Masamichi Imajo

A molecular mechanism that links Hippo signalling to the inhibition of Wnt/β-catenin signalling.

The Hippo signalling pathway has emerged as a key regulator of organ size, tissue homeostasis, and patterning. Recent studies have shown that two effectors in this pathway, YAP/TAZ, modulate Wnt/β‐catenin signalling through their interaction with β‐catenin or Dishevelled, depending on biological contexts. Here, we identify a novel mechanism through which Hippo signalling inhibits Wnt/β‐catenin signalling. We show that YAP and TAZ, the transcriptional co‐activators in the Hippo pathway, suppress Wnt signalling without suppressing the stability of β‐catenin but through preventing its nuclear translocation. Our results show that YAP/TAZ binds to β‐catenin, thereby suppressing Wnt‐target gene expression, and that the Hippo pathway‐stimulated phosphorylation of YAP, which induces cytoplasmic translocation of YAP, is required for the YAP‐mediated inhibition of Wnt/β‐catenin signalling. We also find that downregulation of Hippo signalling correlates with upregulation of β‐catenin signalling in colorectal cancers. Remarkably, our analysis demonstrates that phosphorylated YAP suppresses nuclear translocation of β‐catenin by directly binding to it in the cytoplasm. These results provide a novel mechanism, in which Hippo signalling antagonizes Wnt signalling by regulating nuclear translocation of β‐catenin.

Regulatory mechanisms and functions of MAP kinase signaling pathways

Mitogen‐activated protein kinase (MAPK) pathways play central roles in controlling diverse cellular functions. They are finely regulated by several mechanisms, including scaffolding of their components, and phosphorylation/dephosphorylation and compartmentalization of MAPKs. A number of molecules have been identified as regulators involved in these mechanisms. They modulate the magnitude and the specificity of MAPK signaling, and thereby regulate the wide variety of signaling outputs. Recent studies have identified novel functions of the MAPK signaling pathways. It is becoming clear that strict regulation of the MAPK pathways underlies their manifold functions in numerous biological processes. iubmb Life, 58: 312‐317, 2006

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.

In vivo imaging reveals PKA regulation of ERK activity during neutrophil recruitment to inflamed intestines.

In vivo FRET demonstrates that ERK positively regulates the neutrophil recruitment cascade in the intestine by promoting adhesion and migration.

HOXA5 Counteracts Stem Cell Traits by Inhibiting Wnt Signaling in Colorectal Cancer

Hierarchical organization of tissues relies on stem cells, which either self-renew or produce committed progenitors predestined for lineage differentiation. Here we identify HOXA5 as an important repressor of intestinal stem cell fate in vivo and identify a reciprocal feedback between HOXA5 and Wnt signaling. HOXA5 is suppressed by the Wnt pathway to maintain stemness and becomes active only outside the intestinal crypt where it inhibits Wnt signaling to enforce differentiation. In colon cancer, HOXA5 is downregulated, and its re-expression induces loss of the cancer stem cell phenotype, preventing tumor progression and metastasis. Tumor regression by HOXA5 induction can be triggered by retinoids, which represent tangible means to treat colon cancer by eliminating cancer stem cells.

Human Tribbles homolog 1 functions as a negative regulator of retinoic acid receptor

Tribbles encode an evolutionarily conserved protein family that regulates cell proliferation, motility, metabolism and oncogenic transformation. Emerging evidence suggests that Tribbles function as adaptor or scaffold proteins to facilitate the degradation of their target proteins and to control the activation of various key signaling pathways. In this study, we uncover a novel function of human Tribbles homolog 1 (Trib1) as a regulator of retinoic acid receptor (RAR) signaling. We show that shRNA‐mediated knockdown of Trib1 promotes transcriptional activity of RARs, leading to enhanced expression of endogenous RAR‐target genes. Moreover, our results show that Trib1 directly interacts with RARα and retinoid X receptor‐α (RXRα) through its kinase‐like domain. Consistently, Trib1 colocalizes with RARα and RXRα in the nucleus. Biochemical analyses show that the ligand‐binding domain (LBD) of RARα mediates the interaction with Trib1. Ligand treatment, however, does not affect the binding of Trib1 to RARα/RXRα. Furthermore, a putative LXXLL motif, which is a potential LBD‐binding site and locates in the kinase‐like domain of Trib1, is not required for the binding. These results suggest a unique feature of the binding. Taken together, these results suggest that Trib1 functions as a negative regulator of RARs and shed new light on the molecular mechanisms for nuclear receptor‐mediated transcriptional repression.

Live imaging of extracellular signal-regulated kinase and protein kinase A activities during thrombus formation in mice expressing biosensors based on Förster resonance energy transfer

Essentials Spatiotemporal regulation of protein kinases during thrombus formation remains elusive in vivo. Activities of protein kinases were live imaged in mouse platelets at laser‐ablated arterioles. Protein kinase A was activated in the dislodging platelets at the downstream side of the thrombus. Extracellular signal‐regulated kinase was activated at the core of contracting platelet aggregates.

Identification of Aging-Associated Gene Expression Signatures That Precede Intestinal Tumorigenesis.

Aging-associated alterations of cellular functions have been implicated in various disorders including cancers. Due to difficulties in identifying aging cells in living tissues, most studies have focused on aging-associated changes in whole tissues or certain cell pools. Thus, it remains unclear what kinds of alterations accumulate in each cell during aging. While analyzing several mouse lines expressing fluorescent proteins (FPs), we found that expression of FPs is gradually silenced in the intestinal epithelium during aging in units of single crypt composed of clonal stem cell progeny. The cells with low FP expression retained the wild-type Apc allele and the tissues composed of them did not exhibit any histological abnormality. Notably, the silencing of FPs was also observed in intestinal adenomas and the surrounding normal mucosae of Apc-mutant mice, and mediated by DNA methylation of the upstream promoter. Our genome-wide analysis then showed that the silencing of FPs reflects specific gene expression alterations during aging, and that these alterations occur in not only mouse adenomas but also human sporadic and hereditary (familial adenomatous polyposis) adenomas. Importantly, pharmacological inhibition of DNA methylation, which suppresses adenoma development in Apc-mutant mice, reverted the aging-associated silencing of FPs and gene expression alterations. These results identify aging-associated gene expression signatures that are heterogeneously induced by DNA methylation and precede intestinal tumorigenesis triggered by Apc inactivation, and suggest that pharmacological inhibition of the signature genes could be a novel strategy for the prevention and treatment of intestinal tumors.

Composite regulation of ERK activity dynamics underlying tumour-specific traits in the intestine

Acting downstream of many growth factors, extracellular signal-regulated kinase (ERK) plays a pivotal role in regulating cell proliferation and tumorigenesis, where its spatiotemporal dynamics, as well as its strength, determine cellular responses. Here, we uncover the ERK activity dynamics in intestinal epithelial cells (IECs) and their association with tumour characteristics. Intravital imaging identifies two distinct modes of ERK activity, sustained and pulse-like activity, in IECs. The sustained and pulse-like activities depend on ErbB2 and EGFR, respectively. Notably, activation of Wnt signalling, the earliest event in intestinal tumorigenesis, augments EGFR signalling and increases the frequency of ERK activity pulses through controlling the expression of EGFR and its regulators, rendering IECs sensitive to EGFR inhibition. Furthermore, the increased pulse frequency is correlated with increased cell proliferation. Thus, ERK activity dynamics are defined by composite inputs from EGFR and ErbB2 signalling in IECs and their alterations might underlie tumour-specific sensitivity to pharmacological EGFR inhibition.The ERK signalling pathway regulates homeostasis of the intestinal epithelium. Here the authors identify two modes of ERK activity generated independently from EGFR and ErbB2 receptor and whose balance in cancer is shifted by Wnt pathway activation, resulting in enhanced sensitivity to EGFR inhibitors.

Dynamic ERK signaling regulation in intestinal tumorigenesis

ABSTRACT Extracellular signal-regulated kinase (ERK) plays a critical role in tissue homeostasis and tumorigenesis. By utilizing live imaging approaches, we recently uncovered ERK activity dynamics in the intestinal epithelium. Notably, we showed that ERK activity dynamics are defined by composite regulation from two distinct upstream receptors, and alteration of their functional balance underlies tumor cell-specific traits. Here, we discuss these findings.