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All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM1 7 as the major convertase of epiregulin, transforming growth factor [alpha], amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/[17.sup.-/-] knockout mice corroborated the essential role of adam[17.sup.-/-] in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

Nossuli, Tareck O., Nikolaos G. Frangogiannis, Pascal Knuefermann, Venkatesh Lakshminarayanan, Oliver Dewald, Alida J. Evans, Jacques Peschon, Douglas L. Mann, Lloyd H. Michael, and Mark L. Entman. Brief murine myocardial I/R induces chemokines in a TNF[alpha]-independent manner: role of oxygen radicals. Am J Physiol Heart Circ Physiol 281: H2549-H2558, 2001.--Early chemokine induction in the area at risk of an ischemic-reperfused (I/R) myocardium is first seen in the venular endothelium. Reperfusion is associated with several induction mechanisms including increased extracellular tumor necrosis factor (TNF)-[alpha], reactive oxygen intermediate (ROI) species formation, and adhesion of leukocytes to the venular endothelium. To test the hypothesis that chemokine induction in cardiac venules can occur by ROIs in a TNF-[alpha]-independent manner, and in the absence of leukocyte accumulation, we utilized wild-type (WT) and TNF-[alpha] double-receptor knockout mice (DKO) in a closed-chest mouse model of myocardial ischemia (15 rain) and reperfusion (3 h), in which there is no infarction. We demonstrate that a single brief period of I/R induces significant upregulation of the chemokines macrophage inflammatory protein (MIP) -1[alpha], -1[beta], and -2 at both the mRNA and protein levels. This induction was independent of TNF-[alpha], whereas levels of these chemokines were increased in both WT and DKO mice. Chemokine induction was seen predominantly in the endothelium of small veins and was accompanied by nuclear translocation of nuclear factor-[kappa]B and c-Jun (AP-l) in venular endothelium. Intravenous infusion of the oxygen radical scavenger N-2-mercaptopropionyl glycine (MPG) initiated 15 min before ischemia and maintained throughout reperfusion obviated chemokine induction, but MPG administration after reperfusion had begun had no effect. The results suggest that ROI generation in the reperfused myocardium rapidly induces C-C and C-X-C chemokines in the venular endothelium in the absence of infarction or irreversible cellular injury. macrophage inflammatory protein; endothelium; transgenic animals Received 9 February 2001; accepted in final form 22 August 2001

RANK, a tumor necrosis factor receptor family member, gives critical signals for lymph node and osteoclast development. RANK-deficient mice were generated for the study. The mice had profound osteopetrosis, apparently from a block in osteoclast differentiation. The mice had marked deficiency of spleen B cells.

A deficiency in interleukin-7 (IL-7) receptor among mice causes them to be lymphopenic since the occurrence of defects in cell expansion becomes greater. As a result, the few mature T cells that develop in IL-7R(super -l-) animals are functionally impaired. However, the overexpression of antiapoptosis protein Bcl-2 resolves these defects by blocking positive signals resulting to the death of impaired T cells.

A tumor necrosis factor (TNF) double receptor (TNFR)-knockout mouse model was analyzed to determine the role of endogenous TNF-alpha in the acute-phase response (APR) to systemic or local inflammation. Analysis of TNFR-knockout mice after the intraperitoneal injections of lipopolysaccharide (LPS) indicated the presence of exacerbated fevers compared to normal mice. The response of TNFR-knockout mice to LPS also indicated the role of TNF-alpha in modulating fever during the early phase of the febrile response to high doses of LPS.

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor- [Alpha] converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-[Alpha] (TNF-[Alpha]). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a 'sheddase' during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivo and ex vivo organ explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-[Alpha] or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development. [C] 2001 Academic Press Key Words: TACE; TNF-[Alpha]; EGF; ectodomain shedding; lung branching morphogenesis; lung explant culture; SP-C; vasculogenesis.

Wild type control mice and transgenic mice lacking type 1 tumor necrosis factor receptor (TNFR1) were exposed to both aerosolized lipopolysaccharide (LPS) and Pseudomonas aeruginosa. Results showed that TNFR1-deficient mice exposed to LPS had fewer neutrophils and less macrophage inflammatory protein-2 than control mice. It was also shown that TNFR1 mice had greater lung inflammation after infection with P. aeruginosa than control mice. The results suggest that TNFR1 is needed to attenuate inflammation resulting from bacterial infection but is not needed to increase lung permeability.