Proteasome implication in phorbol ester- and GnRH-induced selective down-regulation of PKC (alpha, epsilon, zeta) in alpha T(3)-1 and L beta T(2) gonadotrope cell lines.
We investigated mechanisms underlying selective down-modulation of PKC isoforms (alpha, epsilon, zeta): 1) during 12-O-tetradecanoyl-phorbol-13 acetate (TPA) (10(-7) M) or GnRH (10(-7) M) desensitization conditions (2- to 6-h treatments) in two gonadotrope cell lines (alpha T(3)-1, L beta T(2)) and 2) in primary pituitary cell cultures from male rats during long-term phorbol ester administration. We demonstrated that, as in alpha T(3)-1 cells, in a more differentiated gonadotrope cell line L beta T(2) the GnRH-receptor coupling (PLC, PLA2, PLD) generated second messengers essential for PKCs activation; the characterized isoforms (alpha, beta II, delta, epsilon, zeta) were selectively and differentially down-regulated by TPA (alpha, beta II, delta, epsilon) or GnRH (delta, epsilon). In whole cell lysates, proteasome inhibitors (proteasome inhibitor I and II, Lactacystin, beta-Lactone, Calpain inhibitor I) prevented in both gonadotrope cell lines the TPA-induced depletion of PKC alpha, epsilon, and the GnRH-elicited PKC epsilon down-regulation; they counteracted in mixed pituitary cell cultures as well, the TPA-evoked PKC alpha, epsilon depletion. In contrast, the inhibitors of calpain(s) and lysosomal proteases (Calpeptin, E64d, Calpain inhibitor II, and PD150606), were ineffective. As shown in alpha T(3)-1 subcellular fractions, proteasome abrogation did not affect membrane translocation of TPA- and GnRH- target isoforms (alpha, epsilon) but, preventing their degradation, favored enzyme accumulation to the membrane compartment. Proteolysis processing of PKCs may be dependent upon their phosphorylated state and/or catalytic activity. Inhibition of PKC catalytic activity (GF109203X, Gö6976), selectively prevented the TPA-evoked PKC alpha depletion in both mixed pituitary cells and alpha T(3)-1 gonadotropes; in alpha T(3)-1 subcellular fractions, PKC alpha inactivation overcame the TPA-evoked isoenzyme degradation by inducing a pronounced membrane accumulation of the isoform without affecting its membrane relocalization. Thus, the proteasome system by adjusting PKC cellular levels, may represent a regulatory proteolytic pathway implicated in the adaptive mechanisms of the time dependent cell responses.
Junoy
Brice
B
Centre National de la Recherche Scientifique UMR 6544, Université de la Méditerranée, Faculté de Médecine, 13916 Marseille, France.
Maccario
Helene
H
Mas
Jean-Louis
JL
Enjalbert
Alain
A
Drouva
Sophia V
SV
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D015153:Blotting, Western; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000235:genetics*; D015536:Down-Regulation / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D006062:Gonadotropins / Q000096:biosynthesis*; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D009097:Multienzyme Complexes / Q000235:genetics*; D010703:Phorbol Esters / Q000494:pharmacology*; D010739:Phospholipase D / Q000378:metabolism; D010766:Phosphorylation; D010902:Pituitary Gland / Q000166:cytology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C / Q000096:biosynthesis* / Q000235:genetics*; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism; D000099221:Protein Kinase C zeta
D016718:Arachidonic Acid; D002154:Calpain; D003546:Cysteine Endopeptidases; D004791:Enzyme Inhibitors; D007987:Gonadotropin-Releasing Hormone; D006062:Gonadotropins; D007295:Inositol Phosphates; D007527:Isoenzymes; D009097:Multienzyme Complexes; D010703:Phorbol Esters; D010739:Phospholipase D; D046988:Proteasome Endopeptidase Complex; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051744:Protein Kinase C-epsilon; D000099221:Protein Kinase C zeta; C497257:Prkce protein, rat