Citations

ABSTRACT Niemann‐Pick disease type C (NPC) is a genetic disorder in which patient cells exhibit lysosomal accumulation of cholesterol and sphingolipids (SLs) caused by defects in either NPC1 or NPC2 proteins. We previously demonstrated that NPC1 human skin fibroblasts overexpressing endosomal Rab proteins (Rab7 or Rab9) showed a correction in the storage disease phenotype. In the current study, we used protein transduction to further investigate Rab9‐mediated reduction of stored lipids in NPC cells. Recombinant human Rab9 fused with the herpes simplex virus VP22 protein fragment was overexpressed, purified, and added to culture medium to induce protein transduction. When VP22‐Rab9 was transduced into NPC1 fibroblasts, nearly all cells showed significant reduction in cellular free cholesterol levels, with no cytotoxicity up to 5 µM. A fraction of the VP22‐Rab9 that was transduced into the cells was shown to bind to rab GDP dissociation inhibitor, suggesting that this pool of VP22‐Rab9 had become prenylated. The reduction in cellular free cholesterol was associated with correction of abnormal intracellular trafficking of BODIPY‐lactosylceramide and an increase of sterols in the culture media. The clearance of lysosomal free cholesterol was also associated with a decrease in LDL‐receptor levels. In addition, we demonstrated reduction of intracellular cholesterol by VP22‐Rab9 transduction in NPC2 fibroblasts and in cultured mouse NPC1 neurons. These observations provide important new information about the correction of membrane traffic in NPC cells by Rab9 overexpression and may lead to new therapeutic approaches for treatment of this disease.

Using quantitative light microscopy and a modified immunoelectron microscopic technique, we have characterized the entry pathway of the cholera toxin binding subunit (CTB) in primary embryonic fibroblasts. CTB trafficking to the Golgi complex was identical in caveolin-1null (Cav1−/−) mouse embryonic fibroblasts (MEFs) and wild-type (WT) MEFs. CTB entry in the Cav1−/− MEFs was predominantly clathrin and dynamin independent but relatively cholesterol dependent. Immunoelectron microscopy was used to quantify budded and surface-connected caveolae and to identify noncaveolar endocytic vehicles. In WT MEFs, a small fraction of the total Cav1-positive structures were shown to bud from the plasma membrane (2% per minute), and budding increased upon okadaic acid or lactosyl ceramide treatment. However, the major carriers involved in initial entry of CTB were identified as uncoated tubular or ring-shaped structures. These carriers contained GPI-anchored proteins and fluid phase markers and represented the major vehicles mediating CTB uptake in both WT and caveolae-null cells.

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2∼8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2∼30–40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.

Internalization of some plasma membrane constituents, bacterial toxins, and viruses occurs via caveolae; however, the factors that regulate caveolar internalization are still unclear. Here, we demonstrate that a brief treatment of cultured cells with natural or synthetic glycosphingolipids (GSLs) or elevation of cholesterol (either by acute treatment with mβ-cyclodextrin/cholesterol or by alteration of growth conditions) dramatically stimulates caveolar endocytosis with little or no effect on other endocytic mechanisms. These treatments also stimulated the movement of GFP-labeled vesicles in cells transfected with caveolin-1-GFP and reduced the number of surface-connected caveolae seen by electron microscopy. In contrast, overexpression of caveolin-1 decreased caveolar uptake, but treatment with GSLs reversed this effect and stimulated caveolar endocytosis. Stimulation of caveolar endocytosis did not occur using ceramide or phosphatidylcholine and was not due to GSL degradation because similar results were obtained using a nonhydrolyzable GSL analog. Stimulated caveolar endocytosis required src kinase and PKC-α activity as shown by i) use of pharmacological inhibitors, ii) expression of kinase inactive src or dominant negative PKCα, and iii) stimulation of src kinase activity upon addition of GSLs or cholesterol. These results suggest that caveolar endocytosis is regulated by a balance of caveolin-1, cholesterol, and GSLs at the plasma membrane.

Abstract CD38/cyclic adenosine diphosphate ribose (cADPR) signaling plays an important role in the regulation of intracellular calcium responses to agonists in a variety of cells, including airway smooth muscle (ASM) cells. The present study was aimed at determining the effect of interleukin (IL)-13, a cytokine implicated in the pathogenesis of asthma, on CD38/cADPR signaling and to ascertain the contribution of CD38/cADPR signaling to IL-13–induced airway hyperresponsiveness. Human ASM cells maintained in culture were exposed to 50 ng/ml IL-13 for 22 h and levels of CD38 expression and intracellular calcium responses to agonists were measured. Treatment of human ASM cells with IL-13 resulted in increased CD38 expression as determined by real-time polymerase chain reaction, Western blot analysis, and indirect immunofluorescence. Increased CD38 expression was reflected as increased ADP-ribosyl cyclase activity in the ASM cell membranes. The net intracellular calcium responses to bradykinin, thrombin, and histamine were significantly (P ≤ 0.05) higher in cells treated with IL-13 compared with controls. Furthermore, 8-bromo-cADPR, a cADPR antagonist, attenuated IL-13–induced augmented intracellular calcium responses to agonists in human ASM cells. These findings indicate that the CD38/cADPR–dependent pathway has a major role in IL-13–induced modulation of calcium signaling in human ASM.

Background— Traditionally, increased pericardial thickness has been considered an essential diagnostic feature of constrictive pericarditis. Although constriction with a normal-thickness pericardium has been demonstrated clinically by noninvasive imaging, the details of clinicopathological correlates have not been described. Methods and Results— A total of 143 patients with proven constriction underwent pericardiectomy at Mayo Clinic between 1993 and 1999. Their baseline characteristics, operative data, and pathological specimens were reviewed retrospectively. The pericardium was of normal thickness (≤2 mm) in 26 patients (18%; group 1) and was thickened (>2 mm) in 117 (82%; group 2). The most common causes of constriction in group 1 included previous cardiac surgery, chest irradiation, previous infarction, and idiopathic disease. There was little difference in symptoms and findings on physical examination between the 2 groups. Microscopically, no patient had an entirely normal pericardium. Histopathological abnormalities in group 1 were mild and focal, including fibrosis, inflammation, calcification, fibrin deposition, and focal noncaseating granulomas. Pericardiectomy was equally effective in relieving symptoms regardless of the presence or absence of increased thickness. Conclusions— Pericardial thickness was not increased in 18% of patients with surgically proven constrictive pericarditis, although the histopathological appearance was focally abnormal in all cases. When clinical, echocardiographic, or invasive hemodynamic features indicate constriction in patients with heart failure, pericardiectomy should not be denied on the basis of normal thickness as demonstrated by noninvasive imaging.