C. Richard Lyttle

The Role of CBP in Estrogen Receptor Cross-Talk with Nuclear Factor-κB in HepG2 Cells

Functional interactions or cross-talk between ligand-activated nuclear receptors and the proinflammatory transcription factor nuclear factor-κB (NF-κB) may play a major role in ligand-mediated modification of diseases processes. In particular, the cardioprotective effects of estrogen replacement therapy are thought to be due in part to the ability of ligand-bound estrogen receptor (ER) to inhibit NF-κB function. In the current study 17β-estradiol-bound ERα interfered with cytokine-induced activation of a NF-κB reporter in HepG2 cells. The estrogen metabolite, 17α-ethinyl estradiol, and the phytoestrogen, genistein, were also effective inhibitors of NF-κB activation, whereas tamoxifen, 4-hydroxytamoxifen, and raloxifene were inactive. This inhibition was reciprocal, as NF-κB interfered with the trans-activation properties of ERα. Ligand-bound ERα did not inhibit NF-κB binding to DNA, but it did decrease the histone acetyltransferase activity required for NF-κB transcriptional activity. Coexpression of the ...

The Pairing of a Selective Estrogen Receptor Modulator, Bazedoxifene, with Conjugated Estrogens as a New Paradigm for the Treatment of Menopausal Symptoms and Osteoporosis Prevention

The menopausal transition is associated with decreased ovarian function and concomitant decline in estrogen production, which may result in physiological effects such as hot flashes, reduced bone mass, and altered lipid profile. It is well established that these unfavorable changes are effectively offset with estrogen therapy (ET) or, in women with a uterus, estrogens in combination with a progestin (hormone therapy). Selective estrogen receptor (ER) modulators (SERMs), which exhibit both ER agonist and antagonist activities depending on the target tissue, have been regarded as offering the potential to provide the benefits of ET and hormone therapy with an improved safety and tolerability profile. To date, no SERM alone has demonstrated an ideal benefit-risk profile for menopausal therapy. The tissue-selective estrogen complex, or the pairing of a SERM with estrogens, may provide an optimal blend of ER agonist and antagonist activities. We evaluated the physiological profile of this novel therapeutic paradigm by using various in vivo models to assess uterine, vasomotor, lipid, and skeletal responses to a tissue-selective estrogen complex partnering bazedoxifene with conjugated estrogens (CE). Bazedoxifene at 3.0 mg/kg effectively antagonized CE-induced uterine stimulation without reversing the positive effects of CE on vasomotor instability. When paired with CE, bazedoxifene at 3.0 mg/kg reduced total cholesterol levels by up to 20% compared with CE alone and significantly increased total bone density relative to control. These preclinical findings showed that the appropriate dose combination of bazedoxifene/CE exhibits positive vasomotor, lipid, and skeletal responses with minimal uterine stimulation.

Developing a SERM: Stringent Preclinical Selection Criteria Leading to an Acceptable Candidate (WAY-140424) for Clinical Evaluation

Abstract: Estrogens are represented by a diverse group of compounds. Within this large family of molecules are tissue‐selective estrogens that have been classified as selective estrogen receptor modulators (SERMs). These compounds are characterized by the fact that they exhibit both estrogen agonist and antagonist activity dependent upon the gene promoter and target tissue being examined. SERMs have been intensively studied over the past decade, especially since one, raloxifene, has been approved for the prevention and treatment of postmenopausal osteoporosis. While not a replacement for hormone replacement therapy (HRT), raloxifene can be an alternative to it and other treatments for osteoporosis. The ideal SERM would provide the positive benefits associated with HRT without the uterine and breast stimulation. Raloxifene does achieve some of the benefits of HRT, specifically on the skeleton and lipid metabolism with no apparent uterine effects, and a potential decreased risk of developing breast cancer associated with raloxifene therapy. However, there are a number of parameters that can be improved. A number of SERMs have been evaluated only to fail in development due to, for the most part, uterine safety issues. In order to develop an improved SERM, a stringent screening process was designed to select compounds that did not stimulate the uterus or breast. At the same time, these new compounds would have a positive impact on the skeleton and lipid metabolism with the additional improvement (over raloxifene) of a neutral effect on hot flashes. Under these strict conditions, WAY‐140424 was developed and, to date, the preclinical pharmacology data have accurately predicted the clinical response demonstrated in phase I and II trials.

Human estrogen receptor regulation in a yeast model system and studies on receptor agonists and antagonists.

An expression system that utilized yeast copper metallothionein promoter and ubiquitin fusion technology to express the human estrogen receptor gene in yeast is described. We have studied the biochemical and transcriptional regulatory properties of the human estrogen receptor. The biochemical properties of the yeast expressed receptors are identical to the receptors isolated from human tissue. Estradiol mediated activation of transcription by the receptor was studied by a reporter beta-galactosidase gene where expression was under the control of estrogen response elements. Using this expression system and a hyperpermeable yeast strain we have studied the effects of various antiestrogens on the regulation of estrogen receptor function. We demonstrate that tamoxifen and ICI 164,384 are capable of binding to the receptor but neither antiestrogen was able to block the estradiol mediated increase in transcription. In fact, both antiestrogens exerted weak agonist activity in this system.

Effects of abaloparatide, a human parathyroid hormone-related peptide analog, on bone mineral density in postmenopausal women with osteoporosis.

CONTEXT Abaloparatide is a novel synthetic peptide analog of parathyroid hormone-related protein (PTHrP) that is currently being developed as a potential anabolic agent in the treatment of postmenopausal osteoporosis. OBJECTIVE This study sought to assess the effects of abaloparatide on bone mineral density (BMD) at the lumbar spine, total hip, and femoral neck in postmenopausal women with osteoporosis. DESIGN Multi-center, multi-national, double-blind placebo controlled trial in which postmenopausal women were randomly assigned to receive 24 weeks of treatment with daily sc injections of placebo, abaloparatide, 20, 40, or 80 μg, or teriparatide, 20 μg. A 24-week extension was also performed in a subset of subjects. PARTICIPANTS Postmenopausal women with osteoporosis (n = 222). MAIN OUTCOME MEASURES BMD by dual-x-ray absorptiometry and biochemical markers of bone turnover. RESULTS At 24 weeks, lumbar spine BMD increased by 2.9, 5.2, and 6.7% in the abaloparatide, 20-, 40-, and 80-μg groups, respectively, and 5.5% in the teriparatide group. The increases in the 40- and 80-μg abaloparatide groups and the teriparatide group were significantly greater than placebo (1.6%). Femoral neck BMD increased by 2.7, 2.2, and 3.1% in abaloparatide, 20-, 40-, and 80-μg groups, respectively, and 1.1% in the teriparatide group. The increase in femoral neck BMD with abaloparatide, 80 μg was significantly greater than placebo (0.8%). Total hip BMD increased by 1.4, 2.0, and 2.6% in the abaloparatide, 20-, 40-, and 80-μg groups, respectively. The total hip increases in the 40- and 80-μg abaloparatide groups were greater than both placebo (0.4%) and teriparatide (0.5%). CONCLUSIONS Compared with placebo, 24 weeks of daily sc abaloparatide increases BMD of the lumbar spine, femoral neck, and total hip in a dose-dependent fashion. Moreover, the abaloparatide-induced BMD increases at the total hip are greater than with the marketed dose of teriparatide. These results support the further investigation of abaloparatide as an anabolic therapy in postmenopausal osteoporosis.

Endometrial synthesis and secretion of complement component-3 by patients with and without endometriosis * †

In the present study we examined complement-3 (C3) synthesis and secretion from early proliferative endometrium of infertile patients with and without endometriosis. One gram of tissue was obtained by endometrial sampling at the time of diagnostic laparoscopy and incubated for 12 to 16 hours in the presence of radioactive methionine. Immunoprecipitation was performed with rabbit antihuman C3 immunoglobulin G and only a single 180-kDa radiolabeled protein (C3) was immunoprecipitated. This protein dissociates into 113- and 69-kDa subunits in the presence of dithiothreitol. The amount of C3 produced and secreted by the endometrium was quantitated as a percentage of counts per minute recovered by immunoprecipitation. Patients with minimal endometriosis produced significantly greater amounts of endometrial C3 than patients with no endometriosis or patients with severe endometriosis.

Increased chemotactic activity of peritoneal fluid in patients with endometriosis

OBJECTIVE Our purpose was to investigate the ability of the peritoneal fluid of patients with endometriosis to induce chemotaxis of neutrophils and macrophages. STUDY DESIGN Peritoneal fluid samples of patients with endometriosis (n = 20), normal fertile controls (n = 12), or patients with medical suppression (n = 8) were evaluated for chemotactic activity. Results of chemotactic activity were analyzed by analysis of variance. RESULTS Peritoneal fluid of patients with endometriosis demonstrated a significantly higher chemotactic activity than that of patients without endometriosis or with medical suppression. Patients who had received medical treatment had the lowest chemotactic activity. (p < 0.001 for endometriosis vs control or treatment patients, p = 0.005 for control group vs treatment group). CONCLUSIONS Patients with endometriosis have a higher chemotactic activity in their peritoneal fluid; prior medical treatment significantly reduces this activity. This chemotactic factor has an estimated weight of 20 kd. The nature and source of this chemotactic factor remains to be determined.

Estrogen regulation of protein synthesis in the immature rat uterus: the analysis of proteins released into the medium during in vitro incubation

Immature rats were treated with estradiol (E2) or other steroids before their uteri were removed and incubated under in vitro conditions in the presence of [35S]methionine. The analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the radiolabeled proteins synthesized and released into the incubation medium demonstrate that E2 regulates the appearance of two proteins. These two proteins have mol wt of 115,000 and 65,000. The concentration of proteins in the medium increases linearly with time, suggesting that they may be secreted. These two proteins were not produced by several other tissues in response to E2 and appear to be specific to the uterus. They also appear to be increased only by estrogens (E2 greater than estrone greater than estriol) and not by other steroids tested. They are increased in response to a single injection within 6 h, and the maximal concentration of proteins occurs approximately 24 h after E2 administration. The protein concentrations have essentially returned to control values by 72 h after hormone injection. The kinetics of the induction is the same for both proteins, suggesting that their increase may be coordinated. Based on the tissue and hormone specificity of the increase in the 115,000- and the 65,000-dalton proteins, they may serve as reliable markers for the study of the uterine response to E2.

Quantitative analysis of gene regulation by seven clinically relevant progestins suggests a highly similar mechanism of action through progesterone receptors in T47D breast cancer cells

Progesterone (P4) is an essential reproductive steroid hormone required for many aspects of female reproductive physiology. Progestins are compounds that demonstrate progesterone-like activity and are used in oral contraception, hormone therapy, and treatment of some reproductive disorders, but differ widely in their chemical structures, potency, and pharmacokinetics. While numerous studies have assessed progestins on specific endpoints, little is known about the activation of global gene expression by progestins. We used Affymetrix GeneChip U133A expression arrays to examine the action of P4 and six clinically relevant synthetic progestins (3-ketodesogestrel, drospirenone, levonorgestrel, medroxyprogesterone acetate, norethindrone acetate, and trimegestone) on the progesterone receptor (PR)-positive T47Dco and the PR-negative T47D-Y breast cancer cell lines. Excluding drospirenone, one or more of the progestins-regulated 329 genes, with 30 genes regulated by at least 2.0-fold by all progestins in the T47Dco cells. The synthetic progestins show a high degree of similarity in their transcriptional responses, and each progestin regulates between 77 and 91% of the genes regulated by P4. Independent quantitative RT-PCR analysis confirmed a similar regulation for S100P, PPL, IL20RA, NET1, ATP1A1, HIG2, and CXCL12 (SDF-1) by all seven progestins. Attempts to find differentially regulated genes by any progestin compared to all other treatments failed, suggesting any differences are quantitative, not qualitative. This analysis demonstrates a high degree of similarity among these progestins on PR-regulated gene expression in T47D cells, suggesting a similar and fairly specific mode of action.

Chemotaxis of macrophages by a peritoneal fluid protein in women with endometriosis

OBJECTIVE To expand on a preliminary study comparing the chemotactic potential of peritoneal fluid (PF) from women with and without endometriosis and to characterize this activity via immunosuppressants and a protease. DESIGN Case control study. SETTING University center. PATIENT(S) Fifty-nine women with endometriosis and 44 without, undergoing laparoscopy. INTERVENTION(S) Collection of PF, endometriotic, ovarian, and endometrial biopsies at laparoscopy. MAIN OUTCOME MEASURE(S) Chemotactic activity of PF was tested via an in vitro assay alone and in the presence of immunosuppressants cyclosporin A (CSA), FK506, rapamycin, and type XVII-b(S-V8) protease and in media incubated with endometriotic, ovarian, or endometrial biopsy specimens. RESULT(S) The PF from women with endometriosis had significantly greater chemotactic activity (cells per well, mean +/- SD) than without endometriosis (142 +/- 39 versus 48 +/- 17). Cyclosporin A significantly inhibited the chemotactic activity of the endometriotic PF; FK506 and rapamycin did not. Incubation of media with endometriotic tissue, but not ovarian or endometrial, for > or = 7 hours displayed chemotactic activity. Protease type XVII-b(S-V8) added to endometriotic PF inhibited this chemotactic activity. CONCLUSION(S) Peritoneal fluid from patients with endometriosis contains a protein chemotactic factor attracting inflammatory cells into the peritoneal cavity, possibly secreted by endometriotic implants. This chemotactic factor may be a member of the immunophilin family because of its inhibition profile.