Galectin-9, a ␤ -galactoside binding lectin, has recently been isolated from murine embryonic kidney. In this study, its biological functions and expression in embryonic, newborn, and adult mice tissues were investigated. By Northern blot analyses, it was found widely distributed and its expression was developmentally regulated. In situ hybridization studies revealed an accentuated expression of galectin-9 in liver and thymus of embryonic mice. In postnatal mice, antigalectin-9 immunoreactivity was observed in various tissues, including thymic epithelial cells. The high expression of galectin-9 in the thymus led us to investigate its role in the clonal deletion of thymocytes. Fusion proteins were generated, which retained lactose-binding activity like the endogenous galectin-9. Galectin-9, at 2.5 M concentration, induced apoptosis in ‫ف‬ 30% of the thymocytes, as assessed by terminal deoxytransferase-mediated dUTP nick end labeling method. The apoptotic effect was dose dependent and lactose inhibitable. At higher concentrations, it induced homotypic aggregation of the thymocytes. Electron microscopy revealed ‫ف‬ 60% of the thymocytes undergoing apoptosis in the presence of galectin-9. By immunofluorescence microscopy, some of the thymocytes undergoing apoptosis had plasmalemmal bound galectin-9. Galectin-9 failed to induce apoptosis in hepatocytes. Taken together, these findings indicate that galectin-9, a developmentally regulated lectin, plays a role in thymocyte-epithelial interactions relevant to the biology of the thymus. ( J. Clin. Invest. 1997. 99:2452-2461.)

Phosphorylation of the cytoplasmic tyrosine residues of the epidermal growth factor receptor (EGFR) upon binding of EGF induces recognition of various intracellular signaling molecules, including Grb2. Here, the reaction kinetics between EGFR and Grb2 was analyzed by visualizing single molecules of Grb2 conjugated to the fluorophore Cy3 (Cy3-Grb2). The plasma membrane fraction was purified from human epithelial carcinoma A431 cells after stimulation with EGF and attached to coverslips. Unitary events of association and dissociation of Cy3-Grb2 on the EGFR in the membrane fraction were observed at different concentrations of Grb2 (0.1-100 nM). The dissociation kinetics could be explained by using a multiple-exponential function with a major (>90%) dissociation rate of 8 s ؊1 and a few minor components, suggesting the presence of multiple bound states. In contrast, the association kinetics could be described by a stretched exponential function, suggesting the presence of multiple reaction channels from many unbound substates. Transitions between the unbound substates were also suggested. Unexpectedly, the rate of association was not proportional to the Grb2 concentration: an increase in Cy3-Grb2 concentration by a factor of 10 induced an increase in the reaction frequency approximately by a factor of three. This effect can compensate for fluctuation of the signal transduction from EGFR to Grb2 caused by variations in the expression level of Grb2 in living cells.EGFR ͉ signal transduction ͉ tyrosine phosphorylation

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Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis. In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development. Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied. Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development. Northern blot analyses revealed a single approximately 4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated. Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros. MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants. Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi. Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants. Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression. The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2. These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.