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.

A human fibroblast cDNA expression library was screened for cDNA clones giving rise to f lat colonies when transfected into v-Ki-ras-transformed NIH 3T3 cells. One such gene, RECK, encodes a membrane-anchored glycoprotein of about 110 kDa with multiple epidermal growth factor-like repeats and serine-protease inhibitor-like domains. While RECK mRNA is expressed in various human tissues and untransformed cells, it is undetectable in tumor-derived cell lines and oncogenically transformed cells. Restored expression of RECK in malignant cells resulted in suppression of invasive activity with concomitant decrease in the secretion of matrix metalloproteinase-9 (MMP-9), a key enzyme involved in tumor invasion and metastasis. Moreover, purified RECK protein was found to bind to, and inhibit the proteolytic activity of, MMP-9. Thus, RECK may link oncogenic signals to tumor invasion and metastasis.Mutations of ras protooncogenes are found in a large variety of human tumors (1). It has been well established that Ras proteins are essential components in various intracellular signaling pathways involved in regulating gene expression and several other aspects of cellular behavior (2). Therefore, it is now important to find targets for these signals relevant to the expression of the malignant phenotype to understand the mechanism of cell transformation and to develop means to cure or prevent cancers.To this end, we have been isolating and characterizing genes that induce flat morphology (or ''flat reversion'') when expressed in a v-Ki-ras-transformed NIH 3T3 cell line, DT (3). The Krev-1 gene (4), also known as rap1A, which encodes a Ras-related protein containing a region identical to the effector domain of Ras, was isolated in a previous study (5) by using a plasmid-based human fibroblast cDNA expression library. Using a similar approach, Cutler et al. (6) isolated another transformation suppressor gene, rsp-1, encoding a leucinerich-repeat protein. Recently, we performed a similar screen of a human fibroblast cDNA expression library constructed with a new phagemid shuttle vector and obtained two cDNA clones exhibiting significant biological activities. One of these, clone CT124, was found to encode a truncated form of the MSX-2 homeobox protein, which induces flat reversion through a dominant-negative mechanism over the endogenous MSX-2 protein (7).Here we describe some properties of the other reversioninducing gene named RECK (reversion-inducing-cysteine-rich protein with Kazal motifs) and its product. This reversioninducing gene is unique in that it encodes an extracellular protein with protease inhibitor-like domains and its expression is suppressed strongly in many tumors and cells transformed by various kinds of oncogenes. Restored expression of the RECK gene inhibits the invasive and metastatic activities of tumor cells. We also found that RECK negatively regulates matrix metalloproteinase-9 (MMP-9) (8) in two ways: suppression of MMP-9 secretion from the cells and direct inhibition of its enzymatic activity. T...

Septins are polymerizing GTP binding proteins required for cortical organization during cytokinesis and other cellular processes. A mammalian septin gene Sept4 is expressed mainly in postmitotic neural cells and postmeiotic male germ cells. In mouse and human spermatozoa, SEPT4 and other septins are found in the annulus, a cortical ring which separates the middle and principal pieces. Sept4-/- male mice are sterile due to defective morphology and motility of the sperm flagellum. In Sept4 null spermatozoa, the annulus is replaced by a fragile segment lacking cortical material, beneath which kinesin-mediated intraflagellar transport stalls. The sterility is rescued by injection of sperm into oocytes, demonstrating that each Sept4 null spermatozoon carries an intact haploid genome. The annulus/septin ring is also disorganized in spermatozoa from a subset of human patients with asthenospermia syndrome. Thus, cortical organization based on circular assembly of the septin cytoskeleton is essential for the structural and mechanical integrity of mammalian spermatozoa.

Human T-cell leukemia virus type I (HTLV-

We report that during cortical development in the mouse embryo, reversion-inducing cysteine-rich protein with Kazal motifs (RECK) critically regulates Notch signaling by antagonizing the ectodomain shedding of Notch ligands, which is mediated by a disintegrin and metalloproteinase domain 10 (ADAM10). In the embryonic brain, RECK is specifically expressed in Nestin-positive neural precursor cells (NPCs). Reck-deficient NPCs undergo precocious differentiation that is associated with downregulated Nestin expression, impaired Notch signaling and defective self-renewal. These phenotypes were substantially rescued either by enhancing Notch signaling or by suppressing endogenous ADAM10 activity. Consequently, we found that RECK regulates the ectodomain shedding of Notch ligands by directly inhibiting the proteolytic activity of ADAM10. This mechanism appeared to be essential for Notch ligands to properly induce Notch signaling in neighboring cells. These findings indicate that RECK is a physiological inhibitor of ADAM10, an upstream regulator of Notch signaling and a critical modulator of brain development.