Inhibition of interleukin 1-stimulated cartilage proteoglycan degradation by a lipophilic inactivator of cysteine endopeptidases

Abstract

Inactivators of cysteine endopeptidases were tested as inhibitors of the cytokine-stimulated release of proteoglycan from cartilage. The test system consisted of bovine nasal septum cartilage maintained in organ culture, and the stimulus was provided by recombinant human interleukin 1 alpha. L-3-Carboxy-2,3-trans-epoxypropionyl-leucylamido-(4-guanidin o)butane (E64) and L-3-carboxy-2,3-trans-epoxypropionyl-leucylamido-(3-methyl)b utane (Ep475) showed no inhibition at concentrations up to 100 microM. In contrast, trans-epoxysuccinyl-leucylamido-(3-methyl)butane ethyl ester (Ep453), a 'prodrug' of Ep475, was an effective inhibitor. The LL-, LD- and DL-isomers gave significant inhibition at 10 microM, and the DD-isomer was inhibitory at 100 microM. None of the isomers had any detectable effect on protein synthesis or glycolysis, and their inhibitory effects were reversible. Iodoacetate inhibited proteoglycan release by a general toxic effect. Our results suggest that cysteine endopeptidase(s) play a part in cytokine-stimulated cartilage breakdown, but that effective inhibitors must pass through membranes.

Participation of intracellular cysteine proteinases, in particular cathepsin B, in degradation of collagen in periosteal tissue explants

“…(4-Amidinophenyl)-methanesulfonylfluoride (APMSF) was from Boehringer Mannheim (Mannheim, Germany). The inhibitors Ep453 and Ep475 (Buttle et al, 1992a) were kind gifts of Dr M. Tamai (Taisho Pharmaceutical Co. Ltd, Tokyo, Japan), and Ca074-Me was prepared as described previously (Buttle et al, 1992b). CA074-Me has the following rate constants (in M-~s -~) for the inactivation of various cysteine proteinases: 120,000 for cathepsin B; <10 for cathepsin H; 20 for cathepsin L; <10 for cathepsin S; and <10 for m-calpain (Buttle et al, 1992b).…”

“…Ep475 is active extracellularly, as it is not able to penetrate the plasma membrane, and Ep453 is active only after entry into the cell and release of an inactivating ethyl ester by cytoplasmic esterases (Buttle et al, 1992a).…”

Inhibition of cartilage proteoglycan release by a specific inactivator of cathepsin b and an inhibitor of matrix metalloproteinases. evidence for two converging pathways of chondrocyte‐mediated proteoglycan degradation

“…Bovine nasal septum cartilage was prepared and cultured as previously described (16). For experiments using 1 p M retinoate rather than 0.3 nM rHuIL-la as stimulant, the cultures were maintained for 5 days with a change of medium on day 2 or day 3, instead of the 48-hour culture period adopted for rHuIL-1 *stimulated explants.…”

“…Recently, the use of type-specific, membrane-permeant inactivators has implicated the lysosomal cysteine proteinases cathepsins B, L, and S in IL-1-stimulated cartilage proteoglycan release (16,17). Only membrane-permeant inactivators or proinactivators were found to be inhibitory (16), which suggests a site of action in a membrane-defined com-partment. The question of which of these lysosomal cysteine proteinases was responsible has remained open.…”

“…Analyses of the products of proteoglycan degradation induced in explants by either agent 16,7) suggest the involvement of a proteinase that cleaves the aggrecan core protein after certain glutamic acid residues. The identity of this enzyme is unknown, and its specificity appears to be unique.…”