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

Abstract

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 immunolabeled 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of vacuolar‐type H+‐ATPase, cathepsin K, matrix metalloproteinase‐9, and receptor activator of NFkB ligand in odontoclasts during physiological root resorption of human deciduous teeth

“…Concerning the subsequent organic matrix degradation, as in osteoclastic bone resorption (Goto et al, 1993(Goto et al, , 1994, lysosomal cysteine proteinase such as cathepsin B was previously immunolocalized in human odontoclasts (Sasaki and Ueno-Matsuda, 1992). In the current obser- Fig.…”

Mouse cathepsin K: cDNA cloning and predominant expression of the gene in osteoclasts, and in some hypertrophying chondrocytes during mouse development

“…Northern analysis of mouse tissues for cathepsin K mRNA. Total RNA was isolated from the following mouse tissues: calvarial bone (lane 1), articular cartilage (2), auricular cartilage (3), nasal cartilage (4), pancreas (5), thymus (6), spleen (7), liver (8), kidney (9), brain (10), testis (11), heart (12), intestine (13), skin (14), lung (15), muscle (16), and eye (17). Denatured RNAs were electrophoresed, transferred by blotting onto the hybridization membrane and hybridized with the pMCatK-I probe (A) and with a probe for 28S rRNA (B).…”

“…belong to at least two different gene superfamilies, the matrix metalloproteinases (MMPs) and the cysteine proteases (cathepsins). Previous immunohistochemical and in situ hybridization data have demonstrated expression of cathepsins B, D, E, L, OC-2 [8][9][10] and MMP-9 (92 kDa gelatinase) [11] in osteoclasts. Cathepsins B, D and L, as well as most MMPs, are also produced in a variety of other tissues, whereas recent data suggest that OC-2, a novel cysteine protease, and MMP-9 are predominantly expressed in osteoclasts [10,11].…”

Aminopeptidases and Dipeptidyl Peptidases Activities in Chicken Bone Tissue.

“…Sannes and colleagues [15] employed MNA substrates for demonstration of dipeptidyl peptidases I and II (DPP-I, DPP-II), and showed DPP-I in osteoblasts, osteocytes, and chondrocytes in proliferating and hypertrophic regions of the growth plate, and DPP-II in osteoblasts, osteocytes, and chondrocytes in the resting zone. In the same experiment, they failed to demonstrate cathepsin B activity in the bone, although it was strongly positive in osteoclasts with other enzyme histochemical [5,20] and immunohistochemical studies [6,9,13,16]. Further, there has been no trial to demonstrate AP-A and DPP-IV activities in the bone.…”

“…However, there have been very few papers addressing histochemical localizations of proteases, with the exception of cathepsins B, L and D [5,6,9,13,15,20]. Lipp [10] first reported that aminopeptidase-M (AP-M) activity was present in osteoclasts, osteoblasts and endothelial cells at the vascular invasion in growth plate using Leu-2-naphthylamide as a substrate.…”