title: Estrogens and androgens inhibit association of RANKL with the pre-osteoblast membrane through post-translational mechanisms.
abstract: We have recently demonstrated that RUNX2 promoted, and 17β-Estradiol (E2) diminished, association of RANKL with the cell membrane in pre-osteoblast cultures. Here we show that, similar to E2, dihydrotestosterone (DHT) diminishes association of RANKL, and transiently transfected GFP-RANKL with the pre-osteoblast membrane without decreasing total RANKL mRNA or protein levels. Diminution of membrane-associated RANKL was accompanied with marked suppression of osteoclast differentiation from co-cultured pre-osteoclasts, even though DHT increased, not decreased, RANKL concentrations in pre-osteoblast conditioned media. A marked decrease in membrane-associated RANKL was observed after 30 min of either E2 or DHT treatment, and near-complete inhibition was observed by 1 hr, suggesting that the diminution of RANKL membrane association was mediated through non-genomic mechanisms. Further indicating dispensability of nuclear action of estrogen receptor, E2-mediated inhibition of RANKL membrane association was mimicked by an estrogen dendrimer conjugate (EDC) that cannot enter the cell nucleus. Finally, the inhibitory effect of E2 and DHT on RANKL membrane association was counteracted by the MMP inhibitor NNGH, and the effect of E2 (and not DHT) was antagonized by the Src inhibitor SU6656. Taken together, these results suggest that estrogens and androgens inhibit osteoblast-driven osteoclastogenesis through non-genomic mechanism(s) that entail, MMP-mediated RANKL dissociation from the cell membrane.
authors.affiliation: Department of Biochemistry and Molecular Medicine , Keck School of Medicine, University of Southern California, Los Angeles, California.|Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California.
authors.forename: Anthony
authors.initials: A
authors.lastname: Martin
authors.affiliation: Department of Biochemistry and Molecular Medicine , Keck School of Medicine, University of Southern California, Los Angeles, California.|Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California.
authors.forename: Jiali
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Biochemistry and Molecular Medicine , Keck School of Medicine, University of Southern California, Los Angeles, California.|Institute for Genetic Medicine, Keck School of Medicine of the University of Southern California, Los Angeles, California.
authors.forename: Jian
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: Aysha B
authors.initials: AB
authors.lastname: Khalid
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Benita
authors.initials: B
authors.lastname: Katzenellenbogen
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: Sung Hoon
authors.initials: SH
authors.lastname: Kim
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois.
authors.forename: John A
authors.initials: JA
authors.lastname: Katzenellenbogen
authors.affiliation: Faculty of Science, Department of Biology, Chulalonkorn University, Bangkok, Thailand.
authors.forename: Suchinda
authors.initials: S
authors.lastname: Malaivijitnond
authors.affiliation: Sackler Faculty of Medicine, Departments of Anatomy and Anthropology and Orthopedic Surgery, Tel Aviv University, Tel Aviv, Israel.
authors.forename: Yankel
authors.initials: Y
authors.lastname: Gabet
authors.affiliation: Department of Orthopaedic Surgery and Biomedical Engineering, University of Tennessee Health Science Center, Memphis, Tennessee.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Krum
authors.affiliation: Department of Biochemistry and Molecular Medicine , Keck School of Medicine, University of Southern California, Los Angeles, California.|Department of Orthopedic Surgery, Keck School of Medicine, University of Southern California, Los Angeles, California.
authors.forename: Baruch
authors.identifier: http://orcid.org/0000-0001-6295-6858
authors.initials: B
authors.lastname: Frenkel
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D018920:Coculture Techniques; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D017077:Culture Media, Conditioned / Q000378:metabolism; D013196:Dihydrotestosterone / Q000494:pharmacology*; D004958:Estradiol / Q000494:pharmacology*; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D020782:Matrix Metalloproteinases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000187:drug effects* / Q000378:metabolism; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects*; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D000067996:RAW 264.7 Cells; D013997:Time Factors; D014162:Transfection; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D017077:Culture Media, Conditioned; D061965:Matrix Metalloproteinase Inhibitors; D047428:Protein Kinase Inhibitors; D053245:RANK Ligand; C493716:Runx2 protein, mouse; C505689:Tnfsf11 protein, mouse; D013196:Dihydrotestosterone; D004958:Estradiol; D019061:src-Family Kinases; D020782:Matrix Metalloproteinases
keywords: MMP; RANKL presentation; Src; membrane-initiated estrogen signaling; sex steroids