title: The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle.
abstract: Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.
authors.affiliation: Diabetes Complications Group, School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia; International Islamic University Malaysia, Kuala Lumpur 53100, Malaysia. Electronic address: muhamad.rostam@rmit.edu.au.
authors.forename: Muhamad A
authors.initials: MA
authors.lastname: Rostam
authors.affiliation: Diabetes Complications Group, School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia. Electronic address: danielle.kamato@rmit.edu.au.
authors.forename: Danielle
authors.initials: D
authors.lastname: Kamato
authors.affiliation: Discipline of Cell Biology, School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia. Electronic address: terry.piva@rmit.edu.au.
authors.forename: Terence J
authors.initials: TJ
authors.lastname: Piva
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Centre, Guangzhou, China; Faculty of Health Sciences, University of Macau, Taipa, Macau, China. Electronic address: whzheng1233@163.com.
authors.forename: Wenhua
authors.initials: W
authors.lastname: Zheng
authors.affiliation: Diabetes Complications Group, School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia; School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, Queensland 4102, Australia. Electronic address: p.little@uq.edu.au.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Little
authors.affiliation: Diabetes Complications Group, School of Health and Biomedical Sciences, RMIT University, Bundoora, Victoria 3083, Australia; Department of Medicine and Immunology, Central and Eastern Clinical School, Monash University, Melbourne, Victoria 3800, Australia. Electronic address: narin.osman@rmit.edu.au.
authors.forename: Narin
authors.initials: N
authors.lastname: Osman
mesh_terms: D002809:Chondroitin Sulfates / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004187:Disaccharides / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006801:Humans; D008954:Models, Biological; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051899:Smad2 Protein / Q000737:chemistry* / Q000378:metabolism*; D013997:Time Factors; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D004187:Disaccharides; D006025:Glycosaminoglycans; D012333:RNA, Messenger; D051899:Smad2 Protein; D016212:Transforming Growth Factor beta; D002809:Chondroitin Sulfates; D019869:Phosphatidylinositol 3-Kinases; D018844:Cyclin-Dependent Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases
keywords: Atherosclerosis; Glycosaminoglycan; Signalling; Smad; Transforming growth factor-β; Vascular smooth muscle