 10), and Tomoregulin-1(TMEFF1) (11). In addition, Cripto has been shown to activate the Ras/Raf/MAPK pathway (12-18) and the phosphatidylin...
Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1-and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.
BackgroundNotch signaling is highly conserved in the metazoa and is critical for many cell fate decisions. Notch activation occurs following ligand binding to Notch extracellular domain. In vitro binding assays have identified epidermal growth factor (EGF) repeats 11 and 12 as the ligand binding domain of Drosophila Notch. Here we show that an internal deletion in mouse Notch1 of EGF repeats 8–12, including the putative ligand binding domain (lbd), is an inactivating mutation in vivo. We also show that maternal and zygotic Notch1lbd/lbd mutant embryos develop through gastrulation to mid-gestation.ResultsNotch1lbd/lbd embryos died at mid-gestation with a phenotype indistinguishable from Notch1 null mutants. In embryonic stem (ES) cells, Notch1lbd was expressed on the cell surface at levels equivalent to wild type Notch1, but Delta1 binding was reduced to the same level as in Notch1 null cells. In an ES cell co-culture assay, Notch signaling induced by Jagged1 or Delta1 was reduced to a similar level in Notch1lbdand Notch1 null cells. However, the Notch1lbd/lbd allele was expressed similarly to wild type Notch1 in Notch1lbd/lbd ES cells and embryos at E8.75, indicating that Notch1 signaling is not essential for the Notch1 gene to be expressed. In addition, maternal and zygotic Notch1 mutant blastocysts developed through gastrulation.ConclusionMouse Notch1 lacking the ligand binding domain is expressed at the cell surface but does not signal in response to the canonical Notch ligands Delta1 and Jagged1. Homozygous Notch1lbd/lbd mutant embryos die at ~E10 similar to Notch1 null embryos. While Notch1 is expressed in oocytes and blastocysts, Notch1 signaling via canonical ligands is dispensable during oogenesis, blastogenesis, implantation and gastrulation.
In a phase II study, dorzagliatin, a novel glucokinase activator (GKA), demonstrated significant HbA1c reduction and safety profile in Chinese T2D patients. Two hundred fifty eight (258) T2D patients were randomly assigned to 5 groups (50mg BID, 75mg BID, 75mg QD, 100mg QD and placebo) for a 12 weeks treatment. Compared with placebo group, HbA1c changes from baseline was significantly lower in the 100mgQD (0.69%, P<0.05), 50mgBID (0.79%, P<0.01) and 75mgBID (1.12%, P<0.001) groups. Post-hoc analyses of the phase II results were conducted to investigate the effects of dorzagliatin on the composite response rate (defined as reaching HbA1c<7.0%, no weight gain and no hypoglycemia), efficacy response β cell functions (HOMA-IR and Disposition Index), and efficacy response in stratified subgroups. Post-hoc analysis showed that the composite response rate was 28.3% (P < 0·05) and 35.4% (P < 0·001), respectively for 50mgBID and 75mgBID groups. It was significantly higher than the placebo group. HOMA-IR and Disposition Index was improved at Week 12 for the 75 mg BID group compared with placebo. Improvement of HOMA-IR was maintained at Week 13, one week after treatment discontinuation. Subjects were stratified into subgroups according to their medical history, disease history, gender, age and BMI. In drug naïve subjects, HbA1c change from baseline was -0.85% in 75mgQD (P<0.05), -0.95% in 100mgQD (P<0.01), -1.04% in 50mgBID (P<0.01), -1.21% in 75mgBID (P<0.001), and -0.17% in placebo group. Significant HbA1c reduction was also observed in 75mgBID (-0.97%, P<0.05) in the subjects who were previously treated with other antidiabetic drugs. No significant trends were observed on HbA1c in subgroups stratified by disease history, gender, age and BMI. The post-hoc analysis demonstrated dorzagliatin provided a comprehensive disease management in T2D patients with benefits of β cell function and insulin resistance improvement in the early stage T2D patients. Disclosure D. Zhu: None. Y. Zhao: None. C. Tang: None. H. Tianxin: None. Y. Li: None. G. Zhao: None. X. Hou: None. Y. Zhang: None. L. Chen: None.
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