Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
Sci Signal ; 12(607)2019 11 12.
Article in English | MEDLINE | ID: mdl-31719172

ABSTRACT

Dysregulated bone morphogenetic protein (BMP) signaling in endothelial cells (ECs) is implicated in vascular diseases such as pulmonary arterial hypertension (PAH). Here, we showed that the transcription factor ATOH8 was a direct target of SMAD1/5 and was induced in a manner dependent on BMP but independent of Notch, another critical signaling pathway in ECs. In zebrafish and mice, inactivation of Atoh8 did not cause an arteriovenous malformation-like phenotype, which may arise because of dysregulated Notch signaling. In contrast, Atoh8-deficient mice exhibited a phenotype mimicking PAH, which included increased pulmonary arterial pressure and right ventricular hypertrophy. Moreover, ATOH8 expression was decreased in PAH patient lungs. We showed that in cells, ATOH8 interacted with hypoxia-inducible factor 2α (HIF-2α) and decreased its abundance, leading to reduced induction of HIF-2α target genes in response to hypoxia. Together, these findings suggest that the BMP receptor type II/ALK-1/SMAD/ATOH8 axis may attenuate hypoxic responses in ECs in the pulmonary circulation and may help prevent the development of PAH.


Subject(s)
Activin Receptors, Type II/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Hypertension, Pulmonary/metabolism , Hypertension, Pulmonary/prevention & control , Hypoxia/metabolism , Signal Transduction , Smad Proteins/metabolism , Activin Receptors, Type II/genetics , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , HEK293 Cells , Humans , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/pathology , Hypoxia/genetics , Hypoxia/pathology , Mice , Mice, Knockout , Smad Proteins/genetics , Zebrafish
2.
Stem Cell Reports ; 6(1): 64-73, 2016 Jan 12.
Article in English | MEDLINE | ID: mdl-26771354

ABSTRACT

Bone morphogenetic protein (BMP) signaling exerts paradoxical roles in pluripotent stem cells (PSCs); it sustains self-renewal of mouse embryonic stem cells (ESCs), while it induces differentiation in other PSCs, including human ESCs. Here, we revisit the roles of BMP-4 using mouse ESCs (mESCs) in naive and primed states. SMAD1 and SMAD5, which transduce BMP signals, recognize enhancer regions together with KLF4 and KLF5 in naive mESCs. KLF4 physically interacts with SMAD1 and suppresses its activity. Consistently, a subpopulation of cells with active BMP-SMAD can be ablated without disturbing the naive state of the culture. Moreover, Smad1/5 double-knockout mESCs stay in the naive state, indicating that the BMP-SMAD pathway is dispensable for it. In contrast, the MEK5-ERK5 pathway mediates BMP-4-induced self-renewal of mESCs by inducing Klf2, a critical factor for the ground state pluripotency. Our study illustrates that BMP exerts its self-renewing effect through distinct functions of different Krüppel-like factors.


Subject(s)
Bone Morphogenetic Protein 4/metabolism , Cell Self Renewal/physiology , Kruppel-Like Transcription Factors/metabolism , Mouse Embryonic Stem Cells/metabolism , Animals , Blotting, Western , Bone Morphogenetic Protein 4/genetics , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Self Renewal/genetics , Gene Expression Regulation, Developmental , HEK293 Cells , Hep G2 Cells , Humans , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , MAP Kinase Signaling System/genetics , MAP Kinase Signaling System/physiology , Mice , Mice, Knockout , Mouse Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/metabolism , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Smad1 Protein/genetics , Smad1 Protein/metabolism , Smad5 Protein/genetics , Smad5 Protein/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism
3.
PLoS One ; 10(3): e0121957, 2015.
Article in English | MEDLINE | ID: mdl-25793894

ABSTRACT

Chondroitin/dermatan sulfate (CS/DS) proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS). Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.


Subject(s)
Chondroitin Sulfates/metabolism , Dermatan Sulfate/analogs & derivatives , Embryonic Development , Sulfotransferases/metabolism , Zebrafish Proteins/metabolism , Zebrafish/embryology , Zebrafish/metabolism , Animals , Chromatography, High Pressure Liquid , Chromatography, Reverse-Phase , Dermatan Sulfate/metabolism , Heparitin Sulfate/metabolism , In Situ Hybridization , Phylogeny , Time Factors
4.
Dev Dyn ; 242(8): 964-75, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23703795

ABSTRACT

BACKGROUND: Chondroitin/dermatan sulfate (CS/DS) proteoglycans present in the extracellular matrix have important structural and regulatory functions. RESULTS: Six human genes have previously been shown to catalyze CS/DS polymerization. Here we show that one of these genes, chpf, is represented by two copies in the zebrafish genome, chpfa and chpfb, while the other five human CS/DS glycosyltransferases csgalnact1, csgalnact2, chpf2, chsy1, and chsy3 all have single zebrafish orthologues. The putative zebrafish CS/DS glycosyltransferases are spatially and temporally expressed. Interestingly, overlapping expression of multiple glycosyltransferases coincides with high CS/DS deposition. Finally, whereas the relative levels of the related polysaccharide HS reach steady-state at around 2 days post fertilization, there is a continued relative increase of the CS amounts per larvae during the first 6 days of development, matching the increased cartilage formation. CONCLUSIONS: There are 7 CS/DS glycosyltransferases in zebrafish, which, based on homology, can be divided into the CSGALNACT, CHSY, and CHPF families. The overlap between intense CS/DS production and the expression of multiple CS/DS glycosyltransferases suggests that efficient CS/DS biosynthesis requires a combination of several glycosyltransferases.


Subject(s)
Chondroitin Sulfates/metabolism , Dermatan Sulfate/metabolism , Glycosyltransferases/metabolism , Zebrafish Proteins/metabolism , Animals , Chondroitin , Glycosyltransferases/classification , Glycosyltransferases/genetics , Phylogeny , Zebrafish , Zebrafish Proteins/classification , Zebrafish Proteins/genetics
5.
J Biol Chem ; 287(40): 33905-16, 2012 Sep 28.
Article in English | MEDLINE | ID: mdl-22869369

ABSTRACT

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation of the common proteoglycan linkage tetrasaccharide were analyzed along with ext2 and extl3 mutants, predicted to have defective HS polymerization. Notably, the effects on HS and CS biosynthesis in the respective mutant strains were shown to differ from what had been hypothesized. In uxs1 and b3gat3 mutant larvae, biosynthesis of CS was shown to be virtually abolished, whereas these mutants still were capable of synthesizing 50% of the HS produced in control larvae. extl3 and ext2 mutants on the other hand were shown to synthesize reduced amounts of hypersulfated HS. Further, extl3 mutants produced higher levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies in the single mutant strains, as well as in ext2;uxs1 double mutants, was conducted. A correlation between HS and CS production and phenotypes was found, such that impaired HS biosynthesis was shown to affect chondrocyte intercalation, whereas impaired CS biosynthesis inhibited formation of the extracellular matrix surrounding chondrocytes.


Subject(s)
Cartilage/metabolism , Chondroitin Sulfates/chemistry , Gene Expression Regulation, Developmental , Heparitin Sulfate/chemistry , Alleles , Animals , Crosses, Genetic , Disease Progression , Female , Genotype , Male , Microscopy, Confocal/methods , Microscopy, Electron, Transmission/methods , Models, Biological , Morphogenesis , Mutation , Pharynx/pathology , Zebrafish
6.
J Biol Chem ; 287(14): 10853-62, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22298785

ABSTRACT

Heparan sulfate proteoglycans, present on cell surfaces and in the extracellular matrix, interact with growth factors and morphogens to influence growth and differentiation of cells. The sulfation pattern of the heparan sulfate chains formed during biosynthesis in the Golgi compartment will determine the interaction potential of the proteoglycan. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes have a key role during biosynthesis, greatly influencing total sulfation of the heparan sulfate chains. The differentiation potential of mouse embryonic stem cells lacking both NDST1 and NDST2 was studied using in vitro differentiation protocols, expression of differentiation markers, and assessment of the ability of the cells to respond to growth factors. The results show that NDST1 and NDST2 are dispensable for mesodermal differentiation into osteoblasts but necessary for induction of adipocytes and neural cells. Gene expression analysis suggested a differentiation block at the primitive ectoderm stage. Also, GATA4, a primitive endoderm marker, was expressed by these cells. The addition of FGF4 or FGF2 together with heparin rescued the differentiation potential to neural progenitors and further to mature neurons and glia. Our results suggest that the embryonic stem cells lacking both NDST1 and NDST2, expressing a very low sulfated heparan sulfate, can take the initial step toward differentiation into all three germ layers. Except for their potential for mesodermal differentiation into osteoblasts, the cells are then arrested in a primitive ectoderm and/or endoderm stage.


Subject(s)
Amidohydrolases/deficiency , Amidohydrolases/metabolism , Cell Differentiation , Embryonic Stem Cells/cytology , Heparitin Sulfate/metabolism , Sulfotransferases/deficiency , Sulfotransferases/metabolism , Adipocytes/cytology , Amidohydrolases/genetics , Animals , Bone Morphogenetic Proteins/metabolism , Carrier Proteins/metabolism , Cell Differentiation/drug effects , Ectoderm/cytology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Embryonic Stem Cells/metabolism , Fibroblast Growth Factors/pharmacology , Gene Knockout Techniques , Heparin/pharmacology , Mesoderm/cytology , Mice , Mutation , Osteoblasts/cytology , Signal Transduction/drug effects , Sulfotransferases/genetics
7.
Arterioscler Thromb Vasc Biol ; 32(5): 1255-63, 2012 May.
Article in English | MEDLINE | ID: mdl-22345168

ABSTRACT

OBJECTIVE: Heparan sulfate proteoglycans regulate key steps of blood vessel formation. The present study was undertaken to investigate if there is a functional overlap between heparan sulfate proteoglycans and chondroitin sulfate proteoglycans during sprouting angiogenesis. METHODS AND RESULTS: Using cultures of genetically engineered mouse embryonic stem cells, we show that angiogenic sprouting occurs also in the absence of heparan sulfate biosynthesis. Cells unable to produce heparan sulfate instead increase their production of chondroitin sulfate that binds key angiogenic growth factors such as vascular endothelial growth factor A, transforming growth factor ß, and platelet-derived growth factor B. Lack of heparan sulfate proteoglycan production however leads to increased pericyte numbers and reduced adhesion of pericytes to nascent sprouts, likely due to dysregulation of transforming growth factor ß and platelet-derived growth factor B signal transduction. CONCLUSIONS: The present study provides direct evidence for a previously undefined functional overlap between chondroitin sulfate proteoglycans and heparan sulfate proteoglycans during sprouting angiogenesis. Our findings provide information relevant for potential future drug design efforts that involve targeting of proteoglycans in the vasculature.


Subject(s)
Endothelium, Vascular/metabolism , Heparan Sulfate Proteoglycans/metabolism , Neovascularization, Pathologic/metabolism , Proteoglycans/metabolism , Vascular Endothelial Growth Factor A/pharmacology , Animals , Blotting, Western , Cell Adhesion/drug effects , Cell Proliferation , Cells, Cultured , Chondroitin , Disease Models, Animal , Endothelium, Vascular/drug effects , Endothelium, Vascular/pathology , Immunohistochemistry , Mice , Neovascularization, Pathologic/chemically induced , Neovascularization, Pathologic/pathology , Signal Transduction/drug effects
8.
J Biol Chem ; 286(52): 44433-40, 2011 Dec 30.
Article in English | MEDLINE | ID: mdl-22049073

ABSTRACT

Deficiency of the heparan sulfate biosynthesis enzyme N-deacetylase/N-sulfotransferase 1 (NDST1) in mice causes severely disturbed heparan sulfate biosynthesis in all organs, whereas lack of NDST2 only affects heparin biosynthesis in mast cells (MCs). To investigate the individual and combined roles of NDST1 and NDST2 during MC development, in vitro differentiated MCs derived from mouse embryos and embryonic stem cells, respectively, have been studied. Whereas MC development will not occur in the absence of both NDST1 and NDST2, lack of NDST2 alone results in the generation of defective MCs. Surprisingly, the relative amount of heparin produced in NDST1(+/-) and NDST1(-/-) MCs is higher (≈30%) than in control MCs where ≈95% of the (35)S-labeled glycosaminoglycans produced is chondroitin sulfate. Lowered expression of NDST1 also results in a higher sulfate content of the heparin synthesized and is accompanied by increased levels of stored MC proteases. A model of the GAGosome, a hypothetical Golgi enzyme complex, is used to explain the results.


Subject(s)
Heparin/biosynthesis , Heparitin Sulfate/biosynthesis , Mast Cells/metabolism , Models, Biological , Sulfotransferases/metabolism , Amidohydrolases/genetics , Amidohydrolases/metabolism , Animals , Cells, Cultured , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Golgi Apparatus/enzymology , Golgi Apparatus/genetics , Heparin/genetics , Heparitin Sulfate/genetics , Mast Cells/cytology , Mice , Mice, Knockout , Peptide Hydrolases/metabolism , Sulfotransferases/genetics
9.
Stem Cells ; 28(2): 191-200, 2010 Feb.
Article in English | MEDLINE | ID: mdl-19937756

ABSTRACT

Embryonic stem (ES) cells continuously decide whether to maintain pluripotency or differentiate. While exogenous leukemia inhibitory factor and BMP4 perpetuate a pluripotent state, less is known about the factors initiating differentiation. We show that heparan sulfate (HS) proteoglycans are critical coreceptors for signals inducing ES cell differentiation. Genetic targeting of NDST1 and NDST2, two enzymes required for N-sulfation of proteoglycans, blocked differentiation. This phenotype was rescued by HS presented in trans or by soluble heparin. NaClO(3) (-), which reduces sulfation of proteoglycans, potently blocked differentiation of wild-type cells. Mechanistically, N-sulfation was identified to be critical for functional autocrine fibroblast growth factor 4 (FGF4) signaling. Microarray analysis identified the pluripotency maintaining transcription factors Nanog, KLF2/4/8, Tbx3, and Tcf3 to be negatively regulated, whereas markers of differentiation such as Gbx2, Dnmt3b, FGF5, and Brachyury were induced by sulfation-dependent FGF receptor (FGFR) signaling. We show that several of these genes are heterogeneously expressed in ES cells, and that targeting of heparan sulfation or FGFR-signaling facilitated a homogenous Nanog/KLF4/Tbx3 positive ES cell state. This finding suggests that the recently discovered heterogeneous state of ES cells is regulated by HS-dependent FGFR signaling. Similarly, culturing blastocysts with NaClO(3) (-) eliminated GATA6-positive primitive endoderm progenitors generating a homogenous Nanog-positive inner cell mass. Functionally, reduction of sulfation robustly improved de novo ES cell derivation efficiency. We conclude that N-sulfated HS is required for FGF4 signaling to maintain ES cells primed for differentiation in a heterogeneous state. Inhibiting this pathway facilitates a more naïve ground state.


Subject(s)
Cell Differentiation/physiology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Heparitin Sulfate/metabolism , Amidohydrolases/genetics , Amidohydrolases/metabolism , Animals , Blotting, Western , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Line, Tumor , Chlorates/pharmacology , Flow Cytometry , Fluorescent Antibody Technique , Homeodomain Proteins/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/metabolism , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Nanog Homeobox Protein , Octamer Transcription Factor-3/metabolism , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/physiology , Sulfotransferases/genetics , Sulfotransferases/metabolism
10.
Dev Cell ; 10(5): 625-34, 2006 May.
Article in English | MEDLINE | ID: mdl-16678777

ABSTRACT

Several receptor tyrosine kinases require heparan sulfate proteoglycans (HSPGs) as coreceptors for efficient signal transduction. We have studied the role of HSPGs in the development of blood capillary structures from embryonic stem cells, a process strictly dependent on signaling via vascular endothelial growth factor receptor-2 (VEGFR-2). We show, by using chimeric cultures of embryonic stem cells defective in either HS production or VEGFR-2 synthesis, that VEGF signaling in endothelial cells is fully supported by HS expressed in trans by adjacent perivascular smooth muscle cells. Transactivation of VEGFR-2 leads to prolonged and enhanced signal transduction due to HS-dependent trapping of the active VEGFR-2 signaling complex. Our data imply that direct signaling via HSPG core proteins is dispensable for a functional VEGF response in endothelial cells. We propose that transactivation of tyrosine kinase receptors by HSPGs constitutes a mechanism for crosstalk between adjacent cells.


Subject(s)
Heparitin Sulfate/pharmacology , Neovascularization, Physiologic/drug effects , Transcriptional Activation/genetics , Vascular Endothelial Growth Factor Receptor-2/metabolism , Amidohydrolases/deficiency , Animals , Cells, Cultured , Chimera/genetics , Collagen/chemistry , Endothelial Cells/cytology , Endothelial Cells/drug effects , Gels/chemistry , Genes, Developmental/genetics , Heparin/pharmacology , Mice , Models, Genetic , Pericytes/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Signal Transduction , Solubility , Stem Cells/cytology , Sulfotransferases/deficiency , Vascular Endothelial Growth Factor A/pharmacology , Vascular Endothelial Growth Factor Receptor-2/deficiency
11.
J Biol Chem ; 279(41): 42355-8, 2004 Oct 08.
Article in English | MEDLINE | ID: mdl-15319440

ABSTRACT

Heparan sulfate structure differs significantly between various cell types and during different developmental stages. The diversity is created during biosynthesis by sulfotransferases, which add sulfate groups to the growing chain, and a C5-epimerase, which converts selected glucuronic acid residues to iduronic acid. All these modifications are believed to depend on initial glucosamine N-sulfation carried out by the enzyme glucosaminyl N-deacetylase/N-sulfotransferase (NDST). Here we report that heparan sulfate synthesized by mouse embryonic stem cells deficient in NDST1 and NDST2 completely lacks N-sulfation but still contains 6-O-sulfate groups, demonstrating that 6-O-sulfation can occur without prior N-sulfation. Reverse transcriptase-PCR analysis indicates that all three identified 6-O-sulfotransferases are expressed by the cells, 6-O-sulfotransferase-1 being the dominating form. The 6-O-sulfated polysaccharide lacking N-sulfate groups also contains N-unsubstituted glucosamine units, raising questions about how these units are generated.


Subject(s)
Amidohydrolases/genetics , Heparitin Sulfate/biosynthesis , Sulfotransferases/genetics , Sulfur/metabolism , Amidohydrolases/physiology , Animals , Blastocyst/metabolism , Carbohydrate Epimerases/chemistry , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , DNA, Complementary/metabolism , Embryo, Mammalian/metabolism , Gene Expression Regulation, Developmental , Genotype , Glucosamine/chemistry , Glucuronic Acid/metabolism , Glycosaminoglycans , Iduronic Acid/metabolism , Mice , Mice, Transgenic , Nitrous Acid/metabolism , Polysaccharides/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/metabolism , Sulfates/chemistry , Sulfotransferases/physiology
12.
Biochim Biophys Acta ; 1573(3): 209-15, 2002 Dec 19.
Article in English | MEDLINE | ID: mdl-12417402

ABSTRACT

Heparan sulfates (HSs) are N- and O-sulfated polysaccharide components of proteoglycans, which are important constituents of the cell surface as well as the extracellular matrix. Heparin, with extensive clinical application as an anticoagulant, is a highly sulfated form of HS present within the granules of connective tissue type mast cells. The diverse functions of HS, which include the modulation of growth factor/cytokine activity, interaction with matrix proteins and binding of enzymes to cell surfaces, depend greatly on the presence of specific, high affinity regions on the chains. N-acetylglucosamine N-deacetylase/N-sulfotransferases, NDSTs, are an important group of enzymes in HS biosynthesis, initiating the sulfation of the polysaccharide chains and thus determining the generation of the high affinity sites. Here, we review the role of the four vertebrate NDSTs in HS biosynthesis as well as their regulated expression. The main emphasis is the phenotypes of mice lacking one or more of the NDSTs.


Subject(s)
Amidohydrolases/metabolism , Gene Expression Regulation, Enzymologic , Heparitin Sulfate/biosynthesis , Sulfotransferases/metabolism , Amidohydrolases/genetics , Animals , Carbohydrate Sequence , Disease Models, Animal , Heparan Sulfate Proteoglycans/metabolism , Humans , Isoenzymes , Mice , Protein Biosynthesis , Sulfotransferases/genetics , Transcription, Genetic
SELECTION OF CITATIONS
SEARCH DETAIL
...