Reactive oxygen species production via NADPH oxidase mediates TGF-beta-induced cytoskeletal alterations in endothelial cells.

Cytoskeletal alterations in endothelial cells have been linked to nitric oxide generation and cell-cell interactions. Transforming growth factor (TGF)-beta has been described to affect cytoskeletal rearrangement in numerous cell types; however, the underlying pathway is unclear. In the present study, we found that human umbilical vein endothelial cells (HUVEC) have marked cytoskeletal alterations with short-term TGF-beta treatment resulting in filipodia formation and F-actin assembly. The cytoskeletal alterations were blocked by the novel TGF-beta type I receptor/ALK5 kinase inhibitor (SB-505124) but not by the p38 kinase inhibitor (SB-203580). TGF-beta also induced marked stimulation of reactive oxygen species (ROS) within 5 min of TGF-beta exposure. TGF-beta stimulation of ROS was mediated by the NAPDH oxidase homolog Nox4 as DPI, an inhibitor of NADPH oxidase, and dominant-negative Nox4 adenovirus blocked ROS production. Finally, inhibition of ROS with ROS scavengers or dominant-negative Nox4 blocked the TGF-beta effect on cytoskeleton changes in endothelial cells. In conclusion, our studies show for the first time that TGF-beta-induced ROS production in human endothelial cells is via Nox4 and that TGF-beta alteration of cytoskeleton in HUVEC is mediated via a Nox4-dependent pathway.

Thrombin and tumor necrosis factor alpha synergistically stimulate tissue factor expression in human endothelial cells: regulation through c-Fos and c-Jun.

Tissue factor is critically important for initiating the activation of coagulation zymogens leading to the generation of thrombin. Quiescent endothelial cells do not express tissue factor on their surface, but many stimuli including cytokines and coagulation proteases can elicit tissue factor synthesis. We challenged human endothelial cells simultaneously with tumor necrosis factor alpha (TNFalpha) and thrombin because many pathophysiological conditions, such as sepsis, diabetes, and coronary artery disease, result in the concurrent presence of circulating inflammatory mediators and activated thrombin. We observed a remarkable synergy in the expression of tissue factor by thrombin plus TNFalpha. This was due to altered regulation of the transcription factors c-Jun and c-Fos. The activation of c-Jun was greater and more sustained than that obtained with either thrombin or TNFalpha alone. Thrombin-stimulated expression of c-Fos was both enhanced and prolonged by the concurrent presence of TNFalpha. These changes support the increased availability of c-Jun/c-Fos AP-1 complexes for mediating transcription at the tissue factor promoter. Transcription factors downstream of the extracellular signal-regulated kinases as well as changes in NFkappaB regulation were not involved in the synergistic increase in tissue factor expression by thrombin and TNFalpha. Thus, concurrent exposure of vascular endothelial cells to cytokines and procoagulant proteases such as thrombin can result in greatly enhanced tissue factor expression on the endothelium, thereby perpetuating the prothrombotic phenotype of the endothelium.

Role for alpha 6 integrin during lens development: Evidence for signaling through IGF-1R and ERK.

We show that alpha 6 integrin function was required for normal lens cell differentiation by using an antisense construct to suppress alpha 6 integrin expression. To elucidate the mechanism by which this integrin functions in the regulation of the lens cell differentiation process, we determined the molecular composition of alpha 6 integrin signaling complexes at distinct stages of differentiation in vivo. Because both alpha 6 integrin and insulin-like growth factor-1 (IGF-1) have been implicated in signaling lens cell differentiation, we examined the possibility that they formed a signaling complex in the embryonic lens. Coprecipitation analysis revealed that alpha 6 integrin/IGF-1 receptor complexes were present and that their association was greatest in the equatorial zone, the region of the embryonic lens in which lens cells proliferate and then initiate their differentiation. These results provide in vivo support for the formation of integrin/growth factor receptor signaling complexes. We also found that extracellular signal-regulated kinase (ERK), a downstream effector of both integrin and growth factor receptor signaling pathways, was associated with the alpha 6 integrin signaling complexes in the embryonic lens. This result was supported by our findings that activated ERK, in addition to its nuclear location, localized to lens cell membranes in specific regions of cell-matrix and cell-cell contact. A connection between integrin ligand engagement and ERK activation was shown in vitro after lens cell attachment to laminin. These results demonstrate that alpha 6 integrin function is required for the early stages of lens cell differentiation most likely through its association with the IGF-1 receptor and the activation of ERK.

Regulation of VE-cadherin linkage to the cytoskeleton in endothelial cells exposed to fluid shear stress.

Endothelial cells exposed to shear stress realigned and elongated in the direction of flow through the coordinated remodeling of their adherens junctions and actin cytoskeleton. The elaborate networks of VE-cadherin complexes in static cultures became more uniform and compact in response to shear. In contrast, the cortical actin present in static cultures was reorganized into numerous stress fiber bundles distributed parallel to the direction of flow. Exposure to shear did not significantly alter the expression of the junctional proteins VE-cadherin, beta-catenin, and alpha-catenin, but the composition of the junctional complexes did change. We detected a marked decrease in the alpha-catenin associated with VE-cadherin complexes in endothelial monolayers subjected to shear. This loss of alpha-catenin, the protein that links beta-catenin-bound cadherin to the actin cytoskeleton, was not due to decreased quantities of beta-catenin associated with VE-cadherin. Instead, the loss of alpha-catenin from the junctional complexes coincided with the increased tyrosine phosphorylation of beta-catenin associated with VE-cadherin. The change in beta-catenin phosphorylation closely correlated with the shear-induced loss of the protein tyrosine phosphatase SHP-2 from VE-cadherin complexes. Thus, the functional interaction of alpha-catenin with VE-cadherin-bound beta-catenin is regulated by the extent of tyrosine phosphorylation of beta-catenin. This, concomitantly, is regulated by SHP-2 associated with VE-cadherin complexes.