NR4A receptors up-regulate the antiproteinase alpha-2 macroglobulin (A2M) and modulate MMP-2 and MMP-9 in vascular smooth muscle cells

Summary

Matrix metalloproteinases (MMPs) are associated with tissue remodelling and repair. In non-vascular tissues, NR4A receptors have been involved in the regulation of MMPs by transcriptional repression mechanisms. Here, we analyse alternative mechanisms involving NR4A receptors in the modulation of MMP activity in vascular smooth muscle cells (VSMC). Lentiviral overexpression of NR4A receptors (NOR-1, Nurr1 and Nur77) in human VSMC strongly decreased MMP-2 and MMP-9 activities (analysed by zymography and DQ-gelatin assays) and protein levels. NR4A receptors also down-regulated MMP-2 mRNA levels. Real-time PCR analysis evidenced that alpha-2-macroglobulin (A2M), but not other MMP inhibitors (TIMP-1 and TIMP-2) were up-regulated in NR4A-transduced cells. Interestingly, A2M was expressed in human vascular tissues including the smooth muscle media layer. While NR4A receptors increased A2M expression and secretion in VSMC, NR4A knockdown significantly reduced basal A2M expression in these cells. The direct transcriptional regulation of the human A2M promoter by NR4A receptors was characterised in luciferase reporter assays, electrophoretic mobility shift assays and by chromatin immunoprecipitation, identifying a NGFI-B response element (NBRE-71/-64) essential for the NR4A-mediated induction. The blockade of A2M partially prevented the reduction of MMPs activity observed in NR4A-transduced cells. Although mouse A2M promoter was unresponsive to NR4A receptors, vascular MMP expression was attenuated in transgenic mice over-expressing human NOR-1 in VSMC challenged with lipopolysaccharide. Our results show that the panproteinase inhibitor A2M is expressed in the vasculature and that NR4A receptors modulate VSMC MMP activity by several mechanisms including the up-regulation of A2M.

Keywords

Matrix metalloproteinases - vascular remodelling - gene expression - transcription factors - smooth muscle cells

• References

• 1 Schober A, Zernecke A. Chemokines in vascular remodelling. Thromb Haemost 2007; 97: 730-737.
• 2 Renna NF, de Las Heras N, Miatello RM. Pathophysiology of vascular remodelling in hypertension. Int J Hypertens 2013; 2013: 808353.
• 3 Chen Q, Jin M, Yang F. et al. Matrix metalloproteinases: inflammatory regulators of cell behaviors in vascular formation and remodelling. Mediators Inflamm 2013; 2013: 928315.
• 4 Ketelhuth DF, Bäck M. The role of matrix metalloproteinases in atherothrombosis. Curr Atheroscler Rep 2011; 13: 162-169.
• 5 Nagase H, Visse R, Murphy G. Structure and function of matrix metalloproteinases and TIMPs. Cardiovasc Res 2006; 69: 562-573.
• 6 Rehman AA, Ahsan H, Khan FH. α-2-Macroglobulin: a physiological guardian. J Cell Physiol 2013; 228: 1665-1675.
• 7 Schmidt B, Mitchell L, Ofosu FA. et al. Alpha-2-macroglobulin is an important progressive inhibitor of thrombin in neonatal and infant plasma. Thromb Haemost 1989; 62: 1074-1077.
• 8 Schaller J, Gerber SS. The plasmin-antiplasmin system: structural and functional aspects. Cell Mol Life Sci 2011; 68: 785-801.
• 9 Ossege LM, Voss B, Müller KM. Expression and secretion of alpha-2-macroglobulin by dust-stimulated alveolar macrophages. Pneumologie 1994; 48: 236-239.
• 10 Boel E, Kristensen T, Petersen CM. et al. Expression of human alpha 2-macroglobulin cDNA in baby hamster kidney fibroblasts: secretion of high levels of active alpha 2-macroglobulin. Biochemistry 1990; 29: 4081-4087.
• 11 Shi DL, Savona C, Gagnon J. et al. Transforming growth factor-beta stimulates the expression of alpha 2-macroglobulin by cultured bovine adrenocortical cells. J Biol Chem 1990; 265: 2881-2887.
• 12 Stouffer GA, LaMarre J, Gonias SL. et al. Activated alpha 2-macroglobulin and transforming growth factor-beta 1 induce a synergistic smooth muscle cell proliferative response. J Biol Chem 1993; 268: 18340-18344.
• 13 Ikari Y, Fujikawa K, Yee KO. et al. Alpha(1)-proteinase inhibitor, alpha(1)-antichymotrypsin, or alpha(2)-macroglobulin is required for vascular smooth muscle cell spreading in three-dimensional fibrin gel. J Biol Chem 2000; 275: 12799-12805.
• 14 Ikari Y, Mulvihill E, Schwartz SM. alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells. J Biol Chem 2001; 276: 11798-11803.
• 15 Martínez-González J, Rius J, Castellό A. et al. Neuron-derived orphan receptor-1 (NOR-1) modulates vascular smooth muscle cell proliferation. Circ Res 2003; 92: 96-103.
• 16 Arkenbout EK, de Waard V, van Bragt M. et al. Protective function of transcription factor TR3 orphan receptor in atherogenesis: decreased lesion formation in carotid artery ligation model in TR3 transgenic mice. Circulation 2002; 106: 1530-1535.
• 17 Bonta PI, van Tiel CM, Vos M. et al. Nuclear receptors Nur77, Nurr1, and NOR-1 expressed in atherosclerotic lesion macrophages reduce lipid loading and inflammatory responses. Arterioscler Thromb Vasc Biol 2006; 26: 2288-2294.
• 18 Nomiyama T, Zhao Y, Gizard F. et al. Deficiency of the NR4A neuron-derived orphan receptor-1 attenuates neointima formation after vascular injury. Circulation 2009; 119: 577-586.
• 19 Rodríguez-Calvo R, Guadall A, Calvayrac O. et al. Over-expression of neuronderived orphan receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury. Hum Mol Genet. 2013; 22: 1949-1959.
• 20 Martínez-González J, Badimon L. The NR4A subfamily of nuclear receptors: new early genes regulated by growth factors in vascular cells. Cardiovasc Res 2005; 65: 609-618.
• 21 Wang Z, Benoit G, Liu J, Prasad S. et al. Structure and function of Nurr1 identifies a class of ligand-independent nuclear receptors. Nature 2003; 423: 555-560.
• 22 Liu D, Jia H, Holmes DI, Stannard A. et al. Vascular endothelial growth factorregulated gene expression in endothelial cells: KDR-mediated induction of Egr3 and the related nuclear receptors Nur77, Nurr1, and Nor 1. Arterioscler Thromb Vasc Biol 2003; 23: 2002-2007.
• 23 Churchman AT, Siow RC. Isolation, culture and characterisation of vascular smooth muscle cells. Methods Mol Biol 2009; 467: 127-138.
• 24 Nomiyama T, Nakamachi T, Gizard F. et al. The NR4A orphan nuclear receptor NOR1 is induced by platelet-derived growth factor and mediates vascular smooth muscle cell proliferation. J Biol Chem 2006; 281: 33467-33476.
• 25 Martorell L, Martínez-González J, Crespo J. et al. Neuron-derived orphan receptor-1 (NOR-1) is induced by thrombin and mediates vascular endothelial cell growth. J Thromb Haemost 2007; 05: 1766-1773.
• 26 Martorell L, Martínez-González J, Rodríguez C. et al. Thrombin and proteaseactivated receptors (PARs) in atherothrombosis. Thromb Haemost 2008; 99: 305-315.
• 27 Zhao Y, Bruemmer D. NR4A orphan nuclear receptors: transcriptional regulators of gene expression in metabolism and vascular biology. Arterioscler Thromb Vasc Biol 2010; 30: 1535-1541.
• 28 Martorell L, Gentile M, Rius J, Rodríguez C. et al. The hypoxia-inducible factor 1/NOR-1 axis regulates the survival response of endothelial cells to hypoxia. Mol Cell Biol 2009; 29: 5828-5842.
• 29 Thakar RG, Cheng Q, Patel S. et al. Cell-shape regulation of smooth muscle cell proliferation. Biophys J 2009; 96: 3423-3432.
• 30 Gruber F, Hufnagl P, Hofer-Warbinek R. et al. Direct binding of Nur77/NAK-1 to the plasminogen activator inhibitor 1 (PAI-1) promoter regulates TNF alpha -induced PAI-1 expression. Blood 2003; 101: 3042-3048.
• 31 Mix KS, Attur MG, Al-Mussawir H. et al. Transcriptional repression of matrix metalloproteinase gene expression by the orphan nuclear receptor NURR1 in cartilage. J Biol Chem 2007; 282: 9492-9504.
• 32 Ohkura N, Ito M, Tsukada T. et al. Structure, mapping and expression of a human NOR-1 gene, the third member of the Nur77/NGFI-B family. Biochim Biophys Acta 1996; 1308: 205-214.
• 33 Wingate AD, Campbell DG, Peggie M. et al. Nur77 is phosphorylated in cells by RSK in response to mitogenic stimulation. Biochem J 2006; 393: 715-724.
• 34 Calvayrac O, Rodríguez-Calvo R, Alonso J. et al. CCL20 is increased in hypercholesterolemic subjects and is upregulated by LDL in vascular smooth muscle cells: role of NF-κB. Arterioscler Thromb Vasc Biol 2011; 31: 2733-2741.
• 35 Castro DS, Hermanson E, Joseph B. et al. Induction of cell cycle arrest and morphological differentiation by Nurr1 and retinoids in dopamine MN9D cells. J Biol Chem 2001; 276: 43277-43284.
• 36 Sottrup-Jensen L, Sand O, Kristensen L. et al. The alpha-macroglobulin bait region. Sequence diversity and localisation of cleavage sites for proteinases in five mammalian alpha-macroglobulins. J Biol Chem 1989; 264: 15781-15789.
• 37 Pei L, Waki H, Vaitheesvaran B. et al. NR4A orphan nuclear receptors are transcriptional regulators of hepatic glucose metabolism. Nat Med 2006; 12: 1048-1055.
• 38 Mohan HM, Aherne CM, Rogers AC. et al. Molecular pathways: the role of NR4A orphan nuclear receptors in cancer. Clin Cancer Res 2012; 18: 3223-3228.
• 39 McMorrow JP, Murphy EP. Inflammation: a role for NR4A orphan nuclear receptors?. Biochem Soc Trans 2011; 39: 688-693.
• 40 Sayegh RA, Tao XJ, Leykin L. et al. Endometrial alpha-2 macroglobulin; localisation by in situ hybridisation and effect on mouse embryo development in vitro. J Clin Endocrinol Metab 1997; 82: 4189-4195.
• 41 He H, McCartney DJ, Wei Q. et al. Characterisation of a murine alpha 2 macroglobulin gene expressed in reproductive and cardiovascular tissue. Biol Reprod 2005; 72: 266-275.
• 42 Bonta PI, Matlung HL, Vos M. et al. Nuclear receptor Nur77 inhibits vascular outward remodelling and reduces macrophage accumulation and matrix metalloproteinase levels. Cardiovasc Res 2010; 87: 561-568.
• 43 You B, Jiang YY, Chen S. et al. The orphan nuclear receptor Nur77 suppresses endothelial cell activation through induction of IkappaBalpha expression. Circ Res 2009; 104: 742-749.
• 44 Saijo K, Winner B, Carson CT. et al. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell 2009; 137: 47-59.
• 45 Harant H, Lindley IJ. Negative cross-talk between the human orphan nuclear receptor Nur77/NAK-1/TR3 and nuclear factor-kappaB. Nucleic Acids Res 2004; 32: 5280-5290.
• 46 Adya R, Tan BK, Chen J. et al. Nuclear factor-kappaB induction by visfatin in human vascular endothelial cells: its role in MMP-2/9 production and activation. Diabetes Care 2008; 31: 758-760.
• 47 Mix KS, McMahon K, McMorrow JP. et al. Orphan nuclear receptor NR4A2 induces synoviocyte proliferation, invasion, and matrix metalloproteinase 13 transcription. Arthritis Rheum 2012; 64: 2126-2136.
• 48 Zhang X, Yan G, Diao Z. et al. NUR77 inhibits the expression of TIMP2 and increases the migration and invasion of HTR-8/SVneo cells induced by CYR61. Placenta 2012; 33: 561-567.
• 49 Zhao Y, Liu Y, Zheng D. Alpha 1-antichymotrypsin/SerpinA3 is a novel target of orphan nuclear receptor Nur 77. FEBS J. 2008; 275: 1025-1038.
• 50 Lerner L, Henriksen MA, Zhang X. et al. STAT3-dependent enhanceosome assembly and disassembly: synergy with GR for full transcriptional increase of the alpha 2-macroglobulin gene. Genes Dev 2003; 17: 2564-2577.