The anti-estrogenic activity of indole-3-carbinol in neonatal rat osteoblasts is associated with the estrogen receptor antagonist 2-hydroxyestradiol.
OBJECTIVE
To gain new insight into the roles of cruciferous vegetable-derived bioactive phytochemicals in bone cells, we investigated the effects of indole-3-carbinol (I3C) on cell proliferation and differentiation in estradiol (E2)-exposed calvarial osteoblasts that were obtained from neonatal rats.


METHODS
Osteoblast activity was assessed by analyzing cellular DNA, cell-associated osteocalcin (OC) levels and alkaline phosphatase (AP) activity. We also examined [(3)H]-estrone (E1) metabolism and estrogen-agonistic and estrogen-antagonistic activities of 2-hydroxy (OH) E1 and 2-OHE2 and their capacity to displace [(3)H]-E2 at ER binding sites using competition studies.


RESULTS
I3C did not affect on cellular DNA, OC levels or AP activity. However, I3C completely inhibited E2-induced increases in cell proliferation and differentiation in neonatal rat osteoblasts. Metabolic studies demonstrated that I3C promoted the conversion of [(3)H]-E1 to 2-OHE1 and 2-OHE2 and those higher rates of conversion (twofold-threefold) were archived when a higher dose of I3C was applied. Proliferation and differentiation studies showed that 2-OHE2 but not 2-OHE1 inhibited E2-induced increases in cell proliferation and differentiation via an ER-mediated mechanism. Likewise, Esr1 was expressed at high level than Esr2. 2-OHE1 showed no activity or affinity for ER.


CONCLUSIONS
This study is the first to show that a bioactive compound derived from cruciferous vegetables, I3C, abolishes the E2-mediated stimulation of cell activities including, proliferation and differentiation, in rat osteoblasts and increases the 2-hydroxylation of E1, resulting in the formation of inactive and anti-estrogenic metabolites. These results suggest that in neonatal rat osteoblasts, the anti-estrogenic effect of I3C is mediated by 2-OHE2 through ER-α.
Department of Reproductive Biology, "Carlos Gual Castro", Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Vasco de Quiroga 15, Sección XVI, 14000, Mexico City, Mexico. juanaenriquez@yahoo.com.mx.
J
J
Enríquez
Genomics of Bone Metabolism Laboratory, Instituto Nacional de Medicina Genómica, 14610, Mexico City, Mexico.
R
R
Velázquez-Cruz
Genomics of Bone Metabolism Laboratory, Instituto Nacional de Medicina Genómica, 14610, Mexico City, Mexico.
A
A
Parra-Torres
Research Support Network, INCMNSZ-Universidad Nacional Autónoma de México, 14000, Mexico City, Mexico.
R
R
Gutiérrez-Sagal
Department of Reproductive Biology, "Carlos Gual Castro", Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán (INCMNSZ), Vasco de Quiroga 15, Sección XVI, 14000, Mexico City, Mexico.
F
F
Larrea
D000818:Animals; D000831:Animals, Newborn; D016588:Anticarcinogenic Agents / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects*; D049109:Cell Proliferation / Q000187:drug effects*; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D004958:Estradiol / Q000031:analogs & derivatives* / Q000494:pharmacology; D004965:Estrogen Antagonists / Q000494:pharmacology*; D065171:Estrogen Receptor Antagonists / Q000494:pharmacology*; D005260:Female; D007211:Indoles / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D060888:Real-Time Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D016588:Anticarcinogenic Agents; D004965:Estrogen Antagonists; D065171:Estrogen Receptor Antagonists; D007211:Indoles; D011960:Receptors, Estrogen; D004958:Estradiol; C001390:2-hydroxyestradiol; C016517:indole-3-carbinol
Estrogen metabolites; Indole-3-carbinol; Osteoblast 2-hydroxylation; Osteoblasts differentiation; Osteoblasts proliferation