Idiopathic infantile arterial calcification (IIAC; OMIM 208000) is characterized by calcification of the internal elastic lamina of muscular arteries and stenosis due to myointimal proliferation. We analyzed affected individuals from 11 unrelated kindreds and found that IIAC was associated with mutations that inactivated ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). This cell surface enzyme generates inorganic pyrophosphate (PP(i)), a solute that regulates cell differentiation and serves as an essential physiologic inhibitor of calcification.

The secretory prohormone chromogranin A (CHGA) is overexpressed in essential hypertension, a complex trait with genetic predisposition, while its catecholamine release-inhibitory fragment catestatin is diminished, and low catestatin predicts augmented adrenergic pressor responses. These findings from studies on humans suggest a mechanism whereby diminished catestatin might increase the risk for hypertension. We generated Chga -/-and humanized mice through transgenic insertion of a human CHGA haplotype in order to probe CHGA and catestatin in vivo.

Inogranic pyrophosphate (PPi) inhibits hydroxyapatite deposition, and mice deficient in the PPi-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) Plasma cell membrane glycoprotein-1 (PC-1) develop peri-articular and arterial calcification in early life. In idiopathic infantile arterial calcification (IIAC), hydroxyapatite deposition and smooth muscle cell (SMC) proliferation occur, sometimes associated with peri-articular calcification. Thus, we assessed PC-1 expression and PPi metabolism in a 25-month-old boy with IIAC and peri-articular calcifications. Plasma PC-1 was <1 ng/ml by enzyme-linked immunosorbent assay in the proband, but 10 to 30 ng/ml in unaffected family members and controls. PC-1 functioned to raise extracellular PPi in cultured aortic SMCs. However, PC-1 was sparse in temporal artery lesion SMCs in the proband, unlike the case for SMCs in atherosclerotic carotid artery lesions of unrelated adults. Proband plasma and explant-cultured dermal fibroblast NTPPPH and PPi were markedly decreased. The proband was heterozygous at the PC-1 locus, and sizes of PC-1 mRNA and polypeptide, and the PC-1 mRNA-coding region sequence were normal in proband fibroblasts. However, immunoreactive PC-1 protein was relatively sparse in proband fibroblasts. Physiological extracellular matrix (ECM) calcification is limited to bones, teeth, and nonarticular (growth) cartilages.1,2 Moreover, ECM calcification must be tightly controlled, because normal calcium and phosphate concentrations in extracellular fluids are near the saturation point for the deposition of basic calcium phosphate crystals in the form of hydroxyapatite, and pathological calcification of the ECM can be observed in any tissue of the body.

Objective. Increased nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity in chondrocytes is associated with cartilage matrix inorganic pyrophosphate (PPi) supersaturation in chondrocalcinosis. This study compared the roles of the transforming growth factor ␤ (TGF␤)-inducible plasma cell membrane glycoprotein-1 (PC-1) and the closely related B10 NTPPPH activities in chondrocyte PPi metabolism.Methods. NTPPPH expression was studied using reverse transcriptase-polymerase chain reaction and Western blotting. Transmembrane PC-1 (tmPC-1), water-soluble secretory PC-1 (secPC-1), and transmembrane B10 were expressed by adenoviral gene transfer or plasmid transfection, and expression of PPi was assessed in cultured articular chondrocytes and immortalized NTPPPH-deficient costal chondrocytes (TC28 cells).Results. PC-1 and B10 messenger RNA were demonstrated in articular cartilages in situ, in untreated cultured normal articular chondrocytes, and in TC28 cells. Expression of tmPC-1 and secPC-1, but not B10, rendered the NTPPPH-deficient TC28 cells able to increase expression of extracellular PPi, with or without addition of TGF␤ (10 ng/ml) to the media. More plasma membrane NTPPPH activity was detected in cells transfected with tmPC-1 than in cells transfected with B10. Furthermore, confocal microscopy with immunofluorescent staining of articular chondrocytes confirmed preferential plasma membrane localization of PC-1, relative to B10. Finally, both PC-1 and B10 increased the levels of intracellular PPi, but PC-1 and B10 appeared to act principally in different intracellular compartments (Golgi and post-Golgi versus pre-Golgi, respectively).Conclusion. PC-1 and B10 NTPPPH activities were not redundant in chondrocytes. Although increased PC-1 and B10 expression caused elevations in intracellular PPi, the major effects of PC-1 and B10 were exerted in distinct subcellular compartments. Moreover, PC-1 (transmembrane and secreted), but not B10, increased the levels of extracellular PPi. Differential expression of PC-1 and B10 could modulate cartilage mineralization in degenerative joint diseases.Articular cartilage chondrocytes have the unique ability to constitutively elaborate relatively large amounts of extracellular inorganic pyrophosphate (PPi), and cartilage is the major source of free PPi in joints (1-3). Moreover, in aging and osteoarthritic (OA) articular cartilage, a dysregulated increase in PPi elaboration in chondrocytes and other changes in the chondrocyte differentiation and matrix composition promote calcium Ms Moffa

Tissue-nonspecific alkaline phosphatase (TNAP) is essential for bone matrix mineralization, but the central mechanism for TNAP action remains undefined. We observed that ATP-dependent (45)Ca precipitation was decreased in calvarial osteoblast matrix vesicle (MV) fractions from TNAP-/- mice, a model of infantile hypophosphatasia. Because TNAP hydrolyzes the mineralization inhibitor inorganic pyrophosphate (PP(i)), we assessed phosphodiesterase nucleotide pyrophosphatase (PDNP/NTPPPH) activity, which hydrolyzes ATP to generate PP(i). Plasma cell membrane glycoprotein-1 (PC-1), but not the isozyme B10 (also called PDNP3) colocalized with TNAP in osteoblast MV fractions and pericellular matrix. PC-1 but not B10 increased MV fraction PP(i) and inhibited (45)Ca precipitation by MVs. TNAP directly antagonized inhibition by PC-1 of MV-mediated (45)Ca precipitation. Furthermore, the PP(i) content of MV fractions was greater in cultured TNAP-/- than TNAP+/+ calvarial osteoblasts. Paradoxically, transfection with wild-type TNAP significantly increased osteoblast MV fraction NTPPPH. Specific activity of NTPPPH also was twofold greater in MV fractions of osteoblasts from TNAP+/+ mice relative to TNAP-/- mice. Thus TNAP attenuates PC-1/NTPPPH-induced PP(i) generation that would otherwise inhibit MV-mediated mineralization. TNAP also paradoxically regulates PC-1 expression and NTPPPH activity in osteoblasts.

GTP cyclohydrolase 1 (GCH1) is rate limiting in the provision of the cofactor tetrahydrobiopterin for biosynthesis of catecholamines and NO. We asked whether common genetic variation at GCH1 alters transmitter synthesis and predisposes to disease. Here we undertook a systematic search for polymorphisms in GCH1, then tested variants' contributions to NO and catecholamine release as well as autonomic function in twin pairs. Renal NO and neopterin excretions were significantly heritable, as were baroreceptor coupling (heart rate response to BP fluctuation) and pulse interval (1/heart rate). Common GCH1 variant C+243T in the 3'-untranslated region (3'-UTRs) predicted NO excretion, as well as autonomic traits: baroreceptor coupling, maximum pulse interval, and pulse interval variability, though not catecholamine secretion. In individuals with the most extreme BP values in the population, C+243T affected both diastolic and systolic BP, principally in females. In functional studies, C+243T decreased reporter expression in transfected 3'-UTRs plasmids. We conclude that human NO secretion traits are heritable, displaying joint genetic determination with autonomic activity by functional polymorphism at GCH1. Our results document novel pathophysiological links between a key biosynthetic locus and NO metabolism and suggest new strategies for approaching the mechanism, diagnosis, and treatment of risk predictors for cardiovascular diseases such as hypertension.

Chromogranins/secretogranins or granins are a class of acidic, secretory proteins that occur in endocrine, neuroendocrine, and neuronal cells. Granins are the precursors of several bioactive peptides and may be involved in secretory granule formation and neurotransmitter/hormone release. Characterization and analysis of chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII) in distant vertebrate species confirmed that CgA and CgB belong to related monophyletic groups, probably evolving from a common ancestral precursor, while SgII sequences constitute a distinct monophyletic group. In particular, selective sequences within these proteins, bounded by potential processing sites, have been remarkably conserved during evolution. Peptides named vasostatin, secretolytin and secretoneurin, which occur in these regions, have been shown to exert various biological activities. These conserved domains may also be involved in the formation of secretory granules in different vertebrates. Other peptides such as catestatin and pancreastatin may have appeared late during evolution. The function of granins as propeptide precursors and granulogenic factors is discussed in the light of recent data obtained in various model species and using knockout mice strains.

Chromogranin A (CHGA/Chga), a proprotein, widely distributed in endocrine and neuroendocrine tissues (not expressed in muscle, liver, and adipose tissues), generates at least four bioactive peptides. One of those peptides, pancreastatin (PST), has been reported to interfere with insulin action. We generated a Chga knock-out (KO) mouse by the targeted deletion of the Chga gene in neuroendocrine tissues. KO mice displayed hypertension, higher plasma catecholamine, and adipokine levels and lower IL-6 and lipid levels compared with wild type mice. Liver glycogen content was elevated, but the nitric oxide (NO) level was diminished. Glucose, insulin, and pyruvate tolerance tests and hyperinsulinemic-euglycemic clamp studies established increased insulin sensitivity in liver but decreased glucose disposal in muscle. Despite higher catecholamine and ketone body levels and muscle insulin resistance, KO mice maintained euglycemia due to increased liver insulin sensitivity. Suppressed mRNA abundance of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase (G6Pase) in KO mice further support this conclusion. PST administration in KO mice stimulated phosphoenolpyruvate carboxykinase and G6Pase mRNA abundance and raised the blood glucose level. In liver cells transfected with G6Pase promoter, PST caused transcriptional activation in a protein kinase C (PKC)-and NO synthase-dependent manner. Thus, PST action may be mediated by suppressing IRS1/ 2-phosphatidylinositol 3-kinase-Akt-FOXO-1 signaling and insulin-induced maturation of SREBP1c by PKC and a high level of NO. The combined effects of conventional PKC and endothelial NO synthase activation by PST can suppress insulin signaling. The rise in blood PST level with age and in diabetes suggests that PST is a negative regulator of insulin sensitivity and glucose homeostasis.Chromogranin A (CHGA/Chga), 2 the index member of the chromogranin/secretogranin protein family (1, 2), is a proprotein that gives rise to biologically active peptides such as the dysglycemic hormone pancreastatin (PST; human CHGA-(250 -301)) (3), the vascular smooth muscle vasodilator vasostatin (human CHGA-(1-76)) (4), and a catecholamine release inhibitory peptide, catestatin (human CHGA-(352-372); bovine Chga-(344 -364)) (5). Several studies, including in vivo analyses of experimental animals (6, 7), showed that PST inhibits insulin release in response to glucose (3), reduces glucose uptake in adipocytes and hepatocytes (8), and triggers glycogenolysis (6). Genetic analysis in humans supports a role for PST in decreasing glucose uptake by ϳ50% (9) while increasing the spillover of free fatty acids but not amino acids. Moreover the PST level is elevated in patients with Type 2 diabetes mellitus (9 -11). Taken together, the data suggest that PST is an important player in metabolism.To further delineate the role of PST in metabolism, we tested whether removal of PST, a negative regulator of insulin action, stabilizes glucose levels in knock-out (KO) mice and protects against metabolic disorders. To t...