TGF-β signalling pathways controlling glycosaminoglycan chain synthesizing enzyme gene expression as a therapeutic target for atherosclerosis

Rostam, M 2016, TGF-β signalling pathways controlling glycosaminoglycan chain synthesizing enzyme gene expression as a therapeutic target for atherosclerosis, Doctor of Philosophy (PhD), Medical Sciences, RMIT University.

Document type: Thesis
Collection: Theses

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Title TGF-β signalling pathways controlling glycosaminoglycan chain synthesizing enzyme gene expression as a therapeutic target for atherosclerosis
Author(s) Rostam, M
Year 2016
Abstract Hyperelongation of glycosaminoglycan (GAG) chains on proteoglycans synthesized by the vascular smooth muscle cells (VSMC) increases lipoprotein binding in the blood vessel wall and in the development of atherosclerotic plaque. GAG chain synthesis occurs via post translational modification of the proteoglycan core protein driven by the concerted action of a wide range of GAG synthesizing enzymes. An in vivo study showed that elevated GAG synthesizing enzyme gene expression contributes to the development of atherosclerosis. In human VSMC, transforming growth factor-β (TGF-β) regulates GAG chain hyperelongation via serine/threonine kinases ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, the involvement of TGF β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate GAG synthesizing enzymes to mediate their effects on proteoglycans was investigated. Experiments were conducted on cultured human VSMC in the presence of TGF β. Quantitative RT-PCR analysis demonstrated the upregulation of xylosyltransferase-1 (XT 1), chondroitin sulfate synthase-1 (ChSy 1), chondroitin polymerising factor (ChPF) and chondroitin sulfotransferase 1 (C4ST 1) mRNA expression. Elevated mRNA expression correlated with the increased protein expression for ChSy 1 and C4ST 1, which was determined by Western blot analysis. Proteoglycan synthesis was assessed by measuring [35S] SO4 incorporation into proteoglycans via the cetylpyridinium chloride precipitation assay. Proteoglycan and GAG chain size were determined by SDS-PAGE and size exclusion chromatography. TGF β stimulation increased proteoglycan synthesis and size indicating GAG chain hyperelongation in VSMC. Investigation on the TGF β signalling pathways using pharmacological experiments revealed that ERK, p38, PI3K and CDK differentially regulated mRNA expression of the enzymes XT 1, ChSy-1 and C4ST 1. Four individual residues (Thr220, Ser245, Ser250 and Ser255) in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of GAG synthesizing enzymes. Further investigation to identify the involvement of multiple CDK isoforms in proteoglycan modification demonstrated that CDK1, CDK2, CDK4, CDK6, CDK7 and CDK9 are present in human VSMC. Studies utilising CDK inhibitors CVT313 (CDK1 and CDK2 inhibitor), PD0332991 (CDK4 and CDK6 inhibitor), roscovitine (CDK2, CDK7, and CDK9 inhibitor) and flavopiridol (CDK1, CDK2, CDK4, CDK6, CDK7 and CDK9 inhibitor) suggested the involvement of these CDKs in biglycan, XT 1, ChSy-1 and C4ST-1 mRNA expression ultimately affecting proteoglycan size. Results also suggest that CDK4 and CDK6 signals exclusively through the Thr220 residue in the Smad2 linker region while the other CDKs (CDK1, CDK2, CDK7 and CDK9) phosphorylate serine residues (Ser245/255) in the TGF β signalling pathway. This study shows that CDKs play a role beyond cell cycling events particulary in proteoglycan GAG chain synthesis and as such they are potential therapeutic targets to prevent atherosclerosis. In conclusion, the current study has identified that the mRNA regulation of three GAG synthesizing enzymes XT 1, ChSy 1 and C4ST 1 in VSMC to involve the signalling pathway from TGF β to Smad2 linker region phosphorylation, exists in these cells. Together, these findings provide a better understanding of the complex pathway that contribute to GAG chain hyperelongation and facilitate the identification of a specific therapeutic target(s) for events important in the early development of atherosclerosis.
Degree Doctor of Philosophy (PhD)
Institution RMIT University
School, Department or Centre Medical Sciences
Subjects Signal Transduction
Biochemistry and Cell Biology not elsewhere classified
Cardiovascular Medicine and Haematology not elsewhere classified
Keyword(s) TGF-β
Cell signalling
Smad linker region
Vascular smooth muscle
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Created: Fri, 25 Nov 2016, 09:51:28 EST by Keely Chapman
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