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1.
Bone ; 75: 96-104, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25701138

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 17ß-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.


Subject(s)
Bone Resorption/metabolism , Core Binding Factor Alpha 1 Subunit/metabolism , Estrogens/metabolism , Osteoblasts/metabolism , Osteoclasts/metabolism , RANK Ligand/metabolism , Animals , Blotting, Western , Cell Differentiation/physiology , Cells, Cultured , Coculture Techniques , Enzyme-Linked Immunosorbent Assay , Female , Mice , Mice, Inbred C57BL , Oligonucleotide Array Sequence Analysis , Osteoblasts/cytology , Osteoclasts/cytology , Polymerase Chain Reaction
2.
J Cell Biochem ; 115(1): 27-33, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23943595

ABSTRACT

The efficacy of glucocorticoids (GCs) in treating a wide range of autoimmune and inflammatory conditions is blemished by severe side effects, including osteoporosis. The chief mechanism leading to GC-induced osteoporosis is inhibition of bone formation, but the role of RUNX2, a master regulator of osteoblast differentiation and bone formation, has not been well studied. We assessed effects of the synthetic GC dexamethasone (dex) on transcription of RUNX2-stimulated genes during the differentiation of mesenchymal pluripotent cells into osteoblasts. Dex inhibited a RUNX2 reporter gene and attenuated locus-dependently RUNX2-driven expression of several endogenous target genes. The anti-RUNX2 activity of dex was not attributable to decreased RUNX2 expression, but rather to physical interaction between RUNX2 and the GC receptor (GR), demonstrated by co-immunoprecipitation assays and co-immunofluorescence imaging. Investigation of the RUNX2/GR interaction may lead to the development of bone-sparing GC treatment modalities for the management of autoimmune and inflammatory diseases.


Subject(s)
Core Binding Factor Alpha 1 Subunit/metabolism , Glucocorticoids/pharmacology , Mesenchymal Stem Cells/cytology , Osteoblasts/drug effects , Alkaline Phosphatase/antagonists & inhibitors , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation/drug effects , Cell Nucleus/metabolism , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Dexamethasone/pharmacology , Mesenchymal Stem Cells/drug effects , Mice , Osteoblasts/cytology , Osteoblasts/metabolism , Pluripotent Stem Cells/cytology , Pluripotent Stem Cells/drug effects , Receptors, Glucocorticoid/metabolism
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