The adhesion molecule L-selectin is cleaved rapidly from the surface of activated leukocytes by tumor necrosis factor-␣ converting enzyme, a cell surface metalloprotease, and also undergoes slower constitutive shedding in unactivated cells. The structural features that render it susceptible to shedding are poorly understood. We therefore analyzed the shedding of a series of mutant and chimeric L-selectin molecules. Although murine L-selectin is cleaved at a specific location in the juxtamembrane region 11 amino acids distal to the cell membrane, this cleavage has little sequence specificity. However, proline substitution at the P2 or P3 position or deletion of the epidermal growth factor (EGF) domain completely blocks the rapid phorbol ester-induced cleavage, but does not affect the slower basal proteolytic shedding. Insertion of the 15-residue membrane-proximal region (MPR) of L-selectin into the heterologous protein B7.2 results in a molecule that undergoes constitutive proteolytic turnover. In contrast, insertion of both the EGF domain and the MPR confers susceptibility to both slow constitutive shedding and the rapid proteolytic cleavage induced by phorbol 12-myristate 13-acetate. These results demonstrate that constitutive and induced L-selectin cleavage are separable processes and that the rapid phorbol ester-induced shedding requires the presence of the EGF domain, a sequence that is remote from the cleavage site.The extracellular domains of many integral membrane glycoproteins undergo proteolytic cleavage and release from the cell surface into the surrounding fluid phase. The biologically significant proteins released in this manner include a number of lymphokines, growth factors, transcription factors, and adhesion molecules (reviewed in Refs. 1-4). Proteolytic cleavage is responsible for the regulated secretion of several cytokines derived from transmembrane precursors (5-8), and the shedding of surface receptors results in the desensitization of responsiveness to various cytokines (9 -12). Membrane protein shedding is also implicated in several disease processes. Inherited mutations in the p55 TNF-␣ 1 receptor lessen its sensitivity to proteolysis, cause its decreased proteolytic clearance from the cell surface, and result in a family of inherited autoinflammatory syndromes (13). Cleavage of the amyloid precursor protein (APP) at the ␤ and ␥ sites, releasing the amyloidogenic A␤ fragment, is implicated in the pathogenesis of Alzheimer's disease, and mutations that increase secretion of this peptide are associated with familial Alzheimer's disease (14 -16). The enzymes responsible for such cleavage are collectively referred to as membrane secretases or sheddases. A common mechanism for the secretion of many of these proteins has been inferred from the ability to inhibit their release with new hydroxamic acid-derived metalloprotease inhibitors (17-21) and by the identification of a mutant cell line that is defective in cleaving multiple proteins (22). More recently, a plasma membrane enzyme responsible ...

Tumor metastasis plays a major role in the morbidity and mortality of cancer patients. Among solid tumors that undergo metastasis, there is often a predilection to metastasize to a particular organ with, for example, prostate cancer preferentially metastasizing to bones and colon cancer preferentially metastasizing to the liver. Although many factors are thought to be important in establishing permissiveness for metastasis, the reasons for organ-specific predilection of each tumor are not understood. Using a B16 murine melanoma model, we tested the hypothesis that organ-specific NK cell subsets play a critical role in organ-specific metastasis of this tumor. Melanoma cells, given intravenously, readily colonized the lungs but not the liver. NK cell depletion (either iatrogenically or by using genetically targeted mice) resulted in substantial hepatic metastasis. Analysis of NK cell subsets, defined by the differential expression of a combination of CD27 and CD11b, indicated a significant difference in the distribution of NK cell subsets in the lung and liver with the mature subset being dominant in the lung and the immature subset being dominant in the liver. Several experimental approaches, including adoptive transfer, clearly indicated that the immature hepatic NK cell subset, CD27+ CD11b–, was protective against liver metastasis; this subset mediated its protection by a perforin-dependent cytotoxic mechanism. In contrast, the more mature NK cell subsets were more efficient at reducing pulmonary tumor load. These data indicate that organ-specific immune responses may play a pivotal role in determining the permissiveness of a given organ for the establishment of a metastatic niche.

A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele. We show that luciferase is highly and uniformly expressed in all tissues examined. Using this mouse model, we describe a facile assay that enables the assessment of the pharmacodynamics of a systemically delivered siRNA formulation. These mice can also be used as universal donors, enabling the efficient and sensitive monitoring of cell trafficking or tissue transplantation. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This strain should generally enhance the ability to rapidly screen, compare and optimize various siRNA formulations for tissue-targeted or -enhanced systemic delivery in a preclinical development setting.