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Quantifying Agonist Activity at G Protein-coupled Receptors
Published on: December 26, 2011
G protein-coupled receptors activate p38 MAPK via a non-canonical TAB1-TAB2- and TAB1-TAB3-dependent pathway in
Neil J Grimsey1, Ying Lin1, Rachan Narala1
1From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093.
Abstract:
Endothelial dysfunction is induced by inflammatory mediators including multiple G protein-coupled receptor (GPCR) agonists. However, the GPCR signaling pathways that promote endothelial dysfunction are incompletely understood. We previously showed that thrombin promotes endothelial barrier disruption through autophosphorylation and activation of p38 mitogen-activated protein kinase (MAPK) via a non-canonical transforming growth factor-β-activated protein kinase-1-binding protein-1 (TAB1) and TAB2-dependent pathway rather than the canonical three-tiered kinase cascade. Here, we sought to determine whether other GPCR agonists stimulate p38 MAPK activation via this non-canonical pathway in human endothelial cells derived from different vascular beds. Using primary human umbilical vein endothelial cells (HUVECs), HUVEC-derived EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs), we found that both non-canonical and canonical p38 activation pathways components are expressed in these various endothelial cell types, including TAB3, a structurally-related TAB2 homolog. Moreover, multiple GPCRs agonists, including thrombin, histamine, prostaglandin E2, and ADP, stimulated robust p38 autophosphorylation, whereas phosphorylation of the upstream MAPKs MAP kinase kinase 3 (MKK3) and MKK6, was virtually undetectable, indicating that non-canonical p38 activation may exist for other GPCRs. Indeed, in EA.hy926 cells, thrombin- and histamine-stimulated p38 activation depended on TAB1-TAB2, whereas in primary HUVECs, both TAB1-TAB2 and TAB1-TAB3 were required for p38 activation. In HDMECs, thrombin-induced p38 activation depended on TAB1-TAB3, but histamine-induced p38 activation required TAB1-TAB2. Moreover, thrombin- and histamine-stimulated interleukin-6 production required both TAB1-TAB2 and TAB1-TAB3 in HUVEC. We conclude that multiple GPCR agonists utilize non-canonical TAB1-TAB2 and TAB1-TAB3-dependent p38 activation to promote endothelial inflammatory responses.
Insights
Multiple G protein-coupled receptor agonists activate p38 MAPK through non-canonical pathways involving TAB1-TAB2 and TAB1-TAB3, promoting endothelial inflammation. This reveals new mechanisms underlying endothelial dysfunction.
Area of Science:
- Molecular Biology
- Cellular Biology
- Immunology
- Cardiovascular Research
Background:
- Endothelial dysfunction, a precursor to cardiovascular diseases, is often triggered by inflammatory mediators acting through G protein-coupled receptors (GPCRs).
- The precise GPCR signaling pathways driving endothelial dysfunction, particularly p38 mitogen-activated protein kinase (MAPK) activation, remain incompletely elucidated.
- Previous research identified a non-canonical pathway for thrombin-induced p38 MAPK activation in endothelial cells, bypassing the conventional three-tiered kinase cascade.
Purpose of the Study:
- To investigate whether various GPCR agonists activate p38 MAPK through the non-canonical pathway in human endothelial cells from diverse vascular beds.
- To identify the specific TAB (T-LAK cell-originated protein kinase inhibitor) proteins involved in non-canonical p38 MAPK activation by different GPCR agonists.
- To determine the role of these non-canonical pathways in mediating GPCR-induced inflammatory responses, such as interleukin-6 production.
Main Methods:
- Utilized primary human umbilical vein endothelial cells (HUVECs), EA.hy926 cells, and human dermal microvascular endothelial cells (HDMECs).
- Assessed p38 MAPK autophosphorylation and phosphorylation of upstream kinases (MKK3, MKK6) following stimulation with GPCR agonists (thrombin, histamine, prostaglandin E2, ADP).
- Investigated the involvement of TAB1, TAB2, and TAB3 in p38 MAPK activation and interleukin-6 production using knockdown or inhibition strategies.
Main Results:
- Multiple GPCR agonists, including thrombin, histamine, prostaglandin E2, and ADP, induced robust p38 MAPK autophosphorylation without significant MKK3/MKK6 phosphorylation, suggesting non-canonical activation.
- Both non-canonical (TAB1/TAB2/TAB3) and canonical p38 pathway components are expressed in different human endothelial cell types.
- Specific TAB protein requirements varied by cell type and agonist: thrombin and histamine primarily used TAB1-TAB2 in EA.hy926 cells, TAB1-TAB2 and TAB1-TAB3 in HUVECs, and TAB1-TAB3 (thrombin) or TAB1-TAB2 (histamine) in HDMECs.
- Both TAB1-TAB2 and TAB1-TAB3 were essential for thrombin- and histamine-stimulated interleukin-6 production in HUVECs.
Conclusions:
- Multiple GPCR agonists can activate p38 MAPK via non-canonical pathways dependent on TAB1-TAB2 and/or TAB1-TAB3 in human endothelial cells.
- These non-canonical pathways play a significant role in mediating endothelial inflammatory responses, such as cytokine production.
- The findings highlight the complexity of GPCR signaling in endothelial cells and identify novel therapeutic targets for inflammatory conditions.
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