Paul H. Schlesinger

Receptor-mediated pinocytosis of mannose glycoconjugates by macrophages: Characterization and evidence for receptor recycling

125I-Mannose--BSA is taken up by alveolar macrophages by receptor-mediated endocytosis. Uptake is macrophage-specific and does not occur in polymorphonuclear leukocytes. Binding (4 degrees C) and uptake (37 degrees C) of 125I--Man--BSA are time- and ligand concentration-dependent [Kuptake = 40 nM; Kd (4 degrees C) = 10 nM]. When adjusted for ligand degradation, ligand uptake is linear with time. Binding saturates at 60 min and requires Ca++. Following binding, ligand remains on the cell surface where it can be released by EGTA and trypsin. Internalization of prebound ligand occurs very rapidly (t 1/2 less than 5 min) when cells are warmed to 37 degrees C. Following internalization of prebound ligand, binding activity is rapidly recovered (t 1/2 less than 5 min). Trypsin treatment (4 degrees C) substantially reduces binding activity (greater than 70% per 30 min). However, binding activity is rapidly recovered in cells treated with trypsin at 4 degrees C by warming to 37 degrees C in the absence of added ligand. Trypsin treatment at 37 degrees C rapidly destroys binding and uptake. On the contrary, 4 degrees C trypsin treatment produces only a modest reduction in subsequent ligand uptake. These results, taken together with the observation that cycloheximide has no effect on ligand uptake, suggest that receptors must be spared from degradation and that reutilization of receptors probably occurs.

BAX-dependent transport of cytochrome c reconstituted in pure liposomes.

elected death signals activate pro-apoptotic BAX, resulting in translocation to mitochondria where it is inserted as an integral, oligomeric membrane protein. Mitochondrial dysfunction follows, with release of cytochrome c and activation of caspases. BAX forms ion-conducting channels in planar lipid bilayers and releases chloride or carboxyfluorescein from artificial liposomes. The ion-transmitting pore formed by BAX can progress to a conductance of ~1.5 nS with low ion selectivity. In some proposed models, BAX interacts with other pore-forming molecules or, alternatively, generally disrupts the outer mitochondrial membrane. Here we show that nanomolar BAX rapidly forms membrane pores that release intravesicular carboxyfluorescein, fluorescein-isothiocyanate-conjugated dextran (FITC– dextran) or FITC–cytochrome c, and that are blocked by dextran molecules of defined size. We also demonstrate concentrationdependent progression from a pore of 10.7 ± 3.2 Å, consisting of two BAX molecules, to one of 22.1 ± 3.9 Å, requiring four BAX molecules, and show that this larger BAX pore is capable of transporting cytochrome c. We analyzed BAX-mediated release of 6-carboxyfluorescein from 200-nm unilamellar vesicles, using recombinant, purified, functionally identical BAX(∆C19) molecules that were monodispersed at pH 7.2 (refs 9, 14–17). Previous studies of the melittin pore have shown that exponential dequenching reflects concentration-dependent activation of oligomeric pores. The exponential kinetics of BAX-pore activation are well described by equation (1), and the fit was not improved by further exponential terms (see Methods):

Bisphosphonates directly inhibit the bone resorption activity of isolated avian osteoclasts in vitro.

Bisphosphonates are useful in treatment of disorders with increased osteoclastic activity, but the mechanism by which bisphosphonates act is unknown. We used cultures of chicken osteoclasts to address this issue, and found that 1-hydroxyethylidenediphosphonic acid (EHDP), dichloromethylidenediphosphonic acid (Cl2MDP), or 3-amino-1-hydroxypropylidene-1,1-diphosphonic acid (APD) all cause direct dose-dependent suppression of osteoclastic activity. Effects are mediated by bone-bound drugs, with 50% reduction of bone degradation occurring at 500 nM to 5 microM of the different agents. Osteoclastic bone-binding capacity decreased by 30-40% after 72 h of bisphosphonate treatment, despite maintenance of cell viability. Significant inhibition of bone resorption in each case is seen only after 24-72 h of treatment. Osteoclast activity depends on ATP-dependent proton transport. Using acridine orange as an indicator, we found that EHDP reduces proton accumulation by osteoclasts. However, inside-out plasma membrane vesicles from osteoclasts transport H+ normally in response to ATP in high concentrations of EHDP, Cl2MDP, or APD. This suggests that the bisphosphonates act as metabolic inhibitors. Bisphosphonates reduce osteoclastic protein synthesis, supporting this hypothesis. Furthermore, [3H]leucine incorporation by the fibroblast, which does not resorb bone, is also diminished by EHDP, Cl2MDP and APD except when co-cultured with bisphosphonate-binding bone particles. Thus, the resorption-antagonizing capacities of EHDP, Cl2MDP and APD reflect metabolic inhibition, with selectivity for the osteoclast resulting from high affinity binding to bone mineral.

Isolation of the galactose-binding lectin that mediates the in vitro adherence of Entamoeba histolytica.

Entamoeba histolytica adheres to human colonic mucus, colonic epithelial cells, and other target cells via a galactose (Gal) or N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin. Blockade of this adherence lectin with Gal or GalNAc in vitro prevents amebic killing of target cells. We have identified and purified the adherence lectin by two methods: affinity columns derivatized with galactose monomers or galactose terminal glycoproteins, and affinity columns and immunoblots prepared with monoclonal antibodies that inhibit amebic adherence. By both methods the adherence lectin was identified as a 170-kD secreted and membrane-bound amebic protein. The surface location of the lectin was confirmed by indirect immunofluorescence. Purified lectin competitively inhibited amebic adherence to target cells by binding to receptors on the target Chinese hamster ovary cells in a Gal-inhibitable manner.

Chloroquine and ammonium ion inhibit receptor-mediated endocytosis of mannose-glycoconjugates by macrophages: Apparent inhibition of receptor recycling

Summary 125I-Mannose-BSA is taken up by macrophages by receptor-mediated pinocytosis. Previous studies indicated that uptake is rapid and proceeds linearly with time in the absence of protein synthesis suggesting that receptors are conserved and recycled. Chloroquine and NH4+ inhibit uptake of 125I-Mannose-BSA and its subsequent digestion by macrophages. Inhibition of uptake is inhibitor-concentration and time dependent. Both binding of ligand (4°) and internalization of pre-bound ligand (4°) following warm-up are unaffected by the inhibitors. However, brief incubation of cells at 37° with inhibitor in the absence of ligand results in a substantial reduction in cell surface binding sites. Our interpretation of these results is that chloroquine and NH4+ inhibit receptor recycling and thereby inhibit ligand uptake.

Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth

The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.

Oxysterols are allosteric activators of the oncoprotein Smoothened

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which plays critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with their function through a specific protein target. We present several lines of evidence that this protein target is the 7-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing their mechanism of action.

Characterization of the Osteoclast Ruffled Border Chloride Channel and Its Role in Bone Resorption

Bone resorption by osteoclasts requires massive transcellular acid transport, which is accomplished by the parallel action of a V-type proton pump and a chloride channel in the osteoclast ruffled border. We have studied the molecular basis for the appearance of acid transport as avian bone marrow mononuclear cells acquire a bone resorptive phenotype in vitro. We demonstrate a critical role for regulated expression of a ruffled border chloride channel as the cells become competent to resorb bone. Molecular characterization of the chloride channel shows that it is related to the renal microsomal chloride channel, p64. In planar bilayers, the ruffled border channel is a stilbene sulfonate-inhibitable, outwardly rectifying chloride channel. A mechanism by which outward rectification of the single channel chloride current could allow efficient regulation of acidification by the channel is discussed.

Wolframin Expression Induces Novel Ion Channel Activity in Endoplasmic Reticulum Membranes and Increases Intracellular Calcium

Wolfram syndrome is an autosomal recessive neuro-degenerative disorder associated with juvenile onset non-autoimmune diabetes mellitus and progressive optic atrophy. The disease has been attributed to mutations in the WFS1 gene, which codes for a protein predicted to possess 9–10 transmembrane segments. Little is known concerning the function of the WFS1 protein (wolframin). Endoglycosidase H digestion, immunocytochemistry, and subcellular fractionation studies all indicated that wolframin is localized to the endoplasmic reticulum in rat brain hippocampus and rat pancreatic islet β-cells, and after ectopic expression in Xenopus oocytes. Reconstitution of wolframin from oocyte membranes into planar lipid bilayers demonstrated that the protein induced a large cation-selective ion channel that was blocked by Mg2+ or Ca2+. Inositol triphosphate was capable of activating channels in the fused bilayers that were similar to channel components induced by wolframin expression. Expression of wolframin also increased cytosolic calcium levels in oocytes. Wolframin thus appears to be important in the regulation of intracellular Ca2+ homeostasis. Disruption of this function may place cells at risk to suffer inappropriate death decisions, thus accounting for the progressive β-cell loss and neuronal degeneration associated with the disease.

Mechanisms balancing skeletal matrix synthesis and degradation

Bone is regulated by evolutionarily conserved signals that balance continuous differentiation of bone matrix-producing cells against apoptosis and matrix removal. This is continued from embryogenesis, where the skeleton differentiates as a solid mass and is shaped into separate bones by cell death and proteolysis. The two major tissues of the skeleton are avascular cartilage, with an extracellular matrix based on type II collagen and hydrophilic proteoglycans, and bone, a stronger and lighter material based on oriented type I collagen and hydroxyapatite. Both differentiate from the same mesenchymal stem cells. This differentiation is regulated by a family of related signals centred on bone morphogenic proteins. Fibroblast growth factors, Indian hedgehog and parathyroid hormone-related protein are important in determining the type of matrix and the relation of skeletal and non-skeletal structures. Removal of mineralized matrix involves apoptosis of matrix cells and differentiation of acid-secreting cells (osteoclasts) from macrophage precursors. Key regulators of matrix removal are signals in the tumour-necrosis-factor family. Osteoclasts dissolve bone by isolating a region of the matrix and secreting HCl and proteinases at that site. Successive cycles of removal and replacement allow growth, repair and remodelling. The signals for bone turnover are predominantly cell-membrane-associated, allowing very specific spatial regulation. In addition to its support function, bone is a reservoir of Ca2+, PO3-(4) and OH-. Secondary modulation of mineral secretion and bone degradation are mediated by humoral signals, including parathyroid hormone and vitamin D, as well as the cytokines that also regulate the underlying cell differentiation.