Toll-like Receptor 4 Stimulates Gene Expression via Smad2 Linker Region Phosphorylation in Vascular Smooth Muscle Cells

Rizwana Afroz; Ying Zhou; Peter J Little; Suowen Xu; Raafat Mohamed; Jennifer Stow; Danielle Kamato

doi:10.1021/acsptsci.9b00113

Toll-like Receptor 4 Stimulates Gene Expression via Smad2 Linker Region Phosphorylation in Vascular Smooth Muscle Cells

ACS Pharmacol Transl Sci. 2020 Mar 11;3(3):524-534. doi: 10.1021/acsptsci.9b00113. eCollection 2020 Jun 12.

Authors

Rizwana Afroz¹, Ying Zhou¹, Peter J Little^{1

2}, Suowen Xu³, Raafat Mohamed^{1

4}, Jennifer Stow⁵, Danielle Kamato^{1

2}

Affiliations

¹ School of Pharmacy, Pharmacy Australia Centre of Excellence, The University of Queensland, Woolloongabba, Queensland 4102, Australia.
² Department of Pharmacy, Xinhua College of Sun Yat-sen University, Tianhe District, Guangzhou 510520, China.
³ Division of Life Science and Medicine, University of Science and Technology of China, Hefei, Anhui China.
⁴ Department of Basic Sciences, College of Dentistry, University of Mosul, Mosul, Iraq.
⁵ Institute of Molecular Bioscience, The University of Queensland, Woolloongabba, Queensland 4102, Australia.

Abstract

Atherosclerosis begins in the vessel wall with the retention of low density lipoproteins to modified proteoglycans with hyperelongated glycosaminoglycan (GAG) chains. Bacterial infections produce endotoxins such as lipopolysaccharide that exacerbate the outcome of atherosclerosis by generating a heightened state of inflammation. Lipopolysaccharide (LPS) via its toll-like receptor (TLR) is well-known for its role in mediating an inflammatory response in the body. Emerging evidence demonstrates that TLRs are involved in regulating vascular functions. In this study we sought to investigate the role of LPS in proteoglycan modification and GAG chain elongation, and we hypothesize that LPS will signal via Smad2 dependent pathways to regulate GAG chain elongation. The in vitro model used human aortic vascular smooth muscle cells. GAG gene expression was assessed by quantitative real-time polymerase chain reaction. Western blotting was performed using whole-cell protein lysates to assess the signaling pathway. LPS via TLR4 stimulates the expression of GAG synthesizing enzymes to an equal extent to traditional cardiovascular agonists. LPS phosphorylates the Smad2 linker region via TAK-1/MAPK dependent pathways which correlated with genes associated with GAG chain initiation and elongation. The well-characterized role of LPS in inflammation and our data on GAG gene expression demonstrates that GAG chain elongation is the earliest marker of the inflammatory cascade in atherosclerosis development.