ALBERT

Description: In mucopolysaccharidosis-I (MPS-I), α-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2–FGFR1-HS interactions, resulting in defective FGF-2–induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS–cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Description: Determining how extracellular matrix (ECM) components influence cytokine-induced growth and differentiation of cells in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel treatment strategies. Bone morphogenetic proteins (BMPs) regulate the growth, differentiation and apoptosis of cells in the brain, bone, bone marrow and diverse tissues. ECM glycosaminoglycans (GAGs) such as heparan sulfate (HS) interact with and influence the biological activity of a number of proteins including BMPs. We examined if heparin, endogenous HS in malignant cells and the structurally abnormal HS accumulated in Hurler cells influence BMP signaling and activity. First we showed using real-time quantitative RT-PCR (qRT-PCR) that the BMP signaling pathway including BMPs 2–7, BMP and activin receptors and Smad-1 and -5 are expressed by SaOS-2 human osteosarcoma cells. Western immunoblotting showed that BMP-4 induced Smad-1 phosphorylation, activation and nuclear translocation. Optimal Smad-1 activation was achieved by 25 ng/ml BMP-4 at 30–60 min, and blocked by the extracellular BMP antagonist chordin. BMP-4 also induced a concentration-dependent increase in alkaline phosphatase activity, indicative of induction of osteogenic differentiation in these malignant cells. Soluble heparin directly inhibited BMP-4 induced Smad-1 phosphorylation, and also markedly augmented the inhibitory effect of chordin. Similar effects were seen with N-desulfated, N-re-acetylated heparin but to a lesser degree than with heparin, indicating that N-sulfation of glucosamine residues in heparin/HS contributes to the effect of GAGs on BMP signaling. Inhibition of sulfation of endogenous GAGs by sodium chlorate augmented BMP-4 mediated increase in alkaline phosphatase, suggesting that endogenous sulfated GAGs themselves block BMP-4 mediated malignant cell differentiation. Because BMPs play a critical role in neurogenesis and osteogenesis, we also examined if GAGs that accumulate in Hurler syndrome impair BMP-4 signaling. Neurological dysfunction and skeletal abnormalities are among the most devastating manifestations of Hurler syndrome, an inborn metabolic disorder due to lack of lysosomal GAG-degrading α-L-iduronidase (IDUA) enzyme that leads to HS and dermatan sulfate GAG accumulation. We recently showed that HS in Hurler syndrome cells are structurally and functionally abnormal, and have impaired capability to bind and mediate FGF-2 signaling (Pan C et al. Blood2005;106:1956–64). In the present study, using Affymetrix microarrays we found that expression of the BMP signaling cascade including BMPs 1–8, -10 and -15, BMP and activin receptors, Smads 1–8, chordin and inhibitors of DNA binding (IDs) 1–4 is equivalent in normal and Hurler bone marrow derived multipotent progenitor cells. In Hurler cells, BMP-4 did induce a concentration- and time-dependent activation and nuclear translocation of Smad-1 (confocal immunofluorescent microscopy). However, BMP-4 activity was significantly enhanced following clearance of the abnormally accumulated GAGs in Hurler cells by recombinant IDUA enzyme, indicating that GAGs in Hurler cells impair BMP-4 activity. Thus, both endogenous GAGs and exogenous (soluble) heparin, via N- and O-sulfated disaccharide residues, inhibit BMP-4 activity. These findings have implications for understanding the pathobiology of diverse diseases, and for developing novel therapeutic agents that may restore BMP signaling and activity.

Description: The characteristic secondary metabolites in tea (theanine, caffeine, and catechins) are important factors contributing to unique tea flavors. However, there has been relatively little research on molecular markers related to these metabolites. Thus, we conducted a genome-wide association analysis of the levels of these tea flavor-related metabolites in three seasons. The theanine, caffeine, and catechin levels in Population 1 comprising 191 tea plant germplasms were examined, which revealed that their heritability exceeded 0.5 in the analyzed seasons, with the following rank order (highest to lowest heritabilities): (+)-catechin 〉 (−)-gallocatechin gallate 〉 caffeine = (−)-epicatechin 〉 (−)-epigallocatechin-3-gallate 〉 theanine 〉 (−)-epigallocatechin 〉 (−)-epicatechin-3-gallate 〉 catechin gallate 〉 (+)-gallocatechin. The SNPs detected by amplified-fragment SNP and methylation sequencing divided Population 1 into three groups and seven subgroups. An association analysis yielded 307 SNP markers related to theanine, caffeine, and catechins that were common to all three seasons. Some of the markers were pleiotropic. The functional annotation of 180 key genes at the SNP loci revealed that FLS, UGT, MYB, and WD40 domain-containing proteins, as well as ATP-binding cassette transporters, may be important for catechin synthesis. KEGG and GO analyses indicated that these genes are associated with metabolic pathways and secondary metabolite biosynthesis. Moreover, in Population 2 (98 tea plant germplasm resources), 30 candidate SNPs were verified, including 17 SNPs that were significantly or extremely significantly associated with specific metabolite levels. These results will provide a foundation for future research on important flavor-related metabolites and may help accelerate the breeding of new tea varieties.