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Antibodies are used in many areas of biomedical and clinical research, but many of these antibodies have not been adequately characterized, which casts doubt on the results reported in many scientific papers. This problem is compounded by a lack of suitable control experiments in many studies. In this article we review the history of the 'antibody characterization crisis', and we document efforts and initiatives to address the problem, notably for antibodies that target human proteins. We also present recommendations for a range of stakeholders - researchers, universities, journals, antibody vendors and repositories, scientific societies and funders - to increase the reproducibility of studies that rely on antibodies., Competing Interests: RK, HV, CL, DH, NP, RM, AL, CS, TE, RT, JT, LF, FF, AB, SL No competing interests declared, AB Co-founder and serving as CEO of SciCrunch Inc, a company that works with publishers to improve the scientific literature; the terms of this agreement have been approved by the COI office at the University of California at San Diego, MR Employed by Addgene, a company that may be affected financially by the opinions reported in this article, HC Employed by Abcam, a for-profit company that sells antibodies and which may be affected financially by the opinions reported in this article, (© 2024, Kahn et al.)

The preclinical research process is a cycle of idea generation, experimentation, and reporting of results. The biomedical research community relies on the reproducibility of published discoveries to create new lines of research and to translate research findings into therapeutic applications. Since 2012, when scientists from Amgen reported that they were able to reproduce only 6 of 53 "landmark" preclinical studies, the biomedical research community began discussing the scale of the reproducibility problem and developing initiatives to address critical challenges. Global Biological Standards Institute (GBSI) released the "Case for Standards" in 2013, one of the first comprehensive reports to address the rising concern of irreproducible biomedical research. Further attention was drawn to issues that limit scientific self-correction, including reporting and publication bias, underpowered studies, lack of open access to methods and data, and lack of clearly defined standards and guidelines in areas such as reagent validation. To evaluate the progress made towards reproducibility since 2013, GBSI identified and examined initiatives designed to advance quality and reproducibility. Through this process, we identified key roles for funders, journals, researchers and other stakeholders and recommended actions for future progress. This paper describes our findings and conclusions., Competing Interests: Competing interests: No competing interests were disclosed.

Low reproducibility rates within life science research undermine cumulative knowledge production and contribute to both delays and costs of therapeutic drug development. An analysis of past studies indicates that the cumulative (total) prevalence of irreproducible preclinical research exceeds 50%, resulting in approximately US$28,000,000,000 (US$28B)/year spent on preclinical research that is not reproducible-in the United States alone. We outline a framework for solutions and a plan for long-term improvements in reproducibility rates that will help to accelerate the discovery of life-saving therapies and cures.

Research advances build upon the validity and reproducibility of previously published data and findings. Yet irreproducibility in basic biologic and preclinical research is pervasive in both academic and commercial settings. Lack of reproducibility has led to invalidated research breakthroughs, retracted articles, and aborted clinical trials. Concerns and requirements for transparent, reproducible, and translatable research are accelerated by the rapid growth of "post-publication peer review," open access publishing, and data sharing that facilitate the identification of irreproducible data/studies; they are magnified by the explosion of high-throughput technologies, genomics, and other data-intensive disciplines. Collectively, these changes and challenges are decreasing the effectiveness of traditional research quality mechanisms and are contributing to unacceptable-and unsustainable-levels of irreproducibility. The global oncology and basic biologic research communities can no longer tolerate or afford widespread irreproducible research. This article discusses (i) how irreproducibility in preclinical research can ultimately be traced to an absence of a unifying life science standards framework, and (ii) makes an urgent case for the expanded development and use of consensus-based standards to both enhance reproducibility and drive innovations in cancer research., (©2014 American Association for Cancer Research.)

Liver X receptors (LXRs) play a critical role in regulating lipid synthesis and transport in numerous tissues. In the skin, activation of LXR induces keratinocyte differentiation and improves epidermal permeability barrier homeostasis. To elucidate the mechanism of LXR action in skin, we mapped its cistrome by identifying LXRβ-RXRα binding sites using ChIP-on-chip in normal human epidermal keratinocytes (NHEKs). The cistrome was integrated with transcription data to obtain a global view of LXR action in keratinocyte biology. Here, we identify 2035 LXRβ-RXRα binding sites containing 4794 LXR response elements in NHEKs and show the presence of consensus heterodimer active regions in genes involved in keratinocyte lipid transport/synthesis and terminal differentiation. Bioinformatics analysis of the cistrome revealed an enrichment of AP1 cis-regulatory motifs in the vicinity of the LXRβ-RXRα binding sites. Importantly, we have demonstrated a direct interaction between LXR and Jun/Fos, indicating that the cooperation between LXR and AP1 may orchestrate keratinocyte differentiation. Finally, we corroborated these results by genome-wide mapping of the c-Fos and c-Jun cistromes in NHEKs, demonstrating that 77% of all the LXRβ-RXRα binding regions show the presence of AP1 motifs at adjacent locations. Our findings provide new insight into the mechanism of LXR action in keratinocyte differentiation, lipid production and barrier formation, further strengthening the validation of LXR as a potential therapeutic target for skin disorders including skin aging, psoriasis, and atopic dermatitis.

The liver X receptors (LXRα and LXRβ) are members of the nuclear receptor superfamily that function as key transcriptional regulators of a number of biological processes, including cholesterol homeostasis, lipid metabolism, and keratinocyte differentiation. Natural ligands that activate LXRs include oxysterol derivatives such as 25-hydroxycholesterol, 27-hydroxycholesterol, 22(R)-hydroxycholesterol, 20(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Related oxysterols, such as 5α,6α-epoxycholesterol (5,6-EC) are present in a number of foods and have been shown to induce atherosclerosis in animal models. Intriguingly, these oxysterols have also been detected in atherosclerotic plaques. Using a variety of biochemical and cellular assays, we demonstrate that 5,6-EC is the first dietary modulator and an endogenous LXR ligand with cell and gene context-dependent antagonist, agonist, and inverse agonist activities. In a multiplexed LXR-cofactor peptide interaction assay, 5,6-EC induced the recruitment of a number of cofactor peptides onto both LXRα and LXRβ and showed an EC(50) of approximately 2 μM in peptide recruitment. Furthermore, 5,6-EC bound to LXRα in a radiolabeled ligand displacement assay (EC(50) = 76 nM), thus demonstrating it to be one of the most potent natural LXRα ligands known to date. Analysis of endogenous gene expression in various cell-based systems indicated the potential of 5,6-EC to antagonize LXR-mediated gene expression. Furthermore, it also induced the expression of some LXR-responsive genes in keratinocytes. These results clearly demonstrate that 5,6-EC is an LXR modulator that may play a role in the development of lipid disorders, such as atherosclerosis, by antagonizing the agonistic action of endogenous LXR ligands.

Selective androgen receptor modulators (SARMs) are androgen receptor (AR) ligands that induce anabolism while having reduced effects in reproductive tissues. In various experimental contexts SARMs fully activate, partially activate, or even antagonize the AR, but how these complex activities translate into tissue selectivity is not known. Here, we probed receptor function using >1000 synthetic AR ligands. These compounds produced a spectrum of activities in each assay ranging from 0 to 100% of maximal response. By testing different classes of compounds in ovariectomized rats, we established that ligands that transactivated a model promoter 40-80% of an agonist, recruited the coactivator GRIP-1 <15%, and stabilized the N-/C-terminal interdomain interaction <7% induced bone formation with reduced effects in the uterus and in sebaceous glands. Using these criteria, multiple SARMs were synthesized including MK-0773, a 4-aza-steroid that exhibited tissue selectivity in humans. Thus, AR activated to moderate levels due to reduced cofactor recruitment, and N-/C-terminal interactions produce a fully anabolic response, whereas more complete receptor activation is required for reproductive effects. This bimodal activation provides a molecular basis for the development of SARMs.

One of the many harmful factors faced by the skin is solar UV radiation, which damages skin by inducing chronic low-grade inflammation through increased expression of proinflammatory cytokines, metalloproteinases (MMPs) and cyclooxygenase-2 (COX-2). Estrogen receptors (ERs) alpha and beta are ligand-dependent transcription factors that are expressed in skin, and an ERbeta agonist has previously shown efficacy in vivo in models of pain and inflammation. Because ERbeta does not carry the breast and uterine proliferation liabilities of ERalpha, we decided to explore the possibility of using ERbeta as a target for photoaging. We show that ERbeta-selective compounds suppressed the expression of cytokines and MMPs in activated keratinocytes and fibroblast-based in vitro models of photoaging. Furthermore, in activated dermal fibroblasts, ERbeta-selective compounds also inhibited COX-2. These activities of ERbeta ligands in skin cells correlated with the expression levels of ERbeta and showed reversal by treatment with a potent synthetic ER antagonist. Furthermore, the pharmacology of ERbeta-selective compound was observed in wild-type but not in skin cells obtained from ERbeta knockout mice. Finally, we demonstrate that a synthetic ERbeta agonist inhibited UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the potential of an ERbeta ligand to regulate multiple pathways underlying the cause of photoaging suggests ERbeta to be a novel therapeutic target for the prevention and treatment of photoaging.

Androgens promote anabolism in the musculoskeletal system while generally repressing adiposity, leading to lean body composition. Circulating androgens decline with age, contributing to frailty, osteoporosis, and obesity; however, the mechanisms by which androgens modulate body composition are largely unknown. Here, we demonstrate that aged castrated rats develop increased fat mass, reduced muscle mass and strength, and lower bone mass. Treatment with testosterone or 5alpha-dihydrotestosterone (DHT) reverses the effects on muscle and adipose tissues while only aromatizable testosterone increased bone mass. During the first week, DHT transiently increased soleus muscle nuclear density and induced expression of IGF1 and its splice variant mechano growth factor (MGF) without early regulation of the myogenic factors MyoD, myogenin, monocyte nuclear factor, or myostatin. A genome-wide microarray screen was also performed to identify potential pro-myogenic genes that respond to androgen receptor activation in vivo within 24 h. Of 24 000 genes examined, 70 candidate genes were identified whose functions suggest initiation of remodeling and regeneration, including the type II muscle genes for myosin heavy chain type II and parvalbumin and the chemokine monocyte chemoattractant protein-1. Interestingly, Axin and Axin2, negative regulators of beta-catenin, were repressed, indicating modulation of the beta-catenin pathway. DHT increased total levels of beta-catenin protein, which accumulated in nuclei in vivo. Likewise, treatment of C2C12 myoblasts with both IGF1Ea and MGF C-terminal peptide increased nuclear beta-catenin in vitro. Thus, we propose that androgenic anabolism involves early downregulation of Axin and induction of IGF1, leading to nuclear accumulation of beta-catenin, a pro-myogenic, anti-adipogenic stem cell regulatory factor.

The use of estrogen-based therapies and the selective estrogen receptor (ER) modulator (SERM), raloxifene, which are approved for postmenopausal osteoporosis, is associated with side effects such as uterine/breast hyperproliferation, thromboembolism, and hot flashes. A combination of a new SERM, bazedoxifene (BZA), and Premarin (conjugated estrogens; CE) is under investigation to mitigate the estrogen/SERM side effects with promising results in Phase III clinical trials. To explore the mechanism of BZA/CE action, we investigated the recruitment of cofactor peptides to ERalpha by components of CE and a mixture containing the 10 major components of CE with or without three different SERMs. Here, we demonstrate differential recruitment of cofactor peptides to ERalpha by the individual CE components using a multiplex nuclear receptor-cofactor peptide interaction assay. We show that estrone and equilin are partial agonists in comparison with 17beta-estradiol in recruiting cofactor peptides to ERalpha. Further, CE was more potent than 17beta-estradiol in mediating ERalpha interaction with cofactor peptides. Interestingly, BZA was less potent than other SERMs in antagonizing the CE-mediated cofactor peptide recruitment to ERalpha. Finally, in accordance with these biochemical findings, 17beta-estradiol and CE, as well as SERM/CE combinations, showed differential gene regulation patterns in MCF-7 cells. In addition, BZA showed antagonism of a unique set of CE-regulated genes and did not down-regulate the expression of a number of CE-regulated genes, the expression of which was effectively antagonized by the other two SERMs. These results indicate that SERMs in combination with CE exhibit differential pharmacology, and therefore, combinations of other SERMs and estrogen preparations may not yield the same beneficial effects that are observed in clinic by pairing BZA with CE.

Liver X receptors (LXRalpha and -beta) are liposensors that exert their metabolic effects by orchestrating the expression of macrophage genes involved in lipid metabolism and inflammation. LXRs are also expressed in other tissues, including skin, where their natural oxysterol ligands induce keratinocyte differentiation and improve epidermal barrier function. To extend the potential use of LXR ligands to dermatological indications, we explored the possibility of using LXR as a target for skin aging. We demonstrate that LXR signaling is down-regulated in cell-based models of photoaging, i.e. UV-activated keratinocytes and TNFalpha-activated dermal fibroblasts. We show that a synthetic LXR ligand inhibits the expression of cytokines and metalloproteinases in these in vitro models, thus indicating its potential in decreasing cutaneous inflammation associated with the etiology of photoaging. Furthermore, a synthetic LXR ligand induces the expression of differentiation markers, ceramide biosynthesis enzymes, and lipid synthesis and transport genes in keratinocytes. Remarkably, LXRbeta-null mouse skin showed some of the molecular defects that are observed in chronologically aged human skin. Finally, we demonstrate that a synthetic LXR agonist inhibits UV-induced photodamage and skin wrinkle formation in a murine model of photoaging. Therefore, the ability of an LXR ligand to modulate multiple pathways underlying the etiology of skin aging suggests that LXR is a novel target for developing potential therapeutics for photoaging and chronological skin aging indications.

Estrogens play a critical role in the regulation of cellular proliferation, differentiation, and apoptosis. Evidence indicates that this regulation is mediated by a complex interface of direct control of gene expression (so-called "genomic action") and by regulation of cell-signaling/phosphorylation cascades (referred to as the "non-genomic", or "extranuclear" action). However, the mechanisms of the non-genomic action of estrogens are not well defined. We have recently described the identification of a novel scaffold protein termed MNAR (modulator of non-genomic action of estrogen receptor), that couples conventional steroid receptors with extranuclear signal transduction pathways, thus potentially providing additional and tissue- or cell-specific level of steroid hormone regulation of cell functions. We have demonstrated that the MNAR is required for ER alpha (ERa) interaction with p60(src) (Src), which leads to activation of Src/MAPK pathway. Our new data also suggest that activation of cSrc in response to E2 leads to MNAR phosphorylation, interaction with p85, and activation of the PI3 and Akt kinases. These data therefore suggest that MNAR acts as an important scaffold that integrates ERa action in regulation of important signaling pathways. ERa non-genomic action has been suggested to play a key role in estrogen-induced cardio-, neuro-, and osteo-protection. Therefore, evaluation of the molecular crosstalk between MNAR and ERa may lead to development of functionally selective ER modulators that can separate between beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS and the "detrimental", proliferative effects in reproductive tissues and organs.

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone-forming osteoblasts and bone-resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen-induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre-osteoclasts. We have characterized a cell-type-specific hormone-inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor-alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre-osteoclasts by the same osteoblast-dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre-osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

By regulating activities and expression levels of key signaling molecules, estrogens control mechanisms that are responsible for crucial cellular functions. Ligand binding to estrogen receptor (ER) leads to conformational changes that regulate the receptor activity, its interaction with other proteins and DNA. In the cytoplasm, receptor interactions with kinases and scaffolding molecules regulate cell signaling cascades (extranuclear/nongenomic action). In the nucleus, estrogens control a repertoire of coregulators and other auxiliary proteins that are associated with ER, which in turn determines the nature of regulated genes and level of their expression (genomic action). The combination of genomic and nongenomic actions of estrogens ultimately confers the cell-type and tissue-type selectivity. Recent studies have revealed some important new insights into the molecular mechanisms underlying ER action, which may help to explain the functional basis of existing selective ER modulators (SERMs) and provide evidence into how ER might be selectively targeted to achieve specific therapeutic goals. In this review, we will summarize some new molecular details that relate to estrogen signaling. We will also discuss some new strategies that may potentially lead to the development of functionally selective ER modulators that can separate between the beneficial, prodifferentiative effects in bone, the cardiovascular system and the CNS as well as the "detrimental," proliferative effects in reproductive tissues and organs., (2007 Wiley-Liss, Inc.)

The orphan nuclear receptor Nurr1 is essential for the development and maintenance of midbrain dopaminergic neurons, the cells that degenerate during Parkinson's disease, by promoting the transcription of genes involved in dopaminergic neurotransmission. Since Nurr1 lacks a classical ligand-binding pocket, it is not clear which factors regulate its activity and how these factors are affected during disease pathogenesis. Since Wnt signaling via beta-catenin promotes the differentiation of Nurr1(+) dopaminergic precursors in vitro, we tested for functional interactions between these systems. We found that beta-catenin and Nurr1 functionally interact at multiple levels. In the absence of beta-catenin, Nurr1 is associated with Lef-1 in corepressor complexes. Beta-catenin binds Nurr1 and disrupts these corepressor complexes, leading to coactivator recruitment and induction of Wnt- and Nurr1-responsive genes. We then identified KCNIP4/calsenilin-like protein as being responsive to concurrent activation by Nurr1 and beta-catenin. Since KCNIP4 interacts with presenilins, the Alzheimer's disease-associated proteins that promote beta-catenin degradation, we tested the possibility that KCNIP4 induction regulates beta-catenin signaling. KCNIP4 induction limited beta-catenin activity in a presenilin-dependent manner, thereby serving as a negative feedback loop; furthermore, Nurr1 inhibition of beta-catenin activity was absent in PS1(-/-) cells or in the presence of small interfering RNAs specific to KCNIP4. These data describe regulatory convergence between Nurr1 and beta-catenin, providing a mechanism by which Nurr1 could be regulated by Wnt signaling.

Androgens play an important role not only in male sexual differentiation, puberty, sexual behavior and spermatogenesis, but also in the maintenance of bone architecture and muscle mass and strength. For decades, steroidal androgens have been used by hypogonadal and aging men as hormone replacement therapy, and abused by prominent athletes as anabolic agents for enhancing physical performance. The use of steroidal androgens is associated with hepatotoxicity, potential for prostate stimulation, virilizing actions and other side effects resulting from their cross-reactivity to related steroid receptors. Therefore, to utilize the therapeutic potential of the androgen receptor for the treatment of indications such as osteoporosis and frailty, several pharmaceutical and biotechnology companies are developing non-steroidal tissue-selective androgen receptor modulators (SARMs) that retain the beneficial properties of natural androgens and exhibit better therapeutic indices. This article reviews the mechanism of androgen action, novel non-steroidal ligands under development and future directions of SARM research for the discovery of novel modulators for frailty and osteoporosis.

Estrogen actions are mediated by a complex interface of direct control of gene expression (the so-called "genomic action") and by regulation of cell signaling/phosphorylation cascades, referred to as the "nongenomic," or extranuclear, action. We have previously described the identification of MNAR (modulator of nongenomic action of estrogen receptor) as a novel scaffold protein that regulates estrogen receptor alpha (ERalpha) activation of cSrc. In this study, we have investigated the role of MNAR in 17beta-estradiol (E2)-induced activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Consistent with our previous results, a direct correlation was established between MNAR expression levels and E2-induced activation of PI3 and Akt kinases. Endogenous MNAR, ERalpha, cSrc, and p85, the regulatory subunit of PI3 kinase, interacted in MCF7 cells treated with E2. The interaction between p85 and MNAR required activation of cSrc and MNAR phosphorylation on Tyr 920. Consequently, the mutation of this tyrosine to alanine (Y920A) abrogated the interaction between MNAR and p85 and the E2-induced activation of the PI3K/Akt pathway, which was required for the E2-induced protection of MCF7 cells from apoptosis. Nonetheless, the Y920A mutant potentiated the E2-induced activation of the Src/MAPK pathway and MCF7 cell proliferation, as observed with the wild-type MNAR. These results provide new and important insights into the molecular mechanisms of E2-induced regulation of cell proliferation and apoptosis.

1alpha,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the biologically active metabolite of vitamin D, mediates its actions via the vitamin D receptor (VDR), a member of the superfamily of steroid/thyroid hormone/retinoid receptors. 1,25-(OH)2D3 is required for calcium and phosphorus homeostasis, and for normal skeletal development as well as maintenance of skeletal architecture. Two VDR ligands, calcitriol (1,25-(OH)2D3) and its synthetic analog alfacalcidol (1alpha-hydroxyvitamin D3), have been approved for the treatment of osteoporosis. However, the use of calcitriol and alfacalcidol is limited by their major side effect, hypercalcemia, which is mediated mainly by VDR activity in the small intestine. In order to identify VDR ligands with less hypercalcemia liability, a number of pharmaceutical companies are pursuing efforts to develop synthetic vitamin D analogs. This review discusses the mechanism of action of vitamin D, and summarizes the currently approved anti-osteoporotic VDR agonists and compounds that are under development. The future directions of vitamin D research for the discovery of novel VDR agonists for osteoporosis are also discussed.

Nuclear receptors represent novel targets for the development of therapeutic agents for the treatment of numerous diseases, including type 2 diabetes, obesity dyslipidemia, atherosclerosis and the metabolic syndrome. There have been many recent advances in the development of new therapeutic agents for a subset of these receptors, including the peroxisome proliferator-activated receptors, the liver X receptors and the farnesoid X receptor. To date, the synthesis of selective modulators that regulate the activity of these receptors has been empirical. However, a detailed understanding of the molecular basis for selective modulation, as well as new insights into the biology of these receptors, might open the door to the rational design of a new generation of therapeutic agents with improved safety and efficacy.

Potent bisphosphonates (BPs) preferentially bind bone at sites of active osteoclastic bone resorption, where they are taken up by the osteoclast and inhibit resorption. We tested the hypothesis that BP affinity to human bone affects antiresorptive potency. [(1)(4)C]-Alendronate binding to human bone was saturable and reversible with an apparent Kd of 72 microM by Scatchard analysis. In competition binding assays, unlabeled alendronate (Ki: 61 microM) was slightly more potent than pyrophosphate (Ki = 156 microM) in blocking [(1)(4)C]-alendronate binding. Likewise, most tested BPs, including etidronate (Ki: 91 microM), ibandronate (116 microM), pamidronate (83 microM), risedronate (85 microM) and zoledronate (81 microM), showed comparable affinities. Interestingly, tiludronate (173 microM; P < 0.05 vs. all other BPs) and especially clodronate (806 microM; P > 0.0001 vs. all other BPs) displayed significantly weaker affinity for bone. The weak affinity of clodronate translated into a requirement for 10-fold higher dosing in in vitro bone resorption assays when bone was pretreated with BP and subsequently washed prior to adding osteoclasts. In stark contrast, neither alendronate nor risedronate lost any efficacy after washing the bone surface. These findings suggest that most clinically tested BPs may have similar affinities for human bone. For those with reduced affinity, this may translate into lower potency that necessitates higher dosing.

Estrogen receptor alpha (ERalpha) serine 118 (Ser118) phosphorylation modulates activation function-1 (AF1) function. Correct positioning of helix 12 promotes agonist-dependent recruitment of cyclin-dependent kinase-7 to catalyze this event. In this study we show robust cyclin-dependent kinase-7-independent, AF2 antagonist-induced Ser118 phosphorylation. Estradiol (E2) and ICI-182,780 (ICI-780) induce Ser118 phosphorylation of wild-type ERalpha and either of two helix 12 mutants, suggesting AF2-independent action, probably via shedding of 90-kDa heat shock protein. With E2 treatment, the predominantly nuclear, phosphorylated ERalpha in COS-1 cells is detergent soluble. Although levels of ICI-780-induced phosphorylation are profound, Ser118-phosphorylated ERalpha is aggregated over the nucleus or in the cytoplasm, fractionating with the cell debris and making detection in cleared lysates improbable. Selective ER modulators (SERMs) elicit a mixed response with phosphorylated ERalpha in both detergent-soluble and -insoluble compartments. Apparent ligand-induced loss of ERalpha protein from cleared lysates is thus due to ligand-induced redistribution into the pellet, not degradation. The COS-1 response to ICI-780 can be mimicked in MCF-7 cells treated with a proteasome inhibitor to block authentic ligand-induced degradation. With SERMs and antagonists, the magnitude of Ser118-phosphorylated receptor redistribution into the insoluble fraction of COS-1 cells correlates with the magnitude of authentic ERalpha degradation in MCF-7 cells. A strong inverse correlation with ligand-induced uterotropism in vivo (P < 0.0001) and direct correlation with AF2-independent transrepression of the matrix metalloprotease-1 promoter in endometrial cells in vitro are seen. These data suggest that ligand-induced Ser118 phosphorylation of ERalpha can be AF2 independent. Furthermore, they identify translocation of Ser118-phosphorylated ERalpha out of the nucleus, leading to cytoplasmic aggregation, as an antagonist pathway that may precede receptor degradation.

Dehydroepiandrosterone (DHEA) exhibits peak adrenal secretion in the fetus at term and around age 30 yr in the adult. Levels then progressively decline, which is associated with decreased levels of testosterone, dihydrotestosterone, and estrogen in peripheral tissues. DHEA supplementation in postmenopausal women increases bone formation and density, an effect mainly attributed to peripheral conversion to sex hormones. In this study, we tested DHEA for direct effects on the androgen (AR) and estrogen (ER) receptors. DHEA bound to AR with a Ki of 1 microM, which was associated with AR transcriptional antagonism on both the mouse mammary tumor virus and prostate-specific antigen promoters, much like the effects of bicalutamide. Unlike bicalutamide, DHEA stimulated, rather than inhibited, LNCaP cell growth, suggesting possible interaction with other hormone receptors. Indeed DHEA bound to ERalpha and ERbeta, with Ki values of 1.1 and 0.5 microM, respectively. Despite the similar binding affinities, DHEA showed preferential agonism of ERbeta with an EC50 of approximately 200 nm and maximal activation at 1 microM. With ERalpha we found 30-70% agonism at 5 microM, depending on the assay. Physiological levels of DHEA are approximately 30 nM and up to 90 nM in the prostate. DHEA at 30 nM is actually sufficient to activate ERbeta transcription to the same degree as estrogen at its circulating concentration, and additive effects are seen when the two were combined. Taken together, DHEA has the potential for physiologically relevant direct activation of ERbeta. With peak levels at term and age 30 yr, there is also a potential for antagonist effects on AR and partial agonism of ERalpha.

The discovery, synthesis, and SAR of chromanes as ER alpha subtype selective ligands are described. X-ray studies revealed that the origin of the ER alpha-selectivity resulted from a C-4 trans methyl substitution to the cis-2,3-diphenyl-chromane platform. Selected compounds from this class demonstrated very potent in vivo antagonism of estradiol in an immature rat uterine weight assay, effectively inhibited ovariectomy-induced bone resorption in a 42 days treatment paradigm, and lowered serum cholesterol levels in ovx'd adult rat models. The best antagonists 8F and 12F also exhibited potent inhibition of MCF-7 cell growth and were shown to be estrogen receptor down-regulators (SERDs).

The androgen receptor (AR) is expressed in the uterus; however, the role of AR in female reproductive physiology is poorly understood. Here we examined the effects of androgens on uterine growth and gene expression in adult ovariectomized rats. Nonaromatizable AR-selective agonists potently stimulate hypertrophy and induce significant myometrial expansion distinct from that induced by 17beta-estradiol (E2). In the endometrium, androgens only modestly increase epithelial cell height and antagonize the trophic effects of E2. To identify underlying mechanisms, global changes in RNA levels 24 h after stimulation with E2 and 5alpha-dihydrotestosterone (DHT) were compared. A total of 491 genes were differentially expressed after E2 treatment, including key regulators of tissue remodeling, cell signaling, metabolism, and gene expression. Of the 164 transcripts regulated by DHT, 86% were also affected by E2, including trophic genes like IGF-I and epithelial secretory genes such as uterocalin. In estrogen receptor (ER)alpha knockout mice, DHT cannot induce uterine growth, suggesting a key role for ERalpha. However, DHT appears not to activate ERalpha directly because DHT induction of IGF-I is blocked by the AR antagonist bicalutamide, and multiple genes regulated directly by ERalpha were not induced by DHT. The similarity between estrogens and androgens instead could reflect general trophic signaling in reproductive tissues because 93 of the 503 genes regulated in the uterus are similarly affected during prostate growth. Thus androgens regulate the trophic environment and architecture of the rodent uterus via a gene expression program that is overlapping but distinct from the estrogen response.

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Requisite levels of intracellular cholesterol and fatty acids are maintained in part by the sterol regulatory element binding proteins (SREBPs). Three major SREBP isoforms exist; SREBP-1a and SREBP-1c are expressed from overlapping mRNAs, whereas SREBP-2 is encoded by a separate gene. The active forms of SREBP-1a and SREBP-1c differ only at their extreme N termini; SREBP-1c lacks 28 aa present in SREBP-1a and instead contains 4 unique aa of its own. While the SREBP-1a and -1c isoforms differentially activate transcription, the molecular basis of this difference is unknown. Here we define the differences between these proteins that confer the enhanced activity of SREBP-1a and demonstrate that this enhancement is a direct result of its avid binding to the coactivator CREB binding protein (CBP) and the mammalian mediator complex. While previous work determined that the C/H1 zinc finger and KIX domains of CBP bind to SREBP-1a, we provide evidence that the interaction with C/H1 is important for gene activation. We further show that the association between the activation domain of SREBP-1 and mediator is through aa 500 to 824 of DRIP150. Finally, we demonstrate the recruitment of mediator to an SREBP-responsive promoter in a sterol-dependent manner.

Farnesoid X receptor (FXR) is a bile acid sensor that regulates the expression of a number of genes the products of which control bile acid and cholesterol homeostasis; however, the role of DRIP205 in FXR-mediated gene regulation remains unexplored. In this study we demonstrate that DRIP205 binds FXR in a ligand-dependent manner in vitro and in vivo. Glutathione S-transferase pull-down assays showed that DRIP205 binds FXR in response to bile acid ligands in a dose-dependent fashion and that the potency of this interaction is associated with the ability of the ligand to activate FXR. In addition, the FXR-DRIP205 interaction required the presence of an intact LXXLL nuclear receptor box 1 (N-terminal) motif of DRIP205. In gel shift assays FXR was also able to recruit DRIP205 in the context of a DNA-bound FXR/RXR (retinoid X receptor) heterodimer. In transient transfection assays, DRIP205 efficiently enhanced a bile acid-activated FXRE-driven reporter gene in a dose-dependent manner in cells overexpressing FXR/RXR, demonstrating that DRIP205 enhances FXR-mediated transactivation. By contrast, an FXRW469A mutant in the activation function 2 domain that does not bind to DRIP205 was unable to activate ligand-stimulated FXR transcription, indicating that DRIP205 is recruited to activation function 2 of FXR. Requirement for the FXR/RXR heterodimer in the DRIP205-FXR interaction was evaluated using an RXR heterodimerization-deficient FXR mutant (FXRL433R). FXRL433R was not able to bind to DRIP205 and failed to enhance an FXRE-driven reporter gene. In addition, DRIP205 was unable to induce FXR-mediated transactivation in the absence of RXR overexpression, indicating that FXR heterodimerization with RXR is required for coactivation by DRIP205. Finally, in HepG2 cells, overexpression or reduction of DRIP205 levels modulated the induction of endogenous FXR target gene mRNA expression by ligand. Together, these results demonstrate that DRIP205 acts as a bona fide coactivator of FXR and underscore the importance of DRIP205 in modulating the bile acid response of FXR target genes.

The androgen receptor (AR) is a member of the steroid receptor superfamily that plays critical roles in the development and maintenance of the male reproductive system and in prostate cancer. Actions of AR are controlled by interaction with several classes of coregulators. In this study, we have identified LATS2/KPM as a novel AR-interacting protein. Human LATS1 and LATS2 are tumor suppressors that are homologs of Drosophila warts/lats. The interaction surface of LATS2 is mapped to the central region of the protein, whereas the AR ligand binding domain is sufficient for this interaction. LATS2 functions as a modulator of AR by inhibiting androgen-regulated gene expression. The mechanism of LATS2-mediated repression of AR activity appears to involve the inhibition of AR NH2- and COOH-terminal interaction. Chromatin immunoprecipitation assays in human prostate carcinoma cells reveal that LATS2 and AR are present in the protein complex that binds at the promoter and enhancer regions of prostate-specific antigen, and overexpression of LATS2 results in a reduction in androgen-induced expression of endogenous prostate-specific antigen mRNA. Immunohistochemistry shows that LATS2 and AR are localized within the prostate epithelium and that LATS2 expression is lower in human prostate tumor samples than in normal prostate. The results suggest that LATS2 may play a role in AR-mediated transcription and contribute to the development of prostate cancer.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

The androgen receptor (AR), when complexed with 5alpha-dihydrotestosterone (DHT), supports the survival and proliferation of prostate cells, a process critical for normal development, benign prostatic hypertrophy, and tumorigenesis. However, the androgen-responsive genetic pathways that control prostate cell division and differentiation are largely unknown. To identify such pathways, we examined gene expression in the ventral prostate 6 and 24 h after DHT administration to androgen-depleted rats. 234 transcripts were expressed significantly differently from controls (p < 0.05) at both time points and were subjected to extensive data mining. Functional clustering of the data reveals that the majority of these genes can be classified as participating in induction of secretory activity, metabolic activation, and intracellular signaling/signal transduction, indicating that AR rapidly modulates the expression of genes involved in proliferation and differentiation in the prostate. Notably AR represses the expression of several key cell cycle inhibitors, while modulating members of the wnt and notch signaling pathways, multiple growth factors, and peptide hormone signaling systems, and genes involved in MAP kinase and calcium signaling. Analysis of these data also suggested that p53 activity is negatively regulated by AR activation even though p53 RNA was unchanged. Experiments in LNCaP prostate cancer cells reveal that AR inhibits p53 protein accumulation in the nucleus, providing a post-transcriptional mechanism by which androgens control prostate cell growth and survival. In summary these data provide a comprehensive view of the earliest events in AR-mediated prostate cell proliferation in vivo, and suggest that nuclear exclusion of p53 is a critical step in prostate growth.

Steroid receptors belong to the nuclear hormone receptor superfamily, which represents one of the important families of drug targets in the pharmaceutical industry. These receptors are generally expressed in a broad range of tissues and play roles in multiple physiological pathways. They represent one of the most complex target classes for drug research as the typical pharmacologically selective, potent, and pure agonist or antagonist may not be sufficient for the development of an optimal compound for therapeutic use. In many cases, the professed compound that is a selective modulator of a steroid hormone receptor that functions as an agonist in one tissue and as an antagonist in another can offer a better therapeutic advantage. In this review, we will selectively summarize members of the steroid hormone receptor subfamily, their therapeutic opportunities, and a glimpse of the methods that can be utilized in the development of drugs that target these receptors.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Loss of genetic material on chromosome 6 has been associated with progression of human melanomas. We showed previously that introducing chromosome 6 into metastatic human melanoma cell lines suppresses metastasis without affecting the ability of the hybrids to form progressively growing tumors. By subtractive hybridization comparing nonmetastatic chromosome 6-containing (neo6/C8161) versus parental (C8161) metastatic cells, the KISS1 metastasis suppressor gene was isolated. However, KISS1 mapped to chromosome 1q32. To identify upstream regulator(s) of (and downstream effectors of) KISS1, microarray hybridization comparing C8161 and neo6/C8161 variants was performed. TXNIP/VDUP1, a thioredoxin-binding protein, was expressed more highly in neo6/C8161 and in nonmetastatic melanomas. Increased TXNIP expression inhibited metastasis and up-regulated KISS1. Surprisingly, TXNIP also mapped to chromosome 1q. PCR karyotyping that refined the region on chromosome 6 identified CRSP3/DRIP130, a transcriptional coactivator, as a metastasis suppressor. CRSP3 transfectant cells had up-regulated KISS1 and TXNIP expression and were suppressed for metastasis. Quantitative real-time reverse-transcription PCR of clinical melanoma samples showed that loss of CRSP3 expression correlated with decreased KISS1 expression and increased metastasis. Thus, we implicated a specific gene on chromosome 6 in the etiology of melanoma metastasis and identified potential up-stream regulators of KISS1 and TXNIP.

The interferon (IFN)-induced signal transduction and transcription activation complex, ISGF3, is assembled from three proteins, STAT1, STAT2, and IRF9. Of these components, STAT2 provides a fundamental and essential transcriptional activation function for ISGF3. In the present study, we show that ISGF3-mediated transcription is dependent on STAT2 interactions with DRIP150, a subunit of the multimeric Mediator coactivator complex. Other Mediator subunits, DRIP77 and DRIP130, were found either to bind STAT2 without augmenting ISGF3 transcriptional activity or to enhance ISGF3 transcription without binding STAT2, but only DRIP150 both enhanced IFN-dependent transcription and coimmunoprecipitated with STAT2. Endogenous DRIP150 and STAT2 were able to interact in solution, and DNA affinity chromatography and chromatin immunoprecipitation assays demonstrated that DRIP150 binds to the mature, activated ISGF3-DNA complex and is recruited to target gene promoters in an IFN-dependent fashion. IFN-dependent recruitment of DRIP130 to an ISGF3 target promoter and SRB10-STAT2 coprecipitation suggest indirect association with a multisubunit Mediator complex. The site of STAT2 interaction was mapped to DRIP150 residues 188 to 566, which are necessary and sufficient for interaction with STAT2. Expression of this DRIP150 fragment, but not DRIP150 fragments outside the STAT2 interaction region, suppressed ISGF3-mediated transcriptional activity in a dominant-negative fashion, suggesting a direct functional role of this domain in mediating STAT2-DRIP150 interactions. These findings indicate that the IFN-activated ISGF3 transcription factor regulates transcription through contact with DRIP150 and implicate the Mediator coactivator complex in IFN-activated gene regulation.

Nuclear receptors (NR) activate transcription by interacting with several different coactivator complexes, primarily via LXXLL motifs (NR boxes) of the coactivator that bind a common region in the ligand binding domain of nuclear receptors (activation function-2, AF-2) in a ligand-dependent fashion. However, how nuclear receptors distinguish between different sets of coactivators remains a mystery, as does the mechanism by which orphan receptors such as hepatocyte nuclear factor 4alpha (HNF4alpha) activate transcription. In this study, we show that HNF4alpha interacts with a complex containing vitamin D receptor (VDR)-interacting proteins (DRIPs) in the absence of exogenously added ligand. However, whereas a full-length DRIP205 construct enhanced the activation by HNF4alpha in vivo, it did not interact well with the HNF4alpha ligand binding domain in vitro. In investigating this discrepancy, we found that the polyamine spermine significantly enhanced the interaction between HNF4alpha and full-length DRIP205 in an AF-2, NR-box-dependent manner. Spermine also enhanced the interaction of DRIP205 with the VDR even in the presence of its ligand, but decreased the interaction of both HNF4alpha and VDR with the p160 coactivator glucocorticoid receptor interacting protein 1 (GR1P1). We also found that GR1P1 and DRIP205 synergistically activated HNF4alpha-mediated transcription and that a specific inhibitor of polyamine biosynthesis, alpha-difluoromethylornithine (DFMO), decreased the ability of HNF4alpha to activate transcription in vivo. These results lead us to propose a model in which polyamines may facilitate the switch between different coactivator complexes binding to NRs.

Two functionally distinct classes of coactivators are recruited by liganded estrogen receptor, the DRIP/Mediator complex and p160 proteins, although the relative dynamics of recruitment is unclear. Previously, we have shown a direct, estradiol-dependent interaction between the DRIP205 subunit of the DRIP complex and the estrogen receptor (ER) AF2 domain. Here we demonstrate the in vivo recruitment of other endogenous DRIP subunits to ER in response to estradiol treatment in MCF-7 cells. To explore the relationship between DRIP and p160 coactivators, we examined the kinetics of coactivator recruitment to the ER target promoter, pS2, by chromatin immunoprecipitation. We observed a cyclic association and dissociation of coactivators with the promoter, with recruitment of p160s and DRIPs occurring in opposite phases, suggesting an exchange between these coactivator complexes at the target promoter.

Following their activation, naive CD4+ T cells can differentiate into one of two effector cell subsets, Th1 and Th2. These two subsets have different cytokine secretion patterns and thus mediate separate arms of the immune response. It has been established that the fat-soluble vitamin D(3) metabolite 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its nuclear receptor, the vitamin D receptor, play an important role in the immune system primarily through the transcriptional inhibition of cytokine genes that either are required for Th1 differentiation or are products of differentiated Th1 cells. Therefore, we wanted to test directly the ability of 1,25(OH)(2)D(3) to alter the Th differentiation process. Our results indicate that 1,25(OH)(2)D(3) inhibits not only the Th1 cytokine IFN-gamma but also the Th2 cytokine IL-4 in naive CD62 ligand+CD4+ T cells during their in vitro polarization. This effect is most dramatic when the ligand is present from the onset of the differentiation process. If the ligand is added after the polarization has ensued, the inhibition is significantly diminished. In activated (CD62 ligand-CD4+) T cells, 1,25(OH)(2)D(3) is still able to inhibit IFN-gamma but has no effect on IL-4 production. Our results also indicate that inhibition of these two cytokines in naive cells by vitamin D receptor and its ligand is neither a result of a cell cycle block nor an inhibition of Th1 or Th2 transcription factor expression but, rather, at least in the case of Th2 differentiation, an attenuation of IL-4 transcription by the receptor.

When UMR-106 osteoblastic cells, LLCPK1 kidney cells, and VDR transfected COS-7 cells were transfected with the rat 24-hydroxylase [24(OH)ase] promoter (-1,367/+74) or the mouse osteopontin (OPN) promoter (-777/+79), we found that the response to 1,25dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] could be significantly enhanced 2- to 5-fold by the protein phosphatase inhibitor, okadaic acid (OA). Enhancement of 1,25-(OH)(2)D(3)-induced transcription by OA was also observed using a synthetic reporter gene containing either the proximal 24(OH)ase vitamin D response element (VDRE) or the OPN VDRE, suggesting that the VDRE is sufficient to mediate this effect. OA also enhanced the 1,25-(OH)(2)D(3)-induced levels of 24(OH)ase and OPN mRNA in UMR osteoblastic cells. The effect of OA was not due to an up-regulation of VDR or to an increase in VDR-RXR interaction with the VDRE. To determine whether phosphorylation regulates VDR-mediated transcription by modulating interactions with protein partners, we examined the effect of phosphorylation on the protein-protein interaction between VDR and DRIP205, a subunit of the vitamin D receptor-interacting protein (DRIP) coactivator complex, using glutathione-S-transferase pull-down assays. Similar to the functional studies, OA treatment was consistently found to enhance the interaction of VDR with DRIP205 3- to 4-fold above the interaction observed in the presence of 1,25-(OH)(2)D(3) alone. In addition, studies were done with the activation function-2 defective VDR mutant, L417S, which is unable to stimulate transcription in response to 1,25-(OH)(2)D(3) or to interact with DRIP205. However, in the presence of OA, the mutant VDR was able to activate 24(OH)ase and OPN transcription and to recruit DRIP205, suggesting that OA treatment may result in a conformational change in the activation function-2 defective mutant that creates an active interaction surface with DRIP205. Taken together, these findings suggest that increased interaction between VDR and coactivators such as DRIP205 may be a major mechanism that couples extracellular signals to vitamin D action.