Abstract
Protein tyrosine kinase (PTK) activity has been implicated in pro-inflammatory gene expression following tumor necrosis factor-α (TNF-α) or interkeukin-1β (IL-1β) stimulation. However, the identity of responsible PTK(s) in cytokine signaling have not been elucidated. To evaluate which PTK is critical to promote the cytokine-induced inflammatory cell adhesion molecule (CAM) expression including VCAM-1, ICAM-1, and E-selectin in human aortic endothelial cells (HAoECs), we have tested pharmacological inhibitors of major PTKs: Src and the focal adhesion kinase (FAK) family kinases - FAK and proline-rich tyrosine kinase (Pyk2). We found that a dual inhibitor of FAK/Pyk2 (PF-271) most effectively reduced all three CAMs upon TNF-α or IL-1β stimulation compared to FAK or Src specific inhibitors (PF-228 or Dasatinib), which inhibited only VCAM-1 expression. In vitro inflammation assays showed PF-271 reduced monocyte attachment and transmigration on HAoECs. Furthermore, FAK/Pyk2 activity was not limited to CAM expression but was also required for expression of various pro-inflammatory molecules including MCP-1 and IP-10. Both TNF-α and IL-1β signaling requires FAK/Pyk2 activity to activate ERK and JNK MAPKs leading to inflammatory gene expression. Knockdown of either FAK or Pyk2 reduced TNF-α-stimulated ERK and JNK activation and CAM expression, suggesting that activation of ERK or JNK is specific through FAK and Pyk2. Finally, FAK/Pyk2 activity is required for VCAM-1 expression and macrophage recruitment to the vessel wall in a carotid ligation model in ApoE−/− mice. Our findings define critical roles of FAK/Pyk2 in mediating inflammatory cytokine signaling and implicate FAK/Pyk2 inhibitors as potential therapeutic agents to treat vascular inflammatory disease such as atherosclerosis.
Highlights
The vascular endothelium plays a key role in directing the immune response to tissues experiencing an inflammatory insult
PF-271 reduced the expression of two other key inflammatory cell adhesion molecule (CAM), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, in human aortic endothelial cells (HAoECs)
These data suggest that focal adhesion kinase (FAK) family kinases are key Protein tyrosine kinase (PTK) affecting tumor necrosis factor-α (TNF-α)-stimulated pro-inflammatory CAM expression
Summary
The vascular endothelium plays a key role in directing the immune response to tissues experiencing an inflammatory insult. Several diseases that affect the endothelium, such as atherosclerosis, hypertension, and diabetes, result in chronic inflammation of the vascular endothelium[1,2] Common vascular insults, such as hypercholesterolemia, high blood pressure, and reactive oxygen species, associated with these diseases lead to activation of endothelial cells (ECs) and resident leukocytes of the vessel wall[2,3,4,5,6,7,8,9]. Apolipoprotein E (ApoE) and low-density lipoprotein receptor (LDLR) knockout mice fed a high fat diet develop chronic inflammation of the vessel wall leading to EC dysfunction, inflammatory CAM expression, and macrophage recruitment[19,20,21] Elevated inflammatory cytokines such as TNF-α and IL-1β have been found in both human and animal atherosclerotic lesions[22,23], and knockdown of these cytokines have alleviated atherosclerosis progression[24,25]. Which PTK(s) plays a major role in these signaling pathways remains unknown
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