Acylated and unacylated ghrelin protect MC3T3-E1 cells against tert-butyl hydroperoxide-induced oxidative injury: pharmacological characterization of ghrelin receptor and possible epigenetic involvement.
Increasing evidence suggests a role for oxidative stress in age-related decrease in osteoblast number and function leading to the development of osteoporosis. This study was undertaken to investigate whether ghrelin, previously reported to stimulate osteoblast proliferation, counteracts tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in MC3T3-E1 osteoblastic cells as well as to characterize the ghrelin receptor (GHS-R) involved in such activity. Pretreatment with ghrelin (10(-7)-10(-11)M) significantly increased viability and reduced apoptosis of MC3T3-E1 cells cultured with t-BHP (250 μM) for three hours at the low concentration of 10(-9)M as shown by MTT assay and Hoechst-33258 staining. Furthermore, ghrelin prevented t-BHP-induced osteoblastic dysfunction and changes in the cytoskeleton organization evidenced by the staining of the actin fibers with Phalloidin-FITC by reducing reactive oxygen species generation. The GHS-R type 1a agonist, EP1572 (10(-7)-10(-11)M), had no effect against t-BHP-induced cytotoxicity and pretreatment with the selective GHS-R1a antagonist, D-Lys(3)-GHRP-6 (10(-7)M), failed to remove ghrelin (10(-9) M)-protective effects against oxidative injury, indicating that GHS-R1a is not involved in such ghrelin activity. Accordingly, unacylated ghrelin (DAG), not binding GHS-R1a, displays the same protective actions of ghrelin against t-BHP-induced cytotoxicity. Preliminary observations indicate that ghrelin increased the trimethylation of lys4 on histones H3, a known epigenetic mark activator, which may regulate the expression of some genes limiting oxidative damage. In conclusion, our data demonstrate that ghrelin and DAG promote survival of MC3T3-E1 cell exposed to t-BHP-induced oxidative damage. Such effect is independent of GHS-R1a and is likely mediated by a common ghrelin/DAG binding site.
Department of Medical Biotechnology and Translational Medicine, Medical Pharmacology Unit, Universita`degli Studi di Milano, Via Vanvitelli, 32, 20129, Milan, Italy.
Elisa
E
Dieci
Lavinia
L
Casati
Francesca
F
Pagani
Fabio
F
Celotti
Valeria
V
Sibilia
D008841:Actin Cytoskeleton / Q000187:drug effects; D000215:Acylation; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D044127:Epigenesis, Genetic; D054439:Ghrelin / Q000378:metabolism / Q000494:pharmacology*; D006657:Histones / Q000378:metabolism; D007211:Indoles; D051379:Mice; D009842:Oligopeptides / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D020011:Protective Agents / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D054440:Receptors, Ghrelin / Q000378:metabolism*; D014364:Tryptophan / Q000031:analogs & derivatives; D020122:tert-Butylhydroperoxide / Q000633:toxicity*
D054439:Ghrelin; D006657:Histones; D007211:Indoles; D009842:Oligopeptides; D020011:Protective Agents; D017382:Reactive Oxygen Species; D054440:Receptors, Ghrelin; C582727:macimorelin; D014364:Tryptophan; D020122:tert-Butylhydroperoxide