Positive and negative elements modulate the promoter of the human liver-specific alpha2-HS-glycoprotein gene.
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The human alpha2-HS-glycoprotein (AHSG) and the 63-kDa rat phosphoprotein (pp63) are homologous plasma proteins that belong to the fetuin family. AHSG and pp63 are involved in important functions such as inhibition of insulin receptor tyrosine kinase activity, inhibition of protease activities, and regulation of calcium metabolism and osteogenesis. Studies of the AHSG proximal promoter performed in vitro in rat and human cells indicate that several NF-1 and C/EBP binding sites exert a positive effect on its transcriptional activity. However, until now, no distal elements have been examined in this gene, in either species. We report that the human AHSG gene promoter acts in a liver-specific manner and is further controlled by three distal, 5'-flanking elements. The negative elements III and I are, respectively, located 5' and 3' of the positive element II. All three elements require the natural context of the human AHSG gene to fully exert their negative or positive effect. Element I harbours a single binding site for NF-1. This nuclear factor thus appears to be able to up- or downregulate the AHSG gene depending on the site it binds to. Elements I, II and possibly III are absent in the rodent Ahsg gene encoding pp63. (+info)
Phosphorylation of human plasma alpha2-Heremans-Schmid glycoprotein (human fetuin) in vivo.
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A fraction of alpha2-Heremans-Schmid (alpha2-HS) glycoprotein (human fetuin) isolated from plasma was phosphorylated at serine-120 and serine-312 as shown by MS and peptide fragment sequencing after tryptic digestion. Serine-312-containing peptides were phosphorylated to 77% as determined from relative peak heights in the mass spectrum, which together with the phosphorylation of serine-120 implies a molar degree of phosphorylation of at least 1. Approximately 20% of the circulating fetuin plasma pool was phosphorylated to approx. 1 mol of phosphate/mol of protein. The remainder did not contain phosphate, resulting in an average phosphorylation degree for the protein in plasma of approx. 0.2 mol/mol. The isolated alpha2-HS glycoprotein was a heterodimer in which the entire C-terminal part of the connecting peptide including threonine-321 was present, but traces of C-terminally trimmed connecting peptide fragments were also found. The short B-chain was O-glycosylated to approx. 40%, whereas the N-glycosylation of asparagine-138 and asparagine-158 seemed to be 100%. This finding, for the first time, that circulating human plasma fetuin is partly phosphorylated, implies that the effects of phosphorylated alpha2-HS glycoprotein on insulin signal transduction seen in different cell systems could be relevant to its physiological function in vivo. (+info)
Genetic mapping and functional studies of a natural inhibitor of the insulin receptor tyrosine kinase: the mouse ortholog of human alpha2-HS glycoprotein.
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Fetuin/alpha2-HS glycoprotein (alpha2-HSG) homologs have been identified in several species including rat, sheep, pig, rabbit, guinea pig, cattle, mouse and human. Multiple physiological roles for these homologs have been suggested, including ability to bind to hydroxyapatite crystals and to specifically inhibit the tyrosine kinase (TK) activity of the insulin receptor (IR). In this study we report the identification, cloning, and characterization of the mouse Ahsg gene and its function as an IR-TK inhibitor. Genomic clones derived from a mouse Svj 129 genomic library were sequenced in order to characterize the intron-exon organization of the mouse Ahsg gene, including an 875 bp subclone containing 154 bp upstream from the transcription start site, the first exon, and part of the first intron. A second genomic subclone harboring a 3.45 kb Bgl II fragment contained exons 2, 3 and 4 in addition to two adjacent elements within the first intron-a repetitive element of the B1 family (92 bp) and a 271 bp tract of (T,C)n*(A,G)n. We have mapped mouse Ahsg at 16 cM adjacent to the Diacylglycerol kinase 3 (Dagk3) gene on chromosome 16 by genotyping interspecific backcross panels between C57BL/6J and Mus spretus. The position is syntenic with human chromosome 3q27, where the human AHSG gene resides. Using recombinant mouse alpha2-HSG expressed from a recombinant baculovirus, we demonstrate that mouse alpha2-HSG inhibits insulin-stimulated IR autophosphorylation and IR-TKA in vitro. In addition, mouse alpha2-HSG (25 microg/ml) completely abolishes insulin-induced DNA synthesis in H-35 rat hepatoma cells. Based on the sequence data and functional analysis, we conclude that the mouse Ahsg gene is the true ortholog of the human AHSG gene. (+info)
alpha 2-HS glycoprotein/fetuin, a transforming growth factor-beta/bone morphogenetic protein antagonist, regulates postnatal bone growth and remodeling.
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Soluble transforming growth factor-beta (TGF-beta)/bone morphogenetic protein (BMP)-binding proteins are widely distributed in mammalian tissues and control cytokine access to membrane signaling receptors. The serum and bone-resident glycoprotein alpha2-HS-glycoprotein/fetuin (ASHG) binds to TGF-beta/BMP cytokines and blocks TGF-beta1 binding to cell surface receptors. Therefore, we examined bone growth and remodeling phenotypes in ASHG-deficient mice. The skeletal structure of Ahsg(-/-) mice appeared normal at birth, but abnormalities were observed in adult Ahsg(-/-) mice. Maturation of growth plate chondrocytes was impaired, and femurs lengthened more slowly between 3 and 18 months of age in Ahsg(-/-) mice. However, bone formation was increased in Ahsg(-/-) mice as indicated by greater cortical thickness, accelerated trabecular bone remodeling, and increased osteoblast numbers on bone surfaces. The normal age-related increase in cortical thickness and bone mineral density was accelerated in Ahsg(-/-) mice and was associated with increased energy required to fracture. Bone formation in response to implanted BMP cytokine extended further from the implant in Ahsg(-/-) compared with Ahsg(+/+) mice, confirming the interaction between ASHG and TGF-beta/BMP cytokines in vivo. Our results demonstrate that ASHG blocks TGF-beta-dependent signaling in osteoblastic cells, and mice lacking ASHG display growth plate defects, increased bone formation with age, and enhanced cytokine-dependent osteogenesis. (+info)
Improved insulin sensitivity and resistance to weight gain in mice null for the Ahsg gene.
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Fetuin inhibits insulin-induced insulin receptor (IR) autophosphorylation and tyrosine kinase activity in vitro, in intact cells, and in vivo. The fetuin gene (AHSG) is located on human chromosome 3q27, recently identified as a susceptibility locus for type 2 diabetes and the metabolic syndrome. Here, we explore insulin signaling, glucose homeostasis, and the effect of a high-fat diet on weight gain, body fat composition, and glucose disposal in mice carrying two null alleles for the gene encoding fetuin, Ahsg (B6, 129-Ahsg(tm1Mbl)). Fetuin knockout (KO) mice demonstrate increased basal and insulin-stimulated phosphorylation of IR and the downstream signaling molecules mitogen-activated protein kinase (MAPK) and Akt in liver and skeletal muscle. Glucose and insulin tolerance tests in fetuin KO mice indicate significantly enhanced glucose clearance and insulin sensitivity. Fetuin KO mice subjected to euglycemic-hyperinsulinemic clamp show augmented sensitivity to insulin, evidenced by increased glucose infusion rate (P = 0.077) and significantly increased skeletal muscle glycogen content (P < 0.05). When fed a high-fat diet, fetuin KO mice are resistant to weight gain, demonstrate significantly decreased body fat, and remain insulin sensitive. These data suggest that fetuin may play a significant role in regulating postprandial glucose disposal, insulin sensitivity, weight gain, and fat accumulation and may be a novel therapeutic target in the treatment of type 2 diabetes, obesity, and other insulin-resistant conditions. (+info)
Correlation of maternal serum fetuin/alpha2-HS-glycoprotein concentration with maternal insulin resistance and anthropometric parameters of neonates in normal pregnancy and gestational diabetes.
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OBJECTIVE: Human fetuin/alpha(2)-HS-glycoprotein (AHSG) is a 49 kDa serum and tissue protein which is a natural inhibitor of insulin receptor signaling. We investigated serum AHSG levels during pregnancy and whether the protein is involved in insulin resistance observed in healthy pregnant women and patients with gestational diabetes. DESIGN: One hundred and four healthy pregnant women and 23 of their neonates, 30 patients with gestational diabetes and their neonates and 30 healthy age-matched non-pregnant females as a control group were investigated in a case-control cross-sectional study. METHODS: Serum AHSG was determined by radial immunodiffusion. RESULTS: We observed an increase of serum AHSG concentration in the second and third trimesters. Gestational diabetes patients had significantly higher AHSG levels than healthy pregnant women and non-pregnant controls. There was a highly significant positive correlation between serum AHSG concentration and indirect parameters of insulin resistance, i.e. tumor necrosis factor-alpha (TNF-alpha), leptin, C-peptide and C-peptide/blood glucose ratio. There was also a negative correlation between maternal AHSG, TNF-alpha, leptin levels and head circumference, body length and body weight of newborns. CONCLUSION: AHSG, TNF-alpha and leptin may contribute to insulin resistance during normal pregnancy and gestational diabetes. AHSG along with these cytokines may also negatively regulate neonatal skeletal development. (+info)
Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles.
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Genetic evidence from mutant mice suggests that alpha(2)-HS glycoprotein/fetuin-A (Ahsg) is a systemic inhibitor of precipitation of basic calcium phosphate preventing unwanted calcification. Using electron microscopy and dynamic light scattering, we demonstrate that precipitation inhibition by Ahsg is caused by the transient formation of soluble, colloidal spheres, containing Ahsg, calcium, and phosphate. These "calciprotein particles" of 30-150 nm in diameter are initially amorphous and soluble but turn progressively more crystalline and insoluble in a time- and temperature-dependent fashion. Solubilization in Ahsg-containing calciprotein particles provides a novel conceptual framework to explain how insoluble calcium precipitates may be transported and removed in the bodies of mammals. Mutational analysis showed that the basic calcium phosphate precipitation inhibition activity resides in the amino-terminal cystatin-like domain D1 of Ahsg. A structure-function analysis of wild type and mutant forms of cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most efficient in inhibiting basic calcium phosphate precipitation. The computer-modeled domain structures suggest that a dense array of acidic residues on an extended beta-sheet of the cystatin-like domain Ahsg-D1 mediates efficient inhibition. (+info)
The serum protein alpha 2-Heremans-Schmid glycoprotein/fetuin-A is a systemically acting inhibitor of ectopic calcification.
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Ectopic calcification is a frequent complication of many degenerative diseases. Here we identify the serum protein alpha2-Heremans-Schmid glycoprotein (Ahsg, also known as fetuin-A) as an important inhibitor of ectopic calcification acting on the systemic level. Ahsg-deficient mice are phenotypically normal, but develop severe calcification of various organs on a mineral and vitamin D-rich diet and on a normal diet when the deficiency is combined with a DBA/2 genetic background. This phenotype is not associated with apparent changes in calcium and phosphate homeostasis, but with a decreased inhibitory activity of the Ahsg-deficient extracellular fluid on mineral formation. The same underlying principle may contribute to many calcifying disorders including calciphylaxis, a syndrome of severe systemic calcification in patients with chronic renal failure. Taken together, our data demonstrate a critical role of Ahsg as an inhibitor of unwanted mineralization and provide a novel therapeutic concept to prevent ectopic calcification accompanying various diseases. (+info)