Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although RhoA plays an important role in cell proliferation and in Ras transformation in fibroblasts and mammary epithelial cells, its role in intestinal epithelial cells (IEC) is unknown. In a previous study (Ray RM, Zimmerman BJ, McCormack SA, Patel TB, and Johnson LR. Am J Physiol Cell Physiol 276: C684-C691, 1999), we showed that polyamine depletion [dl-alpha-difluoromethylornithine (DFMO) treatment] strongly inhibits the proliferation of IEC. In this report, we examined the effect of RhoA on IEC-6 cell proliferation and whether polyamine depletion inhibits cell proliferation in the presence of constitutively active RhoA. Constitutively active RhoA and vector-transfected IEC-6 cell lines were grown in the presence or absence of DFMO, which causes polyamine depletion by inhibiting ornithine decarboxylase, the first rate-limiting step in polyamine synthesis. Constitutively active RhoA significantly increased the rate of cell proliferation. These cells also lost contact inhibition and formed conspicuous foci when they were fully confluent. Decreased p21Waf1/Cip1 expression and increased cyclin-dependent kinase (Cdk2) mRNA levels and activity accompanied the increased proliferation. The inhibition of p21Waf1/Cip1 was independent of p53. There was no activation of the Ras-Raf-MEK-ERK pathway in the RhoA-transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on IEC-6 cell proliferation, focus formation, and Cdk2 expression. The stability of mRNA and protein for Cdk2 and p21Waf1/Cip1 in V14-RhoA cells was not significantly different from that of vector-transfected cells. In conclusion, RhoA activation decreased p21Waf1/Cip1 expression and increased basal and serum-induced ornithine decarboxylase activity, Cdk2 expression, Cdk2 protein, and Cdk2 activity, leading to the stimulation of IEC proliferation and transformation. Polyamine depletion totally prevented RhoA's effect on proliferation by decreasing Cdk2 expression and activity.
 ...
PMID:RhoA stimulates IEC-6 cell proliferation by increasing polyamine-dependent Cdk2 activity. 1281 57

It has been documented that polyamines play a critical role in the regulation of apoptosis in intestinal epithelial cells. We have recently reported that protection from TNF-alpha/cycloheximide (CHX)-induced apoptosis in epithelial cells depleted of polyamines is mediated through the inactivation of a proapoptotic mediator, JNK. In this study, we addressed the involvement of the MAPK pathway in the regulation of apoptosis after polyamine depletion of IEC-6 cells. Polyamine depletion by alpha-difluromethylornithine (DFMO) resulted in the sustained activation of ERK in response to TNF-alpha/CHX treatment. Pretreatment of polyamine-depleted IEC-6 cells with a cell membrane-permeable MEK1/2 inhibitor, U-0126, significantly inhibited TNF-alpha/CHX-induced ERK phosphorylation and significantly increased DNA fragmentation, JNK activity, and caspase-3 activity in response to TNF-alpha/CHX. Moreover, the dose dependency of U-0126-mediated inhibition of TNF-alpha/ CHX-induced ERK phosphorylation correlated with the reversal of the antiapoptotic effect of DFMO. IEC-6 cells expressing constitutively active MEK1 had decreased TNF-alpha/CHX-induced JNK phosphorylation and were significantly protected from apoptosis. Conversely, a dominant-negative MEK1 resulted in high basal activation of JNK, cytochrome c release, and spontaneous apoptosis. Polyamine depletion of the dominant-negative MEK1 cells did not prevent JNK activation or cytochrome c release and failed to confer protection from both TNF-alpha/CHX and camptothecin-induced apoptosis. Finally, expression of a dominant-negative mutant of JNK significantly protected IEC-6 cells from TNF-alpha/CHX-induced apoptosis. These data indicate that polyamine depletion results in the activation of ERK, which inhibits JNK activation and protects cells from apoptosis.
 ...
PMID:Prevention of TNF-alpha-induced apoptosis in polyamine-depleted IEC-6 cells is mediated through the activation of ERK1/2. 1456 73

We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation.
 ...
PMID:MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm. 1549 79

Our previous studies have shown that polyamines are required for normal intestinal mucosal growth and that decreased levels of polyamines inhibit intestinal epithelial cell (IEC) proliferation by stabilizing p53 and other growth-inhibiting proteins. Nucleophosmin (NPM) is a multifunctional protein that recently has been shown to regulate p53 activity. In the present study, we sought to determine whether polyamine depletion increases NPM modulating the stability and transcriptional activity of p53 in a normal IEC-6 intestinal epithelial cell line. Depletion of cellular polyamines by alpha-difluoromethylornithine, the specific inhibitor of polyamine biosynthesis, stimulated expression of the NPM gene and induced nuclear translocation of NPM protein. Polyamine depletion stimulated NPM expression primarily by increasing NPM gene transcription and its mRNA stability, and it induced NPM nuclear translocation through activation of phosphorylation of mitogen-activated protein kinase kinase. Increased NPM interacted with p53 and formed a NPM/p53 complex in polyamine-deficient cells. Inhibition of NPM expression by small interfering RNA targeting NPM (siNPM) not only destabilized p53 as indicated by a decrease in its protein half-life but also prevented the increased p53-dependent transactivation as shown by suppression of the p21 promoter activity. Decreased expression of NPM by siNPM also promoted cell growth in polyamine-deficient cells. These results indicate that 1) polyamine depletion increases expression of the NPM gene and enhances NPM nuclear translocation and 2) increased NPM interacts with and stabilizes p53, leading to inhibition of IEC-6 cell proliferation.
 ...
PMID:Polyamine depletion induces nucleophosmin modulating stability and transcriptional activity of p53 in intestinal epithelial cells. 1587 11

Polyamine depletion prevents apoptosis by increasing serine/threonine phosphorylation leading to either inactivation or activation of pro- and anti-apoptotic proteins, respectively. Despite evidence that protein kinases are regulators of apoptosis, a specific role for protein phosphatases in regulating cell survival has not been established. In this study, we show that polyamine depletion inhibits serine/threonine phosphatase 2A (PP2A). Inhibition of PP2A in cells depleted of polyamines correlated well with increased phosphorylation of Bad at Ser112. Bad Ser112 phosphorylation in response to tumor necrosis factor (TNF)-alpha treatment decreased with time in cells grown in control as well as those grown in the presence of alpha-difluoromethylornithine plus putrescine. However, a sustained increase in the levels of Bad Ser112 phosphorylation was maintained in response to TNF-alpha treatment in cells grown in the presence of alpha-difluoromethylornithine. Inhibition of PP2A by okadaic acid and fostriecin or PP2A small interfering RNA transfection significantly decreased TNF-alpha-induced apoptosis in control and polyamine-depleted cells. Inhibition of PP2A by okadaic acid: 1) increased Bad and Bcl-2 phosphorylation at Ser112 and Ser70, respectively; 2) increased ERK activity; 3) prevented JNK activation; 4) prevented cytochrome c release, and activation of caspases-9 and -3 in response to TNF-alpha. Inhibition of MEK1 by U0126 prevented phosphorylation of Bad at Ser112. These results indicate that polyamines regulate PP2A activity, and inhibition of PP2A in response to polyamine depletion increases steady state levels of Bad and Bcl-2 proteins and their phosphorylation and thereby prevents cytochrome c release, caspase-9, and caspase-3 activation.
 ...
PMID:Protein phosphatase 2A regulates apoptosis in intestinal epithelial cells. 1599 15

Polyamine depletion with the ornithine decarboxylase inhibitor alpha-difluoromethyl ornithine (DFMO), prevents Rac1 activation causing the formation of a thick actin cortex at the cell periphery and inhibits migration of intestinal epithelial cells. In the present study, we demonstrate that MEK activation by EGF increased Rac1 activation, dissociation of intercellular contacts, and migration in both control and polyamine-depleted cells, while U0126, a specific inhibitor of MEK1, prevented disruption of junctions as well as EGF-induced Rac1 activation. Constitutively active MEK1 (CA-MEK) expression altered cell-cell contacts in control and polyamine depleted cells. The expression of constitutively active Rac1 (CA-Rac1) restored beta-catenin to the cell periphery and prevented the formation of actin cortex and caused the appearance of F-actin stress fibers in polyamine-depleted cells. Inhibition of Rac activation by NSC23766, a specific inhibitor of Tiam1, an upstream guanidine nucleotide exchange factor for Rac1, reproduced the beta-catenin localization and actin structure of polyamine-depleted cells. Tiam1 localized more extensively with beta-catenin at the cell periphery in CA-Rac1 cells compared to vector cells. Polyamine depletion decreased the expression of E-cadherin to a greater extent compared to beta-catenin. Subcellular fractionation further confirmed our immuno-localization and western blotting observations. These data suggest that EGF acting through MEK1/ERK to activate Rac1 regulates cell-cell contacts. Thus, decreased migration in polyamine depleted cells may be due to the inhibition of Tiam1 activation of Rac1 and the subsequent decreased expression of beta-catenin and E-cadherin leading to reduced cell-cell contacts.
 ...
PMID:MEK/ERK regulates adherens junctions and migration through Rac1. 1718 46

Intracellular polyamine synthesis is regulated by the enzyme ornithine decarboxylase (ODC), and its inhibition by alpha-difluromethylornithine (DFMO), confers resistance to apoptosis. We have previously shown that DFMO leads to the inhibition of de novo polyamine synthesis, which in turn rapidly activates Src, STAT3 and NF-kappaB via integrin beta3 in intestinal epithelial cells. One mechanism to explain these effects involves the activation of upstream growth factor receptors, such as the epidermal growth factor receptor (EGFR). We therefore hypothesized that EGFR phosphorylation regulates the early response to polyamine depletion. DFMO increased EGFR phosphorylation on tyrosine residues 1173 (pY1173) and 845 (pY845) within 5 min. Phosphorylation declined after 10 min and was prevented by the addition of exogenous putrescine to DFMO containing medium. Phosphorylation of EGFR was concomitant with the activation of ERK1/2. Pretreatment with either DFMO or EGF for 1 h protected cells from TNF-alpha/CHX-induced apoptosis. Exogenous addition of polyamines prevented the protective effect of DFMO. In addition, inhibition of integrin beta3 activity (with RGDS), Src activity (with PP2), or EGFR kinase activity (with AG1478), increased basal apoptosis and prevented protection conferred by either DFMO or EGF. Polyamine depletion failed to protect B82L fibroblasts lacking the EGFR (PRN) and PRN cells expressing either a kinase dead EGFR (K721A) or an EGFR (Y845F) mutant lacking the Src phosphorylation site. Conversely, expression of WT-EGFR (WT) restored the protective effect of polyamine depletion. Fibronectin activated the EGFR, Src, ERKs and protected cells from apoptosis. Taken together, our data indicate an essential role of EGFR kinase activity in MEK/ERK-mediated protection, which synergizes with integrin beta3 leading to Src-mediated protective responses in polyamine depleted cells.
 ...
PMID:EGFR plays a pivotal role in the regulation of polyamine-dependent apoptosis in intestinal epithelial cells. 1782 25

Polyamine-depletion inhibited apoptosis by activating ERK1/2, while, preventing JNK1/2 activation. MKP-1 knockdown by SiRNA increased ERK1/2, JNK1/2, and p38 phosphorylation and apoptosis. Therefore, we predicted that polyamines might regulate MKP1 via MEK/ERK and thereby apoptosis. We examined the role of MEK/ERK in the regulation of MKP1 and JNK, and p38 activities and apoptosis. Inhibition of MKP-1 activity with a pharmacological inhibitor, sanguinarine (SA), increased JNK1/2, p38, and ERK1/2 activities without causing apoptosis. However, pre-activation of these kinases by SA significantly increased camptothecin (CPT)-induced apoptosis suggesting different roles for MAPKs during survival and apoptosis. Inhibition of MEK1 activity prevented the expression of MKP-1 protein and augmented CPT-induced apoptosis, which correlated with increased activities of JNK1/2, caspases, and DNA fragmentation. Polyamine depleted cells had higher levels of MKP-1 protein and decreased JNK1/2 activity and apoptosis. Inhibition of MEK1 prevented MKP-1 expression and increased JNK1/2 and apoptosis. Phospho-JNK1/2, phospho-ERK2, MKP-1, and the catalytic subunit of PP2Ac formed a complex in response to TNF/CPT. Inactivation of PP2Ac had no effect on the association of MKP-1 and JNK1. However, inhibition of MKP-1 activity decreased the formation of the MKP-1, PP2Ac and JNK complex. Following inhibition by SA, MKP-1 localized in the cytoplasm, while basal and CPT-induced MKP-1 remained in the nuclear fraction. These results suggest that nuclear MKP-1 translocates to the cytoplasm, binds phosphorylated JNK and p38 resulting in dephosphorylation and decreased activity. Thus, MEK/ERK activity controls the levels of MKP-1 and, thereby, regulates JNK activity in polyamine-depleted cells.
...
PMID:The mechanism by which MEK/ERK regulates JNK and p38 activity in polyamine depleted IEC-6 cells during apoptosis. 2425 95