Abstract
Although TAK1 has been implicated in inflammation and oxidative stress, its roles in vascular smooth muscle cells (VSMCs) and in response to vascular injury have not been investigated. The present study aimed to investigate the role of TAK1 in modulating oxidative stress in VSMCs and its involvement in neointima formation after vascular injury. Double immunostaining reveals that vascular injury induces a robust phosphorylation of TAK1 (Thr187) in the medial VSMCs of injured arteries in wildtype mice, but this effect is blocked in CD40-deficient mice. Upregulation of TAK1 in VSMCs is functionally important, as it is critically involved in pro-oxidative and pro-inflammatory effects on VSMCs and eventual neointima formation. In vivo, pharmacological inhibition of TAK1 with 5Z-7-oxozeaenol blocked the injury-induced phosphorylation of both TAK1 (Thr187) and NF-kB/p65 (Ser536), associated with marked inhibition of superoxide production, 3-nitrotyrosine, and MCP-1 in the injured arteries. Cell culture experiments demonstrated that either siRNA knockdown or 5Z-7-oxozeaenol inhibition of TAK1 significantly attenuated NADPH oxidase activation and superoxide production induced by CD40L/CD40 stimulation. Co-immunoprecipitation experiments indicate that blockade of TAK1 disrupted the CD40L-induced complex formation of p22phox with p47phox, p67phox, or Nox4. Blockade of TAK1 also inhibited CD40L-induced NF-kB activation by modulating IKKα/β and NF-kB p65 phosphorylation and this was related to reduced expression of proinflammatory genes (IL-6, MCP-1 and ICAM-1) in VSMCs. Lastly, treatment with 5Z-7-oxozeaenol attenuated neointimal formation in wire-injured femoral arteries. Our findings demonstrate previously uncharacterized roles of TAK1 in vascular oxidative stress and the contribution to neointima formation after vascular injury.
Highlights
Transforming growth factor-b (TGF-b)-activated kinase 1 (TAK1), a seine/threonine kinase, was originally identified as a mitogen-activated protein kinase kinase kinase (MAP3K) which can be activated by TGF-b [1]
We found a low level of the phosphorylated TAK1 (Thr187) in uninjured femoral arteries, but the TAK1 phosphorylation was robustly induced in injured arteries from WT mice, notably with strong co-localization with medial SMa-actin-positive SMCs (Figure 1A)
By performing co-immunoprecipitation experiments, we found that treatment of WT vascular smooth muscle cells (VSMCs) with CD40 ligand (CD40L) robustly induced the complex formation of CD40 with both TRAF6 and TAK1, but the TNF receptor-associated factors (TRAFs)-6-TAK1 complex formation was disrupted in CD40deficient VSMCs (Figure 1C)
Summary
Transforming growth factor-b (TGF-b)-activated kinase 1 (TAK1), a seine/threonine kinase, was originally identified as a mitogen-activated protein kinase kinase kinase (MAP3K) which can be activated by TGF-b [1]. TAK1 has been characterized as a key regulator in immune and proinflammatory intracellular signaling pathways [2,3,4]. The activated TAK1 in turn mediates intracellular signaling, via downstream nuclear factor kappaB (NF-kB), p38 MAPK, and c-Jun N-terminal kinase, which may drive inflammatory and oxidative responses in a cell-type specific manner [2,3,7]. The present study aimed to investigate previously uncharacterized roles of TAK1 in vascular oxidative stress and the response to vascular injury. Our findings provide the first description that TAK1 plays a critical role in mediating oxidative stress and proinflammatory phenotype changes in VSMCs and that contributes importantly to neointima formation after vascular injury
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