Localization of cathepsins B,D, and L in the rat osteoclast by immuno-light and -electron microscopy

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and-electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border. Cathepsin L may be more effective in the degradation of bone matrix than cathepsin B.     

Purification and characterization of microsomal and lysosomal beta-glucuronidase from rat liver by use of immunoaffinity chromatography.

Rat liver beta-glucuronidase (EC 3.2.1.31), both from microsomal and lysosomal fractions, were purified about 9500-fold over the homogenate with high yield using affinity chromatography prepared by coupling purified specific immunoglobulin G against rat preputial gland beta-glucuronidase to Sepharose 2B and isoelectric focusing. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of approximately 310000. In dodecylsulfate polyacrylamide gel electrophoresis, the microsomal beta-glucuronidase showed a single band corresponding to a molecular weight of 79000, while the lysosomal beta-glucuronidase had three distinct bands which consisted of one major and two minor bands corresponding to molecular weight of 79000, 74000, and 70000, respectively. A broad pH activity curve with a single optimum at pH 4.4 was observed in both the microsomal and the lysosomal beta-glucuronidases. Immunological gel diffusion technique with rabbit antiserum against rat liver lysosomal beta-glucuronidase revealed that both enzymes had the same or quite similar antigenic determinants.     

Transport of acid phosphatase to lysosomes does not involve passage through the cell surface

In foregoing studies, we reported that LGP107, a major lysosomal membrane glycoprotein in the rat liver, distributes in and circulates continuously throughout the endocytic membrane system (endosomes, lysosomes and plasma membrane), in hepatocytes (1,2). In the present study we examined whether acid phosphatase (APase), an enzyme that is transported to lysosomes as a transmembrane protein, passes through the cell surface during intracellular transport, because transport of newly synthesized APase to lysosomes involves the passage of endosomes containing a ligand which is internalized via receptors on the cell surface and is finally dispatched to lysosomes for degradation (3). When localization of APase in rat hepatocytes was investigated by immunoelectron microscopy, APase was found to be localized in lysosomes and endosomes, but not in coated pits on the cell surface, which are positive for LGP107, and from which antibodies for LGP107 are internalized. Further, unlike LGP107, newly synthesized APase was not detected in plasma membranes isolated from livers of rats given [35S]methionine, and when cultured hepatocytes were exposed to 125I-labeled anti APase IgG at 37 degrees C, there was no transfer of the antibody to lysosomes even after 24 h incubation. Therefore, these results indicate that intracellular movement of APase does not involve cell surface passage in rat hepatocytes, and clearly differs from the recent report that human APase is transported to lysosomes via the cell surface in BHK cells transfected with its cDNA (4).     

Small guanosine triphosphatase Rho/Rho-associated kinase as a novel regulator of intracellular redistribution of lysosomes in invasive tumor cells

To investigate the role of RhoA on the intracellular membrane dynamics of lysosomes in rat hepatoma cells (MM1), we analyzed the localization of lysosomal aspartic proteinase cathepsin D by confocal immunofluorescence microscopy in the dominant active RhoA-transfected cells. Here we show that the transfection of the dominant active form of human small guanosine triphosphatase (GTPase) RhoA in MMI cells, a highly invasive cell line, causes the redistribution and spreading of small punctate structures stained for cathepsin D throughout the cytoplasm. We found that the microtubule organization was markedly different in the two cell lines: uniformly developed and polymerized microtubule filaments were seen in the mock transfectants; however, the dynamic organization of microtubules was less pronounced in the active RhoA transfectants. Furthermore, we found for the first time that a selective inhibitor of Rho-associated kinase (p160ROCK), Y-27632, impeded the subcellular spreading of cathepsin D staining and promoted reclustering of cathepsin D toward the perinuclear region in the active RhoA-transfected cells. To our knowledge, this is the first indication that the RhoA/ROCK-mediated signaling pathway is involved in the intracellular membrane dynamics of lysosomes by regulating the cytoskeletal microtubule organization as well as the actin cytoskeletons.     

Role of fatty acid composition in the development of metabolic disorders in sucrose-induced obese rats

Fatty acids have been shown to be involved in the development of insulin resistance associated with obesity. We used sucrose loading in rats to analyze changes in fatty acid composition in the progression of obesity and the related metabolic disorder. Although rats fed a sucrose diet for 4 weeks had body weights similar to those of control animals, their visceral fat pads were significantly larger, and serum triglyceride levels were higher; however, neither plasma glucose nor insulin levels were significantly higher. After 20 weeks of sucrose loading, body weight and visceral and subcutaneous fat pads had increased significantly compared with those in control rats. Moreover, plasma glucose, insulin, and triglyceride levels were significantly higher. An analysis of individual fatty acid components in the blood and peripheral tissues demonstrated phase- and tissue-dependent changes. After 20 weeks of sucrose loading, palmitoleic acid (16:1 n-7) and oleic acid (18:1 n-9), the major components of monounsaturated fatty acid, showed a ubiquitous increase in plasma and all tissues analyzed. In contrast, linoleic acid (18:2 n-6) and arachidonic acid (20:4 n-6), the major components of polyunsaturated fatty acid in the n-6 family, decreased in plasma and all tissues analyzed. After 4 weeks of sucrose loading, these changes in fatty acid composition were observed only in the liver and plasma and not in fat and muscle. This led us to conclude that elevation of plasma glucose and insulin develop at the late phase of sucrose-induced obesity, when changes in fatty acid composition appear in fat and muscle. Furthermore, changes in fatty acid composition in liver seen after 4 weeks of sucrose loading, when increases in neither plasma glucose nor insulin were detected, suggest that liver may be the initial site of fatty acid imbalance and that aberrations in hepatic fatty acid composition may lead to fatty acid imbalances in other tissues.