Confluence of Vascular Endothelial Cells Induces Cell Cycle Exit by Inhibiting p42/p44 Mitogen-Activated Protein Kinase Activity

Abstract

Like other cellular models, endothelial cells in cultures stop growing when they reach confluence, even in the presence of growth factors. In this work, we have studied the effect of cellular contact on the activation of p42/p44 mitogen-activated protein kinase (MAPK) by growth factors in mouse vascular endothelial cells. p42/p44 MAPK activation by fetal calf serum or fibroblast growth factor was restrained in confluent cells in comparison with the activity found in sparse cells. Consequently, the induction of c-fos, MAPK phosphatases 1 and 2 (MKP1/2), and cyclin D1 was also restrained in confluent cells. In contrast, the activation of Ras and MEK-1, two upstream activators of the p42/p44 MAPK cascade, was not impaired when cells attained confluence. Sodium orthovanadate, but not okadaic acid, restored p42/p44 MAPK activity in confluent cells. Moreover, lysates from confluent 1G11 cells more effectively inactivated a dually phosphorylated active p42 MAPK than lysates from sparse cells. These results, together with the fact that vanadate-sensitive phosphatase activity was higher in confluent cells, suggest that phosphatases play a role in the down-regulation of p42/p44 MAPK activity. Enforced long-term activation of p42/p44 MAPK by expression of the chimera ⌬Raf-1:ER, which activates the p42/p44 MAPK cascade at the level of Raf, enhanced the expression of MKP1/2 and cyclin D1 and, more importantly, restored the reentry of confluent cells into the cell cycle. Therefore, inhibition of p42/p44 MAPK activation by cell-cell contact is a critical step initiating cell cycle exit in vascular endothelial cells.

Fluid shear stress inhibits TNF-α activation of JNK but not ERK1/2 or p38 in human umbilical vein endothelial cells: Inhibitory crosstalk among MAPK family members

“…2). Flow stimulated a small (1.9 Ϯ 0.5-fold) increase in ERK1͞2 activity measured by phospho-ERK1͞2 antibodies as reported (9,13). The magnitude of the increase in ERK1͞2 activity was smaller than shown in Fig.…”

“…Our lab, as well as others, has shown that members of the mitogen-activated protein kinase (MAPK) family are activated by steady laminar flow in a time-and force-dependent manner (7)(8)(9)(10)(11)(12)(13). Recently, two groups showed that steady laminar flow stimulated c-Jun NH 2 -terminal kinases (JNK1 and JNK2) to a greater extent than extracellular signalregulated kinase (ERK)1͞2 (12,14).…”

p70 S6 Kinase-mediated Protein Synthesis Is a Critical Step for Vascular Endothelial Cell Proliferation

“…In endothelial cells, it has been described that PD98059 completely inhibits thymidine incorporation stimulated by vascular endothelial growth factor (36 -38) and affects the mitogenic effect of bFGF (39). Moreover, repression of p42/p44 MAPK activity in confluent endothelial cells is required for the maintenance of the quiescent state (21). In contrast, much less is known about the role of the PI3K-Akt-p70 S6K module in the control of endothelial cell growth.…”

“…Mouse brain capillary endothelial LIBE cells were obtained from Dr. Claesson-Welsh (Ludwig Institute for Cancer Research, Uppsala, Sweden) and were cultivated as previously described (21). Human umbilical vein endothelial cells (HUVEC) were obtained as described previously (22) and were cultivated in EBM-2 supplemented endothelial cell medium (Clonetics, BioWhittaker).…”

Common alterations in gene expression and increased proliferation in recurrent acute myeloid leukemia

“…Increased activation of the RAF/MEK/ERK pathway in relapsed/refractory AML Enhanced activity of the RAF/MEK/ERK pathway is associated with increased mRNA levels of MKP-1, cyclin D1, c-jun, c-fos, and egr-1 (Gille et al, 1992;Rozek and Pfeifer, 1993;Hodge et al, 1998;Vinals and Pouyssegur, 1999). Comparison of expression data from the 10 AML DX bone marrow specimens and the eight AML REL specimens showed a 2.8-fold70.18 increase of MKP-1 in the 'relapse' group.…”