Zinc Deficiency Promotes Calcification in Vascular Smooth Muscle Cells Independent of Alkaline Phosphatase Action and Partly Impacted by Pit1 Upregulation

Inorganic phosphate (Pi) is a critical determinant of calcification, and its concentration is regulated by alkaline phosphatase (ALP) and Pit1. ALP is a key regulator of osteogenic calcification and acts by modulating local inorganic phosphate (Pi) concentrations through hydrolyzing pyrophosphate in the extracellular matrix (ECM). Pit1, a sodium-dependent phosphate transporter, regulates calcification via facilitating phosphate uptake within the cells. To investigate whether zinc differentially regulates osteoblastic and vascular calcifications, we examined ALP activity and Pit1 in osteoblastic and vascular smooth muscle cells (VSMCs). Our findings demonstrate that calcification in osteoblastic MC3T3-E1 cells is decreased via diminished ALP action under zinc deficiency. In contrast, zinc-deficiency-induced calcification in VSMCs is independent of ALP action, as demonstrated by very weak ALP activity and expression in calcified VSMCs. In zinc-deficient A7r5 VSMC, P accumulation increased with increasing Na phosphate concentration (3–7 mM) but not with β-GP treatment, which requires ALP activity to generate Pi. Ca deposition also increased with Na phosphate in a dose-dependent manner; in contrast, β-GP did not affect Ca deposition. In osteoblastic cells, Pit1 expression was not affected by zinc treatments. In contrast, Pit1 expression is highly upregulated in A7r5 VSMC under zinc deficiency. Using phosphonoformic acid, a competitive inhibitor of Pit1, we showed that calcification is inhibited in both A7r5 and MC3T3-E1 cells, indicating a requirement for Pit1 in both calcifications. Moreover, the downregulation of VSMC markers under zinc deficiency was restored by blocking Pit1. Taken together, our results imply that zinc-deficiency-induced calcification in VSMC is independent of ALP action in contrast to osteoblastic calcification. Moreover, Pit1 expression in VSMCs is a target for zinc deficiency and may mediate the inhibition of VSMC marker expression under zinc deficiency.

[1]  I. Kwun,et al.  Depletion of Zinc Causes Osteoblast Apoptosis with Elevation of Leptin Secretion and Phosphorylation of JAK2/STAT3 , 2022, Nutrients.

[2]  M. Barbagallo,et al.  Vitamin D Sources, Metabolism, and Deficiency: Available Compounds and Guidelines for Its Treatment , 2021, Metabolites.

[3]  K. Frosch,et al.  Glucose Metabolism in Osteoblasts in Healthy and Pathophysiological Conditions , 2021, International journal of molecular sciences.

[4]  M. Murshed.  Mechanism of Bone Mineralization. , 2018, Cold Spring Harbor perspectives in medicine.

[5]  Kee-Lung Chang,et al.  Arsenic trioxide affects bone remodeling by effects on osteoblast differentiation and function. , 2012, Bone.

[6]  C. Shanahan,et al.  Arterial Calcification in Chronic Kidney Disease: Key Roles for Calcium and Phosphate , 2011, Circulation research.

[7]  G. Dubyak,et al.  Extracellular pyrophosphate metabolism and calcification in vascular smooth muscle. , 2011, American journal of physiology. Heart and circulatory physiology.

[8]  M. Festing,et al.  Phosphate and vascular calcification: Emerging role of the sodium-dependent phosphate co-transporter PiT-1 , 2010, Thrombosis and Haemostasis.

[9]  M. McKee,et al.  Molecular determinants of extracellular matrix mineralization in bone and blood vessels , 2010, Current opinion in nephrology and hypertension.

[10]  Y. Tintut,et al.  The bone–vascular axis in chronic kidney disease , 2010, Current opinion in nephrology and hypertension.

[11]  E. Golub.  Role of matrix vesicles in biomineralization. , 2009, Biochimica et biophysica acta.

[12]  P. Raggi,et al.  Calcification in atherosclerosis , 2009, Nature Reviews Cardiology.

[13]  T. Akizawa,et al.  Vitamin D and vascular calcification in chronic kidney disease. , 2009, Bone.

[14]  M. Budoff,et al.  Association of serum alkaline phosphatase with coronary artery calcification in maintenance hemodialysis patients. , 2009, Clinical journal of the American Society of Nephrology : CJASN.

[15]  C. Giachelli.  The emerging role of phosphate in vascular calcification. , 2009, Kidney international.

[16]  Xianwu Li,et al.  BMP-2 promotes phosphate uptake, phenotypic modulation, and calcification of human vascular smooth muscle cells. , 2008, Atherosclerosis.

[17]  J. Millán,et al.  Novel Inhibitors of Alkaline Phosphatase Suppress Vascular Smooth Muscle Cell Calcification , 2007, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[18]  Xianwu Li,et al.  Sodium-dependent phosphate cotransporters and vascular calcification , 2007, Current opinion in nephrology and hypertension.

[19]  J. Aubin,et al.  Osteoblast Autonomous Pi Regulation via Pit1 Plays a Role in Bone Mineralization , 2007, Molecular and Cellular Biology.

[20]  R. Levy,et al.  Transforming growth factor-beta1 mechanisms in aortic valve calcification: increased alkaline phosphatase and related events. , 2007, The Annals of thoracic surgery.

[21]  W. C. O'Neill,et al.  Pyrophosphate, alkaline phosphatase, and vascular calcification. , 2006, Circulation research.

[22]  Xianwu Li,et al.  Role of the Sodium-Dependent Phosphate Cotransporter, Pit-1, in Vascular Smooth Muscle Cell Calcification , 2006, Circulation research.

[23]  C. Giachelli.  Vascular calcification: in vitro evidence for the role of inorganic phosphate. , 2003, Journal of the American Society of Nephrology : JASN.

[24]  T. Shigematsu,et al.  [Suppressive effects of bisphosphonates on the vascular calcification in ESRD patients]. , 2002, Clinical calcium.

[25]  H. Koyama,et al.  Induction of Bone-Type Alkaline Phosphatase in Human Vascular Smooth Muscle Cells: Roles of Tumor Necrosis Factor-&agr; and Oncostatin M Derived From Macrophages , 2002, Circulation research.

[26]  R. Terkeltaub,et al.  Tissue-nonspecific alkaline phosphatase and plasma cell membrane glycoprotein-1 are central antagonistic regulators of bone mineralization , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  Y. Nishizawa,et al.  Vascular calcification and inorganic phosphate. , 2001, American journal of kidney diseases : the official journal of the National Kidney Foundation.

[28]  S. C. Garner,et al.  Rickets in cation-sensing receptor-deficient mice: an unexpected skeletal phenotype. , 2001, Endocrinology.

[29]  R. Terkeltaub.  Inorganic pyrophosphate generation and disposition in pathophysiology. , 2001, American journal of physiology. Cell physiology.

[30]  M. McKee,et al.  Phosphate regulation of vascular smooth muscle cell calcification. , 2000, Circulation research.

[31]  David J. Anderson,et al.  Genetic ablation of parathyroid glands reveals another source of parathyroid hormone , 2000, Nature.

[32]  E. Mornet.  Hypophosphatasia: The mutations in the tissue‐nonspecific alkaline phosphatase gene , 2000, Human mutation.

[33]  R. Baron,et al.  Targeted ablation of the vitamin D receptor: an animal model of vitamin D-dependent rickets type II with alopecia. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[34]  A. G. Taylor,et al.  The role of alkaline phosphatase in cartilage mineralization. , 1992, Bone and mineral.

[35]  J. Aubin,et al.  Inorganic phosphate added exogenously or released from beta-glycerophosphate initiates mineralization of osteoid nodules in vitro. , 1992, Bone and mineral.

[36]  Xianwu Li,et al.  Regulation of Vascular Calcification Roles of Phosphate and Osteopontin , 2005 .