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

Abstract

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4 h whereas transactivation via TGFBR1 is delayed for 120 min but also persists for at least 4 h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

Lipopolysaccharide acting via toll-like receptor 4 transactivates the TGF-β receptor in vascular smooth muscle cells

“…The engagement of receptor transactivation markedly expands the range of cellular responses attributable to the index cell surface, usually transmembrane, receptor. The current attributes of transactivation-dependent signalling encompass GPCR transactivation of kinase receptors [25,49,[59][60][61]. Mechanistic studies of GPCR transactivation-dependent signalling define a concrete signalling pathway [23,24,32,49].…”

“…The current attributes of transactivation-dependent signalling encompass GPCR transactivation of kinase receptors [25,49,[59][60][61]. Mechanistic studies of GPCR transactivation-dependent signalling define a concrete signalling pathway [23,24,32,49]. LPS stimulates direct TGFBR1 intermediate phospho-Smad2 carboxy terminal via PI3K/ Akt and MAPK-dependent pathways in HSC-T6 hepatic stellate cells [43].…”

4-Methoxy Sulfonyl Paeonol Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis by Blocking the TGF-β1/Smad, PDGF-BB/MAPK and Akt Signaling Pathways

“…The inhibition of C-terminal phosphorylation is very important for blocking the consequent Smad4 binding and extracellular matrix proteins transcription in the nucleus [48]. The linker region phosphorylation of Smad protein can be regulated by several kinases, such as PI3K/mTORC2, ERK, and JNK [48][49][50][51][52]. In Figure 2, we showed that 4-MSP can decrease the PDGF-BB-induced MEK/ERK, JNK, Akt, and p70S6K phosphorylation.…”

Endothelial Dysfunction and Cardiovascular Disease: History and Analysis of the Clinical Utility of the Relationship

“…We observed a highly specific signalling pathway that regulates GAG chain elongation [ 50 ]. Growth factors, hormones and endotoxins acting via completely different receptor pathways signal specifically via the linker region of Smad2 transcription factor to regulate the genes associated with GAG chain modification [ 52 , 53 , 54 , 55 , 56 , 57 , 58 ]. Furthermore, animal studies provide proof of principle that treating atherosclerosis-prone mice fed a high-fat diet an inhibitor of GAG elongation can prevent at least 50 percent of the lipid deposition [ 46 , 59 ].…”