Hitoshi Kitayama

A ras-related gene with transformation suppressor activity

A 1.8 kb cDNA clone, Krev-1, with revertant-inducing activity on Kirsten sarcoma virus-transformed NIH/3T3 cells, has been isolated from a human fibroblast cDNA expression library. In Krev-1 transfectants, there is a correlation between the levels of specific mRNA and the degrees of suppression of the transformed phenotype. The cDNA encodes a protein of 21,000 daltons that unexpectedly shares around 50% amino acid identities with ras proteins. The Krev-1 homologs are found in mouse, rat, and chicken DNA, and their transcripts are ubiquitously expressed in many rat organs. Thus, the Krev-1 gene seems to play an important role(s) in a wide variety of tissues, and may be involved in the negative growth regulation of certain cell types.

The Membrane-Anchored MMP Inhibitor RECK Is a Key Regulator of Extracellular Matrix Integrity and Angiogenesis

Matrix metalloproteinases (MMPs) are essential for proper extracellular matrix remodeling. We previously found that a membrane-anchored glycoprotein, RECK, negatively regulates MMP-9 and inhibits tumor invasion and metastasis. Here we show that RECK regulates two other MMPs, MMP-2 and MT1-MMP, known to be involved in cancer progression, that mice lacking a functional RECK gene die around E10.5 with defects in collagen fibrils, the basal lamina, and vascular development, and that this phenotype is partially suppressed by MMP-2 null mutation. Also, vascular sprouting is dramatically suppressed in tumors derived from RECK-expressing fibrosarcoma cells grown in nude mice. These results support a role for RECK in the regulation of MMP-2 in vivo and implicate RECK downregulation in tumor angiogenesis.

RECK: A novel suppressor of malignancy linking oncogenic signaling to extracellular matrix remodeling

RECK was first isolated as a transformation suppressor gene by cDNA expression cloning in a mouse fibroblast cell line transformed by an activated RAS oncogene. Subsequently, reduced expression of RECK in transformed cells and cancer cells were demonstrated. Moreover, in several types of tumors, positive correlation between RECK expression and survival of patients have been noted. RECK encodes a GPI-anchored glycoprotein harboring three protease inhibitor-like domains. The RECK protein regulates at least three members of the matrix metalloproteinase (MMP) family, MMP-2, MMP-9, and MT1-MMP, in vitro or in cultured cells. Restored expression of RECK in cancer cell lines results in strong suppression of invasion, metastasis, and tumor angiogenesis. Mice lacking RECK die in utero with reduced integrity of blood vessels, the neural tube, and mesenchymal tissues. In these mice, MMP activity is elevated, and the amount of collagen type I greatly reduced. The RECK null phenotype is partially rescued (half day delay of death and marked recovery of tissue integrity) by MMP-2 null mutation, demonstrating functional interaction between RECK and MMP-2 in vivo and involvement of other target(s) for RECK in the lethal phenotype. These findings indicate that (i) RECK is an important regulator of extracellular matrix remodeling and that (ii) down-regulation of RECK by oncogenic signaling leads to the excessive activation of MMPs thereby promoting malignant behavior of cancer cells such as invasion, metastasis, and angiogenesis.

Association of the Cytoskeletal GTP-binding Protein Sept4/H5 with Cytoplasmic Inclusions Found in Parkinson's Disease and Other Synucleinopathies

α-Synuclein-positive cytoplasmic inclusions are a pathological hallmark of several neurodegenerative disorders including Parkinsons disease, dementia with Lewy bodies, and multiple system atrophy. Here we report that Sept4, a member of the septin protein family, is consistently found in these inclusions, whereas five other septins (Sept2, Sept5, Sept6, Sept7, and Sept8) are not found in these inclusions. Sept4 and α-synuclein can also be co-immunoprecipitated from normal human brain lysates. When co-expressed in cultured cells, FLAG-tagged Sept4 and Myc-tagged α-synuclein formed detergent-insoluble complex, and upon treatment with a proteasome inhibitor, they formed Lewy body-like cytoplasmic inclusions. The tagged Sept4 and α-synuclein synergistically accelerated cell death induced by the proteasome inhibitor, and this effect was further enhanced by expression of another Lewy body-associated protein, synphilin-1, tagged with the V5 epitope. Moreover, co-expression of the three proteins (tagged Sept4, α-synuclein, and synphilin-1) was sufficient to induce cell death. These data raise the possibility that Sept4 is involved in the formation of cytoplasmic inclusions as well as induction of cell death in α-synuclein-associated neurodegenerative disorders.

Tissue Inhibitors of Metalloproteinase 2 Inhibits Endothelial Cell Migration through Increased Expression of RECK

The antiangiogenic function of the tissue inhibitors of metalloproteinases (TIMPs) has been attributed to their matrix metalloproteinase inhibitory activity. Here we demonstrate that TIMP-1 but not Ala+TIMP-1 inhibits both basal and vascular endothelial growth factor (VEGF)-stimulated migration of human microvascular endothelial cells (hMVECs), suggesting that this effect is dependent on direct inhibition of matrix metalloproteinase (MMP) activity. In contrast, TIMP-2 and mutant Ala+TIMP-2, which is devoid of MMP inhibitory activity, block hMVEC migration in response to VEGF-A stimulation. TIMP-2 and Ala+TIMP-2 also suppress basal hMVEC migration via a time-dependent mechanism mediated by enhanced expression of RECK, a membrane-anchored MMP inhibitor, which, in turn, inhibits cell migration. TIMP-2 treatment of hMVECs increases the association of Crk with C3G, resulting in enhanced Rap1 activation. hMVECs stably expressing Rap1 have increased RECK expression and display reduced cell migration compared with those expressing inactive Rap1(38N). RECK-null murine embryo fibroblasts fail to demonstrate TIMP-2–mediated decrease in cell migration despite activation of Rap1. TIMP-2–induced RECK decreases cell-associated MMP activity. Anti-RECK antibody increases MMP activity and reverses the TIMP-2–mediated reduction in cell migration. The effects of TIMP-2 on RECK expression and cell migration were confirmed in A2058 melanoma cells. These results suggest that TIMP-2 can inhibit cell migration via several distinct mechanisms. First, TIMP-2 can inhibit cell migration after VEGF stimulation by direct inhibition of MMP activity induced in response to VEGF stimulation. Secondly, TIMP-2 can disrupt VEGF signaling required for initiation of hMVEC migration. Third, TIMP-2 can enhance expression of RECK via Rap1 signaling resulting in an indirect, time-dependent inhibition of endothelial cell migration.

Plasma membrane recruitment of RalGDS is critical for Ras-dependent Ral activation

In COS cells, Ral GDP dissociation stimulator (RalGDS)-induced Ral activation was stimulated by RasG12V or a Rap1/Ras chimera in which the N-terminal region of Rap1 was ligated to the C-terminal region of Ras but not by Rap1G12V or a Ras/Rap1 chimera in which the N-terminal region of Ras was ligated to the C-terminal region of Rap1, although RalGDS interacted with these small GTP-binding proteins. When RasG12V, Ral and the Rap1/Ras chimera were individually expressed in NIH3T3 cells, they localized to the plasma membrane. Rap1Q63E and the Ras/Rap1 chimera were detected in the perinuclear region. When RalGDS was expressed alone, it was abundant in the cytoplasm. When coexpressed with RasG12V or the Rap1/Ras chimera, RalGDS was detected at the plasma membrane, whereas when coexpressed with Rap1Q63E or the Ras/Rap1 chimera, RalGDS was observed in the perinuclear region. RalGDS which was targeted to the plasma membrane by the addition of Ras farnesylation site (RalGDS-CAAX) activated Ral in the absence of RasG12V. Although RalGDS did not stimulate the dissociation of GDP from Ral in the absence of the GTP-bound form of Ras in a reconstitution assay using the liposomes, RalGDS-CAAX could stimulate it without Ras. RasG12V activated Raf-1 when they were coexpressed in Sf9 cells, whereas RasG12V did not affect the RalGDS activity. These results indicate that Ras recruits RalGDS to the plasma membrane and that the translocated RalGDS induces the activation of Ral, but that Rap1 does not activate Ral due to distinct subcellular localization.

Hypoxia and RAS-signaling pathways converge on, and cooperatively downregulate, the RECK tumor-suppressor protein through microRNAs

Cancer cells show characteristic gene expression profiles. Recent studies support the potential importance of microRNA (miRNA) expression signatures as biomarkers and therapeutic targets. The membrane-anchored protease regulator RECK is downregulated in many cancers, and forced expression of RECK in tumor cells results in decreased malignancy in animal models. RECK is also essential for mammalian development. In this study, we found that RECK is a target of at least three groups of miRNAs (miR-15b/16, miR-21 and miR-372/373); that RECK mutants lacking the target sites for these miRNA show augmented tumor/metastasis-suppressor activities; and that miR-372/373 are upregulated in response to hypoxia through HIF1α and TWIST1, whereas miR-21 is upregulated by RAS/ERK signaling. These data indicate that the hypoxia- and RAS-signaling pathways converge on RECK through miRNAs, cooperatively downregulating this tumor suppressor and thereby promoting malignant cell behavior.

RECK forms cowbell-shaped dimers and inhibits matrix metalloproteinase-catalyzed cleavage of fibronectin

The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.

The membrane-anchored MMP-regulator RECK is a target of myogenic regulatory factors

The membrane-anchored MMP-regulator RECK is down regulated in many solid tumors; the extent of RECK down regulation correlates with poor prognosis. Forced expression of RECK in tumor cells results in suppression of angiogenesis, invasion, and metastasis. Studies on the roles and the mechanisms of regulation of the RECK gene during normal development may therefore yield important insights into how the malignant behaviors of tumor cells arise and how they can be controlled. Our previous studies indicate that mice lacking RECK die around E10.5 with reduced tissue integrity. In the present study, we have found that in later stage wild-type embryos, RECK is abundantly expressed in skeletal muscles, especially in the areas where the myoblast differentiation factor MRF4 is expressed. Consistent with this finding, the RECK-promoter is activated by MRF4 in cultured cells. In contrast, a myoblast determination factor MyoD suppresses the RECK-promoter. Myoblastic cells lacking RECK expression give rise to myotubes at higher efficiency than the cells expressing RECK, indicating that RECK suppresses myotube formation. These findings suggest that MyoD down regulates RECK to facilitate myotube formation, whereas MRF4 up regulates RECK to promote other aspects of myogenesis that require extracellular matrix integrity.

The membrane-anchored metalloproteinase regulator RECK stabilizes focal adhesions and anterior-posterior polarity in fibroblasts

Accumulating evidence indicates that Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a membrane-anchored matrix metalloproteinase regulator, plays crucial roles in mammalian development and tumor suppression. Its mechanisms of action at the single cell level, however, remain largely unknown. In mouse fibroblasts, RECK is abundant around the perinuclear region, membrane ruffles and cell surface. Cells lacking Reck show decreased spreading, ambiguous anterior–posterior (AP) polarity, and increased speed and decreased directional persistence in migration; these characteristics are also found in transformed fibroblasts and fibrosarcoma cells with low RECK expression. RECK-deficient cells fail to form discrete focal adhesions, have increased levels of GTP-bound Rac1 and Cdc42, and a marked decrease in the level of detyrosinated tubulin, a hallmark of stabilized microtubules. RECK-deficient cells also show elevated gelatinolytic activity and decreased fibronectin fibrils. The phenotype of RECK-deficient cells is largely suppressed when the cells are plated on fibronectin-coated substrates. These findings suggest that RECK regulates pericellular extracellular matrix degradation, thereby allowing the cells to form proper cell–substrate adhesions and to maintain AP polarity during migration; this mechanism is compromised in malignant cells.