Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells

McLaughlin, Joseph N.; Mazzoni, Maria Rosa; Cleator, John H.; Earls, Laurie R.; Perdigoto, Ana Luisa; Brooks, Joshua D.; Muldowney, James A.S.; Vaughan, Douglas E.; Hamm, Heidi E.

doi:10.1074/jbc.m500721200

Cited by 60 publications

(43 citation statements)

References 86 publications

(39 reference statements)

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“…However, our conclusions are consistent with 2 previous reports that showed the thrombin-induced upregulation of PAR1. 25,26 The apparent inconsistency may be related to the unique property of PAR1. In contrast to the general G protein-coupled receptors, PAR1 is irreversibly activated by the proteinase agonist.…”

Section: Discussionmentioning

confidence: 99%

Prevention of the Hypercontractile Response to Thrombin by Proteinase-Activated Receptor-1 Antagonist in Subarachnoid Hemorrhage

Yang

Hirano

Maeda

et al. 2007

Stroke

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Background and Purpose-Cerebral vasospasm is one of the major complications of subarachnoid hemorrhage (SAH). Its pathogenesis still remains elusive, and effective therapeutic strategies are yet to be established. We investigated the role of proteinase-activated receptor-1 (PAR1) in the hypercontractile state in SAH. Methods-Rabbit double hemorrhage model was used as a model of SAH. The contractile response to thrombin and the PAR1 expression were evaluated in the isolated rings of basilar artery. Results-Thrombin exhibited only a minor contractile effect in the control, whereas it induced augmented contractions in SAH. The expression of PAR1 was upregulated in SAH. Intracisternal injection of PAR1 antagonist E5555 prevented the enhancement of the contractile responses to thrombin in SAH. The maximal prevention was obtained with 2 g/kg weight/injection. The contractile responses to K ϩ depolarization or endothelin-1 remained unaffected. The upregulation of PAR1 was also prevented by E5555 (2 g/kg weight/injection) to a level similar to that seen in the control. Ex vivo treatment with E5555 (1 mol/L) inhibited the contraction induced by thrombin, whereas it had little effect on the contraction induced by K ϩ depolarization or endothelin-1, in the basilar artery of SAH. E5555 also inhibited the [Ca 2ϩ ] i elevation induced by thrombin, but not trypsin, in cultured smooth muscle cells. Conclusions-PAR1 plays a critical role in upregulating PAR1 itself, thereby enhancing the contractile response to thrombin in SAH. PAR1 could thus be a therapeutic target. However, the usefulness of PAR1 antagonist remains to be investigated in vivo. (Stroke. 2007;38:3259-3265.)

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Section: Discussionmentioning

confidence: 99%

Prevention of the Hypercontractile Response to Thrombin by Proteinase-Activated Receptor-1 Antagonist in Subarachnoid Hemorrhage

Yang

Hirano

Maeda

et al. 2007

Stroke

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“…Expression of PAR1 and PAR3 was monitored in HPAEC by real-time semiquantitative RT-PCR by using PCR SYBR green PCR Master mix (Applied Biosystems) was on a PRISM 7000 Sequence Detection System (Applied Biosystems) with GAPDH as the internal control as described (34). Briefly, after siRNA treatment described above, HPAEC were grown to confluence in six-well plates and total RNA was harvested, reverse-transcribed using oligo-dTs with SuperScript III RT, and real-time semiquantitative analysis was performed.…”

Section: Methodsmentioning

confidence: 99%

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

McLaughlin

Patterson

Malik

2007

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

157

164

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Thrombin activates endothelial cell signaling by cleaving the protease-activated receptor-1 (PAR1). However, the function of the apparently nonsignaling receptor PAR3 also expressed in endothelial cells is unknown. We demonstrate here the crucial role of PAR3 in potentiating the responsiveness of PAR1 to thrombin. We tested the hypothesis that PAR1/PAR3 heterodimerization and its effect in modifying G protein selectivity was responsible for PAR3 regulation of PAR1 sensitivity. Using bioluminescent resonance energy transfer-2, we showed that PAR1 had comparable dimerization affinity for PAR3 as for itself. We observed increased G␣13 coupling between the PAR1/3 heterodimer compared with the PAR1/1 homodimer. Moreover, knockdown of PAR3 moderated the PAR1-activated increase in endothelial permeability. These results demonstrate a role of PAR3 in allosterically regulating PAR1 signaling governing increased endothelial permeability. Because PAR3 is a critical determinant of PAR1 function, targeting of PAR3 may mitigate the effects of PAR1 in activating endothelial responses such as vascular inflammation.T hrombin generation during thrombosis contributes to the pathophysiology of multiple thrombosis-related diseases such as acute lung injury resulting increased endothelial permeability (1), vascular inflammation (1, 2), and edema formation (for recent reviews, see refs. 3 and 4). These complications are the result of thrombin activation of protease-activated receptor-1 (PAR1) (5). Cleavage of the extracellular N terminus of PAR1 initiates signaling (6) by the concomitant activation of heterotrimeric GTP-binding proteins G␣ q and G␣ 12/13 (7-9). The mechanisms by which PAR1 selectively activates G protein signaling remain unknown. Because the nonsignaling receptor PAR3 is also expressed in endothelial cells (10), we surmised that PAR1 and PAR3 are capable of interacting in such manner as to regulate PAR1 signaling. We found that PAR3 directly dimerizes with PAR1 to induce a specific PAR1/G␣ 13 -binding conformation that favors G␣ 13 activation. We propose a model of PAR1 activation involving the interaction with PAR3, which alters the selectivity of PAR1 for G␣ 13 coupling and promotes endothelial barrier dysfunction. PAR3 functions as an essential allosteric modulator of PAR1 signaling through PAR1/3 dimerization and favors a distinct G␣ 13 -activated downstream pathway. Results PAR3 Knockdown in Endothelial Cells Shifts the Potency of ThrombinActivation of PAR1. Endothelial cells express PAR1, -2, and -3, but not PAR4 (10), and of these only PAR1 and PAR3 are thrombinsensitive (11). To address whether PAR3 alters thrombin signaling in endothelial cells, siRNA knockdown was used to reduce thrombin receptor expression in human pulmonary artery endothelial cells (HPAECs). PAR1 and PAR3 suppression was determined by semiquantitative RT-PCR. siRNA against PAR3 did not affect PAR1 expression, whereas it reduced PAR3 expression 8-fold. Conversely, siRNA against PAR1 only modestly affected PAR3 expression, whereas it suppres...

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“…Thrombin has been shown to either upregulate or downregulate the expression of multiple genes in the cultured endothelial cells. 13,14 The expression of the genes related to angiogenesis and cell growth (vascular endothelial growth factor receptor, angiopoietin, platelet derived growth factors), hemostasis (tissue factor, plasminogen activator inhibitor (PAI)-1), cytokines, and chemokines (interleukins-6 and 8, monocyte chemoattractant protein-1), and cell adhesion (vascular cell adhesion molecule [VCAM]-1, intercellular adhesion molecule-1 [ICAM-1], E-selectin) have shown to be upregulated by thrombin. 13 These alterations to gene expression may be linked to the phenotypic conversion of endothelial cells to the proinflammatory phenotype.…”

Section: Par2mentioning

confidence: 99%

“…[5][6][7][8][9][10][11][12] PAR1 in endothelial cells also contributes to angiogenesis, 6,9 in addition to causing an alteration to the expression of multiple genes including cytokines, chemokines, and cell adhesion molecules (Figure). 13,14 In the smooth muscle cells, PAR1 mediates the contraction, cell migration, proliferation, hypertrophy, and production of the extracellular matrix ( Figure). [5][6][7][8][9] Similarly, PAR2 also mediates endothelium-dependent relaxation and angiogenesis in endothelial cells, and mediates the contraction, cell migration, proliferation, hypertrophy and production of the extracellular matrix in the smooth muscle cells.…”

mentioning

confidence: 99%

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

Hirano

2007

ATVB

136

120

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Abstract-Proteinase-activated receptors (PARs) belong to a family of G protein-coupled receptors, thus mediating the cellular effects of proteinases. In the vascular system, thrombin and other proteinases in the coagulation-fibrinolysis system are considered to be the physiologically relevant agonists, whereas PARs are among the most important mechanisms mediating the interaction between the coagulation-fibrinolysis system and the vascular wall. Under physiological conditions, PARs are mainly expressed in endothelial cells, and participate in the regulation of vascular tone, mostly by inducing endothelium-dependent relaxation. PARs in endothelial cells are also suggested to contribute to a proinflammatory phenotypic conversion and an increase in the permeability of vascular lesions. In smooth muscle cells, PARs mediate contraction, migration, proliferation, hypertrophy, and production of the extracellular matrix, thereby contributing to the development of vascular lesions and the pathophysiology of such vascular diseases as atherosclerosis. However, the expression of PARs in the smooth muscle of normal arteries is limited. The upregulation of PARs in the smooth muscle is thus considered to be a key step for PARs to participate in the pathogenesis of vascular lesions. Elucidating the molecular mechanism regulating the PARs expression is therefore important to develop new strategies for the prevention and treatment of vascular diseases. (Arterioscler Thromb Vasc Biol. 2007;27:27-36.)

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Thrombin Modulates the Expression of a Set of Genes Including Thrombospondin-1 in Human Microvascular Endothelial Cells

Cited by 60 publications

References 86 publications

Prevention of the Hypercontractile Response to Thrombin by Proteinase-Activated Receptor-1 Antagonist in Subarachnoid Hemorrhage

Prevention of the Hypercontractile Response to Thrombin by Proteinase-Activated Receptor-1 Antagonist in Subarachnoid Hemorrhage

Protease-activated receptor-3 (PAR3) regulates PAR1 signaling by receptor dimerization

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

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