Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets

Kim, Soochong; Cipolla, Lina; Guidetti, Gianni Francesco; Okigaki, Mitsuhiko; Jin, Jianguo; Torti, Mauro; Kunapuli, Satya P.

doi:10.1074/jbc.m113.461087

Cited by 19 publications

(24 citation statements)

References 48 publications

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“…Increasing evidence has confirmed that the activation of both CaMKII and Pyk2 are closely related to the functions of integrins in many cell types [26, 27]. We observed that blocking integrin β1 with its functional blocking antibody significantly abrogated phosphorylation of Pyk2 at Tyr 402 and CaMKII at Thr 286 in chondrocytes in the application of periodic mechanical stress.…”

Section: Discussionsupporting

confidence: 52%

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Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

Liang

Zeng

Wang

et al. 2017

Cell Physiol Biochem

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Background/Aims: Periodic mechanical stress can promote chondrocyte proliferation and matrix synthesis to improve the quality of tissue-engineered cartilage. Although the integrin β1–ERK1/2 signal cascade has been implicated in periodic mechanical stress-induced mitogenic effects in chondrocytes, the precise mechanisms have not been fully established. The current study was designed to probe the roles of CaMKII and Pyk2 signaling in periodic mechanical stress-mediated chondrocyte proliferation and matrix synthesis. Methods: Chondrocytes were subjected to periodic mechanical stress, proliferation was assessed by direct cell counting and CCK-8 assay; gene expressions were analyzed using quantitative real-time PCR, protein abundance by Western blotting. Results: Mechanical stress, markedly enhanced the phosphorylation levels of Pyk2 at Tyr⁴⁰² and CaMKII at Thr²⁸⁶. Both suppression of Pyk2 with Pyk2 inhibitor PF431396 or Pyk2 shRNA and suppression of CaMKII with CaMKII inhibitor KN-93 or CaMKII shRNA blocked periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis. Additionally, either pretreatment with KN-93 or shRNA targeted to CaMKII prevented the activation of ERK1/2 and Pyk2 under conditions of periodic mechanical stress. Interestingly, in relation to periodic mechanical stress, in the context of Pyk2 inhibition with PF431396 or its targeted shRNA, only the phosphorylation levels of ERK1/2 were abrogated, while CaMKII signal activation was not affected. Moreover, the phosphorylation levels of CaMKII- Thr²⁸⁶ and Pyk2- Tyr⁴⁰² were abolished after pretreatment with blocking antibody against integrinβ1 exposed to periodic mechanical stress. Conclusion: Our results collectively indicate that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis through the integrinβ1–CaMKII–Pyk2–ERK1/2 signaling cascade.

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Section: Discussionsupporting

confidence: 52%

“…ERK1/2 has been widely reported to be downstream of CaMKII and Pyk2 in many cell types under different conditions, and integrins also appear vital for the activation of CaMKII and Pyk2 in several different settings [24-27]. However, these may not be true for all cells.…”

Section: Introductionmentioning

confidence: 85%

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

Liang

Zeng

Wang

et al. 2017

Cell Physiol Biochem

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“…and others have also shown that inhibition of either ERK1/2 or Pyk2 activation results in abolition of thromboxane generation in ADP‐stimulated platelets . Previous studies have shown that 2MeSADP‐induced ERK1/2 phosphorylation occurs independently of outside‐in signaling, and that, rather, MEK1/2, SFK and PI3K proteins regulate ERK1/2 activation . Inhibition of MEK1/2 and PI3K with pharmacological inhibitors prevents 2MeSADP‐mediated secondary platelet aggregation by abolishing thromboxane generation, similarly to PDK1 inhibition .…”

Section: Discussionmentioning

confidence: 97%

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

Manne

Münzer

Badolia

et al. 2018

Journal of Thrombosis and Haemostasis

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Background Platelets are dynamic effector cells with functions that span hemostatic, thrombotic and inflammatory continua. Phosphoinositide-dependent protein kinase 1 (PDK1) regulates protease-activated receptor 4-induced platelet activation and thrombus formation through glycogen synthase kinase3β. However, whether PDK1 also signals through the ADP receptor and its functional importance in vivo remain unknown. Objective To establish the mechanism of PDK1 in ADP-induced platelet activation and thrombosis. Methods We assessed the role of PDK1 on 2MeSADP-induced platelet activation by measuring aggregation, thromboxane generation and phosphorylation events in the presence of BX-795, which inhibits PDK1, or by using platelet-specific PDK1 knockout mice and performing western blot analysis. PDK1 function in thrombus formation was assessed with an in vivo pulmonary embolism model. Results PDK1 inhibition with BX-795 reduced 2-methylthio-ADP (2MeSADP)-induced aggregation of human and murine platelets by abolishing thromboxane generation. Similar results were observed in pdk1 mice. PDK1 was also necessary for the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MEK1/2), extracellular signal-regulated kinase 1/2, and cytosolic phospholipase A2, indicating that PDK1 regulates an upstream kinase in the mitogen-activated protein kinase (MAPK) pathway. We next determined that this upstream kinase is Raf-1, a serine/threonine kinase that is necessary for the phosphorylation of MEK1/2, as pharmacological inhibition and genetic ablation of PDK1 were sufficient to prevent Raf1 phosphorylation. Furthermore, in vivo inhibition or genetic ablation of PDK1 protected mice from collagen/epinephrine-induced pulmonary embolism. Conclusion PDK1 governs thromboxane generation and thrombosis in platelets that are stimulated with 2MeSADP by regulating activation of the MAPK pathway.

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“…These findings indicate that p38 predominantly drives the phosphorylation of cPLA 2 in platelets. In support of this, a number of studies highlight the presence of other pathways that drive thromboxane production in a p38, ERK1/2, and Pyk2 mutually dependent manner [2] [3, 4]. …”

mentioning

confidence: 99%

“…Based on these new and previously published findings [2] [3, 4] [1], we propose the following model incorporating pathways regulating thromboxane generation by all three protein kinases (ERK1/2, p38, Pyk2), upon 2MesADP stimulation of platelets.…”

mentioning

confidence: 99%

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway: reply

Manne

Rondina

2018

Journal of Thrombosis and Haemostasis

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Distinct Role of Pyk2 in Mediating Thromboxane Generation Downstream of Both G12/13 and Integrin αIIbβ3 in Platelets

Cited by 19 publications

References 48 publications

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

Periodic Mechanical Stress INDUCES Chondrocyte Proliferation and Matrix Synthesis via CaMKII-Mediated Pyk2 Signaling

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway

PDK1 governs thromboxane generation and thrombosis in platelets by regulating activation of Raf1 in the MAPK pathway: reply

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