The neuroprotective actions of FK506 binding protein ligands: neuronal survival is triggered by de novo RNA synthesis, but is independent of inhibition of JNK and calcineurin.
The immunosuppressant FK506 displays substantial neuroprotective and neuroregenerative effects. It is not fully understood to which extent these effects depend on the inhibition of the calcineurin phosphatase (PP2B). The present study has re-addressed this issue using Lie120, a novel highly specific inhibitor of calcineurin, which does not block the enzymatic activity of FKBPs or cyclophilins, respectively. We have determined the effect of FK506 (10-500 nM), V-10,367 (a FK506 derivative which does not block calcineurin; 1-5 microM) and Lie120 (a novel specific inhibitor of calcineurin, 0.1-5 microM) on the cellular survival and the pro-degenerative JNK activity of PC12 and Neuro2A cells following application of 200 microM H(2)O(2). FK506 and V-10,367, but not Lie120, protected both cell lines against H(2)O(2)-mediated death, whereas an increase in JNK1 activity was blocked by FK506 and Lie120, but not by V-10,367. Co-incubation of FK506 and V-10,367 with the mRNA synthesis inhibitor actinomycin D abolished the protective effect of FK506 and V-10,367. This antagonization was effective when actinomycin D was applied 30 min or 1 h, but not 2 or 4 h, after H(2)O(2) suggesting that FKBP-ligands confer their neuroprotection by rapid de novo synthesis of (functionally) anti-apoptotic proteins. The search for the corresponding effector genes revealed that the expression of FKBP25, FKBP38 and FKBP52 (analysis by reverse transcription-polymerase chain reaction (RT-PCR) did not change following H(2)O(2) or FK506, and this was also true for the expression of apoptosis-related genes caspase 3, bax, bcl-2 and bcl-xL (analysis by Multiplex-PCR). Summarizing, neuronal protection by FKBP-ligands is not mediated either by calcineurin or by JNK1 in this experimental set-up, whereas the FK506 mediated inhibition of JNK1 is realized by the inhibition of calcineurin, an effective activator of JNK1 in neurons.
Klettner
A
A
Institute of Pharmacology, Christian-Albrechts-University, Hospitalstrasse 4, 24105, Kiel, Germany.
Baumgrass
R
R
Zhang
Y
Y
Fischer
G
G
Bürger
E
E
Herdegen
T
T
Mielke
K
K
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D019703:Calcineurin / Q000502:physiology; D065095:Calcineurin Inhibitors; D053148:Caspase 3; D020169:Caspases / Q000096:biosynthesis / Q000235:genetics; D002470:Cell Survival; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019254:Genes, bcl-2; D006861:Hydrogen Peroxide / Q000494:pharmacology / Q000633:toxicity; D048031:JNK Mitogen-Activated Protein Kinases; D008024:Ligands; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D012313:RNA / Q000096:biosynthesis*; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022021:Tacrolimus Binding Proteins / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein
C494828:Bax protein, rat; C494813:Bcl2l1 protein, rat; D065095:Calcineurin Inhibitors; C451003:Lie120; D008024:Ligands; D009419:Nerve Tissue Proteins; D018696:Neuroprotective Agents; D019384:Nucleic Acid Synthesis Inhibitors; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D013844:Thiazoles; C108850:V 10367; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D003609:Dactinomycin; D012313:RNA; D006861:Hydrogen Peroxide; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; C120817:calcineurin phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D022021:Tacrolimus Binding Proteins; C403485:tacrolimus binding protein 4