Akt Down-Regulates ERK1/2 Nuclear Localization and Angiotensin II-induced Cell Proliferation through PEA-15

Proliferation inhibition of vascular smooth muscle cells (VSMCs) is governed by the activity of a transcription factor network. Krüppel-like factor 4 (Klf4), retinoic acid receptor (RAR␣), and platelet-derived growth factor receptor (PDGFR) are expressed in VSMCs and are components of such a network. However, the relationship among them in the regulation of VSMC proliferation remains unknown. Here, we investigated the mechanisms whereby Klf4 mediates the growth inhibitory effects in VSMCs through RAR␣ and PDGFR␤. We demonstrated that Klf4 directly binds to the 5 regulatory region of RAR␣, down-regulates RAR␣ expression, and specifically inhibits RAR␣-mediated phosphatidylinositol 3-kinase (PI3K) and ERK signaling in cultured VSMCs induced by the synthetic retinoid Am80. Of particular interest, Klf4 inhibits RAR␣ and PDGFR␤ expression while blocking PI3K and ERK signaling induced by Am80 and PDGF-BB, respectively. The anti-proliferative effects of Klf4 on neointimal formation depend largely on PDGFR-mediated PI3K signaling without involvement of the RAR␣-activated signaling pathways. These findings provide a novel mechanism for signal suppression and growth inhibitory effects of Klf4 in VSMCs. Moreover, the results of this study suggest that Klf4 is one of the key mediators of retinoid actions in VSMCs.The regulation of differentiation and proliferation of vascular smooth muscle cells (VSMCs) 2 is known to be critical in blood vessel formation during embryogenesis and in pathological states such as atherogenesis, restenosis, and hypertension. There is considerable interest in identifying extracellular signals, signal transduction molecules, and transcription factors that are involved in the regulation of VSMC proliferation and differentiation (1, 2). Many different environmental cues are known to affect VSMC proliferation and differentiation, including endothelial cell-VSMC interactions, VSMC-matrix contacts, mechanical forces, and various cytokines such as platelet-derived growth factor BB (PDGF-BB), activin, and transforming growth factor-␤1 (3, 4). The Krüppel-like factors (Klfs) are DNA-binding transcriptional regulators that regulate a diverse array of cellular processes, including development, differentiation, proliferation, and apoptosis. Members of this family bind similar CACCC elements on DNA (5). Klf4 was first identified as being highly expressed in epithelial cells, and gain/ loss of function studies in vivo have confirmed a critical role for this factor in epithelial cell differentiation (6). Subsequent work has underlined the importance of Klf4 in a wide range of cellular processes, such as endothelial pro-inflammatory activation, macrophage gene expression, tumor cell development, and stem cell biology (7). Klf4 was also isolated as a Klf expressed in the vasculature and could be induced by shear stress (8). Klf4 has been shown to repress the transforming growth factor-␤-dependent increase in VSMC differentiation markers, including those for ␣-smooth muscle actin and SM22␣ (1). A recent study sh...

Section: Discussionsupporting

confidence: 67%

Krüppel-like Factor 4 Inhibits Proliferation by Platelet-derived Growth Factor Receptor β-mediated, Not by Retinoic Acid Receptor α-mediated, Phosphatidylinositol 3-Kinase and ERK Signaling in Vascular Smooth Muscle Cells

Zheng

Han

Bernier

et al. 2009

“…The outcomes of signaling via the mitogen-activated protein kinase pathway are determined by the regulation of ERK nuclear translocation (5,10,44), which is required for activation of many transcription factors. The duration and extent of translocation is dependent on the type of stimulus, and ERK nuclear translocation is required to induce specific responses in the form of gene expression (45).…”

Section: Discussionmentioning

confidence: 99%

“…MEK binds to inactive ERK in resting cells (8). The natural regulation of ERK translocation has also been demonstrated by differential expression of cytoplasmic anchors such as PEA15 (9,10) or Sef (11) or expression of nuclear anchors such as DUSP5 (12) or Vanishing (13). It has been shown that the stimulation-induced nuclear accumulation of ERK requires the synthesis of short lived nuclear anchors (14).…”

mentioning

confidence: 99%

ERK Nuclear Translocation Is Dimerization-independent but Controlled by the Rate of Phosphorylation

Lidke

Huang

Post

et al. 2010

Upon activation, ERKs translocate from the cytoplasm to the nucleus. This process is required for the induction of many cellular responses, yet the molecular mechanisms that regulate ERK nuclear translocation are not fully understood. We have used a mouse embryo fibroblast ERK1-knock-out cell line expressing green fluorescent protein (GFP)-tagged ERK1 to probe the spatiotemporal regulation of ERK1. Real time fluorescence microscopy and fluorescence correlation spectroscopy revealed that ERK1 nuclear accumulation increased upon serum stimulation, but the mobility of the protein in the nucleus and cytoplasm remained unchanged. Dimerization of ERK has been proposed as a requirement for nuclear translocation. However, ERK1-⌬4, the mutant shown consistently to be dimerization-deficient in vitro, accumulated in the nucleus to the same level as wild type (WT), indicating that dimerization of ERK1 is not required for nuclear entry and retention. Consistent with this finding, energy migration Förster resonance energy transfer and fluorescence correlation spectroscopy measurements in living cells did not detect dimerization of GFP-ERK1-WT upon activation. In contrast, the kinetics of nuclear accumulation and phosphorylation of GFP-ERK1-⌬4 were slower than that of GFP-ERK1-WT. These results indicate that the differential shuttling behavior of the mutant is a consequence of delayed phosphorylation of ERK by MEK rather than dimerization. Our data demonstrate for the first time that a delay in cytoplasmic activation of ERK is directly translated into a delay in nuclear translocation.Stimulation of numerous cell surface receptors leads to activation of the Raf/MEK 7 /ERK signaling pathway. In this kinase cascade, Raf phosphorylates only MEK, and MEK phosphorylates only ERK, whereas ERK is able to phosphorylate many substrates in nearly all cell compartments (1). Noncatalytic activation of a few partners by c-Raf is well documented, but the biological outcomes of the ERK pathway are predominantly driven by the kinase activity, as evidenced, for example, by chemical inhibition (reviewed in Ref.2). ERK is primarily located in the cytoplasm of resting cells, although overexpression results in cytoplasmic and nuclear localization (3). It has long been recognized that in the course of physiological signal transduction, ERK accumulates in the nucleus after acute stimulation of the cell (3, 4). Nuclear translocation of ERK is required for cell cycle entry. Thus, retention of ERK in the cytoplasm alters neither ERK kinase activity nor phosphorylation of cytoplasmic substrates, whereas ERK-dependent transcription and cell proliferation are blocked (5). It has been demonstrated that ERK phosphorylates the Phe-Gly nucleoporins Nup50, Nup153, and Nup154, reducing importin-␤-mediated nucleocytoplasmic transport (6). This observation would expand the role of ERK nuclear entry to include the regulation of nucleocytoplasmic transport of certain classes of proteins while crossing the nucleopore.MEK functions as the cytoplasmic anchor for ERK su...

“…Nevertheless, PEA15 seems to be an excellent convergence point for ERK1/2 and Akt signaling pathways that needs to be fully addressed. This fact is even more interesting if we consider the implication of PEA15 in biological processes such as response to angiotensin (25) or cell motility and invasion (30,31). But the interference of E1a with the ERK1/2 signaling pathway is also due to the up-regulation of the FIGURE 5.…”

Section: Discussionmentioning

confidence: 99%

“…The stability and functionality of the protein seem to be dependent on the phosphorylation at two critical residues, Ser 104 and Ser 116 (24). This last residue, Ser 116 , is a natural phosphoacceptor of Akt activity with implications in the stability and functionality of the protein (25,26). Considering the inhibitory effect of E1a in the Akt signaling pathway (11,12), we decided to evaluate if E1a was affecting PEA15 stability through interference with Akt.…”

Section: E1a Blocks V-h-ras-induced Erk1/2 Activation and Promotes Itmentioning

confidence: 99%

E1a Gene Expression Blocks the ERK1/2 Signaling Pathway by Promoting Nuclear Localization and MKP Up-regulation

Callejas‐Valera

Guinea-Viniegra

Ramírez-Castillejo

et al. 2008

In response to oncogenic signals, cells have developed safe mechanisms to avoid transformation through activation of a senescence program. Upon v-H-Ras overexpression, normal cells undergo senescence through several cellular processes, including activation of the ERK1/2 pathway. Interestingly, the E1a gene from adenovirus 5 has been shown to rescue cells from senescence by a yet unknown mechanism. We investigated whether E1a was able to interfere with the ERK1/2 signaling pathway to rescue cells from v-H-Ras-mediated senescence. Our results show that, E1a overexpression blocks v-H-Ras-mediated ERK1/2 activation by two different and concomitant mechanisms. E1a through its ability to interfere with PKB/Akt activation induces the down-regulation of the PEA15 protein, an ERK1/2 nuclear export factor, leading to nuclear accumulation of ERK1/2. In addition to this, we show that E1a increases the expression of the inducible ERK1/2 nuclear phosphatases (MAPK phosphatases) MKP1/DUSP1 and DUSP5, which leads to ERK1/2 dephosphorylation. We confirmed our observations in the human normal diploid fibroblasts IMR90, in which we could also show that an E1a mutant, unable to bind retinoblastoma protein (pRb), cannot rescue cells from v-H-Ras-induced senescence. In conclusion, E1a is able to rescue from Ras-induced senescence by affecting ERK1/2 localization and phosphorylation.