Robel Getachew

TGF-β stimulates biglycan synthesis via p38 and ERK phosphorylation of the linker region of Smad2

Transforming growth factor (TGF)-β treatment of human vascular smooth-muscle cells increases the expression of biglycan and causes marked elongation of its glycosaminoglycan (GAG) chains. We investigated the role of MAP kinases and Smad transcription factors in this response. TGF-β-stimulated phosphorylation of p38, ERK, and JNK as well as Smad2 at both its carboxy terminal (phospho-Smad2C) and in the linker region (phospho-Smad2L). Pharmacological inhibition of ERK and p38 blocked TGF-β-mediated GAG elongation and expression of biglycan whereas inhibition of JNK had no effect. Inhibition of ERK and p38 but not JNK attenuated the effect of TGF-β to increase phospho-Smad2L. High levels of phospho-Smad2L were detected in a nuclear fraction of TGF-β treated cells. Thus, MAP kinase signaling through ERK and p38 and via phosphorylation of the linker region of Smad2 mediates the effects of TGF-β on biglycan synthesis in vascular smooth-muscle cells.

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

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 β (TGF-β) 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-β stimulated and SB431542 inhibited the phosphorylation of Smad2/3. In human VSMC, TGF-β increased [35S]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-β-mediated [35S]sulfate incorporation with 92% inhibition at 3 μm. Two different p38 MAP kinase inhibitors, SB203580 and SB202190, but not the inactive analogue SB202474, concentration-dependently blocked TGF-β-mediated [35S]sulfate incorporation. TGF-β increased [3H]glucosamine incorporation into glycosaminoglycans by 180% and [35S]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-β to increase proteoglycan to LDL binding. TGF-β 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.

Thrombin stimulation of proteoglycan synthesis in vascular smooth muscle is mediated by protease-activated receptor-1 transactivation of the transforming growth factor beta type I receptor

Growth factors modify the structure of the glycosaminoglycan (GAG) chains on biglycan leading to enhanced LDL binding. G-protein receptor-coupled agonists such as thrombin, signal changes the structure of proteoglycans produced by vascular smooth muscle cells (VSMCs). One component of classical G-protein-coupled receptor (GPCR) signaling invokes transactivation of protein tyrosine kinase receptors such as the epidermal growth factor receptor. Serine/threonine receptor growth factors such as transforming growth factor-(TGF)-β are potent activators of proteoglycan synthesis. We have used the model of proteoglycan synthesis to demonstrate that the signaling paradigm of GPCR signaling can be extended to include the transactivation of serine/threonine receptor, specifically the TGF-β type I receptor (TβRI) also known as activin-like kinase (ALK) V. Thrombin stimulated elongation of GAG chains and increased proteoglycan core protein expression and these responses were blocked by the TβRI antagonist, SB431542 and TβRI siRNA knockdown, as well as several protease-activated receptor (PAR)-1 antagonists. The canonical downstream response to TGF-β is increased C-terminal phosphorylation of the transcription factor Smad2 generating phospho-Smad2C (phosphorylation of Smad2 C-terminal region). Thrombin stimulated increased phospho-Smad2C levels, and the response was blocked by SB431542 and JNJ5177094. The proteolytically inactive thrombin mimetic thrombin-receptor activating peptide also stimulated an increase in cytosolic phospho-Smad2C. Signaling pathways for growth factor regulated proteoglycan synthesis represent therapeutic targets for the prevention of atherosclerosis, but the novel finding of a GPCR-mediated transactivation of a serine/threonine growth factor receptor almost certainly has implications well beyond the synthesis of proteoglycans.

PDGF β-Receptor Kinase Activity and ERK1/2 Mediate Glycosaminoglycan Elongation on Biglycan and Increases Binding to LDL

The initiation of atherosclerosis involves the subendothelial retention of lipoproteins by proteoglycans (PGs). Structural characteristics of glycosaminoglycan (GAG) chains on PGs influence lipoprotein binding and are altered adversely by platelet-derived growth factor (PDGF). The signaling pathway for PDGF-mediated GAG elongation via the PDGF receptor (PDGFR) was investigated. In human vascular smooth muscle cells, PDGF significantly increased (35)S-sulfate incorporation into PGs and GAG chain size. PGs from PDGF-stimulated cells showed increased binding low-density lipoprotein (P < 0.001) in gel mobility shift assays. Knockdown of PDGFRbeta using small interfering RNA demonstrated that PDGF mediated changes in PGs via PDGFRbeta. GAG synthesis and hyperelongation was blocked by inhibition of receptor tyrosine kinase autophosphorylation site Tyr857 activity using Ki11502 or imatinib. Downstream signaling to GAG hyperelongation was mediated through ERK MAPK and not phosphatidylinositol-3 kinase or phospholipase Cgamma. In high-fat-fed apolipoprotein E(-/-) mice, inhibition of PDGFRbeta activity by imatinib reduced aortic total lipid staining area by 35% (P < 0.05). Inhibition of PDGFRbeta tyrosine kinase activity leads to inhibition of GAG synthesis on vascular PGs and aortic lipid area in vivo. PDGFRbeta and its signaling pathways are potential targets for novel therapeutic agents to prevent the earliest stages atherosclerosis.

Endothelin-1 Stimulation of Proteoglycan Synthesis in Vascular Smooth Muscle is Mediated by Endothelin Receptor Transactivation of the Transforming Growth Factor-β Type I Receptor

We utilized human vascular smooth muscle cells to address the question if a G-protein-coupled receptor, the endothelin (ET) receptor, could transactivate a serine/threonine kinase receptor, specifically the transforming growth factor (TGF)-β receptor, TβRI. Functionality of the interaction was addressed by studying endothelin-1-stimulated proteoglycan synthesis. Signaling molecules were assessed by Western blotting and proteoglycan synthesis by [35S]sulfate and 35S-met/cys incorporation and molecular size by SDS-PAGE. Endothelin-1 treatment led to a time- and concentration-dependent increase in cytosolic phosphoSmad2C, which was inhibited by the mixed endothelin receptor antagonist bosentan and the TβRI antagonist SB431542. Endothelin-1 treatment led to a time-dependent increase in nuclear phosphoSmad2C. Endothelin-1-stimulated proteoglycan synthesis was partially inhibited (40%) by SB431542 and completely blocked by bosentan. The effect of endothelin-1 to stimulate an increase in glycosaminoglycan size on biglycan was also blocked in a concentration-dependent manner by SB431542. These data extend the current paradigm of G-protein coupled receptor signaling to include the transactivation of the serine kinase receptor for TGF-β (TβRI). This response should be considered in the context of response to endothelin-1, and the options for therapeutically targeting endothelin-1 are accordingly broadened to include downstream signaling otherwise associated with TGF-β receptor activation.

Thrombin mediated proteoglycan synthesis utilizes both protein tyrosine kinase and serine/threonine kinase receptor transactivation in vascular smooth muscle cells

Background: GPCR transactivation of PTKRs and TGF-βRs mediates proteoglycan synthesis in human VSMC. Results: Transactivation of TGF-βRs is integrin-dependent, and inhibition of both transactivation pathways blocks proteoglycan synthesis. Conclusion: GPCR utilize transactivation pathways and not classical signaling in proteoglycan synthesis. Significance: GPCR transactivation of receptor kinase pathways may be broader and more significant than previously recognized. G protein-coupled receptor signaling is mediated by three main mechanisms of action; these are the classical pathway, β-arrestin scaffold signaling, and the transactivation of protein-tyrosine kinase receptors such as those for EGF and PDGF. Recently, it has been demonstrated that G protein-coupled receptors can also mediate signals via transactivation of serine/threonine kinase receptors, most notably the transforming growth factor-β receptor family. Atherosclerosis is characterized by the development of lipid-laden plaques in blood vessel walls. Initiation of plaque development occurs via low density lipoprotein retention in the neointima of vessels due to binding with modified proteoglycans secreted by vascular smooth muscle cells. Here we show that transactivation of protein-tyrosine kinase receptors is mediated by matrix metalloproteinase triple membrane bypass signaling. In contrast, serine/threonine kinase receptor transactivation is mediated by a cytoskeletal rearrangement-Rho kinase-integrin system, and both protein-tyrosine kinase and serine/threonine kinase receptor transactivation concomitantly account for the total proteoglycan synthesis stimulated by thrombin in vascular smooth muscle. This work provides evidence of thrombin-mediated proteoglycan synthesis and paves the way for a potential therapeutic target for plaque development and atherosclerosis.

G protein coupled receptor transactivation: extending the paradigm to include serine/threonine kinase receptors

The current paradigm of G protein coupled receptor signaling involves a classical pathway being the activation of phospholipase C and the generation of 1,4,5-inositol trisphosphate, signaling through β-arrestin scaffold molecules and the transactivation of tyrosine kinase growth factor receptors. Transactivation greatly expands the range of signaling pathways and responses attributable to the receptor. Recently it has been revealed that G protein coupled receptor agonists can also transactivate the serine/threonine kinase cell surface receptor for transforming growth factor-β (Alk5). This leads to the generation of carboxyl terminal phosphorylated Smad2 which is the immediate downstream product of the activated Alk5. Thus, the current paradigm of G protein coupled signaling can be expanded to include the transactivation of the serine kinase receptor Alk5. These insights expand the possibilities for outcomes of therapeutically targeting GPCRs where more substantive and prolonged actions such as the synthesis of extracellular matrix may be affected.

TGF-β stimulates biglycan core protein synthesis but not glycosaminoglycan chain elongation via Akt phosphorylation in vascular smooth muscle.

Transforming growth factor-β (TGF-β) can mediate proteoglycan synthesis via Smad and non-Smad signalling pathways in vascular smooth muscle (VSM). We investigated whether TGF-β-mediated proteoglycan synthesis is via PI3K/Akt. TGF-β induced a rapid phosphorylation of Akt that continued upto 4 h. Akt phosphorylation was blocked by Akt1/2 inhibitor SN30978; however, it did not block Smad2 phosphorylation at either the carboxy or linker regions indicating that TGF-β-mediated Akt phosphorylation is independent of Smad2 signalling. The role of Akt in TGF-β-mediated proteoglycan synthesis was investigated. Treatment with SN30978 showed a concentration-dependent decrease in TGF-β-mediated [35S]-sulphate and [35S]-Met/Cys incorporation into secreted proteoglycans; however, SDS-PAGE showed no change in biglycan size. In TGF-β-treated cells, biglycan mRNA levels increased by 40–100% in 24 h and was significantly blocked by SN30978. Our findings demonstrate that Akt is a downstream signalling component of TGF-β-mediated biglycan core protein synthesis but not glycosaminoglycan chain hyper-elongation in VSM.

Growth factor-mediated hyper-elongation of glycosaminoglycan chains on biglycan requires transcription and translation

The mechanism through which growth factors cause glycosaminoglycan (GAG) hyper-elongation is unclear. We have investigated the role of transcription and translation on the GAG hyper-elongation effect of plateletderived growth factor (PDGF) in human vascular smooth muscle cells (VSMCs). To determine if the response involves specific signalling pathways or the process of GAG hyper-elongation we have also investigated the effects of epidermal growth factor (EGF), transforming growth factor-β (TGF-β) and thrombin. We report that both actinomycin D and cycloheximide completely abolished the ability of PDGF to stimulate radiosulphate incorporation and GAG elongation into secreted proteoglycans, and to increase the size of xyloside GAGs. Blocking de novo protein synthesis completely prevented the action of all growth factors tested to elongate GAG chains. These results lay a foundation for further investigation into the genes and proteins implicated in this response.

Smad2-dependent glycosaminoglycan elongation in aortic valve interstitial cells enhances binding of LDL to proteoglycans

OBJECTIVE Calcific aortic valve disease is a progressive condition that shares some common pathogenic features with atherosclerosis. Transforming growth factor-β1 is a recognized mediator of atherosclerosis and is expressed in aortic valve lesions. Transforming growth factorβ1 stimulates glycosaminoglycan elongation of proteoglycans that is associated with increased lipid binding. We investigated the presence of transforming growth factor-β1 and downstream signaling intermediates in diseased human aortic valves and the effects of activated transforming growth factor-β1 receptor signaling on aortic valve interstitial cell proteoglycan synthesis and lipid binding as a possible mechanism for the initiation of the early lesion of calcific aortic valve disease. METHODS AND RESULTS Diseased human aortic valve leaflets demonstrated strong immunohistochemical staining for transforming growth factor-β1 and phosphorylated Smad2/3. In primary porcine aortic valve interstitial cells, Western blots showed that transforming growth factor-β1 stimulated phosphorylation in both the carboxy and linker regions of Smad2/3, which was inhibited by the transforming growth factor-β1 receptor inhibitor SB431542. Gel electrophoresis and size exclusion chromatography demonstrated that SB431542 decreased transforming growth factor-β1-mediated [(35)S]-sulfate incorporation into proteoglycans in a dose-dependent manner. Further, in proteoglycans derived from transforming growth factor-β1-treated valve interstitial cells, gel mobility shift assays demonstrated that inhibition of transforming growth factor-β1 receptor signaling resulted in decreased lipid binding. CONCLUSIONS Classic transforming growth factor-β1 signaling is present in human aortic valves in vivo and contributes to the modification of proteoglycans expressed by valve interstitial cells in vitro. These findings suggest that transforming growth factor-β1 may promote increased low-density lipoprotein binding in the early phases of calcific aortic valve disease.