Miki Ebisuya

The duration, magnitude and compartmentalization of ERK MAP kinase activity: mechanisms for providing signaling specificity

ERK MAP kinase signaling plays a pivotal role in diverse cellular functions, including cell proliferation, differentiation, migration and survival. One of the central questions concerning this signaling is how activation of the same protein kinase, ERK, elicits distinct cellular outcomes. Recent progress has demonstrated that differences in the duration, magnitude and subcellular compartmentalization of ERK activity generate variations in signaling output that regulate cell fate decisions. Furthermore, several molecules have been identified as spatial, temporal or strength-controlling regulators of ERK activity. Signaling by various extracellular stimuli thus could be modulated by these regulators to give qualitative and quantitative differences in ERK activity, which are then interpreted by the cells as determinants for appropriate responses.

Ripples from neighbouring transcription.

Transcriptional initiation of each gene is assumed to be independently controlled in mammals. On the other hand, recent large-scale transcriptome analyses have shown that the genome is pervasively transcribed, such that the most of its DNA gives rise to RNAs. This raises the question of whether it is possible to pinpoint and activate a particular locus without perturbing numerous neighbouring transcripts. Here we show that intensive transcription at one locus frequently spills over into its physical neighbouring loci. Rapid induction of immediate-early genes (IEGs) in response to growth factor stimulation is accompanied by co-upregulation of their neighbouring genes. Profiling the primary transcripts in the nucleus with whole-genome tiling arrays delineated simultaneous activation of transcription centred on IEGs. Even in surrounding intergenic regions, transcriptional activation took place at the same time. Acetylation levels of histone H3 and H4 are elevated along with the IEG induction and neighbouring co-upregulation. Inhibition of the mitogen-activated protein kinase (MAPK) pathway or the transcription factor SRF suppresses all transcriptional upregulation. These results suggest that transcriptional activation has a ripple effect, which may be advantageous for coordinated expression.

Continuous ERK Activation Downregulates Antiproliferative Genes throughout G1 Phase to Allow Cell-Cycle Progression

BACKGROUND The ERK family of MAP kinase plays a critical role in growth factor-stimulated cell-cycle progression from G0/G1 to S phase. It has been suggested that sustained activation, but not transient activation, of ERK is necessary for inducing S phase entry. Although the essential role of ERK MAP kinase in growth factor-stimulated gene expression, especially expression of immediate-early genes, is well established, it has remained unclear how ERK activity duration affects the promotion of G1 phase progression to S phase. RESULTS We have found that inhibition of ERK activation by the MEK inhibitor or dominant-negative MEK1 even immediately before the onset of S phase leads to the cessation of S phase entry. Our analyses reveal that there are ERK-dependent downregulated genes, whose expression levels return to their original levels rapidly after ERK inactivation, and that their downregulation mostly requires AP-1 activity. Remarkably, microinjection experiments demonstrate that many of the downregulated genes act as antiproliferative genes during G1 phase and that their forced expression to the levels before growth factor stimulation even in late G1 phase blocks S phase entry. CONCLUSIONS Thus, continuous ERK activation downregulates antiproliferative genes until the onset of S phase to allow successful G1 phase progression. This mechanism may also work as a fail-safe mechanism, which prevents inappropriate stimuli that induce transient ERK activation from causing S phase entry.

ERK Activation Propagates in Epithelial Cell Sheets and Regulates Their Migration during Wound Healing

In epithelial cell movements, which occur during wound healing or embryonic morphogenesis, sheets of cells move together as a unit. Molecular mechanisms that regulate this sheet movement have been largely unknown, although cell locomotion or movement mechanisms for individual cells, such as for fibroblastic cells, have been extensively studied. Here, we show that, during wound healing, sheets of MDCK epithelial cells migrate coordinately as a unit, and wound-induced activation of ERK MAP kinase (ERK1/2) propagates in cell sheets in accordance with the cell sheet movement. Inhibition of ERK1/2 activation by specific MEK inhibitors or by expressing dominant-negative ERK2 results in marked inhibition of the sheet movement during wound healing, and inhibition of the cell sheet movement by disrupting actin cytoskeleton suppresses propagation of ERK1/2 activation. These results indicate that cell movement and ERK1/2 activation form a positive feedback loop, which facilitates cell sheet migration. Moreover, we find that Src family kinase inhibitors suppress both cell migration and propagation of ERK1/2 activation, suggesting that Src family kinase may participate in this feedback loop. Interestingly, neither cell sheet migration as a unit nor migration-dependent propagation of ERK1/2 activation occurs during wound healing in fibroblastic 3Y1 cells. Thus, our results identify specific requirements of ERK1/2 MAP kinase for epithelial cell sheet movement.

Dual role of YAP and TAZ in renewal of the intestinal epithelium.

The rapidly self-renewing intestinal epithelium represents an exquisite model for stem cell biology. So far, genetic studies in mice have uncovered crucial roles for several signalling pathways in the tissue. Here we show, by using intestine-specific gene transfer (iGT), that Hippo signalling effectors, YAP and TAZ, promote both the proliferation of intestinal stem/progenitor cells and their differentiation into goblet cells. These functions of YAP/TAZ are regulated by the upstream Hippo pathway kinases MST1/2 and LATS1/2. Moreover, we identify TEADs and Klf4 as partner transcription factors of YAP/TAZ in the proliferation and differentiation processes, respectively. These results indicate that Hippo signalling plays a dual role in renewal of the intestinal epithelium through the regulation of two different processes, stem/progenitor cell proliferation and differentiation into goblet cells, using two different types of transcription factor. Moreover, iGT should provide a robust platform to elucidate molecular mechanisms of intestinal epithelium self-renewal.

ERK5 Regulates Muscle Cell Fusion through Klf Transcription Factors

In skeletal muscle differentiation, muscle-specific genes are regulated by two groups of transcription factors, the MyoD and MEF2 families, which work together to drive the differentiation process. Here, we show that ERK5 regulates muscle cell fusion through Klf transcription factors. The inhibition of ERK5 activity suppresses muscle cell fusion with minimal effects on the expression of MyoD, MEF2, and their target genes. Promoter analysis coupled to microarray assay reveals that Klf-binding motifs are highly enriched in the promoter regions of ERK5-dependent upregulated genes. Remarkably, Klf2 and Klf4 expression are also upregulated during differentiation in an ERK5-dependent manner, and knockdown of Klf2 or Klf4 specifically suppresses muscle cell fusion. Moreover, we show that Sp1 transcription factor links ERK5 to Klf2/4, and that nephronectin, a Klf transcriptional target, is involved in muscle cell fusion. Therefore, an ERK5/Sp1/Klf module plays a key role in the fusion process during skeletal muscle differentiation.

ABL1 regulates spindle orientation in adherent cells and mammalian skin.

Despite the growing evidence for the regulated spindle orientation in mammals, a systematic approach for identifying the responsible genes in mammalian cells has not been established. Here we perform a kinase-targeting RNAi screen in HeLa cells and identify ABL1 as a novel regulator of spindle orientation. Knockdown of ABL1 causes the cortical accumulation of Leu-Gly-Asn repeat-enriched-protein (LGN), an evolutionarily conserved regulator of spindle orientation. This results in the LGN-dependent spindle rotation and spindle misorientation. In vivo inactivation of ABL1 by a pharmacological inhibitor or by ablation of the abl1 gene causes spindle misorientation and LGN mislocalization in mouse epidermis. Furthermore, ABL1 directly phosphorylates NuMA, a binding partner of LGN, on tyrosine 1774. This phosphorylation maintains the cortical localization of NuMA during metaphase, and ensures the LGN/NuMA-dependent spindle orientation control. This study provides a novel approach to identify genes regulating spindle orientation in mammals and uncovers new signalling pathways for this mechanism.

A Fasting-Responsive Signaling Pathway that Extends Life Span in C. elegans

Intermittent fasting is one of the most effective dietary restriction regimens that extend life span in C. elegans and mammals. Fasting-stimulus responses are key to the longevity response; however, the mechanisms that sense and transduce the fasting stimulus remain largely unknown. Through a comprehensive transcriptome analysis in C. elegans, we find that along with the FOXO transcription factor DAF-16, AP-1 (JUN-1/FOS-1) plays a central role in fasting-induced transcriptional changes. KGB-1, one of the C. elegans JNKs, acts as an activator of AP-1 and is activated in response to fasting. KGB-1 and AP-1 are involved in intermittent fasting-induced longevity. Fasting-induced upregulation of the components of the SCF E3 ubiquitin ligase complex via AP-1 and DAF-16 enhances protein ubiquitination and reduces protein carbonylation. Our results thus identify a fasting-responsive KGB-1/AP-1 signaling pathway, which, together with DAF-16, causes transcriptional changes that mediate longevity, partly through regulating proteostasis.

Revolving movement of a dynamic cluster of actin filaments during mitosis

Arp2/3 actin filament nucleating complex drives circumnavigation of cortical actin clusters during mitosis.

Regulation of pluripotency in male germline stem cells by Dmrt1

Spermatogonial stem cells (SSCs) present the potential to acquire pluripotency under specific culture conditions. However, the frequency of pluripotent cell derivation is low, and the mechanism of SSC reprogramming remains unknown. In this study, we report that induction of global DNA hypomethylation in germline stem (GS) cells (cultured SSCs) induces pluripotent cell derivation. When DNA demethylation was triggered by Dnmt1 depletion, GS cells underwent apoptosis. However, GS cells were converted into embryonic stem (ES)-like cells by double knockdown of Dnmt1 and p53. This treatment down-regulated Dmrt1, a gene involved in sexual differentiation, meiosis, and pluripotency. Dmrt1 depletion caused apoptosis of GS cells, but a combination of Dmrt1 and p53 depletion also induced pluripotency. Functional screening of putative Dmrt1 target genes revealed that Dmrt1 depletion up-regulates Sox2. Sox2 transfection up-regulated Oct4 and produced pluripotent cells. This conversion was enhanced by Oct1 depletion, suggesting that the balance of Oct proteins maintains SSC identity. These results suggest that spontaneous SSC reprogramming is caused by unstable DNA methylation and that a Dmrt1-Sox2 cascade is critical for regulating pluripotency in SSCs.