Miki Hieda

Membrane-anchored growth factors, the epidermal growth factor family: beyond receptor ligands.

The epidermal growth factor (EGF) family and the EGF receptor (EGFR, ErbB) tyrosine kinase family have been spearheading the studies of signal transduction events that determine cell fate and behavior in vitro and in vivo. The EGFR family and their signaling pathways are giving us tremendous advantages in developing fascinating molecular target strategies for cancer therapy. Currently, two important types of EGFR inhibitors are in clinical use: neutralizing antibodies of EGFR or ErbB2, and synthetic small compounds of tyrosine kinase inhibitors designed for receptors. On the other hand, basic research of the EGF family ligands presents new challenges as membrane‐anchored growth factors. All members of the EGF family have important roles in development and diseases and are shed from the plasma membrane by metalloproteases. The ectodomain shedding of the ligands has emerged as a critical component in the functional transactivation of EGFRs in interreceptor cross‐talk in response to various shedding stimulants such as G‐protein coupled receptor agonists, growth factors, cytokines, and various physicochemical stresses. Among the EGFR‐ligands, heparin‐binding EGF‐like growth factor (HB‐EGF) is a prominent ligand in our understanding of the pathophysiological roles of ectodomain shedding in cancer, wound healing, cardiac diseases, etc. Here we focus on ectodomain shedding of the EGF family ligands, especially HB‐EGF by disintegrin and metalloproteases, which are not only key events of receptor cross talk, but also novel intercellular signaling by their carboxy‐terminal fragments to regulate gene expression directly. (Cancer Sci 2008; 99: 214–220)

Localized Regulation of Axonal RanGTPase Controls Retrograde Injury Signaling in Peripheral Nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.

Importin α can migrate into the nucleus in an importin β‐ and Ran‐independent manner

A classical nuclear localization signal (NLS)‐containing protein is transported into the nucleus via the formation of a NLS‐substrate/importin α/β complex. In this study, we found that importin α migrated into the nucleus without the addition of importin β, Ran or any other soluble factors in an in vitro transport assay. A mutant importin α lacking the importin β‐binding domain efficiently entered the nucleus. Competition experiments showed that this import pathway for importin α is distinct from that of importin β. These results indicate that importin α alone can enter the nucleus via a novel pathway in an importin β‐ and Ran‐independent manner. Furthermore, this process is evolutionarily conserved as similar results were obtained in Saccharomyces cerevisiae. Moreover, the import rate of importin α differed among individual nuclei of permeabilized cells, as demonstrated by time‐lapse experiments. This heterogeneous nuclear accumulation of importin α was affected by the addition of ATP, but not ATPγS. These results suggest that the nuclear import machinery for importin α at individual nuclear pore complexes may be regulated by reaction(s) that require ATP hydrolysis.

Membrane-anchored growth factor, HB-EGF, on the cell surface targeted to the inner nuclear membrane

Heparin-binding EGF-like growth factor (HB-EGF) is synthesized as a type I transmembrane protein (proHB-EGF) and expressed on the cell surface. The ectodomain shedding of proHB-EGF at the extracellular region on the plasma membrane yields a soluble EGF receptor ligand and a transmembrane-cytoplasmic fragment (HB-EGF-CTF). The cytoplasmic domain of proHB-EGF (HB-EGF-cyto) interacts with transcriptional repressors to reverse their repressive activities. However, how HB-EGF-cyto accesses transcriptional repressors is yet unknown. The present study demonstrates that, after exposure to shedding stimuli, both HB-EGF-CTF and unshed proHB-EGF translocate to the nuclear envelope. Immunoelectron microscopy and digitonin-permeabilized cells showed that HB-EGF-cyto signals are at the inner nuclear membrane. A short sequence element within the HB-EGF-cyto allows a transmembrane protein to localize to the nuclear envelope. The dominant-active form of Rab5 and Rab11 suppressed nuclear envelope targeting. Collectively, these data demonstrate that membrane-anchored HB-EGF is targeted to the inner nuclear membrane via a retrograde membrane trafficking pathway.

The carboxyl-terminal fragment of pro-HB-EGF reverses Bcl6-mediated gene repression.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4–6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6.

Cancer‐associated upregulation of histone H3 lysine 9 trimethylation promotes cell motility in vitro and drives tumor formation in vivo

Global histone modification patterns correlate with tumor phenotypes and prognostic factors in multiple tumor types. Recent studies suggest that aberrant histone modifications play an important role in cancer. However, the effects of global epigenetic rearrangements on cell functions remain poorly understood. In this study, we show that the histone H3 lysine 9 (H3K9) methyltransferase SUV39H1 is clearly involved in regulating cell migration in vitro. Overexpression of wild‐type SUV39H1, but not enzymatically inactive SUV39H1, activated migration in breast and colorectal cancer cells. Inversely, migration was reduced by knockdown of SUV39H1 or chemical inhibition by chaetocin. In addition, H3K9 trimethylation (H3K9me3) was specifically increased in invasive regions of colorectal cancer tissues. Moreover, the presence of H3K9me3 positively correlated with lymph node metastasis in colorectal cancer patients. Furthermore, overexpression of SUV39H1 drove tumorigenesis in mouse, resulting in a considerable decrease in survival rate. These data indicate that H3K9 trimethylation plays an important role in human colorectal cancer progression, possibly by promoting collective cell invasion.

Loss of histone H4K20 trimethylation predicts poor prognosis in breast cancer and is associated with invasive activity.

IntroductionLoss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.MethodsLevels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.ResultsH4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.ConclusionsH4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.

Plasma-membrane-anchored growth factor pro-amphiregulin binds A-type lamin and regulates global transcription.

Amphiregulin (AR), a member of the EGF family, is synthesized as a type I transmembrane protein precursor (proAR) and expressed on the cell surface. Shedding of proAR yields a transmembrane-cytoplasmic fragment (AR-CTF), as well as a soluble AR. Here we demonstrate that the proAR-shedding stimuli trigger endocytosis of both AR-CTF and un-shed proAR. ProAR translocates from the plasma membrane to the inner nuclear membrane, whereas AR-CTF is translocated to the lysosome via retrograde membrane trafficking. Nuclear envelope localization of proAR involves truncation of the C-terminus, which subsequently activates the ER-retrieval signal. The truncated form of proAR interacts with A-type lamin and is retained at the inner nuclear membrane. Heterochromatin formation is then induced and global transcription is transiently suppressed. This study gives new insight into epigenetic chromatin organization in mammalian cells: a plasma-membrane-anchored growth factor is targeted to the inner nuclear membrane where it participates in dynamic chromatin organization and control of transcription.

Measuring histone and polymerase dynamics in living cells.

Publisher Summary The dynamics of proteins can be monitored in living cells after tagging them with the green fluorescent protein (GFP). A construct encoding GFP fused to the protein of interest is expressed in a cell, so the resulting fluorescent hybrid can be seen directly. Mutant GFPs with altered fluorescence are available (e.g., enhanced cyan and yellow fluorescent proteins—ECFPs and EYFPs), and one—PAGFP—is photoactivated by irradiation with 413-nm light so its fluorescence (488-nm excitation) increases 100-fold. Therefore, it is possible to monitor histone and polymerase dynamics in real time, using these GFP tags and photobleaching techniques such as fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). This chapter presents the studies on the dynamics of the core histones and RNA polymerase II to illustrate these techniques.

Nuclear Import of the U1A Splicesome Protein Is Mediated by Importin α/β and Ran in Living Mammalian Cells

Abstract U1A is a component of the uracil-rich small nuclear ribonucleoprotein. The molecular mechanism of nuclear import of U1A was investigated in vivo and in vitro. When recombinant deletion mutants of U1A are injected into the BHK21 cell cytoplasm, the nuclear localization signal (NLS) of U1A is found in the N-terminal half of the central domain (residues 100–144 in mouse U1A). In an in vitro assay, it was found that the U1A-NLS accumulated in only a portion of the nuclei in the absence of cytosolic extract. In contrast, the addition of importin α/β and Ran induced the uniform nuclear accumulation of U1A-NLS in all cells. Furthermore, U1A was found to bind the C-terminal portion of importin α. In addition, the in vitro nuclear migration of full-length U1A was found to be exclusively dependent on importin α/β and Ran. Moreover, in living cells, the full-length U1A accumulated in the nucleus in a Ran-dependent manner, and nuclear accumulation was inhibited by the importin β binding domain of importin α. These results suggest that the nuclear import of U1A is mediated by at least two distinct pathways, an importin α/β and Ran-dependent and an -independent pathway in permeabilized cells, and that the latter pathway may be suppressed in intact cells.