Asymmetric allostery in estrogen receptor-α homodimers drives responses to the ensemble of estrogens in the hormonal milieu.

The estrogen receptor-α (ER) is thought to function only as a homodimer but responds to a variety of environmental, metazoan, and therapeutic estrogens at subsaturating doses, supporting binding mixtures of ligands as well as dimers that are only partially occupied. Here, we present a series of flexible ER ligands that bind to receptor dimers with individual ligand poses favoring distinct receptor conformations-receptor conformational heterodimers-mimicking the binding of two different ligands. Molecular dynamics simulations showed that the pairs of different ligand poses changed the correlated motion across the dimer interface to generate asymmetric communication between the dimer interface, the ligands, and the surface binding sites for epigenetic regulatory proteins. By examining the binding of the same ligand in crystal structures of ER in the agonist vs. antagonist conformers, we also showed that these allosteric signals are bidirectional. The receptor conformer can drive different ligand binding modes to support agonist vs. antagonist activity profiles, a revision of ligand binding theory that has focused on unidirectional signaling from the ligand to the coregulator binding site. We also observed differences in the allosteric signals between ligand and coregulator binding sites in the monomeric vs. dimeric receptor, and when bound by two different ligands, states that are physiologically relevant. Thus, ER conformational heterodimers integrate two different ligand-regulated activity profiles, representing different modes for ligand-dependent regulation of ER activity.

An ADIOL-ERß-CtBP transrepression pathway negatively regulates microglia-mediated inflammation.

Microglia and astrocytes play essential roles in the maintenance of homeostasis within the central nervous system, but mechanisms that control the magnitude and duration of responses to infection and injury remain poorly understood. Here, we provide evidence that 5-androsten-3ß,17ß-diol (ADIOL) functions as a selective modulator of estrogen receptor (ER)ß to suppress inflammatory responses of microglia and astrocytes. ADIOL and a subset of synthetic ERß-specific ligands, but not 17ß-estradiol, mediate recruitment of CtBP corepressor complexes to AP-1-dependent promoters, thereby repressing genes that amplify inflammatory responses and activate Th17 T cells. Reduction of ADIOL or ERß expression results in exaggerated inflammatory responses to TLR4 agonists. Conversely, the administration of ADIOL or synthetic ERß-specific ligands that promote CtBP recruitment prevents experimental autoimmune encephalomyelitis in an ERß-dependent manner. These findings provide evidence for an ADIOL/ERß/CtBP-transrepression pathway that regulates inflammatory responses in microglia and can be targeted by selective ERß modulators.

Structural and Molecular Mechanisms of Cytokine-Mediated Endocrine Resistance in Human Breast Cancer Cells.

Human breast cancers that exhibit high proportions of immune cells and elevated levels of pro-inflammatory cytokines predict poor prognosis. Here, we demonstrate that treatment of human MCF-7 breast cancer cells with pro-inflammatory cytokines results in ERα-dependent activation of gene expression and proliferation, in the absence of ligand or presence of 4OH-tamoxifen (TOT). Cytokine activation of ERα and endocrine resistance is dependent on phosphorylation of ERα at S305 in the hinge domain. Phosphorylation of S305 by IKKß establishes an ERα cistrome that substantially overlaps with the estradiol (E2)-dependent ERα cistrome. Structural analyses suggest that S305-P forms a charge-linked bridge with the C-terminal F domain of ERα that enables inter-domain communication and constitutive activity from the N-terminal coactivator-binding site, revealing the structural basis of endocrine resistance. ERα therefore functions as a transcriptional effector of cytokine-induced IKKß signaling, suggesting a mechanism through which the tumor microenvironment controls tumor progression and endocrine resistance.

Resistance to FOXM1 inhibitors in breast cancer is accompanied by impeding ferroptosis and apoptotic cell death.

PURPOSE: Cancer treatments often become ineffective because of acquired drug resistance. To characterize changes in breast cancer cells accompanying development of resistance to inhibitors of the oncogenic transcription factor, FOXM1, we investigated the suppression of cell death pathways, especially ferroptosis, in FOXM1 inhibitor-resistant cells. We also explored whether ferroptosis activators can synergize with FOXM1 inhibitors and can overcome FOXM1 inhibitor resistance. METHODS: In estrogen receptor-positive and triple-negative breast cancer cells treated with FOXM1 inhibitor NB73 and ferroptosis activators dihydroartemisinin and JKE1674, alone and in combination, we measured suppression of cell viability, motility, and colony formation, and monitored changes in gene and protein pathway expressions and mitochondrial integrity. RESULTS: Growth suppression of breast cancer cells by FOXM1 inhibitors is accompanied by increased cell death and alterations in mitochondrial morphology and metabolic activity. Low doses of FOXM1 inhibitor strongly synergize with ferroptosis inducers to reduce cell viability, migration, colony formation, and expression of proliferation-related genes, and increase intracellular Fe+2 and lipid peroxidation, markers of ferroptosis. Acquired resistance to FOXM1 inhibition is associated with increased expression of cancer stem-cell markers and proteins that repress ferroptosis, enabling cell survival and drug resistance. Notably, resistant cells are still sensitive to growth suppression by low doses of ferroptosis activators, effectively overcoming the acquired resistance. CONCLUSION: Delineating changes in viability and cell death pathways that can overcome drug resistance should be helpful in determining approaches that might best prevent or reverse resistance to therapeutic targeting of FOXM1 and ultimately improve patient clinical outcomes.

Dual-mechanism estrogen receptor inhibitors.

Efforts to improve estrogen receptor-α (ER)-targeted therapies in breast cancer have relied upon a single mechanism, with ligands having a single side chain on the ligand core that extends outward to determine antagonism of breast cancer growth. Here, we describe inhibitors with two ER-targeting moieties, one of which uses an alternate structural mechanism to generate full antagonism, freeing the side chain to independently determine other critical properties of the ligands. By combining two molecular targeting approaches into a single ER ligand, we have generated antiestrogens that function through new mechanisms and structural paradigms to achieve antagonism. These dual-mechanism ER inhibitors (DMERIs) cause alternate, noncanonical structural perturbations of the receptor ligand-binding domain (LBD) to antagonize proliferation in ER-positive breast cancer cells and in allele-specific resistance models. Our structural analyses with DMERIs highlight marked differences from current standard-of-care, single-mechanism antiestrogens. These findings uncover an enhanced flexibility of the ER LBD through which it can access nonconsensus conformational modes in response to DMERI binding, broadly and effectively suppressing ER activity.

Targeting the oncogenic transcription factor FOXM1 to improve outcomes in all subtypes of breast cancer.

FOXM1 (Forkhead box M1) is an oncogenic transcription factor that is greatly upregulated in breast cancer and many other cancers where it promotes tumorigenesis, and cancer growth and progression. It is expressed in all subtypes of breast cancer and is the factor most associated with risk of poor patient survival, especially so in triple negative breast cancer (TNBC). Thus, new approaches to inhibiting FOXM1 and its activities, and combination therapies utilizing FOXM1 inhibitors in conjunction with known cancer drugs that work together synergistically, could improve cancer treatment outcomes. Targeting FOXM1 might prove especially beneficial in TNBC where few targeted therapies currently exist, and also in suppressing recurrent advanced estrogen receptor (ER)-positive and HER2-positive breast cancers for which treatments with ER or HER2 targeted therapies that were effective initially are no longer beneficial. We present these perspectives and future directions in the context of what is known about FOXM1, its regulation, and its key roles in promoting cancer aggressiveness and metastasis, while being absent or very low in most normal non-regenerating adult tissues. We discuss new inhibitors of FOXM1 and highlight FOXM1 as an attractive target for controlling drug-resistant and difficult-to-suppress breast cancers, and how blocking FOXM1 might improve outcomes for patients with all subtypes of breast cancer.

Effective combination treatments for breast cancer inhibition by FOXM1 inhibitors with other targeted cancer drugs.

PURPOSE: Few targeted treatment options currently exist for patients with advanced, often recurrent breast cancers, both triple-negative breast cancer (TNBC) and hormone receptor-positive breast cancer. Forkhead box M1 (FOXM1) is an oncogenic transcription factor that drives all cancer hallmarks in all subtypes of breast cancer. We previously developed small-molecule inhibitors of FOXM1 and to further exploit their potential as anti-proliferative agents, we investigated combining FOXM1 inhibitors with drugs currently used in the treatment of breast and other cancers and assessed the potential for enhanced inhibition of breast cancer. METHODS: FOXM1 inhibitors alone and in combination with other cancer therapy drugs were assessed for their effects on suppression of cell viability and cell cycle progression, induction of apoptosis and caspase 3/7 activity, and changes in related gene expressions. Synergistic, additive, or antagonistic interactions were evaluated using ZIP (zero interaction potency) synergy scores and the Chou-Talalay interaction combination index. RESULTS: The FOXM1 inhibitors displayed synergistic inhibition of proliferation, enhanced G2/M cell cycle arrest, and increased apoptosis and caspase 3/7 activity and associated changes in gene expression when combined with several drugs across different pharmacological classes. We found especially strong enhanced effectiveness of FOXM1 inhibitors in combination with drugs in the proteasome inhibitor class for ER-positive and TNBC cells and with CDK4/6 inhibitors (Palbociclib, Abemaciclib, and Ribociclib) in ER-positive cells. CONCLUSION: The findings suggest that the combination of FOXM1 inhibitors with several other drugs might enable dose reduction in both agents and provide enhanced efficacy in treatment of breast cancer.

Estradiol promotes cell survival and induces Greb1 expression in granulosa cell tumors of the ovary through an ERα-dependent mechanism.

Granulosa cell tumor (GCT) is a form of ovarian tumor characterized by its tendency to recur years after surgical ablation. Little is known about the mechanisms involved in GCT development and progression. GCTs can produce estradiol (E2), but whether this hormone could play a role in this cancer through its nuclear receptors, i.e. ERα and ERß, remains unknown. Here, we addressed this issue by cell-based and molecular studies on human GCTs and GCT cell lines. Importantly, we observed that E2 significantly increased the growth of GCT cells by promoting cell survival. The use of selective agonists of each type of receptor, together with Esr1 (ERα) or Esr2 (ERß)-deleted GCT cells, revealed that E2 mediated its effects through ERα-dependent genomic mechanisms and ERß/ERα-dependent extra-nuclear mechanisms. Notably, the expression of Greb1, a prototypical ER target gene, was dose-dependently upregulated by E2 specifically through ERα in GCT cells. Accordingly, using GCTs from patients, we found that GREB1 mRNA abundance was positively correlated to intra-tumoral E2 concentrations. Tissue microarray analyses showed that there were various combinations of ER expression in primary and recurrent GCTs, and that ERα expression persisted only in combination with ERß in ~40% of recurrent tumors. Altogether, this study demonstrates that E2 can promote the progression of GCTs, with a clear dependence on ERα. In addition to demonstrating that GCTs can be classified as a hormone-related cancer, our results also highlight that the nature of ER forms present in recurrent GCTs could underlie the variable efficiency of endocrine therapies. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Exploration of alcohol-enhanced Cu-mediated radiofluorination toward practical labeling.

Copper-mediated nucleophilic radiofluorination using boronic precursors is a promising, general method to label aromatic compounds with [18 F]fluoride. However, in various reports, large amounts of precursor (60 µmol) were needed to achieve high radiochemical conversions (RCCs), which is neither ideal nor practical for the preparation of 18 F radiopharmaceuticals. To investigate this matter, we studied alcohol-enhanced Cu-mediated nucleophilic radiofluorination using a variety of model reactions in which we varied the concentration of [18 F]fluoride (no carrier added or isotope diluted) and the amount of precursor, base, and Cu(OTF)2 (Py)4 . We found that lower amounts of precursors (e.g., 15 µmol) could be used and that the amount of base (e.g., K2 CO3 or KHCO3 ) played a critical and limiting role in the labeling reactions. Greater than one-equivalent of base and sufficient amounts of precursors and Cu(OTf)2 (Py)4 were required to achieve good to high RCCs. The RCCs were also dependent on the overall concentration of the labeling reactions, with low reaction volumes and high concentrations of reagents being preferred. Our findings will help to improve the design of radiolabeling protocols using alcohol-enhanced copper-mediated radiofluorination of boronic precursors for the preparation of 18 F labeled radiopharmaceuticals and other radiohalogen-labeled compounds.

Contrasting activities of estrogen receptor beta isoforms in triple negative breast cancer.

PURPOSE: Triple negative breast cancer (TNBC), an aggressive subtype of breast cancer, lacks the three major receptors for predicting outcome or targeting therapy. Hence, our aim was to evaluate the potential of estrogen receptor beta (ERß) as a possible endocrine therapy target in TNBC. METHODS: The expression and prognostic effect of ERß isoforms were analyzed using TCGA breast tumor data, and the expression of ERß isoform mRNA and protein in TNBC cell lines was assayed. Endogenous ERß2 and ERß5 were knocked down with siRNA, and ERß2, ERß5, and ERß1 were upregulated using a doxycycline-inducible lentiviral system. Cell proliferation, migration and invasion, and specific gene expressions were evaluated. RESULTS: ERß2 and ERß5 were the predominant endogenous forms of ERß in TNBC tumors and cell lines. High ERß2 predicted worse clinical outcome. Knockdown of endogenous ERß2/ERß5 in cell lines suppressed proliferation, migration and invasion, and downregulated proto-oncogene survivin expression. ERß2/ERß5 upregulation did the reverse, increasing survivin and these cell activities. ERß1 was barely detectable in TNBC cell lines, but its upregulation reduced survivin, increased tumor suppressor expression (E-cadherin and cystatins), and suppressed proliferation, migration and invasion in both ligand-independent and dependent manners, suggesting the possible translational benefit of ERß ligands. CONCLUSIONS: ERß2/ERß5 and ERß1 exhibit sharply contrasting activities in TNBC cells. Our findings imply that delineating the absolute amounts and relative ratios of the different ERß isoforms might have prognostic and therapeutic relevance, and could enable better selection of optimal approaches for treatment of this often aggressive form of breast cancer.

Increase in chemokine CXCL1 by ERß ligand treatment is a key mediator in promoting axon myelination.

Estrogen receptor ß (ERß) ligands promote remyelination in mouse models of multiple sclerosis. Recent work using experimental autoimmune encephalomyelitis (EAE) has shown that ERß ligands induce axon remyelination, but impact peripheral inflammation to varying degrees. To identify if ERß ligands initiate a common immune mechanism in remyelination, central and peripheral immunity and pathology in mice given ERß ligands at peak EAE were assessed. All ERß ligands induced differential expression of cytokines and chemokines, but increased levels of CXCL1 in the periphery and in astrocytes. Oligodendrocyte CXCR2 binds CXCL1 and has been implicated in normal myelination. In addition, despite extensive immune cell accumulation in the CNS, all ERß ligands promoted extensive remyelination in mice at peak EAE. This finding highlights a component of the mechanism by which ERß ligands mediate remyelination. Hence, interplay between the immune system and central nervous system may be responsible for the remyelinating effects of ERß ligands. Our findings of potential neuroprotective benefits arising from the presence of CXCL1 could have implications for improved therapies for multiple sclerosis.

Suppression of breast cancer metastasis and extension of survival by a new antiestrogen in a preclinical model driven by mutant estrogen receptors.

PURPOSE: Many human breast tumors become resistant to endocrine therapies and recur due to estrogen receptor (ERα) mutations that convey constitutive activity and a more aggressive phenotype. Here, we examined the effectiveness of a novel adamantyl antiestrogen, K-07, in suppressing the growth of breast cancer metastases containing the two most frequent ER-activating mutations, Y537S and D538G, and in extending survival in a preclinical metastatic cancer model. METHODS: MCF7 breast cancer cells expressing luciferase and Y537S or D538G ER were injected into NOD-SCID-gamma female mice, and animals were treated orally with the antiestrogen K-07 or control vehicle. Comparisons were also made with the antiestrogen Fulvestrant. The development of metastases was monitored by in vivo bioluminescence imaging with phenotypic characterization of the metastases in liver and lung by immunohistochemical and biochemical analyses. RESULTS: These breast cancer cells established metastases in liver and lung, and K-07 treatment reduced the metastatic burden. Mice treated with K-07 also survived much longer. By day 70, only 28% of vehicle-treated mice with mutant ER metastases were alive, whereas all K-07-treated D538G and Y537S mice were still alive. K-07 also markedly reduced the level of metastatic cell ER and the expression of ER-regulated genes. CONCLUSION: The antiestrogen K-07 can reduce in vivo metastasis of breast cancers and extend host survival in this preclinical model driven by constitutively active mutant ERs, suggesting that this compound may be suitable for further translational examination of its efficacy in suppression of metastasis in breast cancers containing constitutively active mutant ERs.

Diffusion tensor imaging identifies aspects of therapeutic estrogen receptor ß ligand-induced remyelination in a mouse model of multiple sclerosis.

Diffusion tensor imaging (DTI) has been shown to detect white matter degeneration in multiple sclerosis (MS), a neurodegenerative autoimmune disease that presents with diffuse demyelination of the central nervous system. However, the utility of DTI in evaluating therapeutic remyelination has not yet been well-established. Here, we assessed the ability of DTI to distinguish between remyelination and neuroprotection following estrogen receptor ß ligand (Indazole chloride, IndCl) treatment, which has been previously shown to stimulate functional remyelination, in the cuprizone (CPZ) diet mouse model of MS. Adult C57BL/6 J male and female mice received a normal diet (control), demyelination-inducing CPZ diet (9wkDM), or CPZ diet followed by two weeks of a normal diet (i.e., remyelination period) with either IndCl (RM + IndCl) or vehicle (RM + Veh) injections. We evaluated tissue microstructure of the corpus callosum utilizing in vivo and ex vivo DTI and immunohistochemistry (IHC) for validation. Compared to control mice, the 9wkDM group showed decreased fractional anisotropy (FA), increased radial diffusivity (RD), and no changes in axial diffusivity (AD) both in vivo and ex vivo. Meanwhile, RM + IndCl groups showed increased FA and decreased RD ex vivo compared to the RM + Veh group, in accordance with the evidence of remyelination by IHC. In conclusion, the DTI technology used in the present study can identify some changes in myelination and is a valuable translational tool for evaluating MS pathophysiology and therapeutic efficacy.

Full antagonism of the estrogen receptor without a prototypical ligand side chain.

Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.

Inhibiting androgen receptor nuclear entry in castration-resistant prostate cancer.

Clinical resistance to the second-generation antiandrogen enzalutamide in castration-resistant prostate cancer (CRPC), despite persistent androgen receptor (AR) activity in tumors, highlights an unmet medical need for next-generation antagonists. We have identified and characterized tetra-aryl cyclobutanes (CBs) as a new class of competitive AR antagonists that exhibit a unique mechanism of action. These CBs are structurally distinct from current antiandrogens (hydroxyflutamide, bicalutamide, and enzalutamide) and inhibit AR-mediated gene expression, cell proliferation, and tumor growth in several models of CRPC. Conformational profiling revealed that CBs stabilize an AR conformation resembling an unliganded receptor. Using a variety of techniques, it was determined that the AR-CB complex was not recruited to AR-regulated promoters and, like apo AR, remains sequestered in the cytoplasm, bound to heat shock proteins. Thus, we have identified third-generation AR antagonists whose unique mechanism of action suggests that they may have therapeutic potential in CRPC.

Copper-Mediated Nucleophilic Radiobromination of Aryl Boron Precursors: Convenient Preparation of a Radiobrominated PARP-1 Inhibitor.

The copper-mediated nucleophilic radiobromination of aryl boron precursors with a radiobromide ion is a novel radiolabeling method that is efficient and robust. High radiochemical conversion (RCC) was observed using a variety of solvents, temperatures and catalysts. The reaction is also clean and is feasible for purification to obtain high chemical and radiochemical purity. This method provides a very useful route for the preparation of radiobrominated pharmaceuticals, including a radiobromine labeled PARP-1 inhibitor, and it is a valuable addition to the family of copper-mediated radiolabeling processes.

Non-nuclear estrogen receptor alpha activation in endothelium reduces cardiac ischemia-reperfusion injury in mice.

Steroid hormone receptors including estrogen receptors (ER) classically function as ligand-regulated transcription factors. However, estrogens also elicit cellular effects through binding to extra-nuclear ER (ERα, ERß, and G protein-coupled ER or GPER) that are coupled to kinases. How extra-nuclear ER actions impact cardiac ischemia-reperfusion (I/R) injury is unknown. We treated ovariectomized wild-type female mice with estradiol or an estrogen-dendrimer conjugate (EDC), which selectively activates extra-nuclear ER, or vehicle interventions for two weeks. I/R injury was then evaluated in isolated Langendorff perfused hearts. Two weeks of treatment with estradiol significantly decreased infarct size and improved post-ischemic contractile function. Similarly, EDC treatment significantly decreased infarct size and increased post-ischemic functional recovery compared to vehicle-treated hearts. EDC also caused an increase in myocardial protein S-nitrosylation, consistent with previous studies showing a role for this post-translational modification in cardioprotection. In further support of a role for S-nitrosylation, inhibition of nitric oxide synthase, but not soluble guanylyl cyclase blocked the EDC mediated protection. The administration of ICI182,780, which is an agonist of G-protein coupled estrogen receptor (GPER) and an antagonist of ERα and ERß, did not result in protection; however, ICI182,780 significantly blocked EDC-mediated cardioprotection, indicating participation of ERα and/or ERß. In studies determining the specific ER subtype and cellular target involved, EDC decreased infarct size and improved functional recovery in mice lacking ERα in cardiomyocytes. In contrast, protection was lost in mice deficient in endothelial cell ERα. Thus, extra-nuclear ERα activation in endothelium reduces cardiac I/R injury in mice, and this likely entails increased protein S-nitrosylation. Since EDC does not stimulate uterine growth, in the clinical setting EDC-like compounds may provide myocardial protection without undesired uterotrophic and cancer-promoting effects.

17ß-Estradiol Dysregulates Innate Immune Responses to Pseudomonas aeruginosa Respiratory Infection and Is Modulated by Estrogen Receptor Antagonism.

Females have a more severe clinical course than males in terms of several inflammatory lung conditions. Notably, females with cystic fibrosis (CF) suffer worse outcomes, particularly in the setting of Pseudomonas aeruginosa infection. Sex hormones have been implicated in experimental and clinical studies; however, immune mechanisms responsible for this sex-based disparity are unknown and the specific sex hormone target for therapeutic manipulation has not been identified. The objective of this study was to assess mechanisms behind the impact of female sex hormones on host immune responses to P. aeruginosa We used wild-type and CF mice, which we hormone manipulated, inoculated with P. aeruginosa, and then examined for outcomes and inflammatory responses. Neutrophils isolated from mice and human subjects were tested for responses to P. aeruginosa We found that female mice inoculated with P. aeruginosa died earlier and showed slower bacterial clearance than males (P < 0.0001). Ovariectomized females supplemented with 17ß-estradiol succumbed to P. aeruginosa challenge earlier than progesterone- or vehicle-supplemented mice (P = 0.0003). 17ß-Estradiol-treated ovariectomized female mice demonstrated increased lung levels of inflammatory cytokines, and when rendered neutropenic the mortality difference was abrogated. Neutrophils treated with 17ß-estradiol demonstrated an enhanced oxidative burst but decreased P. aeruginosa killing and earlier cell necrosis. The estrogen receptor (ER) antagonist ICI 182,780 improved survival in female mice infected with P. aeruginosa and restored neutrophil function. We concluded that ER antagonism rescues estrogen-mediated neutrophil dysfunction and improves survival in response to P. aeruginosa ER-mediated processes may explain the sex-based mortality gap in CF and other inflammatory lung illnesses, and the ER blockade represents a rational therapeutic strategy.

Estrogens and androgens inhibit association of RANKL with the pre-osteoblast membrane through post-translational mechanisms.

We have recently demonstrated that RUNX2 promoted, and 17ß-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.