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Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated Into Chondrocytes Generates Pi but Not Ppi

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Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated Into Chondrocytes Generates Pi but Not Ppi International Journal of Molecular Sciences Article Hydrolysis of Extracellular ATP by Vascular Smooth Muscle Cells Transdifferentiated into Chondrocytes Generates Pi but Not PPi Rene Buchet 1,* , Camille Tribes 1, Valentine Rouaix 1, Bastien Doumèche 1 , Michele Fiore 1 , Yuqing Wu 2, David Magne 1 and Saida Mebarek 1 1 Institute for Molecular and Supramolecular Chemistry and Biochemistry, Université Lyon 1, French National Centre for Scientific Research, F-69622 Lyon, France; [email protected] (C.T.); [email protected] (V.R.); [email protected] (B.D.); michele.fi[email protected] (M.F.); [email protected] (D.M.); [email protected] (S.M.) 2 State Key Laboratory of Supramolecular Structure and Materials, Institute of Theoretical Chemistry, Jilin University, Changchun 130012, China; [email protected] * Correspondence: [email protected] Abstract: (1) Background: Tissue non-specific alkaline phosphatase (TNAP) is suspected to induce atherosclerosis plaque calcification. TNAP, during physiological mineralization, hydrolyzes the min- eralization inhibitor inorganic pyrophosphate (PPi). Since atherosclerosis plaques are characterized by the presence of necrotic cells that probably release supraphysiological concentrations of ATP, we explored whether this extracellular adenosine triphosphate (ATP) is hydrolyzed into the mineraliza- Citation: Buchet, R.; Tribes, C.; tion inhibitor PPi or the mineralization stimulator inorganic phosphate (Pi), and whether TNAP is Rouaix, V.; Doumèche, B.; Fiore, M.; involved. (2) Methods: Murine aortic smooth muscle cell line (MOVAS cells) were transdifferentiated Wu, Y.; Magne, D.; Mebarek, S. into chondrocyte-like cells in calcifying medium, containing ascorbic acid and β-glycerophosphate. Hydrolysis of Extracellular ATP by ATP hydrolysis rates were determined in extracellular medium extracted from MOVAS cultures Vascular Smooth Muscle Cells during their transdifferentiation, using 31P-NMR and IR spectroscopy. (3) Results: ATP and PP Transdifferentiated into i hydrolysis by MOVAS cells increased during transdifferentiation. ATP hydrolysis was sequential, Chondrocytes Generates Pi but Not PPi. Int. J. Mol. Sci. 2021, 22, 2948. yielding adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine without https://doi.org/10.3390/ijms22062948 any detectable PPi. The addition of levamisole partially inhibited ATP hydrolysis, indicating that TNAP and other types of ectonucleoside triphoshatediphosphohydrolases contributed to ATP hy- Academic Editors: Hidenori Koyama, drolysis. (4) Conclusions: Our findings suggest that high ATP levels released by cells in proximity to Catalina Carrasco-Pozo and vascular smooth muscle cells (VSMCs) in atherosclerosis plaques generate Pi and not PPi, which may Christian Jung exacerbate plaque calcification. Received: 1 February 2021 Keywords: ATP; alkaline phosphatase; aortic smooth muscle cell; calcification; chondrocyte; plaque Accepted: 11 March 2021 calcification; kinetics; MOVAS; nucleotide; pyrophosphate Published: 14 March 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in 1. Introduction published maps and institutional affil- iations. Atherosclerosis calcification is a predictor of cardiovascular mortality [1]. Vulner- able plaques are characterized by a thin fibrous cap, macrophage infiltration, a large necrotic core, intraplaque hemorrhage, and calcification [2–6]. During atherosclerosis plaque calcification in mice, vascular smooth muscle cells (VSMCs) transdifferentiate into chondrocyte-like cells, which then induce calcification in the vascular walls [7–9]. Like Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. osteoblasts and growth plate chondrocytes, these cells express bone/cartilage-specific Runt- This article is an open access article related transcription factor 2 (RUNX2), triggering the expression of bone/cartilage-specific distributed under the terms and proteins, such as osteocalcin (encoded by Bglap)[10,11]. Moreover, these cells have in- conditions of the Creative Commons creased tissue non-specific alkaline phosphatase (TNAP) activity and produce extracellular Attribution (CC BY) license (https:// vesicles which accumulate calcium-phosphate crystals [11–13]. TNAP (E.C. 3.1.3.1) is a creativecommons.org/licenses/by/ phosphomonoesterase with a broad range of substrates in vitro including nucleotides and 4.0/). pyrophosphate (Figure1). Int. J. Mol. Sci. 2021, 22, 2948. https://doi.org/10.3390/ijms22062948 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 2948 2 of 16 Int. J. Mol. Sci. 2021, 22, 2948 cles which accumulate calcium-phosphate crystals [11–13]. TNAP (E.C. 3.1.3.1) is a2 phos- of 16 phomonoesterase with a broad range of substrates in vitro including nucleotides and py- rophosphate (Figure 1). FigureFigure 1. 1.Extracellular Extracellular adenosineadenosine triphosphatetriphosphate (ATP)(ATP) isis hydrolyzed hydrolyzed by by ectonucleotide ectonucleotide pyrophos-pyrophos- phatasephatase phosphodiesterase phosphodiesterase (ENPP1)(ENPP1) toto form form PP PPi iand and adenosine adenosine monophosphate monophosphate (AMP). (AMP). AMP AMP is is furtherfurther hydrolyzed hydrolyzed by by CD73 CD73 or or tissue tissue non-specific non-specific alkaline alkaline phosphatase phosphatase (TNAP) (TNAP) to to form form adenosine adenosine andand P Pi.i. ATP ATP and and adenosine adenosine diphosphate diphosphate (ADP) (ADP) can can also also be be hydrolyzed hydrolyzed by by TNAP, TNAP, CD39 CD39 (ENTPD1), (ENTPD1), oror other other types types of of ectonucleoside ectonucleoside triphoshatediphosphohydrolases. triphoshatediphosphohydrolases. TheThehydrolysis hydrolysis of of PP PPi,i, which which forms two Pi, is mainly catalyzed by TNAP. ATP can be transported from intracellular to extracel- forms two Pi, is mainly catalyzed by TNAP. ATP can be transported from intracellular to extracellular lular media via connexin, pannexin, or ATP anion channels, while PPi is transported via progressive media via connexin, pannexin, or ATP anion channels, while PPi is transported via progressive ankylosis protein homolog (ANKH). Eventually, large quantities of nucleotides are also released ankylosis protein homolog (ANKH). Eventually, large quantities of nucleotides are also released from from necrotic cells. necrotic cells. TNAPTNAP isis thethe keykey enzymeenzyme during calcification calcification since since it it hydrolyzes hydrolyzes extracellular extracellular pyro- py- phosphate (PPi), a strong inhibitor of apatite formation [14]. Extracellular PPi originates rophosphate (PPi), a strong inhibitor of apatite formation [14]. Extracellular PPi originates from: (i) intracellular PPi transported into the extracellular medium by progressive anky- from: (i) intracellular PPi transported into the extracellular medium by progressive anky- losislosis protein protein homolog homolog (ANKH) (ANKH) [15[15];]; and and (ii) (ii) from from the the hydrolysis hydrolysis of of extracellular extracellular ATP ATP by by ectonucleotide pyrophosphatase phosphodiesterase (ENPP1) to produce PPi and AMP ectonucleotide pyrophosphatase phosphodiesterase (ENPP1) to produce PPi and AMP (Figure 1) [16]. Under physiological conditions, normal levels of extracellular PPi and (Figure1)[ 16]. Under physiological conditions, normal levels of extracellular PPi and otherother calcification calcification inhibitors, inhibitors, including including osteopontin, osteopontin, matrix-Gla matrix-Gla proteins, proteins, and and Fetuin Fetuin A, are A, sufficientare sufficient to prevent to prevent vascular vascular calcification calcification by inhibiting by inhibiting the formation the formation of apatite. of apatite. However, How- i underever, under pathological pathological conditions, conditions, where where the PP thei concentration PP concentration can decrease, can decrease, for example for exam- in theple case in the of systemiccase of systemic ENPP1 deficiency ENPP1 deficiency (Generalized (Generalized Arterial Calcification Arterial Calcification of Infancy), of it In- is sufficientfancy), it tois inducesufficient vascular to induce calcification vascular [calcification17]. Therefore, [17]. the Therefore, Pi/PPi ratio the regulates Pi/PPi ratio apatite regu- formationlates apatite [18 ,formation19]. Besides [18,19]. PPi, ATP Besides and ADPPPi, ATP can alsoand inhibitADP can apatite also formationinhibit apatite [20]. Asfor- amation result of[20]. its As ability a result to hydrolyze of its ability PP ito, TNAP hydrolyze is a putativePPi, TNAP drug is a target putative to treat drug vascular target to calcificationtreat vascular [21 calcification]. Under physiological [21]. Under conditions, physiological the conditions, concentration the of concentration extracellular of ATP ex- istracellular found to beATP in theis found 10–1000 to nMbe in range the [1022–,23100].0 Upon nM range atherosclerosis [22,23]. Upon around atherosclerosis a threefold (plus)around increase a threefold in extracellular (plus) increase ATP in is extracellular released from ATP the is cells released [24]. from Necrotic the cells cells [24]. release Ne- highcrotic quantities cells release of ATPhigh thatquantities act as of danger-signal ATP that act recruiting as danger leukocytes-signal recruiting [25]. ATPleukocytes is ac- tively[25]. ATP released is actively from activated released or from stressed activated cells [ 26or, 27stressed] and passively cells [26,27] from and necrotic passively cells from [28]
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