Smad and p38 MAP Kinase-mediated Signaling of Proteoglycan Synthesis in Vascular Smooth Muscle

Harsha Dadlani,Mandy L Ballinger,Narin Osman,Robel Getachew,Peter J Little

doi:10.1074/jbc.m703125200

Abstract

Atherosclerosis is the underlying pathological process of most cardiovascular disease. A critical component of the "response to retention" hypothesis of atherogenesis is proteoglycan/low density lipoprotein (LDL) binding. Transforming growth factor beta (TGF-beta) is present in atherosclerotic lesions, regulates vascular smooth muscle cell (VSMC) proteoglycan synthesis via an unknown signaling pathway, and increases proteoglycan/LDL binding. This pathway was investigated using the activin receptor-like kinase 5 (ALK5) inhibitor SB431542 and inhibitors of p38 MAP kinase as a possible downstream or alternative mediator. TGF-beta stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-beta increased [(35)S]sulfate incorporation into proteoglycans associated with a 19% increase in glycosaminoglycan (GAG) chain size by size exclusion chromatography. SB431542 caused a concentration-dependent decrease in TGF-beta-mediated [(35)S]sulfate incorporation with 92% inhibition at 3 mum. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-beta-mediated [(35)S]sulfate incorporation. TGF-beta increased [(3)H]glucosamine incorporation into glycosaminoglycans by 180% and [(35)S]Met/Cys incorporation into proteoglycan core proteins by 35% with both effects completely inhibited by SB431542. Blocking both Smad2/3 and p38 MAP kinase pathways prevented the effect of TGF-beta to increase proteoglycan to LDL binding. TGF-beta mediates its effects on proteoglycan synthesis in VSMCs via the ALK5/Smad2/3 phosphorylation pathway as well as via the p38 MAP kinase signaling cascade. Further studies of downstream pathways controlling proteoglycan synthesis may identify potential therapeutic targets for the prevention of atherosclerosis and cardiovascular disease.

Highlights

Nakashima et al [6] have recently provided definitive evidence that human atherosclerosis commences with the deposition of lipid in the outer area of diffuse intimal thickenings; they show that this retention and deposition are associated with expression of the proteoglycan, biglycan
A TGF-␤-activated Smad2/3 phosphorylation pathway is present in human internal mammary artery-derived vascular smooth muscle cell (VSMC) and, along with p38 MAP kinase signaling, is responsible for the TGF-␤-mediated elongation of GAG chains on proteoglycans
TGF-␤ stimulates the synthesis of elongated GAG chains on VSM proteoglycans [7, 30]

Summary

Introduction

Nakashima et al [6] have recently provided definitive evidence that human atherosclerosis commences with the deposition of lipid in the outer area of diffuse intimal thickenings; they show that this retention and deposition are associated with expression of the proteoglycan, biglycan. Separation of proteoglycans on an SDS-polyacrylamide gel showed an increase in electrophoretic mobility of biglycan in the presence of either 10 ␮M SB203580 or SB202190 relative to TGF-␤ treatment alone (Fig. 2B, lane 3 versus lanes 4/5 and 6/7), indicating that inhibition of p38 MAP kinase activity by either inhibitor is sufficient to interfere with GAG synthesis in VSMC.

Results

Conclusion