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Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

ABSTRACT: cAMP has been found to play a role in mediating the negative regulation of cell motility, although its underlying molecular mechanism remains poorly understood. By using CHO (Chinese-hamster ovary) cells that express the EP2 subtype of PGE2 (prostaglandin E2) receptors, we provide evidence that an increase in cellular cAMP content leads to inhibition of cellular Rac activity, which serves as a mechanism for this negative regulation. In CHO cells expressing EP2, but not in vector control cells, PGE2 dose-dependently inhibited chemotaxis towards IGF-I (insulin-like growth factor-I), which is a Rac-dependent process, with the maximal 75% inhibition observed at 10(-8) M PGE2. EP2 stimulation failed to inhibit tyrosine phosphorylation either of IGF-I receptor or IRS-1 (insulin receptor substrate-1), or activation of phosphoinositide 3-kinase or Akt in response to IGF-I, but potently and dose-dependently inhibited IGF-I-induced activation of cellular Rac activity and membrane ruffling. However, PGE2 failed to inhibit Val12-Rac-induced membrane ruffling. Similar to the case of CHO cells, PGE2 inhibited PDGF (platelet-derived growth factor)-induced Rac activation and chemotaxis in vascular smooth muscle cells endogenously expressing EP2. The inhibitory effects of PGE2 on IGF-I-induced chemotaxis, membrane ruffling and Rac activation were faithfully reproduced by a low concentration of forskolin, which induced a comparable extent of cAMP elevation as with 10(-8) M PGE2, and were potentiated by isobutylmethylxanthine. The protein kinase A inhibitor Rp isomer of adenosine 3',5'-cyclic monophosphorothioate reduced PGE2 inhibition of Rac activation and chemotaxis. These results indicate that EP2 mediates Rac inhibition through a mechanism involving cAMP and protein kinase A, thereby inhibiting membrane ruffling and chemotaxis.

SUBMITTER: Nagasawa SY

PROVIDER: S-EPMC1134749 | biostudies-other | 2005 Feb

REPOSITORIES: biostudies-other

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Project description:Hypoxia is a major driving force in vascularization and vascular remodeling. Pharmacological inhibition of prolyl hydroxylases (PHDs) leads to an oxygen-independent and long-lasting activation of hypoxia-inducible factors (HIFs). Whereas effects of HIF-stabilization on transcriptional responses have been thoroughly investigated in endothelial cells, the molecular details of cytoskeletal changes elicited by PHD-inhibition remain largely unknown. To investigate this important aspect of PHD-inhibition, we used a spheroid-on-matrix cell culture model.Microvascular endothelial cells (glEND.2) were organized into spheroids. Migration of cells from the spheroids was quantified and analyzed by immunocytochemistry. The PHD inhibitor dimethyloxalyl glycine (DMOG) induced F-actin stress fiber formation in migrating cells, but only weakly affected microvascular endothelial cells firmly attached in a monolayer. Compared to control spheroids, the residual spheroids were larger upon PHD inhibition and contained more cells with tight VE-cadherin positive cell-cell contacts. Morphological alterations were dependent on stabilization of HIF-1? and not HIF-2? as shown in cells with stable knockdown of HIF-? isoforms. DMOG-treated endothelial cells exhibited a reduction of immunoreactive Rac-1 at the migrating front, concomitant with a diminished Rac-1 activity, whereas total Rac-1 protein remained unchanged. Two chemically distinct Rac-1 inhibitors mimicked the effects of DMOG in terms of F-actin fiber formation and orientation, as well as stabilization of residual spheroids. Furthermore, phosphorylation of p21-activated kinase PAK downstream of Rac-1 was reduced by DMOG in a HIF-1?-dependent manner. Stabilization of cell-cell contacts associated with decreased Rac-1 activity was also confirmed in human umbilical vein endothelial cells.Our data demonstrates that PHD inhibition induces HIF-1?-dependent cytoskeletal remodeling in endothelial cells, which is mediated essentially by a reduction in Rac-1 signaling.

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Inhibition of Rac activation as a mechanism for negative regulation of actin cytoskeletal reorganization and cell motility by cAMP.

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