Endocytosis of tartrate-resistant acid phosphatase by osteoblast-like cells is followed by inactivation of the enzyme.

Tartrate-resistant acid phosphatase (TRACP) is generally used as a marker of osteoclasts. Yet, other bone-associated cells, such as osteoblasts and osteocytes, may also express activity of this enzyme. Osteoblasts containing TRACP activity are seen particularly in the vicinity of areas of bone resorption, suggesting that osteoclasts somehow induce TRACP activity in osteoblasts. In a recent study, we found that osteoblast-like cells appeared to have the capacity to endocytose TRACP released by osteoclast precursors. In the present study, we investigated the endocytosis of TRACP in more detail as well as the fate of the endocytosed enzyme. We found that incubation of osteoblast-like cells with TRACP-coated beads resulted in attachment of a high number of beads to the cells. After culturing osteoblast-like cells with medium conditioned by blood monocytes that contain TRACP, activity of the enzyme was found in the cells. Following replacement of the medium by normal medium that did not contain TRACP, a decrease in the level of TRACP activity in osteoblast-like cells occurred. Our data strongly suggest that osteoblast-like cells recognize TRACP released by osteoclast precursors and that upon endocytosis inactivation of the enzyme occurs. We propose that uptake of the enzyme is important for the control of enzyme activity, thereby preventing degradation of matrix constituents.

The bone lining cell: its role in cleaning Howship's lacunae and initiating bone formation.

In this study we investigated the role of bone lining cells in the coordination of bone resorption and formation. Ultrastructural analysis of mouse long bones and calvariae revealed that bone lining cells enwrap and subsequently digest collagen fibrils protruding from Howship's lacunae that are left by osteoclasts. By using selective proteinase inhibitors we show that this digestion depends on matrix metalloproteinases and, to some extent, on serine proteinases. Autoradiography revealed that after the bone lining cells have finished cleaning, they deposit a thin layer of a collagenous matrix along the Howship's lacuna, in close association with an osteopontin-rich cement line. Collagenous matrix deposition was detected only in completely cleaned pits. In bone from pycnodysostotic patients and cathepsin K-deficient mice, conditions in which osteoclastic bone matrix digestion is greatly inhibited, bone matrix leftovers proved to be degraded by bone lining cells, thus indicating that the bone lining cell "rescues" bone remodeling in these anomalies. We conclude that removal of bone collagen left by osteoclasts in Howship's lacunae is an obligatory step in the link between bone resorption and formation, and that bone lining cells and matrix metalloproteinases are essential in this process.

Early effects of high doses of retinol (vitamin A) on the in situ cellular metabolism in rat liver.

Understanding of the possible toxicity associated with hypervitaminosis A becomes increasingly important in view of the popularity of vitamin A supplementation. Hypervitaminosis A for many years may eventually lead to hepatocellular damage. In the present study, rats were treated for 7 days with high doses of retinol to study the early effects on the metabolism of different types of liver cells using (enzyme) histochemistry, immunohistochemistry and electron microscopy. Excessive intake of vitamin A activates Kupffer cells and induces accumulation of lipid droplets in fat-storing cells as well as proliferation of these cells. Moreover, it affects the metabolic heterogeneity in the liver lobules, but does not lead to apparent cell damage. Based on the changes in marker enzymes for different metabolic processes, it is concluded that the capacity for breakdown of purines, the antioxidant capacity, the potential for phagocytosis and the regulation of ammonia levels were largely decreased. Increased alkaline phosphatase activity in hepatocytes pointed to an activated process of transport of retinol esters over the bile canalicular membrane. The possible causes of these metabolic changes have been described in the discussion.

In situ substrate specificity and ultrastructural localization of polyamine oxidase activity in unfixed rat tissues.

Data concerning the substrate specificity and the exact intracellular localization of the polyamine-catabolizing enzyme polyamine oxidase are conflicting. Biochemical studies have shown that N1-acetylation of spermine and spermidine dramatically increases the specificity of these compounds for peroxisomal polyamine oxidase to produce spermidine and putrescine, respectively. On the other hand, polyamine oxidase activity was demonstrated histochemically both in peroxisomes and in cytoplasm of several tissues, using spermidine and/or spermine as substrate. To elucidate the in situ substrate specificity of polyamine oxidase and the localization of its activity, enzyme activity was detected in rat liver, kidney, and duodenum at the light and electron microscopic levels. For this purpose, unfixed cryostat sections were applied to avoid changes in enzyme activity owing to chemical fixation. Spermine, spermidine, their N1-acetylated forms, and putrescine were used as substrates, and cerium ions as capturing agent for H2O2. Control reactions were performed in the absence of substrate or in the presence of substrate and specific oxidase inhibitors. At the light microscopic level, final reaction product specifically generated by polyamine oxidase activity was found exclusively in a granular form in hepatocytes, epithelial cells of proximal tubules of the kidney, and epithelial cells of duodenal villi with N1-acetylspermidine or N1-acetylspermine as substrates. Final reaction product was not observed in any of the tissues after incubation in the presence of putrescine, spermidine, or spermine. Formation of specific final reaction product was prevented by incubation in the presence of a specific polyamine oxidase inhibitor, but it was not affected by a diamine oxidase inhibitor. Ultrastructural studies revealed that polyamine oxidase activity is localized exclusively to the matrix of peroxisomes of kidney and liver and to microperoxisomes of the duodenum. The localization patterns obtained with unfixed tissues are in agreement with biochemical data. Strong intraperoxisomal, interperoxisomal, and intercellular heterogeneity in polyamine oxidase activity was found in all tissues investigated.

The digestion of phagocytosed collagen is inhibited by the proteinase inhibitors leupeptin and E-64.

Using morphometric methods the effects of the thiol-proteinase inhibitors leupeptin and E-64 on the digestion of intracytoplasmic collagen fibrils were studied in cultured mouse bone explants. Both drugs caused a dose-dependent increase of lysosomal structures containing cross-banded collagen fibrils (CCV) in periosteal fibroblasts. After an incubation period of 48 hours, leupeptin (in a concentration of 65 microM) caused a thirty-fold increase in the volume fraction of CCV. This effect proved to be reversible following upon the withdrawal of the drug. Since the leupeptin-related accumulation of intracellular collagen fibrils was not significantly inhibited by alpha, alpha dipyridyl (a drug that interferes with collagen fibril formation), it is thought unlikely that the fibrils represented newly synthesized collagen. This view is further substantiated by data obtained from explants incubated in the presence of the phagocytosis-inhibiting agent cytochalasin B. This compound completely inhibited the leupeptin-related accumulation of CCV. The data strongly suggest that collagen fibrils found in cytoplasmic vacuoles of periosteal fibroblasts represent collagen taken up by phagocytosis, the integrity of cytoplasmic actin filament systems is a prerequisite for phagocytosis of collagen to occur, and thiol-proteinases, such as cathepsin B, L, and/or N, play an essential role in the digestion of internalized collagen.