Search

Reck encodes a membrane-anchored glycoprotein implicated in the regulation of extracellular metalloproteinases, Notch-signaling, and Wnt7-signaling and shown to play critical roles in embryogenesis and tumor suppression. Precise mechanisms of its actions in vivo, however, remain largely unknown. By homologous recombination, we generated a new Reck allele, ReckCreERT2 (MGI symbol: Reck ). This allele is defective in terms of Reck function but expected to induce loxP-mediated recombination in the cells committed to express Reck. Similarity in the expression patterns of the ReckCreERT2 transgene and the endogenous Reck gene was confirmed in five tissues. In the adult hippocampus, induction of Reck expression after transient cerebral ischemia could be demonstrated using this allele. These results indicate the utility of this Cre-driver allele in further studies.

It is difficult for flying robots with a conventional PID controller to fly stably with external disturbances such as wind. Thus, a flight control method that can change the control parameters of a conventional PID controller according to the external disturbances is described in this paper. The control parameters of the PID controller are automatically adjusted based on a radial basis function neural network (RBFNN). The experimental results show that the control method is capable of effectively dealing with external disturbances.

The extracellular serine protease inhibitor serpinE2 is overexpressed in breast cancer and has been shown to foster metastatic spread. Here, we investigated the hypothesis that serpinE2 creates tumor-promoting conditions in the tumor microenvironment (TME) by affecting extracellular matrix remodeling. Using two different breast cancer models, we show that blocking serpinE2, either by knock-down (KD) in tumor cells or in response to a serpinE2 binding antibody, decreases metastatic dissemination from primary tumors to the lungs. We demonstrate that in response to serpinE2 KD or antibody treatment there are dramatic changes in the TME. Multiphoton intravital imaging revealed deposition of a dense extracellular collagen I matrix encapsulating serpinE2 KD or antibody-treated tumors. This is accompanied by a reduction in the population of tumor-promoting macrophages, as well as a decrease in chemokine ligand 2, which is known to affect macrophage abundance and polarization. In addition, TIMP-1 secretion is increased, which may directly inhibit matrix metalloproteases critical for collagen degradation in the tumor. In summary, our findings suggest that serpinE2 is required in the extracellular milieu of tumors where it acts in multiple ways to regulate tumor matrix deposition, thereby controlling tumor cell dissemination.

Effects of two small G-proteins, Rap1 and Ras, on the sodium channel activity in NG108-15 cells were studied using sindbis virus-mediated gene transfer. When an activated Rap1A mutant (Rap1-12V, the activated mutant of Rap1 carrying glycine to valine substitution at codon 12) or a dominant-negative H-Ras mutant (Ras-17N, carrying serine to asparagine substitution at codon 17) was expressed in differentiated NG108-15 cells, the proportion of cells generating action potential decreased and the amplitudes of sodium current diminished. This effect was sensitive to an inhibitor of protein kinase A. The effects of a cyclic AMP (cAMP) analog (dibutyl cAMP) on sodium current in these cells were biphasic: inhibitory at lower concentrations (

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.

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.

The inhibitory effects of vitamins A and K toward P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined in the reconstituted system containing the microsomal fraction prepared from the recombinant Saccharomyces cerevisiae cells producing rat P4501A1 and yeast NADPH-P450 reductase. On vitamins A, all-trans-retinol, all-trans-retinal, all-trans-retinoic acid and retinol-palmitate showed competitive inhibition with K(i) values of 0.068, 0.079, 2.6 and 2.0 microM, respectively. Judging from the K(i) values, the inhibitory effects of those vitamins A appear to have physiological significance on the basis of their contents in liver, lung and kidney. On vitamins K, vitamin K(1) showed competitive inhibition with K(i) value of 24 microM, while vitamin K(2) showed noncompetitive inhibition with K(i) value of 60 microM. Judging from these K(i) values together with the contents of these vitamins K in liver, the inhibitory effects of the vitamins K are not as significant as those of vitamins A. These results suggest that the ingestion of enough amounts of vitamins A from foods might lead to the inhibition of the activity of P4501A1 which is known to be induced by smoking, drugs such as omeprazole and lansoprazole, and environmental pollutants like dioxins.

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The V max of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of V max were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.

Background Developmental angiogenesis proceeds through multiple morphogenetic events including sprouting, intussusception, and pruning. Mice lacking the membrane-anchored metalloproteinase regulator Reck die in utero around embryonic day 10.5 with halted vascular development; however, the mechanisms by which this phenotype arises remain unclear. Results We found that Reck is abundantly expressed in the cells associated with blood vessels undergoing angiogenesis or remodelling in the uteri of pregnant female mice. Some of the Reck-positive vessels show morphological features consistent with non-sprouting angiogenesis. Treatment with a vector expressing a small hairpin RNA against Reck severely disrupts the formation of blood vessels with a compact, round lumen. Similar defects were found in the vasculature of Reck-deficient or Reck conditional knockout embryos. Conclusions Our findings implicate Reck in vascular remodeling, possibly through non-sprouting angiogenesis, in both maternal and embyornic tissues.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are bioactive lysophospholipids that affect various cellular processes through G protein-coupled receptors. In our current study, we found by in situ hybridization that E11.5 mouse embryos strongly expressed the LPA receptor subtype LPA(1) in cartilaginous bone primordia and the surrounding mesenchymal cells. However, despite their wide-ranging actions, the roles of lysophospholipids in chondrogenesis remain poorly understood. The mouse clonal cell line ATDC5 undergoes a sequential differentiation of chondroprogenitor cells in vitro. Undifferentiated and differentiated ATDC5 cells express LPA(1) and other lysophospholipid receptors including S1P receptor S1P(1) and S1P(2). Taking advantage of this cell model, we studied the effects of LPA on the activities of chondroprogenitor cells. LPA markedly stimulates both DNA synthesis and the migration of ATDC5 chondroprogenitor cells in culture, whereas S1P suppresses the migration of these cells. Treatment with Ki16425, an LPA(1)- and LPA(3)-specific receptor antagonist, suppressed the fetal bovine serum-stimulated migration of ATDC5 cells by almost 80%. These results indicate that LPA plays an important role in the activation of chondroprogenitor cells.

Extracellular matrix (ECM) undergoes continuous remodeling during mammalian development. Although involvement of matrix metalloproteinases (MMPs) in ECM degradation has been well documented, how this process is regulated to allow proper ECM accumulation remains unclear. We previously showed the involvement of a membrane-anchored MMP regulator, RECK (reversion-inducing cysteine-rich protein with Kazal motifs), in vascular development in mice. Here we report that Reck mRNA can be detected in developing cartilage in E13.5∼16.5 mouse embryos and is progressively upregulated during differentiation of a chondrogenic cell line ATDC5 in vitro. In the early phase of ATDC5 differentiation, RECK expression stays low, multiple MMPs are upregulated, and there is ECM degradation at the sites of cellular condensation. In the later phase, RECK is upregulated inside the expanding cartilaginous nodules where type II collagen is accumulated while active ECM degradation persists along the rim of the nodules. Constitutive RECK expression suppressed initial cellular condensation, whereas RECK knockdown suppressed the later ECM accumulation in the cartilaginous nodules. These results suggest that RECK expression at the right place (in the core of the nodules) and at the right time (only in the later phase) is important for proper chondrogenesis and that RECK, together with MMPs, plays a crucial role in regulating dynamic processes of tissue morphogenesis.

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.

Purpose: RECK, a membrane-anchored regulator of matrix metalloproteinases (MMPs), is widely expressed in healthy tissue, whereas it is expressed at lower levels in many tumor-derived cell lines. Studies in mice and cultured cells have shown that restoration of RECK expression inhibits tumor invasion, metastasis, and angiogenesis. However, the clinical relevance of these findings remains to be fully documented. Here we examined the expression of RECK and one of its targets, MMP-9, in colorectal cancer tissue. Experimental Design: The RECK and MMP-9 expression levels in colorectal cancer samples from 53 patients were determined by immunohistochemical techniques. The expression level of each protein was scored, and the patients were divided into two groups based on these scores. In 33 cases, we performed gelatin zymography to estimate the degree of MMP-2 and MMP-9 activation. Microvessel density and vascular endothelial growth factor (VEGF) expression were also evaluated histologically. Results: RECK protein was detected in 30 of 53 (56.6%) specimens. Importantly, patients with tumors expressing relatively high levels of RECK (high-RECK group) had a significantly lower risk of recurrence than did patients with tumors expressing relatively low levels of RECK (low-RECK group; P = 0.011). Moreover, RECK-dominant (RECK score ≥ MMP-9 score) patients showed a significantly lower incidence of recurrence than did MMP-9-dominant patients (P = 0.0003). Multivariate analysis revealed that the RECK/MMP-9 balance was an independent prognostic factor (P = 0.0122). The expression of VEGF and microvessel density were inversely correlated with the level of RECK expression. Conclusions: RECK/MMP-9-balance is an informative prognostic indicator for colorectal cancer. Our data also suggest that RECK suppresses tumor angiogenesis, probably by limiting the availability of VEGF in tumor tissues.

The matrix metalloproteinase (MMP) family (approximately 25 members in mammals) has been implicated in extracellular matrix remodeling associated with embryonic development, cancer formation and progression, and various other physiological and pathological events. Inactivating mutations in individual matrix metalloproteinase genes in mice described so far, however, are nonlethal at least up to the first few weeks after birth, suggesting functional redundancy among MMP family members. Here, we report that mice lacking two MMPs, MMP-2 (nonmembrane type) and MT1-MMP (membrane type), die immediately after birth with respiratory failure, abnormal blood vessels, and immature muscle fibers reminiscent of central core disease. In the absence of MMP-2 and MT1-MMP, myoblast fusion in vitro is also significantly retarded. These findings suggest functional overlap in mice between the two MMPs with distinct molecular natures.

Rap1A, first identified as a suppressor of transformed phenotype induced by an activated ras oncogene, is abundantly expressed in the brain. Its neurophysiological function, however, is poorly understood. When an activated Rap1A mutant (Rap1-12V) or a dominant negative H-Ras mutant (Ras-17N) was expressed in CA1 neurons in cultured hippocampal slices using the sindbis virus-mediated gene transfer technique, NMDA receptor current in response to Schaffer collateral stimulation was suppressed. Expression of activated H-Ras mutant (Ras-12V) resulted in the elevation of both NMDA receptor current and AMPA receptor current. These results implicate counteracting functions of Ras and Rap1 in the regulation of NMDA receptor-mediated synaptic transmission and a positive regulatory role of Ras in AMPA receptor-mediated synaptic transmission.

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.

Memo is a conserved protein that was identified as an essential mediator of tumor cell motility induced by receptor tyrosine kinase activation. Here we show that Memo null mouse embryonic fibroblasts (MEFs) are impaired in PDGF-induced migration and this is due to a defect in sphingosine-1-phosphate (S1P) signaling. S1P is a bioactive phospholipid produced in response to multiple stimuli, which regulates many cellular processes. S1P is secreted to the extracellular milieu where it exerts its function by binding a family of G-protein coupled receptors (S1PRs), causing their activation in an autocrine or paracrine manner. The process, termed cell-autonomous S1PR signaling, plays a role in survival and migration. Indeed, PDGF uses cell-autonomous S1PR signaling to promote cell migration; we show here that this S1P pathway requires Memo. Using vascular endothelial cells (HUVECs) with Memo knock-down we show that their survival in conditions of serum-starvation is impaired. Furthermore, Memo loss in HUVECs causes a reduction of junctional VE-cadherin and an increase in sprout formation. Each of these phenotypes is rescued by S1P or S1P agonist addition, showing that Memo also plays an important role in cell-autonomous S1PR signaling in endothelial cells. We also produced conventional and endothelial cell-specific conditional Memo knock-out mouse strains and show that Memo is essential for embryonic development. Starting at E13.5 embryos of both strains display bleeding and other vascular problems, some of the phenotypes that have been described in mouse strains lacking S1PRs. The essential role of Memo in embryonic vascular development may be due in part to alterations in S1P signaling. Taken together our results show that Memo has a novel role in the S1P pathway and that Memo is needed to promote cell-autonomous S1PR activation.

Inhibitory effects of detergents Triton X-100 and Chaps on 7-ethoxycoumarin O-deethylation activity were examined in the recombinant microsomes containing both rat CYP1A1 and yeast NADPH-P450 reductase (the mixed system) and their fused enzyme (the fused system). Triton X-100 showed competitive inhibition in both mixed and fused systems with K(i) values of 24.6 and 21.5 microM, respectively. These results strongly suggest that Triton X-100 binds to the substrate-binding pocket of CYP1A1. These K(i) values are far below the critical micelle concentration of Triton X-100 (240 microM). Western blot analysis revealed no disruption of the microsomal membrane by Triton X-100 in the presence of 0-77 microM Triton X-100. On the other hand, Chaps gave distinct inhibitory effects to the mixed and fused systems. In the fused system, a mixed-type inhibition was observed with K(i) and K(i)' values of 1.2 and 5.4 mM of Chaps, respectively. However, in the mixed system, multiple inhibition modes by Chaps were observed. Western blot analysis revealed that the solubilized fused enzyme by Chaps preserved the activity whereas the solubilized CYP1A1 and NADPH-P450 reductase reductase showed no activity in the mixed system. Thus, the comparison of the mixed and fused systems appears quite useful to elucidate inhibition mechanism of detergents.