Transforming growth factor–β1 mediated CHST11 and CHSY1 mRNA expression is ROS dependent in vascular smooth muscle cells

Abstract

Transforming growth factor (TGF)-β1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-β1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-β1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-β1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-β1 mediated Smad2 linker region phosphorylation. TGF-β1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-β1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-β1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-β1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation. KeywordsAtherosclerosis . Nox . Reactive oxygen species . Mitogen activated protein kinases . Glycosaminoglycan Abbreviations CHST11 Chondroitin 4-Ο-sulfotransferase 1 CHSY1 Chondroitin synthase 1 DPI Diphenyleneiodonium ERK Extracellular signal-regulated kinase GAG Glycosaminoglycan JNK C-Jun N-terminal kinase MAPKs Mitogen activated protein kinases. Nox Nicotinamide adenine dinucleotide phosphate oxidase ROS Reactive oxygen species TGFBR1 Transforming growth factor-β receptor type 1 TGF-β1 Transforming growth factor β1 VSMCs Vascular smooth muscle cells * Hossein Babaahmadi-Rezaei

Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation

“…Recent observations by our lab show that in keratinocytes, the GPCR agonist thrombin transactivates the EGFR to phosphorylate the Smad2 linker region [27]. Smad2 linker region phosphorylation drives proteoglycan synthesis [22] and GAG gene expression [23][24][25]…”

GPCR transactivation signalling in vascular smooth muscle cells: role of NADPH oxidases and reactive oxygen species

“…Pharmacological inhibition of ROS/Nox with NAC, DPI and apocynin has no effect on carboxy terminal phosphorylation of Smad2 (data not published). However, DPI and apocynin prevent TGF-β-induced phosphorylation of Smad2 linker region (22). Transfection of human pulmonary artery SMCs with dominant negative Smad2 and Smad3 blocked Nox4 gene expression and ROS production caused by TGF-β, suggesting that TGF-β triggers Nox4-derived ROS generation via the Smad2/3 pathway (104).…”

“…MAPKs are downstream components of TGF-β signalling (106,107). In human VSMCs, TGF-β mediated ROS production leads to the activation of MAPK, ERK and p38 (22). Antioxidants, NAC and catalase, suppress ROS production by TGF-β and inhibit the phosphorylation of ERK1/2 and p38 in rat renal epithelial cells, resulting in the prevention of TGF-β-induced epithelial-mesenchymal transition (23).…”

“…Reactive oxygen species (ROS) are highly chemically reactive species arising from multiple metabolic and enzymatic sources inside all cells (20). ROS play a role in S/TKR-and PTKR-mediated signalling pathways (21,22,23) and in the GPCR transactivation of growth factor receptors (24,25). Therefore, understanding the role of ROS in GPCR transactivation signalling of both S/TKR and PTKR may reveal a common therapeutic target for all GPCR transactivation-dependent signalling.…”

Unifferentiated Connective Tissue Dysplasia as a Potential Predictor of Arterial Hypertension Development in Patients With Type 2 Diabetes Mellitus

“…Such a proinflammatory condition affects the function of functionally active cells (pericytes, smooth muscle cells, fibroblasts), which worsens reparative processes in vessels and tissues, including angiogenesis [12]. One of the mechanisms of the pathological effect of hyperglycemia is also thought to be the increase in the activity of prophylactic factors, in particular, TGF-β, which is a modulator of proteoglycan synthesis in the extracellular matrix [12][13]. is The increase in excretion of type IV collagen in patients with diabetes, with diabetic nephropathy and nephropathy of another genesis serves as an evidence of connective tissue metabolic abnormalities role in the extracellular matrix [14][15].…”