Embryos deficient in the morphogen Sonic hedgehog (Shh) or the endocytic receptor megalin exhibit common neurodevelopmental abnormalities. Therefore, we have investigated the possibility that a functional relationship exists between the two proteins. During embryonic development, megalin was found to be expressed along the apical surfaces of neuroepithelial cells and was coexpressed with Shh in the ventral floor plate of the neural tube. Using enzyme-linked immunosorbent assay, homologous ligand displacement, and surface plasmon resonance techniques, it was found that the aminoterminal fragment of Shh (N-Shh) bound to megalin with high affinity. Megalin-expressing cells internalized NShh through a mechanism that was inhibited by antagonists of megalin, viz. anti-receptor-associated protein and anti-megalin antibodies. Heparin also inhibited NShh endocytosis, implicating proteoglycans in the internalization process, as has been described for other megalin ligands. Use of chloroquine to inhibit lysosomal proteinase activity showed that N-Shh endocytosed via megalin was not efficiently targeted to the lysosomes for degradation. The ability of megalin-internalized N-Shh to bypass lysosomes may relate to the finding that the interaction between N-Shh and megalin was resistant to dissociation with low pH. Together, these findings show that megalin is an efficient endocytic receptor for NShh. Furthermore, they implicate megalin as a new regulatory component of the Shh signaling pathway.

The Pim protein kinases are frequently overexpressed in prostate cancer and certain forms of leukemia and lymphoma. 5-(3-Trifluoromethylbenzylidene)thiazolidine-2,4-dione (4a) was identified by screening to be a Pim-1 inhibitor and was found to attenuate the autophosphorylation of tagged Pim-1 in intact cells. Although 4a is a competitive inhibitor with respect to ATP, a screen of approximately 50 diverse protein kinases demonstrated that it has high selectivity for Pim kinases. Computational docking of 4a to Pim-1 provided a model for lead optimization, and a series of substituted thiazolidine-2,4-dione congeners was synthesized. The most potent new compounds exhibited IC50s of 13 nM for Pim-1 and 2.3 µM for Pim-2. Additional compounds in the series demonstrated selectivities of more than 2500-fold and 400-fold for Pim-1 or Pim-2, respectively, while other congeners were essentially equally potent toward the two isozymes. Overall, these compounds are new Pim kinase inhibitors that may provide leads to novel anticancer agents.

The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu- or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.Most current anticancer drugs kill actively dividing cells by the induction of apoptosis (Fulda, 2009). In addition to this "classical" cancer chemotherapy, approaches that block molecular pathways involved in tumor cell proliferation and therapies that induce alternative cell death pathways are of interest for drug development (Ricci and Zong, 2006). Apoptotic cell death involves a series of events leading to characteristic changes in cell morphology, including loss of cell membrane asymmetry, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and activation of caspases (Ricci and Zong, 2006). Unfortunately, cancer cells often acquire resistance to agents that activate the apoptotic pathway (Fulda, 2009). Therefore, alternative cell death pathways are being examined for exploitation in cancer chemotherapy (Ricci and Zong, 2006).Autophagy is a reversible catabolic adaptive process responsible for the degradation of long-lived proteins and cell survival during starvation and/or growth factor deprivation (Kundu and Thompson, 2008). During autophagy, parts of the c...

Cubilin has recently been shown to function as an endocytic receptor for high density lipoproteins (HDL). The lack of apparent transmembrane and cytoplasmic domains in cubilin raises questions as to the means by which it can mediate endocytosis. Since cubilin has been reported to bind the endocytic receptor megalin, we explored the possibility that megalin acts in conjunction with cubilin to mediate HDL endocytosis. While megalin did not bind to HDL, delipidated HDL, or apoA-I, it was found to copurify with cubilin isolated by HDL-Sepharose affinity chromatography. Cubilin and megalin exhibited coincident patterns of mRNA expression in mouse tissues including the kidney, ileum, thymus, placenta, and yolk sac endoderm. The expression of both receptors in yolk sac endoderm-like cells was inducible by retinoic acid treatment but not by conditions of sterol depletion. Suppression of megalin activity or expression by treatment with either megalin antibodies or megalin antisense oligodeoxynucleotides resulted in inhibition of cubilin-mediated endocytosis of HDL. Furthermore, megalin antisense oligodeoxynucleotide treatment resulted in reduced cell surface expression of cubilin. These data demonstrate that megalin acts together with cubilin to mediate HDL endocytosis and further suggest that megalin may play a role in the intracellular trafficking of cubilin.Cubilin is a recently identified receptor that mediates binding and endocytosis of intrinsic factor-vitamin B 12 complex (1), immunoglobulin light chains (2), and high density lipoproteins (HDL) 1 (3, 4). The primary sequence of the ϳ460-kDa cubilin polypeptide lacks a discernible membrane-spanning element (1, 5). However, a sequence located in the amino-terminal region having amphipathic helical characteristics has been implicated in plasma membrane anchoring/association (6). It is not clear how such a "peripheral" association allows interaction of cubilin with cytosolic components of the endocytic machinery. A plausible hypothesis is that an integral membrane protein(s) functions in concert with cubilin to facilitate the endocytic activities. The principal candidate for such an accessory protein is megalin (also known as gp330 and LRP-2), a member of the low density lipoprotein receptor family. Megalin binds cubilin and colocalizes with it in apical endocytic invaginations and endosomes of renal proximal tubule cells and yolk sac epithelial cells (1). In the present study, the role of megalin in cubilin-mediated endocytosis of HDL is examined. EXPERIMENTAL PROCEDURESProteins-Human apolipoprotein A-I (apoA-I) was obtained from Dr. Bryan Brewer (Molecular Disease Branch, NHLBI, National Institutes of Health, Bethesda, MD). Megalin was purified from porcine kidney as described previously (7).Lipoproteins-Human DiI-HDL was purchased from Biomedical Technologies. Human HDL (density ϭ 1.063-1.21 g/ml) and LDL were provided by Dr. Bryan Brewer. Delipidated HDL (apoHDL) was prepared as described (8). DiI-HDL and HDL were depleted of apoE-HDL and other heparin-binding par...

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