Biomedical subjects

S F Arnold

Publications and source records attributed to S F Arnold.

32 records · Page 2Linked to original sources

PubMed1996

Kinetic analysis of the interaction of human estrogen receptor with an estrogen response element.

The kinetics of the interaction between recombinant human estrogen receptor and chicken vitellogenin gene II estrogen response element (ERE) were determined by ERE-Sepharose chromatography. The association constant of the interaction between the ERE and the human estrogen receptor was dependent on receptor concentration, estradiol binding and temperature. The highest association constant (80-100 x 10(6)M-1) was measured for the estradiol-bound receptor prepared at 25 degrees C and at concentrations higher than 7 nM. At high receptor concentrations (>7 nM) the binding mechanism of estradiol to the receptor was positive cooperative, indicating receptor homodimerization. At lower concentrations the binding mechanism was partially cooperative and the association constant of the liganded receptor was significantly lower. The binding mechanism at 4 degrees C was cooperative as well, and the association constants were similarly dependent upon receptor concentration, but were 50% lower than the receptor prepared at 25 degrees C. The association constant of the unliganded receptor was 4- to 5-fold lower than that of the liganded receptor at 25 degrees C. These data suggest that in addition to estradiol-induced conformational changes in the receptor, the receptor dimers are subjected to temperature-dependent changes, which further increase their affinity for an ERE.

Animals↗

PubMed1996

Differential interaction of natural and synthetic estrogens with extracellular binding proteins in a yeast estrogen screen.

We have used the yeast estrogen (YES) consisting of the human estrogen receptor and a reporter containing two estrogen response elements linked to the lacZ gene to evaluate the interaction between ovarian, phyto-, and synthetic estrogens with extracellular binding proteins. YES was incubated with charcoal-stripped human serum, human sex hormone-binding globulin, or human alpha-fetoprotein in the presence of concentrations of various estrogens that induced a 100% estrogenic response, as measured by beta-galactosidase activity. The activity of estradiol and coumestrol, a phytoestrogen, was reduced 75% with physiological levels of serum, sex hormone-binding globulin, or alpha-fetoprotein. The beta-galactosidase activity of genistein, another phytoestrogen, also decreased with extracellular proteins but to a lower extent than estradiol. In contrast, the activity of the synthetic estrogens diethylstilbestrol, kepone, and p,'p-DDD was only minimally reduced with extracellular proteins. These results indicate a potential fundamental difference in the interaction of estrogens from diverse sources with extracellular binding proteins. This suggests that the capacity for various estrogens to induce estrogen-associated responses is in part regulated by their affinity for extracellular bindings proteins.

Adult↗

PubMed1996

Synergistic signals in the environment.

Numerous endogenous signals (such as hormones and growth factors) or environmental signals (including chemicals or temperature) contribute to determining the overall biological response produced by cells. Some combinations of endogenous or environmental signals produce synergistic activity. This commentary examines the different types of interactions between signals that contribute to synergy at the biological level.

Animals↗

PubMed1996

Identification of environmental chemicals with estrogenic activity using a combination of in vitro assays.

Environmental chemicals that function as estrogens have been suggested to be associated with an increase in disease and dysfunctions in animals and humans. To characterize chemicals that may act as estrogens in humans, we have compared three in vitro assays which measure aspects of human estrogen receptor (hER)-mediated estrogenicity. Chemicals were first tested for estrogen-associated transcriptional activity in the yeast estrogen screen (YES). This was created by expressing hER and two estrogen response elements linked to the lacZ gene in yeast. Second, chemicals that were tested in YES were then assayed for direct interaction with hER in a competition binding assay. Third, chemicals were tested in the estrogen-responsive MCF-7 human breast cancer cell line transiently transfected with a plasmid containing two estrogen response elements linked to the luciferase gene. Together, these assays have identified two metabolites of DDT, o,p'-DDD and p,p'-DDD, that have estrogenic activity. Interestingly, previous studies had reported that the DDD metabolites were nonestrogenic in whole animal models. Alachlor, the most frequently used herbicide in the United States, cis-nonachlor, and trans-nonachlor displayed weak estrogenic activity in the combined assays. The antifungal agent benomyl had no estrogenic activity. We propose that a combination of in vitro assays can be used in conjunction with whole animal models for a more complete characterization of chemicals with estrogenic activity.

Binding, Competitive↗

PubMed1996

Interaction of environmental chemicals with the estrogen and progesterone receptors from the oviduct of the American alligator.

Reports of reproductive abnormalities in the American alligator from Lake Apopka, Florida, have been linked to a spill of DDT and other pesticides suspected of having hormonelike activity. To determine whether environmental chemicals had the potential to function as exogenous hormones in the American alligator, we examined the ability of chemicals to bind the estrogen receptor (aER) and progesterone receptor (aPR) in a protein extract prepared from the oviduct of the alligator. In competition binding assays with [3H]17 beta-estradiol, some DDT metabolites showed inhibition of [3H]17 beta-estradiol binding to aER. A combination of DDTs demonstrated an additive decrease in [3H]17 beta-estradiol binding to aER. Modern-use chemicals such as alachlor, trans-nonachlor, endosulfan, and atrazine also competed with [3H]17 beta-estradiol for binding to the aER. To test the effect of chemicals identified in alligator eggs from Lake Apopka on [3H]17 beta-estradiol binding, we mixed these chemicals at concentrations measured in eggs in the competition binding assay. 2,2-bis(4-chlorophenyl)-N-(methoxymethyl)acetamide (p,p'-DDD) and trans-nonachlor, both found in Lake Apopka, interacted with aER, whereas others such as chlordane and toxaphene did not. Surprisingly, combinations of these chemicals decreased [3H]17 beta-estradiol binding in a greater than additive manner. To assess the ability of chemicals to interact with aPR, we performed commpetition binding assays with the synthetic progestin [3H]R5020. Most of the chemicals tested did not reduce [3H]R5020 binding to aPR, whereas endosulfan, alachlor, and kepone inhibited binding. These results provide the first evidence that environmental chemicals bind the aER and aPR from the American alligator, supporting the hypothesis that the reported reproductive abnormalities may be related to the modulation of endocrine-related responses. The findings that combinations of chemicals demonstrated a greater than additive interaction with the aER and some chemicals bind to the aPR in the competition binding assay are novel. This suggests that interactions of these chemicals with the endocrine system are complex.

Alligators and Crocodiles↗

PubMed1996

A yeast estrogen screen for examining the relative exposure of cells to natural and xenoestrogens.

Xenoestrogens, such as o,p'-DDT and octyl phenol (OP), have been associated with reproductive abnormalities in various wildlife species. Xenoestrogens mimic the natural estrogen 17 beta-estradiol and compete for binding to the estrogen receptor. Even though the affinity of o,p'-DDT and OP for the estrogen receptor is approximately 1000-fold lower than 17 beta-estradiol, the actions of xenoestrogens could be enhanced if their bioavailability in serum were greater than 17 beta-estradiol. To test this hypothesis, the yeast estrogen screen (YES) was created by expressing human estrogen receptor (hER) and two estrogen response elements (ERE) linked to the lacZ gene. The beta-galactosidase activity of the YES system was significantly increased after treatment with 17 beta-estradiol or the xenoestrogens diethylstilbestrol (DES), o,p'-DDT, and OP but not with vehicle, antiestrogen ICI 164,384, dexamethasone, or testosterone. To determine whether serum proteins affected the bioavailability of natural estrogens compared to xenoestrogens, albumin, sex hormone binding globulin (SHBG), or charcoal-stripped serum were added to the YES system and beta-galactosidase activity assayed. Albumin and SHBG decreased beta-galactosidase activity in the presence of estradiol to a greater extent than DES, o,p'-DDT, and OP. Human and alligator charcoal-stripped serum were also effective at selectively reducing beta-galactosidase activity in the presence of estradiol compared to xenoestrogens. Human serum was more effective than alligator serum in reducing beta-galactosidase activity in the presence of xenoestrogens, indicating that serum may serve as a biomarker for sensitivity to xenoestrogens. Selective binding of 17 beta-estradiol by proteins in serum indicates that certain xenoestrogens may exert greater estrogenicity than originally predicted. The estrogenic potency of a compound involves its binding affinity, bioavailability in serum, and persistence in the environment. Our data demonstrate the utility of the YES system for identifying and characterizing environmental estrogens.

Albumins↗

PubMed1995

Phosphorylation of tyrosine 537 on the human estrogen receptor is required for binding to an estrogen response element.

We report here that the phosphorylation of tyrosine 537 on the human estrogen receptor (hER) controls the receptor's dimerization and DNA binding ability. The DNA-binding form of both the hER from human MCF-7 mammary carcinoma cells and the hER overexpressed in Sf9 insect cells was isolated using estrogen response element (ERE) affinity chromatography. Western blot analyses demonstrated that the DNA-binding form of the hER from MCF-7 or Sf9 cells was (i) phosphorylated at tyrosine 537, (ii) localized in the nucleus of estradiol-treated MCF-7 cells with an apparent molecular mass of 67 kDa, and (iii) hyperphosphorylated at serine residue(s). The non-DNA-binding form of the hER was (i) devoid of phosphorylation at tyrosine 537, (ii) cytosolic with an apparent molecular mass of 66 kDa, and (iii) hypophosphorylated at serine residue(s). The dephosphorylation of the purified hER at phosphotyrosine 537 with a tyrosine phosphatase eliminated binding to an ERE in a gel mobility shift assay. The binding of the tyrosine-dephosphorylated hER to an ERE was restored by the rephosphorylation of tyrosine 537 with Src family tyrosine kinases, p60c-src or p56lck. Mutation of tyrosine 537 to phenylalanine confirmed that the phosphorylation of tyrosine 537 is necessary for the hER to bind an ERE. An anti-hER antibody restored the binding of the tyrosine-dephosphorylated hER to an ERE, indicating that the bivalent anti-hER antibody brought together the two inactive hER monomers. A far-Western blot confirmed that phosphotyrosine 537 is required for hER homodimerization. These experiments establish that dimerization of the hER and DNA binding are regulated by phosphorylation at tyrosine 537. This is the first demonstration of the regulation of dimerization of a steroid hormone receptor by phosphorylation. These results are significant since p60c-src is overexpressed in estrogen-dependent breast cancers and may act to enhance the activity of the hER.

Amino Acid Sequence↗

PubMed1995

An antiestrogen: a phosphotyrosyl peptide that blocks dimerization of the human estrogen receptor.

We have previously identified tyrosine-537 as a constitutively phosphorylated site on the human estrogen receptor (hER). A 12-amino acid phosphotyrosyl peptide containing a selected sequence surrounding tyrosine-537 was used to investigate the function of phosphotyrosine-537. The phosphotyrosyl peptide completely blocked the binding of the hER to an estrogen response element (ERE) in a gel mobility shift assay. Neither the nonphosphorylated tyrosyl peptide nor an unrelated phosphotyrosyl peptide previously shown to inhibit the signal transducers and activators of transcription factor (STAT) blocked binding of the hER to the ERE. The hER phosphotyrosyl peptide was shown by molecular sizing chromatography to dissociate the hER dimer into monomers. The hER specifically bound the 32P-labeled phosphotyrosyl peptide, indicating that the inhibition of ERE binding was caused by the phosphotyrosyl peptide binding directly to the hER and blocking dimerization. These data suggest that the phosphorylation of tyrosine-537 is a necessary step for the formation of the hER dimer. In addition, we propose that the dimerization of the hER occurs by a previously unrecognized Src homology 2 domain (SH2)-like phosphotyrosyl coupling mechanism. Consequently, the phosphotyrosyl peptide represents a class of antagonists that inhibits estrogen action by a mechanism other than interacting with the receptor's hormone binding site.

Amino Acid Sequence↗

PubMed1995

In vivo and in vitro phosphorylation of the human estrogen receptor.

We report here that the human estrogen receptor (hER) overexpressed in Sf9 insect cells is phosphorylated similarly to hER from the human MCF-7 mammary carcinoma cell line. The recombinant and native hER labeled to steady-state with [32P]phosphate were purified to homogeneity using specific DNA-affinity chromatography followed by SDS-gel electrophoresis. Resolution of the hER tryptic digests by reverse phase-high performance liquid chromatography revealed that five [32P]phosphopeptides from the hER expressed in the Sf9 cells had retention times identical to five of the seven [32P]phosphopeptides from the hER in MCF-7 cells. Uniquely, a dephosphorylation of a single 32P-labeled peptide occurred in response to estradiol treatment of MCF-7 cells. In vitro protein kinase assays with the purified recombinant hER revealed that the DNA-dependent protein kinase (DNA-PK) phosphorylated the receptor and induced a decrease in the receptor's mobility as demonstrated by SDS-gel electrophoresis. In contrast, protein kinases A and C did not phosphorylate the purified recombinant hER. These results suggest that in the process of becoming transcriptionally active the estrogen receptor undergoes a dephosphorylation after estrogen-binding and subsequent phosphorylations, in part by the DNA-PK.

Animals↗

PubMed1995

Phosphorylation of the human estrogen receptor by mitogen-activated protein kinase and casein kinase II: consequence on DNA binding.

We determined the amino acid and radiolabel sequences of tryptic [32P]phosphopeptides of the purified human estrogen receptor (hER) from MCF-7 cells and Sf9 cells. Serine 118 was identified as a site that was phosphorylated independently of estradiol-binding in MCF-7 cells. Proline is on the carboxy terminus of serine 118, which suggests that the serine-proline may be a consensus phosphorylation site motif for either the mitogen-activated protein (MAP) kinase or p34cdc2 kinase. MAP kinase selectively phosphorylated the recombinant hER in vitro on serine 118 independent of estradiol-binding, whereas p34cdc2 did not phosphorylate the hER. We demonstrated previously that serine 167 of the hER was phosphorylated in an estradiol-dependent manner. We therefore compared the consequence of hER phosphorylation at serine 118 by MAP kinase and phosphorylation at serine 167 by casein kinase II on the receptor's affinity for specific DNA binding. The binding of the hER to an estrogen response element was not altered by phosphorylation with MAP kinase at serine 118 but was significantly increased when phosphorylated at serine 167 by casein kinase II. These data suggest that phosphorylation of the hER by MAP kinase(s) pathways may influence receptor action by a mechanism other than the estradiol-dependent phosphorylation of hER by casein kinase II.

Adenosine Triphosphate↗

PubMed1995

Phosphorylation of the human estrogen receptor on tyrosine 537 in vivo and by src family tyrosine kinases in vitro.

Its reactivity to the antiphosphotyrosine 4G10 monoclonal antibody by Western blot analysis demonstrated that the human estrogen receptor (hER) from human MCF-7 cells and the recombinant hER expressed in Sf9 insect cells were phosphorylated on tyrosine(s). Reverse phase-HPLC separation of a tryptic digest of the 32P-labeled purified hER from Sf9 and MCF-7 cells followed by amino acid and radiolabel sequencing revealed that tyrosine-537 was phosphorylated. The phosphorylation on tyrosine-537 was independent of estradiol treatment of MCF-7 cells, indicating that tyrosine-537 is a basal phosphorylation site. Two src family tyrosine kinases, p60c-src and p56lck, phosphorylated the purified recombinant hER on tyrosine-537 in vitro. In addition, two tyrosine phosphatases, protein tyrosine phosphatase-1B and src homology-2 protein tyrosine phosphatase-1, dephosphorylated phosphotyrosine-537 of the hER in vitro. These data suggest that tyrosine phosphorylation of the hER is regulated by potentially oncogenic tyrosine kinases and phosphatases that may modulate the function of ER in normal and/or abnormal cell growth.

Animals↗

PubMed1994

Serine 167 is the major estradiol-induced phosphorylation site on the human estrogen receptor.

Serine 167 has been identified by radiolabel and amino acid sequencing as the major estrogen-induced phosphorylation site on the human estrogen receptor (hER) from human MCF-7 mammary carcinoma cells. The phosphorylation of the hER on serine 167 was estrogen-dependent, increasing 4-fold upon estradiol treatment of MCF-7 cells and accounted for almost half of the total [32P]phosphate incorporated into the recombinant hER from Sf9 insect cells and the native hER from MCF-7 cells. Casein kinase II was found to phosphorylate the purified recombinant hER on serine 167 in vitro. In addition, estradiol binding enhanced by 2-fold the phosphorylation of the purified recombinant hER by casein kinase II in vitro. Western blot analysis and [32P]phosphate incorporation confirmed the presence of casein kinase II in Sf9 cells. These results demonstrate that the hER is phosphorylated on serine 167 by casein kinase II in a hormone-dependent manner.

Animals↗

PubMedDate not supplied

Weak estrogenic activity from continuous-release pellets.

In the process of evaluating a proprietary compound for weak estrogenic activity, two different types of dosing regimens were used, repeated daily dosing (three times/d) and continuous-release pellets. In studies using the proprietary compound, rats that were dosed via intraperitoneal injection showed no estrogenic responses, while those receiving the test compound via continuous-release pellets displayed several estrogenic responses. Because of the conflicting results, the control pellets were evaluated for estrogenic activity in the same battery of tests using the same number of pellets. In studies using only the control pellets, several estrogenic responses were observed including increased uterine weight, uterine stromal cell proliferation, estrous conversion, uterine progesterone receptor content, and decreased uterine estrogen receptor content. Animals receiving no pellet implant showed no estrogenic responses. In addition, a methylene chloride/DMSO extract of the control pellets promoted expression of a reporter gene controlled by the estrogen receptor and demonstrated competition with 17 beta-estradiol for binding to the human estrogen receptor. It is concluded that component(s) of the control pellets possess weak estrogenic activity.

Animals↗

PubMedDate not supplied

Cellular bioavailability of natural hormones and environmental contaminants as a function of serum and cytosolic binding factors.

Environmental contaminants have been reported to function as hormone mimics in various wildlife species. To investigate a potential mechanism for the interaction of contaminants with the endocrine system, we evaluated the cellular bioavailability of numerous chemicals. Hormone binding proteins from oviductal cytosol of the American alligator (Alligator mississippiensis) and yellow-bellied turtle (Trachemys scripta) were used in competitive binding assays with [3H] 17 beta-estradiol. Most of the environmental contaminants, and the potent, synthetic estrogen diethylstilbestrol (DES), did not interact with the cytosolic binding proteins. Among the compounds tested, o,p'-DDT and toxaphene exhibited the greatest affinity for the binding proteins. The functional consequence of the apparent lack of interaction of most contaminants with binding proteins was studied in a strain of yeast containing the human estrogen receptor (YES assay). The activation of YES with estradiol was reduced 30% in the presence of a physiological concentration (0.01 mg/mL) of human sex hormone binding globulin (SHBG), a hormone binding protein found in the blood. In contrast, the activity of DES was not inhibited by 0.01 mg/mL SHBG. Interestingly, ethinyl estradiol, a major component of contraceptives, did not appear to appreciably interact with SHBG in the YES system. Together, these data suggest that cytosolic and circulating binding proteins bind many environmental contaminants with much less affinity than native steroids. Therefore, such contaminants may be more hormonally active than previously hypothesized.

Alligators and Crocodiles↗