ENHANCEMENT OF NEURITE OUTGROWTH FOLLOWING CALPAIN INHIBITION IS MEDIATED BY PROTEIN-KINASE-C

We examined the interdependence of calpain and protein kinase C (PKC) activities on neurite outgrowth in SH-SY-5Y human neuroblastoma cells. SH-SY-5Y cells elaborated neurites when deprived of serum or after a specific thrombin inhibitor, hirudin, was added to serum-containing me dium. The extent of neurite outgrowth under these conditions was enhan ced by treatment of cells with the cell-permeant cysteine protease inh ibitors N-acetyl-leucyl-leucyl-norleucinal (''C1'') and calpeptin or b y the phospholipid-mediated intracellular delivery of either a recombi nant peptide corresponding to a conserved inhibitory sequence of human calpastatin or a neutralizing anti-calpain antisera. Calpain inhibiti on in intact cells was confirmed by immunoblot analysis showing inhibi tion of calpain autolysis and reduced proteolysis of the known calpain substrates fodrin and microtubule-associated protein 1. The above inh ibitory peptides and antiserum did not induce neurites in medium conta ining serum but lacking hirudin, suggesting that increased surface pro tein adhesiveness is a prerequisite for enhancement of neurite outgrow th by calpain inhibition. Treatment of cells with the PKC inhibitor H7 , staurosporine, or sphingosine induced neurite outgrowth independentl y of serum concentration. Because calpain is thought to regulate PKC a ctivity, we examined this potential interrelationship during neurite o utgrowth. Simultaneous treatment with calpain and PKC inhibitors did n ot produce additive or synergistic effects on neurite outgrowth. PKC a ctivation by 2-O-tetradecanoylphorbol 13-acetate (TPA) prevented and r eversed both neurite initiation by serum deprivation and its enhanceme nt by calpain inhibitors. Treatment of cells with the calpain inhibito r C1 retarded PKC down-regulation following TPA treatment. Cell-free a nalyses demonstrated the relative specificity of various protease and kinase inhibitors for calpain and PKC and confirmed the ability of mil limolar calcium-requiring calpain to cleave the SH-SY-5Y PKC regulator y subunit from the catalytic subunit, yielding a free catalytic subuni t (protein kinase M). These findings suggest that the influence of PKC on neurite outgrowth is downstream from that of surface adhesiveness and calpain activity.