bromochloroacetic-acid and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

The autoimmune blistering skin disease pemphigus vulgaris (PV) is caused primarily by autoantibodies against desmosomal cadherins. It was reported that apoptosis can be detected in pemphigus skin lesions and that apoptosis can be induced by PV-IgG in cultured keratinocytes. However, the role of apoptosis in PV pathogenesis is unclear at present. In this study, we provide evidence that apoptosis is not required for acantholysis in PV. In skin lesions from two PV patients, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positivity, but not cleaved caspase-3, was detected in single keratinocytes in some lesions but was completely absent in other lesions from the same patients. In cultures of human keratinocytes (HaCaT and normal human epidermal keratinocytes), PV-IgG from three different PV patients caused acantholysis, fragmented staining of Dsg 3 staining, and cytokeratin retraction in the absence of nuclear fragmentation, TUNEL positivity, and caspase-3 cleavage and hence in the absence of detectable apoptosis. To further rule out the contribution of apoptotic mechanisms, we used two different approaches that are effective to block apoptosis induced by various stimuli. Inhibition of caspases by z-VAD-fmk as well as overexpression of Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory proteins FLIP(L) and FLIP(S) to inhibit receptor-mediated apoptosis did not block PV-IgG-induced effects, indicating that apoptosis was not required. Taken together, we conclude that apoptosis is not a prerequisite for skin blistering in PV but may occur secondary to acantholysis.

Topics: Acantholysis; Amino Acid Chloromethyl Ketones; Anoikis; Apoptosis; Biopsy; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 3; Cell Line; Cell Nucleus Shape; Cysteine Proteinase Inhibitors; Desmoglein 3; DNA Fragmentation; Humans; Immunoglobulin G; Immunohistochemistry; In Situ Nick-End Labeling; Keratinocytes; Keratins; Pemphigus; Transfection

In epithelial cells, cell-cell and cell-matrix junctions, desmosomes and hemidesmosomes, provide anchorage sites for the keratin-intermediate filaments. The plakin proteins desmoplakin (DP), plectin, and periplakin represent intracellular constituents of these adhesion junctions. In staurosporine-treated apoptotic HaCaT cells, DP, plectin, and periplakin became cleaved coordinately with the elimination of keratins 10 and 14, while involucrin, actin, and keratin 18 displayed considerable stability. The caspase inhibitor zVAD-fmk prevented both the cell detachment and protein cleavage, indicating the function of caspases in these events. Closer examination in vitro revealed that while caspases 2 and 4 most efficiently cleaved DP, and plectin served as a target for caspases 3 and 7, periplakin as well as keratins were cleaved by caspase 6. The involvement of multiple caspases in the destruction of epithelial cell integrity ensures the efficient elimination of cytoskeleton, but also provides specificity for selectively targeting individual adhesion molecules.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Caspases; Cell Adhesion; Cell Nucleus; Cytoskeletal Proteins; Cytoskeleton; Desmoplakins; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Epithelial Cells; Humans; Immunoblotting; Intermediate Filament Proteins; Keratinocytes; Keratins; Plakins; Plectin; Poly(ADP-ribose) Polymerases; Recombinant Proteins; Staurosporine; Time Factors

The effect of mechanical injury on chondrocyte viability and matrix degradation was studied. It was proposed that mechanical injury to human cartilage explants results in chondrocyte apoptosis with associated loss of glycosaminoglycans.. Full thickness human cartilage explants, 5 mm in diameter were subjected to a single static mechanical stress of 14 MPa for 500 ms under radially unconfined compression. Glycosaminoglycan (GAG) release and percentage of cells undergoing apoptosis were measured at 96 h after injury. To establish the time course of apoptosis, explants were subjected to 30% strain and cultured for varying intervals up to 7 days after injury. A group of loaded explants were also treated with the broad spectrum caspase inhibitor z-Vad.fmk after injury.. Internucleosomal DNA fragmentation as one indicator of apoptosis was observed in 34% (S.D.+/-11) of chondrocytes at 96 h in response to mechanical loading at 14 MPa, compared to 4% (S.D.+/-2) in the non-loaded explants. Evidence for cell death induction via apoptosis was also obtained by electron microscopy and caspase cleavage of cytokeratin. GAG release was also higher for the loaded explants, mean 1.9% (S.D.+/-0.14) of total GAG content, compared to control explants, mean 0.8% (S.D.+/-0.28). The percentage of apoptotic cells also correlated with the level of GAG release into the culture media. The percentage of apoptotic chondrocytes demonstrated a progressive increase from 6 h to 7 days post-injury. When loaded explants were cultured in z-Vad.fmk after injury, a 50% reduction in apoptosis rates was seen.. These results demonstrate that mechanical injury induces chondrocyte apoptosis and release of GAG from the matrix. The time course suggests that a therapeutic window may exist where apoptosis could be inhibited. This potentially identifies a new approach to chondroprotection.

Topics: Adolescent; Adult; Amino Acid Chloromethyl Ketones; Apoptosis; Cartilage, Articular; Caspase Inhibitors; Caspases; Cells, Cultured; Chondrocytes; DNA Fragmentation; Enzyme Inhibitors; Glycosaminoglycans; Humans; Keratins; Linear Models; Microscopy, Electron; Middle Aged; Statistics, Nonparametric; Time Factors