In mucopolysaccharidosis-I (MPS-I IntroductionHurler syndrome (mucopolysaccharidosis [MPS] type I) is an inborn error of glycosaminoglycan (GAG) metabolism caused by deficiency of ␣-L-iduronidase required for heparan sulfate (HS) and dermatan sulfate (DS) degradation. 1-3 HS and DS accumulation leads to cell and organ dysfunction. While progressive neuropsychologic dysfunction is a hallmark of Hurler and related storage diseases, 4 its cellular and molecular mechanisms remain undefined.The fibroblast growth factor (FGF) cytokine family affects cell growth, migration, differentiation, and neuroectodermal development. 5 FGF-2, a prototypical member involved in tissue morphogenesis and neurogenesis, 6 has 2 kinds of cell-surface receptors. Specific, high-affinity FGF receptors (FGFRs) possessing intrinsic tyrosine kinase activity mediate cellular responses, while lowaffinity receptors composed of HS proteoglycans (HSPGs) act as extracellular FGF-2 reservoirs and coreceptors. 7 Although its mechanism of formation is unclear, the FGF-2-FGFR-HSPG complex is necessary for mitogenesis and optimal biologic response to FGF-2. 8,9 Cell-type-specific and developmentally regulated HSPG synthesis regulates cell growth and differentiation by modulating extracellular signaling molecule activity. [10][11][12] Specificity of interactions between HSPG and signaling molecules depends on HSPG sequence, location, and 3-dimensional structure. 11,13 The importance of HSPG in FGF signaling is underscored by their role in neurogenesis, axonal guidance, and synapse formation. 12,14 FGF-2 also protects neurons against apoptosis and promotes neuronal survival. 15 While GAGs synthesized by cultured Hurler fibroblasts are thought to be structurally normal, 16 GAGs that accumulate in a tissue-specific manner 17 consist of a combination of large, normally sized GAGs and partially degraded, smaller chains (oligosaccharides). 18,19 How accumulated oligosaccharides cause abnormal tissue development and function remains uncertain. Our previous studies on normal hematopoiesis suggest that a high concentration of small, abnormally sulfated HS chains (such as may be present in Hurler syndrome) are detrimental to orderly stem-cell growth and Supported by the U.S. Department of Veterans Affairs (P.G.), NIH 1-R03-HD-41 411-01 and NIH 1-R01-NS-48 606-01 (P.G.), the Children's Cancer Research Fund (P.G., C.P.), and the University of Minnesota Medical School.C. Pan and S.E.S. designed and performed research, analyzed data, and wrote the manuscript; M.S.N., J.J.B., and E.J.S. performed research and analyzed data; M.R. and L.K. isolated and cultured multipotent progenitor cells; R.Z. and S.B.S. designed research; C. Peters provided bone marrow samples and designed research; P.G. designed research, analyzed data, and wrote the manuscript. For personal use only. on May 9, 2018. by guest www.bloodjournal.org From differentiation. [20][21][22] We hypothesize that abnormal size and sulfation of accumulated HS oligosaccharides leads to aberrant functional properti...

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

Bone morphogenetic proteins (BMPs) and their endogenous antagonists are important for brain and bone development and tumor initiation and progression. Heparan sulfate (HS) proteoglycans (HSPG) modulate the activities of BMPs and their antagonists. How glycosaminoglycans (GAGs) influence BMP activity in various malignancies and in inherited abnormalities of GAG metabolism, and the structural features of GAGs essential for modulation of BMP signaling, remain incompletely defined. We examined whether chemically modified soluble heparins, the endogenous HS in malignant cells and the HS accumulated in Hurler syndrome cells influence BMP-4 signaling and activity. We show that both exogenous (soluble) and endogenous GAGs modulate BMP-4 signaling and activity, and that this effect is dependent on specific sulfate residues of GAGs. Our studies suggest that endogenous sulfated GAGs promote the proliferation and impair differentiation of malignant human cells, providing the rationale for investigating whether pharmacological agents that inhibit GAG synthesis or function might reverse this effect. Our demonstration of impairment of BMP-4 signaling by GAGs in multipotent stem cells in human Hurler syndrome identifies a mechanism that might contribute to the progressive neurological and skeletal abnormalities in Hurler syndrome and related mucopolysaccharidoses. bone morphogenetic proteins; glycosaminoglycans; mucopolysaccharidosis I; osteosarcoma; stem cells DETERMINING HOW COMPONENTS of the extracellular matrix (ECM) such as proteoglycans influence cytokine-induced cell growth and differentiation in malignancies and other diseases is critical for understanding disease pathophysiology and for developing novel therapeutic strategies (42).Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-␤ (TGF-␤) superfamily of extracellular signaling molecules that regulate the growth, differentiation, and apoptosis of cells in the brain, bone, bone marrow, and diverse tissues (2, 3). The Ͼ15 known BMPs are grouped into subfamilies based on homology within the mature domains, and BMP signaling is initiated by binding to specific type I and type II transmembrane serine/threonine kinase receptors (25). Type I receptors activated by ligand-bound type II receptors phosphorylate R-Smads (Smad-1, Smad-5, and presumably, Smad-8) anchored to the cell membrane, which then complex with Co-Smads (Smad-4), translocate to the nucleus, and regulate gene expression in cooperation with other transcription factors. The activity of R-Smads is further influenced by inhibitory Smads (I-Smads-6 and -7) (25).It is increasingly recognized that ECM and cell surface heparan sulfate (HS) proteoglycans (HSPG) are critical determinants of the biological activity of BMPs and their endogenous antagonists in vivo and in vitro (6, 9, 11, 12, 27-29, 34, 37). As with other proteins, it is believed that specific sulfated residues in the glycosaminoglycan (GAG) side chains of HSPG bind to BMPs and their antagonists and thereby modulate receptor-...

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.