Generation and characterization of a mouse line carrying Reck-CreERT2 knock-in allele.

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.

Involvement of the Reck tumor suppressor protein in maternal and embryonic vascular remodeling in mice.

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 embyonic tissues.

Stimulatory actions of lysophosphatidic acid on mouse ATDC5 chondroprogenitor cells.

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.

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.

Serpin E2 promotes breast cancer metastasis by remodeling the tumor matrix and polarizing tumor associated macrophages.

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.

Mutations in two matrix metalloproteinase genes, MMP-2 and MT1-MMP, are synthetic lethal in mice.

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.

The membrane-anchored matrix metalloproteinase (MMP) regulator RECK in combination with MMP-9 serves as an informative prognostic indicator for colorectal cancer.

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 > or = 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.

Memo has a novel role in S1P signaling and is [corrected] crucial for vascular development.

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.

Dual effects of the membrane-anchored MMP regulator RECK on chondrogenic differentiation of ATDC5 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 approximately 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.

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.