title: Estrogens antagonize RUNX2-mediated osteoblast-driven osteoclastogenesis through regulating RANKL membrane association.
abstract: In addition to its thoroughly investigated role in bone formation, the osteoblast master transcription factor RUNX2 also promotes osteoclastogenesis and bone resorption. Here we demonstrate that 17β-estradiol (E2), strongly inhibits RUNX2-mediated osteoblast-driven osteoclastogenesis in co-cultures. Towards deciphering the underlying mechanism, we induced premature expression of RUNX2 in primary murine pre-osteoblasts, which resulted in robust differentiation of co-cultured splenocytes into mature osteoclasts. This was attributable to RUNX2-mediated increase in RANKL secretion, determined by ELISA, as well as to RUNX2-mediated increase in RANKL association with the osteoblast membrane, demonstrated using confocal fluorescence microscopy. The increased association with the osteoblast membrane was recapitulated by transiently expressed GFP-RANKL. E2 abolished the RUNX2-mediated increase in membrane-associated RANKL and GFP-RANKL, as well as the concomitant osteoclastogenesis. RUNX2-mediated RANKL cellular redistribution was attributable in part to a decrease in Opg expression, but E2 did not influence Opg expression either in the presence or absence of RUNX2. Diminution of RUNX2-mediated osteoclastogenesis by E2 occurred regardless of whether the pre-osteoclasts were derived from wild type or estrogen receptor alpha (ERα)-knockout mice, suggesting that activated ERα inhibited osteoblast-driven osteoclastogenesis by acting in osteoblasts, possibly targeting RUNX2. Indeed, microarray analysis demonstrated global attenuation of the RUNX2 response by E2, including abrogation of Pstpip2 expression, which likely plays a critical role in membrane trafficking. Finally, the selective ER modulators (SERMs) tamoxifen and raloxifene mimicked E2 in abrogating the stimulatory effect of osteoblastic RUNX2 on osteoclast differentiation in the co-culture assay. Thus, E2 antagonizes RUNX2-mediated RANKL trafficking and subsequent osteoclastogenesis. Targeting RUNX2 and/or downstream mechanisms that regulate RANKL trafficking may lead to the development of improved SERMs and possibly non-hormonal therapeutic approaches to high turnover bone disease.
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: martinan@usc.edu.
authors.forename: Anthony
authors.initials: A
authors.lastname: Martin
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: silviaxiong0923@gmail.com.
authors.forename: Jian
authors.initials: J
authors.lastname: Xiong
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: tkoromila@yahoo.gr.
authors.forename: Theodora
authors.initials: T
authors.lastname: Koromila
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA.
authors.forename: Jie S
authors.initials: JS
authors.lastname: Ji
authors.affiliation: Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: schan061@ucr.edu.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chang
authors.affiliation: Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: yaesamso@usc.edu.
authors.forename: Yae S
authors.initials: YS
authors.lastname: Song
authors.affiliation: Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: mill252@usc.edu.
authors.forename: Jonathan L
authors.initials: JL
authors.lastname: Miller
authors.affiliation: Metabolic Disorders Research, Amgen Inc., 1 Amgen Center Dr, Thousand Oaks, CA 91320, USA. Electronic address: ehan@amgen.com.
authors.forename: Chun-Ya
authors.initials: CY
authors.lastname: Han
authors.affiliation: Metabolic Disorders Research, Amgen Inc., 1 Amgen Center Dr, Thousand Oaks, CA 91320, USA. Electronic address: paulk@amgen.com.
authors.forename: Paul
authors.initials: P
authors.lastname: Kostenuik
authors.affiliation: UCLA and Orthopaedic Hospital Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, David Geffen School of Medicine, UCLA, 10833 Le Conte Ave, Los Angeles, CA 90095, USA. Electronic address: smirand5@uthsc.edu.
authors.forename: Susan A
authors.initials: SA
authors.lastname: Krum
authors.affiliation: Department of Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: chimgee@usc.edu.
authors.forename: Nyam-Osor
authors.initials: NO
authors.lastname: Chimge
authors.affiliation: Department of Anatomy and Anthropology, Sackler Faculty of Medicine, Tel Aviv University, P.O. Box 39040, Tel Aviv 69978, Israel. Electronic address: yagabet@gmail.com.
authors.forename: Yankel
authors.initials: Y
authors.lastname: Gabet
authors.affiliation: Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Department of Orthopaedic Surgery, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA; Institute for Genetic Medicine, Keck School of Medicine, University of Southern California, 1795 Zonal Ave, Los Angeles, CA 90033, USA. Electronic address: frenkel@usc.edu.
authors.forename: Baruch
authors.initials: B
authors.lastname: Frenkel
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001862:Bone Resorption / Q000378:metabolism*; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D004967:Estrogens / Q000378:metabolism*; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D053245:RANK Ligand / Q000378:metabolism*
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D004967:Estrogens; D053245:RANK Ligand; C493716:Runx2 protein, mouse; C505689:Tnfsf11 protein, mouse
keywords: Postmenopausal osteoporosis; Protein trafficking; Secretion; TRAP