The MEK/ERK pathway acts upstream of NF kappa B1 (p50) homodimer activity and Bcl-2 expression in a murine B-cell lymphoma cell line. MEK inhibition restores radiation-induced apoptosis

In a previously published report (Kurland, J. F., Kodym, R., Story, M. D., Spurgers, K. B., McDonnell, T. J., and Meyn, R. E. (2001) J. Biol. Chem. 276, 45380-45386), we described the NF kappa B status for two murine B-cell lymphoma cell lines, LY-as (apoptosis-sensitive) and LY-ar (apoptosis-refractory) and provided evidence that NF kappa B1 (p50) homodimers contribute to the expression of Bcl-2 in the LY-ar line. In the present study, we investigated the upstream signals leading to p50 homodimer activation and Bcl-2 expression. We found that in LY-ar cells, ERK1 and ERK2 were constitutively phosphorylated, whereas LY-as cells had no detectable ERK1 or ERK2 phosphorylation. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK1 and ERK2, a reversal of nuclear p50 homodimer DNA binding, and a decrease in Bcl-2 protein expression. Similarly, activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with tumor necrosis factor-alpha, an I kappa B kinase activator, did not alter the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an I kappa B kinase-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. We conclude that the MEK/ERK pathway acts upstream of p50 homodimer activity and Bcl-2 expression in this B-cell lymphoma cell system and suggest that the use of MEK inhibitors could be useful clinically in combination with ionizing radiation to treat lymphoid malignancies.     

Retinoic acid increases amount of phosphorylated raf; Ectopic expression of cFMS reveals that retinoic acid-induced differentiation is more strongly dependent on ERK2 signaling than induced go arrest is

Summary Retinoic acid is known to cause the myeloid differentiation and G1/0 cell cycle arrest of HL-60 cells in a process that requires mitogen-activated protein/extracellular signal regulated kinase (MEK)-dependent extracellular signal regulated kinase (ERK)2 activation. It has also been shown that ectopic expression of cFMS, a platelet-derived growth factor (PDGF)-family transmembrane tyrosine kinase receptor, enhances retinoic acid-induced differentiation and G1/0 arrest. The mechanism of how the retinoic acid and cFMS signaling pathways intersect is not known. The present data show that the ectopic expression of cFMS results in the differential loss of sensitivity of retinoic acid-induced differentiation or G1/0 arrest to inhibition of ERK2 activation. PD98059 was used to inhibit MEK and consequently ERK2. In wild-type HL-60 cells, PD98059 blocked retinoic acid-induced differentiation; but in cFMS stable transfectants, PD98059 only attenuated the induced differentiation, with the resulting response resembling that of retinoic acid-treated wild-type HL-60. In wild-type HL-60, PD98059 greatly attenuated the retinoic acid-induced G1/0 arrest allied with retinoblastoma (RB) hypophosphorylation; but in cFMS stable transfectants, PD98059 had no inhibitory effect on RB hypophosphorylation and G1/0 arrest. This differential sensitivity to PD98059 and uncoupling of retinoic acid-induced differentiation and G1/0 arrest in cFMS transfectants is associated with changes in mitogen-activated protein kinase signaling molecules. The cFMS transfectants had more activated ERK2 than did the wild-type cells, which surprisingly was not attributable to enhanced mitogen-activated protein-kinase-kinase-kinase (RAF) phosphorylation. Retinoic acid increased the amount of activated ERK2 and phosphorylated RAF in both cell lines. But PD98059 eliminated detectable ERK2 activation, as well as inhibited RAF phosphorylation, in untreated and retinoic acid-treated wild-type HL-60 and cFMS transfectants, consistent with MEK or ERK feedback-regulation of RAF, in all four cases. Since PD98059 blocks the cFMS-conferred enhancement of the retinoic acid-induced differentiation, but not growth arrest, the data indicate that cFMS-enhanced differentiation acts primarily through MEK and ERK2, but cFMS-enhanced G1/0 arrest allied with RB hypophosphorylation depends on another cFMS signal route, which by itself can effect G1/0 arrest without activated ERK2. Ectopic expression of cFMS and differential sensitivity to ERK2 inhibition thus reveal that retinoic acid-induced HL-60 cell differentiation and G1/0 arrest are differentially dependent on ERK2 and can be uncoupled. A significant unanticipated finding was that retinoic acid caused a MEK-dependent increase in the amount of phosphorylated RAF. This increase may help sustain prolonged ERK2 activation.     

Oligonol, an oligomerized lychee fruit-derived polyphenol, activates the Ras/Raf-1/MEK1/2 cascade independent of the IL-6 signaling pathway in rat primary adipocytes

Oligonol is a lychee fruit-derived low-molecular form of polyphenol. In this study, the effect of Oligonol on the mitogen activated-protein kinase (MAPK) signaling pathway in primary adipocytes was investigated to examine the mechanism underlying the enhanced levels of phosphorylated extracellular-signaling regulatory kinase1/2 (ERK1/2) that accompany an in vitro increase in lipolysis. Oligonol significantly elevated the levels of activated Ras and the phosphorylation of Raf-1 and MAPK/ERK kinase1/2 (MEK1/2) with no increase in pan-Raf-1 and -MEK1/2 proteins. The increase in phosphorylation of Raf-1 and MEK1/2 with Oligonol was inhibited completely by pretreatment with GW5074, a selective Raf-1 inhibitor, or PD98059, a selective MEK1/2 inhibitor. IL-6 also activated the MAPK signaling pathway in adipocytes through the association with its receptor. IL-6-induced phosphorylation of Raf-1 and MEK1/2 was significantly inhibited by pretreatment with the IL-6 receptor antibody. Under such a condition, however, the levels of phosphorylated Raf-1 and MEK1/2 with Oligonol still remained significantly higher, and there was a significant decrease in secretion of IL-6 from adipocytes, compared with untreated control cells. These results suggest that Oligonol activates the Ras/Raf-1/MEK1/2 signaling pathway, independent of the IL-6 signaling pathway, leading to activation of ERK1/2 proteins in primary adipocytes.     

cAMP potentiates H(2)O(2)-induced ERK1/2 phosphorylation without the requirement for MEK1/2 phosphorylation

In Jurkat T lymphocytes, hydrogen peroxide (H(2)O(2)) potentiates the phosphorylation level of extracellular signal-regulated kinase 1 and 2 (ERK1/2) caused by T cell receptor (TCR) stimulation with anti-CD3 and anti-CD28 or anti-CD3 alone. Submillimolar concentrations of H(2)O(2)-induced phosphorylation of ERK1/2 and MAP/ERK kinase 1 and 2 (MEK1/2) without antigenic stimulation. H(2)O(2) also induced the electrophoretic mobility shift of Lck from 56 to 60 kDa. The MEK inhibitor, PD98059 attenuated ERK1/2 and MEK1/2 phosphorylation, as well as the migration shift of Lck induced by H(2)O(2). The phospholipase C (PLC) inhibitor, U73122, and EGTA reduced the phosphorylation of both ERK1/2 and MEK1/2 induced by H(2)O(2). Interestingly, an increase of intracellular cAMP level with forskolin or 8-(4-chlorophenylthio)-cAMP augmented ERK1/2 phosphorylation by H(2)O(2), while inhibiting MEK1/2 phosphorylation by H(2)O(2). These results demonstrate an alternative pathway that results in augmentation of ERK1/2 phosphorylation without concomitant MEK1/2 phosphorylation in T cells.     

The mitogen-activated protein kinase extracellular signal-regulated kinase 1/2 pathway is involved in formyl-methionyl-leucyl-phenylalanine-induced p47phox phosphorylation in human neutrophils

Phosphorylation of p47 phagocyte oxidase, (p47(phox)), one of the NADPH oxidase components, is essential for the activation of this enzyme and for superoxide production. p47(phox) is phosphorylated on multiple serine residues, but the kinases involved in this process in vivo remain to be characterized. We examined the role of extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinase in p47(phox) phosphorylation. Inhibition of ERK1/2 activation by PD98059, a specific inhibitor of ERK kinase 1/2, inhibited the fMLP-induced phosphorylation of p47(phox). However, PD98059 weakly affected PMA-induced p47(phox) phosphorylation, even though ERK1/2 activation was abrogated. This effect was confirmed using U0126, a second ERK kinase inhibitor. Unlike PD98059 and U0126, the p38 mitogen-activated protein kinase inhibitor SB203580 did not inhibit the phosphorylation of p47(phox) induced either by fMLP or by PMA. Two-dimensional phosphopeptide mapping analysis showed that, in fMLP-induced p47(phox) phosphorylation, PD98059 affected the phosphorylation of all the major phosphopeptides, suggesting that ERK1/2 may regulate p47(phox) phosphorylation either directly or indirectly via other kinases. In PMA-induced p47(phox) phosphorylation, GF109203X, a protein kinase C inhibitor, strongly inhibits p47(phox) phosphorylation. However, in fMLP-induced p47(phox) phosphorylation, PD98059 and GF109203X partially inhibited the phosphorylation of p47(phox) when tested alone, and exerted additive inhibitory effects on p47(phox) phosphorylation when tested together. These results show for the first time that the ERK1/2 pathway participates in the phosphorylation of p47(phox). Furthermore, they strongly suggest that p47(phox) is targeted by several kinase cascades in intact neutrophils activated by fMLP and is therefore a converging point for ERK1/2 and protein kinase C.     

Histamine activates tyrosine hydroxylase in bovine adrenal chromaffin cells through a pathway that involves ERK1/2 but not p38 or JNK

In bovine adrenal chromaffin cells (BACC) histamine promotes a rapid increase in the intracellular levels of Ca2+ together with the release of catecholamines and the phosphorylation of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH). In this study we investigated the role of the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinases (ERK1/2), stress activated protein kinase (p38) and Jun N-terminal kinases (JNK) on the histamine-induced activation and phosphorylation of TH. We found that in BACC histamine produced a rapid, long lasting and histamine type-1 (H1) receptor-dependent increase in the phosphorylation levels of ERK1/2, p38 and JNK which was accompanied by a H1 receptor-dependent increase in TH activity. This increase in TH activity was partially blocked by the MEK1/2 inhibitor U0126 but was unaffected by the p38 antagonist SB203580 or the JNK blocker JNKI1. To study the effect of MAPK inhibition on histamine-induced TH phosphorylation, we generated phospho-specific antibodies against the different phosphorylated forms of TH. Treatment with U0126 totally inhibited the histamine-induced phosphorylation of TH at Ser31, without affecting the phosphorylation of either Ser40 or Ser19. Neither SB203580 nor JNKI1 treatments produced any significant modification of the histamine-induced TH phosphorylation. Our data support the hypothesis that the up-regulation of the ERK1/2 pathway, but not that of p38 or JNK, promoted by histamine is involved in the phosphorylation of TH at Ser31 and that this phosphorylation event is required for the full activation of this enzyme.     

Plasma membrane oestrogen receptor mediates neuroprotection against beta-amyloid toxicity through activation of Raf-1/MEK/ERK cascade in septal-derived cholinergic SN56 cells

Rapid oestrogen neuroprotection against beta-amyloid peptide (Abeta)-induced toxicity, a main feature of Alzheimer's disease, may be partially initiated at the plasma membrane. However, the mechanism by which this oestrogen effect occurs is unknown. In a septal murine cell line (SN56), we observed that short exposures to either 17beta-oestradiol (E2) or membrane impermeant E2 bound to horseradish peroxidase (E-HRP) induced a biphasic stimulation of extracellular-signal regulated protein kinase (ERK1/2) phosphorylation, with peak inductions detected around 4-8 min in the early phase and a second maximum around 8 h after treatment. ERK1/2 phosphorylation was abolished by ERK1/2 kinase (MEK) inhibitors PD98059 and U0126. Interestingly, PD98059 was also shown to block rapid E2-related prevention of death in cells exposed to Abeta fragment 1-40 (Abeta1-40) for 24 h. In contrast, no neuroprotective effects were obtained when MEK inhibitor was used to selectively abolish the late phosphorylation phase. Furthermore, both ERK1/2 activation and E2-associated protection were blocked by an inhibitor of Raf-1 kinase. Raf-1 may be involved in these effects because oestrogen caused the rapid serine 338 (Ser338) phosphorylation of this protein. In addition, the oestrogen receptor (ER) antagonist ICI 182,780 was also observed to block ERK1/2 phosphorylation. We propose a novel mechanism in SN56 cells by which rapid effects of oestrogen leading to neuroprotection are signalled through Raf-1/MEK/ERK1/2 pathway, possibly by activation of a membrane-related ER.