calpain and Chediak-Higashi-Syndrome

We have reported previously that the abnormally down-regulated protein kinase C (PKC) causes cellular dysfunction observed in natural killer (NK) cells, polymorphonuclear leucocytes (PMNs) and fibroblasts from beige mouse, an animal model of Chediak-Higashi syndrome (CHS). Here we show that the abnormal down-regulation of PKC activity also occurs in Epstein-Barr (EB) virus-transformed cell lines from CHS patients. When CHS cell lines were stimulated with concanavalin A (Con A) for 20 min, the membrane-bound PKC activity declined markedly, whereas that in control cell lines increased. We found that E-64-d, which protects PKC from calpain-mediated proteolysis, reversed the declined PKC activity and corrected the increased Con A cap formation to almost normal levels in CHS cell lines. We confirmed that the dysregulation of PKC activity also occurred in peripheral blood mononuclear leucocytes (PBMC) from CHS patients and that E-64-d corrected both the declined PKC activity and increased Con A cap formation. E-64-d also corrected the reduced lysosomal elastase and cathepsin G activity in CHS cell lines. In contrast, chelerythrin, a specific inhibitor of PKC, and C2-ceramide, which promotes PKC breakdown induced by calpain, increased Con A cap formation and inhibited both elastase and cathepsin G activity in normal cell lines. Moreover, we found that ceramide production in CHS cell lines increased significantly after Con A stimulation, which coincides with our previous observation in fibroblasts from CHS mice. These results suggest an association between ceramide-induced PKC down-regulation and the cellular dysfunctions in CHS.

Topics: Calpain; Cathepsin G; Cathepsins; Cell Line, Transformed; Ceramides; Chediak-Higashi Syndrome; Child; Concanavalin A; Cysteine Proteinase Inhibitors; Down-Regulation; Drug Antagonism; Female; Humans; Leucine; Leukocytes, Mononuclear; Lysosomes; Male; Pancreatic Elastase; Protein Kinase C; Receptor Aggregation; Serine Endopeptidases; Sphingomyelin Phosphodiesterase

Protein kinase C (PKC) plays an essential role in intracellular signal transduction for various cell functions, including concanavalin A (Con A)-induced cap formation. This enzyme is known to be proteolysed by calpain, which is a Ca2(+)-dependent thiol proteinase. As reported previously, in polymorphonuclear leukocytes (PMNs) from beige mouse, the model of Chediak-Higashi syndrome, Con A-induced cap formation significantly increased compared with that in normal mouse. However, after pretreatment of beige PMNs with the thiol proteinase inhibitors leupeptin or E-64, the capping decreased to normal levels. Meanwhile, Con A-induced the translocation of PKC from the cytosolic to membrane fraction within 5 min in both mice, which is essential to the activation of this enzyme. However, after the translocation, an abnormal rapid decline in membrane-bound PKC activity was noted in beige mouse PMNs. Both leupeptin and E-64 also corrected the rapid decline in PKC activity observed in the beige mouse. These findings suggest that the normalization of Con A cap formation in beige mouse PMNs by the thiol proteinase inhibitors is associated with the correction of abnormality in PKC activity.

Topics: Animals; Calpain; Chediak-Higashi Syndrome; Concanavalin A; Disease Models, Animal; Down-Regulation; Immunologic Capping; Mice; Mice, Inbred C57BL; Neutrophils; Protein Kinase C

Protein kinase C (PKC) is essential in intracellular signal transduction for various cell functions including natural killer (NK) cell activity. This enzyme is hydrolysed by calpain, which is Ca2+-dependent thiol proteinase. We showed here that in NK activity-deficient beige (bg/bg) mouse, the model of Chediak-Higashi syndrome, the translocated membrane-bound PKC activity declined rapidly in NK cell-enriched lymphocytes after TPA stimulation. However, the rapid decline was abolished by the pretreatment of cells with leupeptin (a thiol and serine proteinase inhibitor) or E64 (a thiol proteinase inhibitor). Furthermore, these reagents improved the impaired NK cell activity in beige mouse whereas they did not affect NK cell activity in C57BL/6 (+/+) and the heterozygous (+/bg) mice. Meanwhile, TPA stimulation induced only low levels in NK cytotoxic factors (NKCF) release from beige NK cells, but these reagents augmented the lowered NKCF release. These results suggest that the improvement of impaired NK cell activity in beige mouse by the thiol proteinase inhibitors may be due to the elimination of abnormal rapid down-regulation of PKC, resulting in the augmentation of the lowered PKC activity.

Topics: Animals; Calpain; Chediak-Higashi Syndrome; Cysteine Proteinase Inhibitors; Cytotoxicity, Immunologic; Disease Models, Animal; Killer Cells, Natural; Killer Factors, Yeast; Leucine; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Protein Biosynthesis; Protein Kinase C; Proteins; Tetradecanoylphorbol Acetate