Rabbit Anti-Human ENPP1 Polyclonal antibody

Calcific aortic valve disease is the most common heart valve disorder. So far, there is no medical treatment for calcific aortic valve disease. The expression of ectonucleotidases, which metabolize nucleotides into phosphate products, may influence the calcification of the aortic valve. In this study, we investigated if the administration of an ectonucleotidase inhibitor, ARL67156 (6-N,N-Diethyl-D-beta,gamma-dibromomethyleneATP trisodium salt), may prevent the calcification of the aortic valve in the warfarin-induced mineralization rat model. Male Wistar rats were treated with warfarin or warfarin +ARL67156 for 28 days. All rats had comprehensive Doppler-echocardiographic studies at 28 day. A gene profiling of ectonucleotidases expressed in aortas of rats was documented by quantitative real-time PCR. The amount of calcium was determined by quantitative method and von Kossa staining. Ex vivo cultures of rat aortas were also used to further assess the effect of ARL67156 on the calcifying process and Akt signaling. Mineralization of the aorta/aortic valve was documented in warfarin-treated rats and was accompanied by the development of aortic stenosis. These changes were paralleled by an increased of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). Administration of the ectonucleotidase inhibitor, ARL67156 prevented the development of aortic stenosis by lowering the level of apoptosis and mineralization of the aortic valve/aorta. In addition, ARL67156 normalized the level of pAkt, an important kinase involved in the survival pathway. Inhibition of ectonucleotidase activity prevented the development of calcific aortic valve disease in a rat model. On that account, ectonucleotidase may represent a novel target in the treatment of calcific aortic valve disease. (C) 2012 Elsevier B.V. All rights reserved.

To examine the roles of FGF and ERK MAPK signaling in osteocyte differentiation and function, we performed microarray analyses using the osteocyte cell line MLO-Y4. This experiment identified a number of mineralization-related genes that were regulated by FGF2 in an ERK MAPK-dependent manner. Real-time PCR analysis indicated that FGF2 upregulates Ank, Enpp1, Mgp, Slc20a1, and Dmp1 in MLO-Y4 cells. Consistent with this observation, the selective FGF receptor inhibitor PD173074 decreased Ank, Enpp1, Slc20a1, and Dmp1 mRNA expression in mouse calvaria in organ culture. Since Dmp1 plays a central role in osteocyte differentiation and mineral homeostasis, we further analyzed FGF regulation of Dmp1. Similar to FGF2, FGF23 upregulated Dmp1 expression in MLO-Y4 cells in the presence of Klotho. Furthermore, increased extracellular phosphate levels partially inhibited FGF2-induced upregulation of Dmp1 mRNA expression, suggesting a coordinated regulation of Dmp1 expression by FGF signaling and extracellular phosphate. In MLO-Y4 osteocytes and in MC3T3E1 and primary calvaria osteoblasts, U0126 strongly inhibited both basal expression of Dmp1 mRNA and FGF2-induced upregulation. Consistent with the in vitro observations, real-time PCR and immunohistochemical analysis showed a strong decrease in Dmp1 expression in the skeletal elements of ERK1 (-/-) ; ERK2 (flox/flox) ; Prx1-Cre mice. Furthermore, scanning electron microscopic analysis revealed that no osteocytes with characteristic dendritic processes develop in the limbs of ERK1 (-/-) ; ERK2 (flox/flox) ; Prx1-Cre mice. Collectively, our observations indicate that FGF signaling coordinately regulates mineralization-related genes in the osteoblast lineage and that ERK signaling is essential for Dmp1 expression and osteocyte differentiation.