Asleep at the switch: MEK kinases control transit to gliogenesis in developing cortex.

In this issue of Neuron, Li et al. (2012) show that the neuron/glia cell fate switch of cortical progenitors is regulated by MEK1 and MEK2. The observations resonate with recent studies on the genesis of low-grade astrocytomas and highlight neuronal support functions of astrocytes in the postnatal brain.

MEK Is a Key Regulator of Gliogenesis in the Developing Brain.

We have defined functions of MEK in regulating gliogenesis in developing cerebral cortex using loss- and gain-of-function mouse genetics. Radial progenitors deficient in both Mek1 and Mek2 fail to transition to the gliogenic mode in late embryogenesis, and astrocyte and oligodendroglial precursors fail to appear. In exploring mechanisms, we found that the key cytokine-regulated gliogenic pathway is attenuated. Further, the Ets transcription family member Etv5/Erm is strongly regulated by MEK and Erm overexpression can rescue the gliogenic potential of Mek-deleted progenitors. Remarkably, Mek1/2-deleted mice surviving postnatally exhibit cortices almost devoid of astrocytes and oligodendroglia and exhibit neurodegeneration. Conversely, expression of constitutively active MEK1 leads to a major increase in numbers of astrocytes in the adult brain. We conclude that MEK is essential for acquisition of gliogenic competence by radial progenitors and that levels of MEK activity regulate gliogenesis in the developing cortex.

c-Raf, but not B-Raf, is essential for development of K-Ras oncogene-driven non-small cell lung carcinoma.

We have investigated the role of individual members of the Raf/Mek/Erk cascade in the onset of K-Ras oncogene-driven non-small cell lung carcinoma (NSCLC). Ablation of Erk1 or Erk2 in K-Ras oncogene-expressing lung cells had no significant effect due to compensatory activities. Yet, elimination of both Erk kinases completely blocked tumor development. Similar results were obtained with Mek kinases. Ablation of B-Raf had no significant effect on tumor development. However, c-Raf expression was absolutely essential for the onset of NSCLC. Interestingly, concomitant elimination of c-Raf and B-Raf in adult mice had no deleterious consequences for normal homeostasis. These results indicate that c-Raf plays a unique role in mediating K-Ras signaling and makes it a suitable target for therapeutic intervention.

MAPK-activated protein kinase 2 differentially regulates plasmodium falciparum glycosylphosphatidylinositol-induced production of tumor necrosis factor-{alpha} and interleukin-12 in macrophages.

Proinflammatory responses induced by Plasmodium falciparum glycosylphosphatidylinositols (GPIs) are thought to be involved in malaria pathogenesis. In this study, we investigated the role of MAPK-activated protein kinase 2 (MK2) in the regulation of tumor necrosis factor-alpha (TNF-alpha) and interleukin (IL)-12, two of the major inflammatory cytokines produced by macrophages stimulated with GPIs. We show that MK2 differentially regulates the GPI-induced production of TNF-alpha and IL-12. Although TNF-alpha production was markedly decreased, IL-12 expression was increased by 2-3-fold in GPI-stimulated MK2(-/-) macrophages compared with wild type (WT) cells. MK2(-/-) macrophages produced markedly decreased levels of TNF-alpha than WT macrophages mainly because of lower mRNA stability and translation. In the case of IL-12, mRNA was substantially higher in MK2(-/-) macrophages than WT. This enhanced production is due to increased NF-kappaB binding to the gene promoter, a markedly lower level expression of the transcriptional repressor factor c-Maf, and a decreased binding of GAP-12 to the gene promoter in MK2(-/-) macrophages. Thus, our data demonstrate for the first time the role of MK2 in the transcriptional regulation of IL-12. Using the protein kinase inhibitors SB203580 and U0126, we also show that the ERK and p38 pathways regulate TNF-alpha and IL-12 production, and that both inhibitors can reduce phosphorylation of MK2 in response to GPIs and other toll-like receptor ligands. These results may have important implications for developing therapeutics for malaria and other infectious diseases.

Selective role for Mek1 but not Mek2 in the induction of epidermal neoplasia.

The Ras/Raf/Mek/Erk mitogen-activated protein kinase pathway regulates fundamental processes in normal and malignant cells, including proliferation, differentiation, and cell survival. Mutations in this pathway have been associated with carcinogenesis and developmental disorders, making Mek1 and Mek2 prime therapeutic targets. In this study, we examined the requirement for Mek1 and Mek2 in skin neoplasia using the two-step 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate (DMBA/TPA) skin carcinogenesis model. Mice lacking epidermal Mek1 protein develop fewer papillomas than both wild-type and Mek2-null mice following DMBA/TPA treatment. Mek1 knockout mice had smaller papillomas, delayed tumor onset, and half the tumor burden of wild-type mice. Loss of one Mek1 allele, however, did not affect tumor development, indicating that one Mek1 allele is sufficient for normal papilloma formation. No difference in TPA-induced hyperproliferation, inflammation, or Erk activation was observed between wild-type, conditional Mek1 knockout, and Mek2-null mice, indicating that Mek1 findings were not due to a general failure of these processes. These data show that Mek1 is important for skin tumor development and that Mek2 cannot compensate for the loss of Mek1 function in this setting.

Map2k1 and Map2k2 genes contribute to the normal development of syncytiotrophoblasts during placentation.

The mammalian genome contains two ERK/MAP kinase kinase genes, Map2k1 and Map2k2, which encode dual-specificity kinases responsible for ERK/MAP kinase activation. In the mouse, loss of Map2k1 function causes embryonic lethality, whereas Map2k2 mutants survive with a normal lifespan, suggesting that Map2k1 masks the phenotype due to the Map2k2 mutation. To uncover the specific function of MAP2K2 and the threshold requirement of MAP2K proteins during embryo formation, we have successively ablated the Map2k gene functions. We report here that Map2k2 haploinsufficiency affects the normal development of placenta in the absence of one Map2k1 allele. Most Map2k1(+/-)Map2k2(+/-) embryos die during gestation because of placenta defects restricted to extra-embryonic tissues. The impaired viability of Map2k1(+/-)Map2k2(+/-) embryos can be rescued when the Map2k1 deletion is restricted to the embryonic tissues. The severity of the placenta phenotype is dependent on the number of Map2k mutant alleles, the deletion of the Map2k1 allele being more deleterious. Moreover, the deletion of one or both Map2k2 alleles in the context of one null Map2k1 allele leads to the formation of multinucleated trophoblast giant (MTG) cells. Genetic experiments indicate that these structures are derived from Gcm1-expressing syncytiotrophoblasts (SynT), which are affected in their ability to form the uniform SynT layer II lining the maternal sinuses. Thus, even though Map2k1 plays a predominant role, these results enlighten the function of Map2k2 in placenta development.

MEK ablation in MCF-7 cells blocks DNA synthesis induced by serum, but not by estradiol or growth factors.

The role of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling in estrogen receptor positive (ER(+)) MCF-7 breast carcinoma cells is not well understood. We depleted MEK by cotransfection of MEK1 and MEK2 siRNA duplexes in a MCF-7 derived line (MCF-7/ lacZ, ML-20) and determined its effect on serum, 17beta-estradiol (E(2)), and growth factor induced DNA synthesis. MEK knockdown did not decrease fetal bovine serum-induced DNA synthesis in ML-20 cells although it did inhibit DNA synthesis induced by estrogen-stripped calf serum (CCS) suggesting that MEK activation plays an important role in growth signaling induced by serum components other than estrogen. Consistent with this notion, MEK knockdown only modestly decreased DNA synthesis induced by E(2)-supplemented CCS medium in ML-20 cells. Similarly, MEK knockdown only caused moderate decreases in DNA synthesis induced by fibroblast growth factor-1 (FGF-1) or heregulin-beta1 (HRGbeta1) in this media. Also, there were only minimal effects of MEK knockdown in cells treated with growth factor-supplemented serum-free medium. Although MEK depletion inhibited ERK1/2 phosphorylation induced by CCS in these cells, that induced by growth factor supplemented CCS media was relatively unaffected. Similarly, ERK1/2 phosphorylation induced by growth factor-supplemented serum-free media was also relatively unaffected by MEK depletion. These results suggest that pathways regulating DNA synthesis induced by serum in MCF-7 cells are significantly more dependent on constitutive MEK levels than that induced by E(2) or growth factors.

MEK1 and MEK2, different regulators of the G1/S transition.

The ERK cascade is activated by hormones, cytokines, and growth factors that result in either proliferation or growth arrest depending on the duration and intensity of the ERK activation. Here we provide evidence that the MEK1/ERK module preferentially provides proliferative signals, whereas the MEK2/ERK module induces growth arrest at the G1/S boundary. Depletion of either MEK subtype by RNA interference generated a unique phenotype. The MEK1 knock down led to p21cip1 induction and to the appearance of cells with a senescence-like phenotype. Permanent ablation of MEK1 resulted in reduced colony formation potential, indicating the importance of MEK1 for long term proliferation and survival. MEK2 deficiency, in contrast, was accompanied by a massive induction of cyclin D expression and, thus, CDK4/6 activation followed by nucleophosmin hyperphosphorylation and centrosome over-amplification. Our results suggest that the two MEK subtypes have distinct ways to contribute to a regulated ERK activity and cell cycle progression.