Inhibition of Orai1-mediated Ca(2+) entry is a key mechanism of the antiproliferative action of sirolimus in human arterial smooth muscle.
Sirolimus (rapamycin) is used in drug-eluting stent strategies and proved clearly superior in this application compared with other immunomodulators such as pimecrolimus. The molecular basis of this action of sirolimus in the vascular system is still incompletely understood. Measurements of cell proliferation in human coronary artery smooth muscle cells (hCASM) demonstrated a higher antiproliferative activity of sirolimus compared with pimecrolimus. Although sirolimus lacks inhibitory effects on calcineurin, nuclear factor of activated T-cell activation in hCASM was suppressed to a similar extent by both drugs at 10 μM. Sirolimus, but not pimecrolimus, inhibited agonist-induced and store-operated Ca(2+) entry as well as cAMP response element binding protein (CREB) phosphorylation in human arterial smooth muscle, suggesting the existence of an as-yet unrecognized inhibitory effect of sirolimus on Ca(2+) signaling and Ca(2+)-dependent gene transcription. Electrophysiological experiments revealed that only sirolimus but not pimecrolimus significantly blocked the classical stromal interaction molecule/Orai-mediated, store-operated Ca(2+) current reconstituted in human embryonic kidney cells (HEK293). A link between Orai function and proliferation was confirmed by dominant-negative knockout of Orai in hCASM. Analysis of the effects of sirolimus on cell proliferation and CREB activation in an in vitro model of arterial intervention using human aorta corroborated the ability of sirolimus to suppress stent implantation-induced CREB activation in human arteries. We suggest inhibition of store-operated Ca(2+) entry based on Orai channels and the resulting suppression of Ca(2+) transcription coupling as a key mechanism underlying the antiproliferative activity of sirolimus in human arteries. This mechanism of action is specific for sirolimus and not a general feature of drugs interacting with FK506-binding proteins.
Institute of Biophysics, Medical University of Graz, Graz, Austria;
Sarah
S
König
Sara
S
Browne
Bernhard
B
Doleschal
Michaela
M
Schernthaner
Michael
M
Poteser
Heinrich
H
Mächler
Eric
E
Wittchow
Marlen
M
Braune
Martin
M
Muik
Christoph
C
Romanin
Klaus
K
Groschner
D001011:Aorta / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D015220:Calcium Channels / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects*; D002317:Cardiovascular Agents / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects*; D003331:Coronary Vessels / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D055786:Gene Knockout Techniques; D057809:HEK293 Cells; D006801:Humans; D006965:Hyperplasia; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D050778:NFATC Transcription Factors / Q000378:metabolism; D000071740:ORAI1 Protein; D010766:Phosphorylation; D020123:Sirolimus / Q000494:pharmacology*; D015607:Stents / Q000009:adverse effects*; D016559:Tacrolimus / Q000031:analogs & derivatives / Q000494:pharmacology; D013997:Time Factors; D046509:Tissue Culture Techniques; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection
C115163:CREB1 protein, human; D015220:Calcium Channels; D002317:Cardiovascular Agents; D017362:Cyclic AMP Response Element-Binding Protein; D050778:NFATC Transcription Factors; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C117268:pimecrolimus; D020123:Sirolimus; D016559:Tacrolimus
CREB; NFAT; calcium signaling; coronary restenosis; sirolimus