IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways

Abstract

Stimulation of microvascular endothelial cells with interleukin (IL)-8 leads to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibited by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXCR2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indicating that the CXCR2 couples to G(i). In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibits Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-mediated formation of stress fibers was prevented by dominant negative Rho. Dominant negative Rac on the other hand initially translocated to actin-rich filopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endothelial cells, using different signal transduction cascades. The retraction of endothelial cells due to activation of the CXCR2 may contribute to the increased vascular permeability observed in acute inflammation and during the angiogenic response.

Chemokines in tumor angiogenesis and metastasis

“…Because most of the G protein coupled receptors can activate a variety of effector pathways via various G protein subunits, considerable heterogeneity exists in signaling pathways leading to ERK1/2 phosphorylation and subsequent activation of transcription factors [33]. In addition, recent data suggests that signals dependent on G proteins mediated through Rho and Rac pathways result in actin polymerization, reconstitution of adhesion molecules and other cellular components leading to cell migration [198].…”

Functional expression of CXCR4 (CD184) on small-cell lung cancer cells mediates migration, integrin activation, and adhesion to stromal cells

“…Actin-polymerization assay was performed as described earlier (Barak et al, 1980;Schraufstatter et al, 2001). Briefly, NCI-H82 and NCI-H446 SCLC cells were seeded on Labtech-eightwell-chamber slides precoated with 0.01% poly-L-lysine Solution (Sigma, St Louis, MO, USA).…”

KSHV-GPCR and CXCR2 transforming capacity and angiogenic responses are mediated through a JAK2-STAT3-dependent pathway

“…As previously shown (Schraufstatter et al, 2001) Next, we tried to test for branching/sprouting in transfected NIH 3T3 cells. It has been described before that maligne transformed NIH 3T3 are also able to form tube structures (Van Rompaey et al, 2002) by which aggressive tumor cells generate nonendothelial cell-lined channels delimited by extracellular matrix.…”

“…Polymerized actin was visualized as previously described (Schraufstatter et al, 2001). Briefly, NIH 3T3 cells stably transfected with CXCR2, D138V, or KSHV-GPCR were seeded at low density on coverslips and grown in DMEM containing 10% CS.…”

“…In addition, recent investigations have shown that activation of the CXCR2 expressed on microvascular endothelial cells is responsible for the angiogenic response to IL-8 (Addison et al, 2000;Schraufstatter et al, 2001).…”