Debra F. Skafar

Reproductive Factors, Hormone Use, Estrogen Receptor Expression and Risk of Non Small-Cell Lung Cancer in Women

PURPOSE Estrogen receptor (ER) expression in lung tumors suggests that estrogens may play a role in the development of lung cancer. We evaluated the role of hormone-related factors in determining risk of non-small-cell lung cancer (NSCLC) in women. We also evaluated whether risk factors were differentially associated with cytoplasmic ER-alpha and/or nuclear ER-beta expression-defined NSCLC in postmenopausal women. PATIENTS AND METHODS Population-based participants included women aged 18 to 74 years diagnosed with NSCLC in metropolitan Detroit between November 1, 2001 and October 31, 2005. Population-based controls were identified through random digit dialing, matched to patient cases on race and 5-year age group. Interview data were analyzed for 488 patient cases (241 with tumor ER results) and 498 controls. RESULTS Increased duration of hormone replacement therapy (HRT) use in quartiles was associated with decreased risk of NSCLC in postmenopausal women (odds ratio = 0.88; 95% CI, 0.78 to 1.00; P = .04), adjusting for age, race, pack-years, education, family history of lung cancer, current body mass index, years exposed to second-hand smoke in the workplace, and obstructive lung disease history. Among postmenopausal women, ever using HRT, increasing HRT duration of use in quartiles, and increasing quartiles of estrogen use were significant predictors of reduced risk of NSCLC characterized as ER-alpha and/or ER-beta positive. None of the hormone-related variables were associated with nuclear ER-alpha- or ER-beta-negative NSCLC. CONCLUSION These findings suggest that postmenopausal hormone exposures are associated with reduced risk of ER-alpha- and ER-beta-expressing NSCLC. Understanding tumor characteristics may direct development of targeted treatment for this disease.

Mutations targeted to a predicted helix in the extreme carboxyl-terminal region of the human estrogen receptor-α alter its response to estradiol and 4-hydroxytamoxifen

The human estrogen receptor-α, a member of the nuclear receptor superfamily, is a ligand-regulated transcriptional modulator. Because comparatively little is known about the extreme carboxyl-terminal region of the estrogen receptor (F domain), we used secondary structure prediction to design mutations that delete the F domain (S554stop), disrupt a possible turn (G556L/G557L), and alter a predicted helix (S559A/E562A, Q565P), and we evaluated the effects of these mutations on hormone binding and transcription activation in response to estradiol and the mixed agonist/antagonist 4-hydroxytamoxifen. Mutations that deleted the F domain (S554stop) or targeted the predicted helix (S559A/E562A, Q565P) greatly reduced or eliminated the agonist activity of 4-hydroxytamoxifen. Deleting the F domain increased the affinity of the receptor for estradiol and decreased the antagonist activity of 4-hydroxytamoxifen. The Q565P mutant exhibited a non-cooperative hormone-binding mechanism, as well as an impaired response to estradiol and increased antagonist activity of 4-hydroxytamoxifen. Our results show that mutations in the F domain alter not only the response to estradiol, the affinity for hormone, and the interaction between receptor subunits but can uncouple the agonist and antagonist activities of 4-hydroxytamoxifen. These results suggest that the F domain modulates the activity of the estrogen receptor-α by multiple mechanisms.

Interleukin-1beta-induced nitric oxide production in rat aortic endothelial cells: inhibition by estradiol in normal and high glucose cultures.

Expression of inducible nitric oxide synthase (iNOS) and the resultant increased nitric oxide (NO) production are associated with septic shock, atherosclerosis, and cytokine-induced vascular injury. Estrogen is known to impact vascular injury and vascular tone, in part through regulation of NO production. In the current study, we examined the effect of physiological concentrations of estradiol on interleukin-1beta (IL-1beta)-induced NO production in rat aortic endothelial cells (RAECs). 17Beta-estradiol significantly decreased IL-1beta-induced iNOS protein levels and reduced NO production in RAECs. High glucose (25 mM) elevated the increase in IL-1beta-induced iNOS protein and NO production. Nevertheless, estradiol still inhibited IL-1beta-induced iNOS and NO production even in the presence of high glucose. These data suggest that estradiol may exert its beneficial effects in part by inhibiting induction of endothelial iNOS, a possible mechanism for the protective effect of estradiol against diabetes-associated cardiovascular complications.

The multifunctional estrogen receptor-alpha F domain

The members of the nuclear receptor superfamily act as transcriptional regulatory factors and exhibit a multidomain structure characterized as domains A-E/F. This review focuses on a small, relatively understudied region at the extreme carboxy-terminus of the estrogen receptor (ER) alpha, the F domain. The F domain contributes to differences in the activity of ER alpha and beta subtypes; it is required for tamoxifen’s agonist activity on an estrogen response element, and it modifies the receptor’s interactions with coregulators including steroid receptor coactivator-1. The differences between the F domains of the ER alpha and beta subtypes and among the other members of the nuclear hormone receptor superfamily may offer opportunities for selective control of the activity of these proteins.

Understanding the human estrogen receptor-alpha using targeted mutagenesis

The estrogen receptor-alpha is a wonderfully complex protein important in normal biology, breast cancer, and as a target for anti-cancer agents. We are using the available structures of the hERalpha as well as secondary structure predictions to guide site-directed mutagenesis in order to test the importance of specific interactions and regions in the ligand-regulated activity of the protein. In one area of interest, we are investigating the role of the F domain in the ligand-stimulated activity of the hERalpha. Results from our laboratory and others suggest that the F domain modulates the activity of the hERalpha. In order to better understand the role of the F domain in the hERalpha, we have constructed mutants within this region. Mutations within a predicted alpha-helical region alter the response of the ER to estradiol (E2), eliminate or impair the agonist activity of 4-hydroxytamoxifen (4-OHT), and alter the ability of E2 to overcome 4-OHTs antagonist activity. Deleting the F domain increases the affinity of the receptor for E2; by contrast, mutating a residue in the middle of the predicted helix to a proline does not alter the affinity for E2, but does change the binding mechanism from a positive cooperative to a noncooperative interaction. These and other results show the F domain exhibits substantial functional complexity, and support the idea that this domain modulates the activity of the hERalpha. In a second area of interest, we are investigating the role of hydrophobic and hydrogen-bonding interactions at the start of helix 12 in the activity of the hERalpha. Leucine-536 (L536) has been proposed to participate in hydrophobic interactions that form part of a capping motif stabilizing the start of helix 12. When mutated, the resulting receptors exhibit a reduced response, or even an inverted response, to E2 and 4-OHT on both ERE-driven and AP-1-driven promoters. Interestingly, these mutated receptors also exhibit altered interactions with probes that recognize the agonist-bound and 4-OHT-bound conformations of the ERalpha. Thus, L536 couples the binding of ligand with the conformation of the receptor. Overall, these results show that combining structure-based hypotheses with functional tests of the ERs activity can identify regions and interactions that are important in the ligand-stimulated activity of the protein.

Formation of a powerful capping motif corresponding to start of "Helix 12" in agonist-bound estrogen receptor-α contributes to increased constitutive activity of the protein

The human estrogen receptor α (hERα), a ligand-activated transcription factor, provides an excellent system for study of the conversion of a protein from inactive to active states. It binds to many different ligands which leads which to the expression or the suppression of genes in a cell-specific and promoter-specific manner, and a multitude of mutations have been identified that modify the activity of the receptor. Helix 12 is a key α-helix in the hormone-binding domain of the hERα that is directly involved in transcription activation. In this report, tyrosine 537 has been identified as the Ncap residue of helix 12 in the structures of the agonist-bound hERα ligand-binding domain. A capping motif has been identified in the sequence of the hERα that corresponds to the start of helix 12 in the fully active, agonist-bound conformation of the receptor. Analysis of the literature indicates that, with one exception, substitution of amino acids at position 537, which occur more frequently at the Ncap than tyrosine, is correlated with increased constitutive activity of the hERα. The results are consistent with the hypothesis that formation of a powerful capping motif that corresponds to the start of helix 12 in the agonist-bound receptor contributes to activation of the hERα. This is the first proposed example in which mutations that alter helix capping would lead to the constitutive activation of a protein. This hypothesis could therefore provide a novel mechanism through which mutations play a role in pathological processes.

Hydrocortisone modulates the effect of estradiol on endothelial nitric oxide synthase expression in human endothelial cells.

The interaction between hydrocortisone and estradiol on the regulation of endothelial nitric oxide synthase (eNOS) expression was investigated in human umbilical vein endothelial cells (HUVECs). Following incubation in medium containing dextran-coated-charcoal-stripped serum (DCC-stripped medium) for 4 days, incubation of HUVECs with 0.1 nM estradiol for 24 hr in the absence of hydrocortisone increased levels of eNOS mRNA measured by ribonuclease protection assay above control (0 nM estradiol). 2 microM hydrocortisone applied for 24 hr preceding and during estradiol application inhibited the estradiol-elicited increase in eNOS mRNA levels, reducing mRNA levels from 134% +/- 14% of control to 85% +/- 5% of control. Significant (ANOVA, p<0.01) reductions of estradiol-mediated increases of mRNA levels occurred over a range of hydrocortisone concentrations (10 nM, p<0.05; 2 microM, p<0.05; n=3-12). In the presence of 2 microM hydrocortisone, 10 nM estradiol significantly reduced eNOS mRNA levels to 59% +/- 3% of control. The ability of hydrocortisone to block or reverse the estradiol-mediated increase in eNOS mRNA levels may provide a link between elevated hydrocortisone levels and decreased NO production, potentially contributing to the development of hypertension and cardiovascular disease in vivo and antagonizing cardioprotective effects of estrogens.

Modulation of nuclear receptor activity by the F domain

The F domain located at the C-terminus of proteins is one of the least conserved regions of the estrogen receptors alpha and beta, members of the nuclear hormone receptor superfamily. Indeed, many members of the superfamily lack the F domain. However, when present, removing the F domain entirely or mutating it alters transactivation, dimerization, and the responses to agonist and antagonist ligands. This review focuses on the functions of the F domain of the estrogen receptors, particularly in relation to other members of the superfamily.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells

ABSTRACT During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2s effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Dimerization of the RU486-bound calf uterine progesterone receptor

Dimerization of the RU486-bound progesterone receptor was studied by measuring the Hill coefficient of RU486 binding in calf uterine cytosol at receptor concentrations between 0.3 and 15 nM. The limiting value of the Hill coefficient at high receptor concentrations was 1.38 +/- 0.01. The limiting value of the Hill coefficient at low receptor concentrations was 1.05 +/- 0.03. The dimerization constant, defined as the concentration of receptor at which the Hill coefficient was midway between the limiting values, was 2.6 +/- 0.1 nM. In contrast, the dimerization constant of the progesterone-bound receptor, which was measured using the same approach, is 7 nM [Skafar, D. F. Biochemistry 30 (1991) 6148-6154]. The results presented here support and quantify the observation that the RU486-bound progesterone receptor will dimerize at lower receptor concentrations than the progesterone-bound receptor.