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 preferential interaction of proteins with solvent components was measured in aqueous lactose and glucose systems by using a high precision densimeter. In all cases, the protein was preferentially hydrated; i.e., addition of these sugars to an aqueous solution of the protein resulted in an unfavorable free-energy change. This effect was shown to increase with an increase in protein surface area, explaining the protein stabilizing action of these sugars and their enhancing effect of protein associations. Correlation of the preferential interaction parameter with the effect of the sugars on the surface tension of water, i.e., their positive surface tension increment, has led to the conclusion that the surface free energy perturbation by sugars plays a predominant role in their preferential interaction with proteins. Other contributing factors are the exclusion volume of the sugars and the chemical nature of the protein surface.

The preferential interactions of lysozyme with solvent components and the effects of solvent additives on its stability were examined for several neutral osmolytes: L-proline, L-serine, gamma-aminobutyric acid, sarcosine, taurine, alpha-alanine, beta-alanine, glycine, betaine, and trimethylamine N-oxide. It was shown that all these substances stabilize the protein structure against thermal denaturation and (except for trimethylamine N-oxide for which interaction measurements could not be made) are strongly excluded from the protein domain, rendering unlikely their direct binding to proteins. On the other hand, valine, not known as an osmolyte, had no stabilizing effect, although it induced a large protein-preferential hydration. A possible explanation is given for the use of these substances as osmotic-pressure-regulating agents in organisms living under high osmotic pressure.

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...

The 2;5 chromosomal translocation is frequently associated with anaplastic large cell lymphomas (ALCLs). The translocation creates a fusion gene consisting of the alk (anaplastic lymphoma kinase) gene and the nucelophosmin (npm) gene: the 3' half of alk derived from chromosome 2 is fused to the 5' portion of npm from chromosome 5. A recent study shows that the product of the npm-alk fusion gene is oncogenic. To help understand how the npm-alk oncogene transform cells, it is important to investigate the normal biological function of the alk gene product, ALK. Here, we show molecular cloning of cDNAs for both the human and mouse ALK proteins. The deduced amino acid sequences reveal that ALK is a novel receptor protein-tyrosine kinase having a putative transmembrane domain and an extracellular domain. These sequences are absent in the product of the transforming npm-alk gene. ALK shows the greatest sequence similarity to LTK (leukocyte tyrosine kinase) whose biological function is presently unknown. RNA blot hybridization analysis of various tissues reveals that the alk mRNA is dominantly detected in the brain and spinal cord. Immunoblotting with anti-ALK antibody shows that ALK is highly expressed in the neonatal brain. Furthermore, RNA in situ hybridization analysis shows that the alk mRNA is dominantly expressed in neurons in speci®c regions of the nervous system such as the thalamus, mid-brain, olfactory bulb, and ganglia of embryonic and neonatal mice. These data suggest that ALK plays an important role(s) in the development of the brain and exerts its e ects on speci®c neurons in the nervous system.