Matrilysin is a matrix metalloproteinase expressed in the tumor cells of greater than 80% of intestinal adenomas. The majority of these intestinal tumors are associated with the accumulation of b-catenin, a component of the cadherin adhesion complex and, through its association with the T Cell Factor (Tcf) DNA binding proteins, a regulator in the Wnt signal transduction pathway. In murine intestinal tumors, matrilysin transcripts show striking overlap with the accumulation of b-catenin protein. The matrilysin promoter is upregulated as much as 12-fold by b-catenin in colon tumor cell lines in a manner inversely proportional to the endogenous levels of b-catenin/Tcf complex and is dependent upon a single optimal Tcf-4 recognition site. Coexpression of the Ecadherin cytoplasmic domain blocked this induction and reduced basal promoter activity in every colon cancer cell line tested. Inactivation of the Tcf binding site increased promoter activity and overexpression of the Tcf factor, LEF-1, signi®cantly downregulated matrilysin promoter activity, suggesting that b-catenin transactivates the matrilysin promoter by virtue of its ability to abrogate Tcf-mediated repression. Because genetic ablation of matrilysin decreases tumor formation in multiple intestinal neoplasia (Min) mice, we propose that regulation of matrilysin production by b-catenin accumulation is a contributing factor to intestinal tumorigenesis.

Matrix metalloproteinases (MMPs) are highly expressed in the human endometrium during menstruation, and these enzymes participate in the cyclic destruction and regeneration characteristic of the primate endometrium. To examine hormonal regulation of MMPs in vivo, we evaluated MMP expression and localization in the endometrium of ovariectomized rhesus macaques under various hormonal conditions. Although all MMPs were up-regulated by progesterone (P4) withdrawal, their expression declined spontaneously after menstruation in the absence of P4. Of 7 MMPs examined, only matrilysin and stromelysin-3 were suppressed any further when P4 levels were experimentally re-elevated. MMP expression was confined to the upper functionalis zone during menstruation, but after menstrual breakdown was complete, matrilysin and the tissue inhibitor of MMPs, TIMP-1, shifted expression from the functionalis to the basalis zone in the absence of both estradiol and P4. The spiral arteries in the functionalis, but not the basalis, were intense foci of MMP and TIMP-1 expression. Menstruation and MMP expression after P4 withdrawal were similar in both the presence and absence of estradiol. In sum, endometrial MMPs in vivo are strongly up-regulated by P4 withdrawal, but zone-specific tissue gradients greatly influence the pattern and degree of MMP expression.

Agmatinase, which hydrolyzes agmatine to putrescine and urea, not only represents a potentially important mechanism for regulating the biological effects of agmatine in mammalian cells but also represents an alternative to ornithine decarboxylase for polyamine biosynthesis. We have isolated a full-length cDNA encoding human agmatinase whose function was confirmed by complementation in yeast. The single-copy human agmatinase gene located on chromosome 1 encodes a 352-residue protein with a putative mitochondrial targeting sequence at the NH(3)-terminus. Human agmatinase has about 30% identity to bacterial agmatinases and <20% identity to mammalian arginases. Residues required for binding of Mn(2+) at the active site in bacterial agmatinase and other members of the arginase superfamily are fully conserved in human agmatinase. Agmatinase mRNA is most abundant in human liver and kidney but also is expressed in several other tissues, including skeletal muscle and brain. Its expression in human liver is induced during hepatitis B virus infection, suggesting that agmatinase may play a role in the pathophysiology of this disease.

Enhancement of tyrosine phosphorylation in cells by the application of pervanadate, an extremely potent phosphotyrosine phosphatase inhibitor, provokes the rapid metalloprotease-dependent cleavage of ErbB-4, a transmembrane receptor tyrosine kinase. The pervanadate-induced proteolysis occurs in NIH 3T3 cells expressing transfected human ErbB-4 and in several cell lines that express endogenous ErbB-4. One product of this proteolytic event is a membrane-anchored molecule of approximately 80 kDa, which is heavily tyrosine phosphorylated and which possesses tyrosine kinase catalytic activity toward an exogenous substrate in vitro. This response to pervanadate is not dependent on protein kinase C activation, which has previously been demonstrated to also activate ErbB-4 cleavage. Hence, the pervanadate and 12-O-tetradecanoylphorbol-13-acetate-induced proteolytic cleavage of ErbB-4 seem to proceed by different mechanisms, although both require metalloprotease activity. Moreover, pervanadate activation of ErbB-4 cleavage, but not that of 12-O-tetradecanoylphorbol-13-acetate , is blocked by the oxygen radical scavenger pyrrolidine dithiocarbomate. A second phosphotyrosine phosphatase inhibitor, phenylarsine oxide, also stimulates a similar cleavage of ErbB-4 but, unlike pervanadate, is not sensitive to pyrrolidine dithiocarbomate. Last, pervanadate is shown to stimulate the proteolytic cell surface processing of a second and unrelated transmembrane molecule: the precursor for amphiregulin, an epidermal growth factor-related molecule. Amphiregulin cleavage by pervanadate occurred in the absence of a cytoplasmic domain and tyrosine phosphorylation of this substrate.

To examine the role of matrilysin (MAT), an epithelial cell-specific matrix metalloproteinase, in the normal development and function of reproductive tissues, we generated transgenic animals that overexpress MAT in several reproductive organs. Three distinct forms of human MAT (wild-type, active, and inactive) were placed under the control of the murine mammary tumor virus promoter/enhancer. Although wild-type, active, and inactive forms of the human MAT protein could be produced in an in vitro culture system, mutations of the MAT cDNA significantly decreased the efficiency with which the MAT protein was produced in vivo. Therefore, animals carrying the wild-type MAT transgene that expressed high levels of human MAT in vivo were further examined. Mammary glands from female transgenic animals were morphologically normal throughout mammary development, but displayed an increased ability to produce ␤-casein protein in virgin animals. In addition, beginning at approximately 8 mo of age, the testes of male transgenic animals became disorganized with apparent disintegration of interstitial tissue that normally surrounds the seminiferous tubules. The disruption of testis morphology was concurrent with the onset of infertility. These results suggest that overexpression of the matrix-degrading enzyme MAT alters the integrity of the extracellular matrix and thereby induces cellular differentiation and cellular destruction in a tissue-specific manner. INTRODUCTIONThe matrix metalloproteinases (MMPs) 1 are a family of extracellular proteases thought to be responsible for normal matrix remodeling and pathological tissue destruction by virtue of their ability to catabolize extracellular matrix components (Birkedael-Hansen et al., 1993; Hulboy et al., 1997 for review). The expression pattern of MMPs in normal tissues suggests that they are particularly involved in the remodeling associated with reproductive processes, including menstruation, trophoblast invasion, mammary gland morphogenesis, and involution of the uterus, mammary gland, and prostate . Studies with natural and synthetic inhibitors of MMPs (Brannstrom et al., 1988;Butler et al., 1991;Talhouk et al., 1992;Marbaix et al., 1996) and recent experiments that utilized genetically altered mice that express altered MMP substrates (Liu et al., 1995) or lack specific MMP family members (Rudolph-Owen et al., 1997) have confirmed the importance of MMPs in reproductive processes in several systems.Sixteen MMP family members have been described (reviewed in Hulboy et al., 1997; see also Cossins et al., 1996;Puente et al., 1996). All MMPs described have three essential domains: a signal sequence or predomain to direct secretion from the cell, a pro sequence to maintain latency, and a catalytic domain containing the critical zinc-binding site. Matrilysin (MAT, uterine (Birkedael-Hansen et al., 1993 for review). This and other domains found in specific MMP family members generate diversity by modifying properties such as substrate specificity, interaction with endogenous inh...

The expression patterns of matrix metalloproteinase (MMP) family members during the murine estrous cycle and postpartum uterine involution were analyzed, and the consequence of removing specific MMPs during uterine functions was determined using mice deficient in either matrilysin (MAT) or stromelysin-1 (STR-1). In wild-type animals, MAT, STR-1, STR-2, STR-3, and gelatinase A were consistently expressed during the most active phases of the estrous cycle, estrus and proestrus. The messenger RNA for these MMPs as well as collagenase-3 and the tissue inhibitors of metalloproteinases were also expressed during uterine involution, as determined by Northern analysis and in situ hybridization. Notably, MAT, STR-2, and collagenase-3 messenger RNA levels were elevated at early times of involution and rapidly decreased with time, whereas the transcripts for other MMPs remained elevated throughout the involution process. Involution proceeded normally in mice lacking MAT or STR-1; however, the expression of STR-1 and STR-2 was dramatically up-regulated in MAT nullizygous mice, and the expression of MAT and STR-2 was moderately up-regulated in STR-1-deficient animals. We conclude that the concerted action of several MMPs is likely to play an important role in the remodeling of the postpartum uterus, and that mechanisms that compensate for the loss of a specific MMP during this process appear to exist.

MLN944 (XR5944) is a novel bis-phenazine that has demonstrated exceptional efficacy against a number of murine and human tumor models. The drug was reported originally as a dual topoisomerase I/II poison, but a precise mechanism of action for this compound remains to be determined. Several lines of evidence, including the marginal ability of MLN944 to stabilize topoisomerase-dependent cleavage, and the sustained potency of MLN944 in mammalian cells with reduced levels of both topoisomerases, suggest that other activities of the drug exist. In this study, we show that MLN944 intercalates into DNA, but has no effect on the catalytic activity of either topoisomerase I or II. MLN944 displays no significant ability to stimulate DNA scission mediated by either topoisomerase I or II compared with camptothecin or etoposide, respectively. In addition, yeast genetic models also point toward a topoisomerase-independent mechanism of action. To examine cell cycle effects, synchronized human HCT116 cells were treated with MLN944, doxorubicin, camptothecin, or a combination of the latter two to mimic a dual topoisomerase poison. MLN944 treatment was found to induce a G1 and G2 arrest in cells that is unlike the typical G2-M arrest noted with known topoisomerase poisons. Finally, transcriptional profiling analysis of xenograft tumors treated with MLN944 revealed clusters of regulated genes distinct from those observed in irinotecan hydrochloride (CPT-11)-treated tumors. Taken together, these findings suggest that the primary mechanism of action of MLN944 likely involves DNA binding and intercalation, but does not appear to involve topoisomerase inhibition.

We present a new computational method for identifying regulated pathway components in transcript profiling (TP) experiments by evaluating transcriptional activity in the context of known biological pathways. We construct a graph representing thousands of protein functional relationships by integrating knowledge from public databases and review articles. We use the notion of distance in a graph to define pathway neighborhoods. The pathways perturbed in an experiment are then identified as the subgraph induced by the genes, referred to as activity centers, having significant density of transcriptional activity in their functional neighborhoods. We illustrate the predictive power of this approach by performing and analyzing an experiment of TP53 overexpression in NCI-H125 cells. The detected activity centers are in agreement with the known TP53 activation effects and our independent experimental results. We also apply the method to a serum starvation experiment using HEY cells and investigate the predicted activity of the transcription factor MYC. Finally, we discuss interesting properties of the activity center approach and its possible applications beyond the comparison of two experiments.