Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.
In osteoclasts, TRACP co-localized with cathepsin K in transcytotic vesicles and was activated by cathepsin K in vitro, suggesting that TRACP may degrade organic matrix components in transcytotic vesicles in an event regulated by cathepsin K.Introduction: TRACP is an enzyme with unknown biological function. In addition to its phosphatase activity, TRACP is capable of generating reactive oxygen species (ROS). Bone-resorbing osteoclasts contain large amounts of TRACP, and transgenic animal models suggest that TRACP has a role in bone resorption. Osteoclasts resorb bone by secreting acid and lysosomal enzymes such as cathepsin K into an extracellular resorption lacuna between the cell membrane and bone surface. Matrix degradation products are then endocytosed, transcytosed, and secreted through a functional secretory domain in the basolateral membrane facing bone marrow.
Materials and Methods:We have studied intracellular localization of TRACP in osteoclasts with antibodies against various known endosomal and lysosomal proteins using confocal microscopy. We also studied co-localization of TRACP with cathepsin K and endocytosed bone matrix components and the effect of cathepsin K digestion on the ROS generating activity of TRACP in vitro. Results: Double-staining experiments of TRACP with endosomal and lysosomal markers showed that, although some endosomal staining was detected, TRACP was not present in lysosomes. However, TRACP was present in transcytotic vesicles, where it co-localized with cathepsin K. Cathepsin K digestion of TRACP in vitro increased the phosphatase activity by 5.6-fold and the ROS generating activity by 2.0-fold. Conclusions: These results suggest that cathepsin K may activate the ROS-generating activity of TRACP in transcytotic vesicles of resorbing osteoclasts, the ROS being targeted to finalize degradation of organic bone matrix components during their transcytosis.
Resistance to apoptosis is a critical feature of neoplastic cells. Galectin-1 is an endogenous carbohydrate-binding protein that induces death of leukemia and lymphoma cells, breast cancer cells, and the LNCaP prostate cancer cell line, but not other prostate cancer cell lines. To understand the mechanism of galectin-1 sensitivity of LNCaP cells compared with other prostate cancer cells, we characterized glycan ligands that are important for conferring galectin-1 sensitivity in these cells, and analyzed expression of glycosyltransferase genes in galectin-1-sensitive, prostate-specific antigen-positive (PSA + ) LNCaP cells compared with a galectin-1-resistant PSA À LNCaP subclone. We identified one glycosyltransferase, core 2 N-acetylglucosaminyltransferase, which is downregulated in galectin-1-resistant PSA À LNCaP cells compared with galectin-1-sensitive PSA + LNCaP cells. Intriguingly, this is the same glycosyltransferase required for galectin-1 susceptibility of T lymphoma cells, indicating that similar O-glycan ligands on different polypeptide backbones may be common death trigger receptors recognized by galectin-1 on different types of cancer cells. Blocking O-glycan elongation by expressing A2,3-sialyltransferase 1 rendered LNCaP cells resistant to galectin-1, showing that specific O-glycans are critical for galectin-1 susceptibility. Loss of galectin-1 susceptibility and synthesis of endogenous galectin-1 has been proposed to promote tumor evasion of immune attack; we found that galectin-1-expressing prostate cancer cells killed bound T cells, whereas LNCaP cells that do not express galectin-1 did not kill T cells. Resistance to galectin-1-induced apoptosis may directly contribute to the survival of prostate cancer cells as well as promote immune evasion by the tumor. [Cancer Res 2007;67(13):6155-62]
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