Biomedical subjects

C R Lyttle

Publications and source records attributed to C R Lyttle.

At least 37 records · Page 2Linked to original sources

PubMed1992

Leukocyte chemotactic activity of cyclophilin.

During the purification of eosinophil chemotactic factors synthesized by the uterus in response to estrogen we isolated a protein having an N-terminal (15 amino acids) sequence identical to that of rat cyclophilin. Our data demonstrate that cyclophilin, a cytosolic protein isolated from bovine thymocytes, which specifically binds the immunosuppressive drug cyclosporin A, as well as recombinant human cyclophilin, displays eosinophil chemotactic activity. In addition to its chemotactic activity, cyclophilin stimulated the release of peroxidase activity from eosinophils. Maximal chemotactic activity of cyclophilin was achieved at a concentration of approximately 10 nM. At similar concentrations cyclophilin was also able to stimulate the migration of neutrophils. This chemotactic activity could be prevented by the addition of cyclosporin A, but not by a nonimmunosuppressive analog (1-fur-furyl-cyclosporin A) at similar concentrations. This chemotactic activity may represent an additional mechanism by which immunosuppressive drugs function to prevent tissue rejection.

Amino Acid Isomerases↗

PubMed1992

Hormonal regulation of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase in rat liver cytosol.

In rat liver, dihydrodiol dehydrogenase and 3 alpha-hydroxysteroid dehydrogenase activities are catalyzed by the same protein. This study shows that estrogen and growth hormone can induce the enzyme in hypophysectomized rats. This implies that estrogen can exert an effect on hepatic steroid and carcinogen metabolism in the absence of the anterior pituitary.

3-Hydroxysteroid Dehydrogenases↗

PubMed1992

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.

Amino Acid Sequence↗

PubMed1992

Progesterone and RU486 regulation of uterine complement C3 after prior induction with estradiol.

Previous results demonstrated that progesterone (P4) given simultaneously with estradiol (E2) prevented stimulation by E2 of complement C3 expression in the immature rat uterus. Northern blot analysis revealed that simultaneous administration of P4 was able to prevent the E2-stimulated increase in C3 mRNA concentration in the luminal epithelial cells. The purpose of the present investigation was to determine whether progesterone modulates C3 expression after the gene has been induced by prior administration of E2 and also to determine the reversibility of this effect by the concomitant administration of RU38486, 17 beta-hydroxy-11 beta-[4-dimethylaminophenyl]estra-4,9,-dien-3-one (RU486). This regulation was studied by examination of protein synthesis as well as mRNA concentrations. Immature 21-day-old female rats were treated with E2 for 2 days (1 microgram/day), followed 24 h later by P4 (500 micrograms) or vehicle. Uteri were removed 6, 9, and 18 h after progesterone treatment and the radiolabeled secreted proteins were analyzed by SDS-PAGE and immunoprecipitation using a goat anti-rat C3 antibody. In animals treated with vehicle, E2-stimulated C3 synthesis remained elevated at 6 and 9 h and returned to control values by 18 h. In contrast, the administration of P4 resulted in a decrease in C3 synthesis at 6 and 9 h with the greatest decrease observed at 9 h. Similar results were obtained when C3 mRNA concentrations were examined. E2-stimulated C3 mRNA concentrations were decreased in rats treated with progesterone compared to those treated with vehicle alone.2

Animals↗

PubMed1992

Estrogen receptors in fracture healing.

Estrogen has profound effects on the regulation of bone metabolism, but its role in fracture healing is unknown. Several recent reports have documented the presence of estrogen receptors in vitro and in diseased tissue. The authors investigated estrogen receptors in nonneoplastic skeletal tissues by immunohistochemical and radioligand binding techniques. Using a fibular osteotomy model of fracture healing in New Zealand rabbits, radioligand binding detected specific, high affinity, saturable estradiol binding sites at low levels throughout fracture healing, with a trend towards a bimodal distribution. Peaks occurred three and 16 days after osteotomy. No estrogen receptor was found by either method in later fracture callus, growth plate, or periosteum. These findings suggest a possible role for estrogen in the early inductive phase and later phase of endochondral ossification in fracture healing.

Animals↗

PubMed1991

Progesterone augments proliferation induced by epidermal growth factor in a feline mammary adenocarcinoma cell line.

Steroid hormones and peptide growth factors promote growth and development of normal mammary tissues and some types of breast cancer. Ovarian steroids may influence mammary growth directly or indirectly. The epidermal growth factor (EGF) family of proteins may also regulate mammary growth. These two pathways may function independently of each other or they may act in concert, with steroids inducing transcription of genes that encode growth factors or growth factor receptors. We used a feline mammary adenocarcinoma cell line (K12) to address whether there was an interrelation between progesterone (PGN) and EGF-associated growth pathways. K12 cells responded to EGF by a dose-dependent increase in proliferation. PGN or promegestone (R5020, a synthetic progestagen) alone did not stimulate K12 growth, but when EGF and PGN, or EGF and R5020 were combined, they were synergistic. This synergistic response was abrogated by the PGN receptor antagonist RU486 or by antibodies that blocked binding of EGF to its receptor. K12 cells expressed characteristic double-affinity EGF receptors, as well as p185 (a functionally and structurally related protein, product of the neu gene) on their surface. PGN receptors were also found on intact cells and in cleared cytosols. Stimulation of K12 cells by PGN or by R5020 induced a two- to threefold increase in the number of high-affinity surface EGF receptors after 24 h. Stimulation of these cells by PGN also affected the relative levels of phosphorylation of the EGF receptor and p185 within minutes, but not of other cellular phosphoproteins. Our results show that PGN enhances the EGF-induced growth of K12 cells and suggest that this effect may be mediated at least partly via an increase in the number or function of high-affinity EGF receptors.

Adenocarcinoma↗

PubMed1991

Complement C3 synthesis, peroxidase activity and eosinophil chemotaxis in the rat uterus: effect of estradiol and testosterone.

Treatment of immature rats with estradiol (E2) produced a large increase in uterine peroxidase activity which was accompanied by an increase in eosinophil chemotactic factor (ECF-U). The synthesis of complement C3 was also induced in the uterus and the amount of this 180 kDa protein was determined both by immunoprecipitation and after separation by polyacrylamide gel electrophoresis. Testosterone (T) did not produce an increase in any of these parameters although it antagonized the estrogen-induced increase in uterine peroxidase activity and these effects were more pronounced in estrogen-primed animals. This antagonism was prevented by the antiandrogen, flutamide. Testosterone showed little effect on eosinophil chemotactic activity and did not inhibit the E2-stimulated synthesis of C3. The results with T were supported by the lack of any significant effect by flutamide which antagonizes receptor-mediated androgenic events. These findings are discussed in relation to the action of other types of hormonal steroids (progesterone, dexamethasone) in inhibiting these estrogen-induced molecular changes in the rat uterus and contribute to our understanding of steroid-steroid interaction and the regulation of uterine function.

Animals↗

PubMed1991

Ontogeny of the production of an estrogen-regulated eosinophil chemotactic factor in the rat uterus.

The ontogeny of an estrogen-induced eosinophil chemotactic activity in the uterus after estrogen administration and its regulation by progesterone was studied in neonatal rats at 3, 7, 10, and 13 days of age. These results were compared with the increase in peroxidase activity and the secretion of complement component C3 by the uterus. No chemotactic activity was observed in animals younger than 14 days, and after this age the estrogen regulation of the response was also prevented by the co-administration of progesterone. The secretion of complement C3 appeared earlier, at 10 days of age. It was also observed that the uterus of 3-7-day-old rats responded to estrogen by increasing the secretion of two proteins of 36 and 110 kDa. No further regulation or synthesis was observed with increasing age for these two proteins.

Aging↗

PubMed1991

Estradiol down-regulation of the rat uterine estrogen receptor.

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.

Animals↗

PubMed1991

The effect of estradiol on the production and secretion of complement component 3 by the rat uterus and surgically induced endometriotic tissue.

We have recently demonstrated that glandular epithelial cells isolated from human endometriotic tissue synthesize and secrete complement component 3 (C3). Furthermore, because C3 is capable of producing many of the immunological activities known to be associated with human endometriosis, we studied the production, secretion, and regulation of C3 using the rat model for endometriosis. Endometriosis was surgically induced in 20 adult female rats. The animals were then ovariectomized and one half were treated with estradiol (E2) for 3 days. Uterine luminal epithelial cells synthesized and secreted C3 only after E2 administration, whereas the uteri from control animals did not produce C3. In contrast, the ectopic endometrium from control animals produced and secreted C3, and this expression was strongly upregulated by in vivo E2 administration. We conclude that surgically induced endometriosis in the rat has properties biochemically independent from the intact uterus and may serve as a useful model to further investigate the regulation of C3 synthesis from human endometriosis.

Animals↗

PubMed1990

Antagonism of estrogen- and antiestrogen-induced uterine complement component C3 expression by ICI 164,384.

The uterus of the immature rat synthesizes and secretes complement component C3 in response to estradiol treatment. This response occurs in the uterine epithelial cells and is also stimulated by several antiestrogens including tamoxifen and LY117018. The administration of a new antiestrogen ICI 164,384 blocked the estradiol as well as the antiestrogen-stimulated increases in uterine weight, epithelial cell height, C3 synthesis and C3 mRNA. ICI 164,384 demonstrated no agonist properties in terms of epithelial cell response as determined by C3 expression.

Animals↗

PubMed1990

Estrogen regulation of placental alkaline phosphatase gene expression in a human endometrial adenocarcinoma cell line.

A human endometrial tumor (Ishikawa) cell line in culture responded to estradiol stimulation, as measured by growth and alkaline phosphatase activity. These effects were similar whether the medium was enriched with serum or was serum-free. Estradiol increased placental alkaline phosphatase activity 2-3-fold over control in these Ishikawa cells. The mechanism for this increase appeared to be at the level of transcription, at least in part, since there was an increase in the concentration of placental alkaline phosphatase mRNA. The administration of tamoxifen or 4-hydroxytamoxifen was unable to antagonize the estradiol-stimulated alkaline phosphatase enzyme activity or mRNA expression. The administration of tamoxifen alone had no effect on alkaline phosphatase enzyme activity, but tamoxifen did stimulate the steady state concentration of alkaline phosphatase mRNA. In contrast, a new antiestrogen, ICI 164,384, was able to antagonize both of these estradiol-stimulated effects.

Adenocarcinoma↗

PubMed1990

Progesterone regulation of estradiol-induced rat uterine secretory protein, complement C3.

Previously, using DNA sequencing, Northern and Southern analysis, and immunohistochemical data, we identified an estrogen-stimulated secretory protein as the third component of complement (C3). In this study, we demonstrate that progesterone modulated the estradiol regulation of C3 in immature rats as well as during the normal reproductive cycle. C3 was most abundant during estrus and reached its lowest concentration in diestrus. Immunoprecipitations reveal that progesterone prevented the estradiol-stimulated increase in radiolabeled C3 both in the media and tissue. The mechanism for the progesterone inhibition of estrogen-stimulated C3 appeared to be at the level of transcription or possibly mRNA stability since progesterone blocked the estradiol-stimulated increase in C3 mRNA.

Animals↗

PubMed1990

The stimulation of uterine complement component C3 gene expression by antiestrogens.

We have previously demonstrated that complement component C3 is regulated by estradiol in the rat uterus. The antiestrogens tamoxifen, LY117018, and LY156758 exert both agonist and antagonist effects on the immature rat uterus. In this study, these three antiestrogens also stimulated an increase in the synthesis and secretion of C3. The combination of LY117018 and estradiol did not increase C3 to a greater extent than LY117018 alone, which suggests a similar mechanism of regulation. The regulation may be transcriptional since both estradiol and tamoxifen increase the concentration of C3 mRNA. Results of in situ hybridization revealed that the increase in C3 mRNA occurred in the luminal epithelial cells. Although the induction by estradiol and the antiestrogens was similar in most aspects, the time course for tamoxifen-stimulated synthesis differed from estradiol in that the time required to achieve maximal concentrations of C3 was delayed by 12 h with tamoxifen. This pattern did not appear to be related to the time it took to convert tamoxifen to 4-hydroxytamoxifen since the C3 response for these antiestrogens were identical. The antiestrogen-stimulated increase in C3 synthesis and mRNA concentration was prevented by the co-administration of progesterone lending support to the hypothesis that the antiestrogens regulate C3 synthesis via a mechanism similar to estrogen.

Animals↗

PubMed1990

Glucocorticoid and progestin regulation of eosinophil chemotactic factor and complement C3 in the estrogen-treated rat uterus.

To study the steroid regulation of estradiol-induced uterine eosinophil chemotactic factor activity (ECF-U) in the rat, we injected immature female rats with combinations of estradiol, dexamethasone, progesterone, and the antihormones RU-486 and ketoconazole. An in vitro chemotactic system using blind well chambers and employing eosinophil-differentiated HL-60 promyelocytic cells was used as a bioassay for ECF-U activity. Dexamethasone and progesterone both antagonized the effects of estradiol on stimulation of ECF-U activity; neither hormone induced ECF-U activity when given alone. RU-486 and ketoconazole were able to block the inhibitory effects of dexamethasone and progesterone. The effects of dexamethasone and progesterone appear to be mediated through their specific nuclear receptors and are not due to direct effects on the chemotactic cell. Comparison of the steroid regulation of ECF-U activity with the estradiol-induced synthesis and secretion of complement component C3 showed that progesterone antagonized the effects of estradiol in both systems, whereas dexamethasone was antagonistic on ECF-U, but not on the secretion of C3. Taken together these results suggest that estradiol's effects in the rat uterus may be modulated by other steroids in at least two systems, bringing into question the common practice of studying uterine steroid actions by evaluation of a single protein or mRNA.

Animals↗

PubMed1990

Chemotactic capabilities of HL-60 human myeloid leukemia cells differentiated to eosinophils.

HL-60 human myeloid leukemia cells can be induced to differentiate into cells with many of the morphological, biochemical, and functional features of mature neutrophils, monocyte/macrophages, and eosinophils. HL-60 neutrophils are known to respond to several chemotactic compounds, but similar data do not exist for HL-60 eosinophils. We studied the chemotactic capabilities of eosinophil-differentiated HL-60 cells using a blind-well chamber technique. HL-60 eosinophils responded to the specific eosinophilotactic tetrapeptides Ala-Gly-Ser-Glu, Val-Gly-Asp-Glu, and Val-Gly-Ser-Glu; to the formylated tripeptide N-formyl-L-Met-L-Leu-L-Phe (fMLP); and to extracts estrogen-treated rat uteri, which contain an eosinophilotactin. Concentration optima for chemotaxis of HL-60 eosinophils were similar to those for normal human and rodent eosinophils. Neutrophil-differentiated HL-60 cells responded only to fMLP, whereas undifferentiated HL-60s showed no chemotactic ability. The time course of development of chemotactic competence paralleled other developmental changes in HL-60 eosinophilic differentiation, suggesting a common control mechanism. We conclude that the ability to respond to specific eosinophilotactic compounds is a feature of HL-60 cells that have differentiated to eosinophils and that this cell line may be used as a model system in studies of eosinophil chemotaxis.

Cell Differentiation↗

PubMed1990

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.

Complement C3↗

PubMed1989

Estrogen regulation of tissue-specific expression of complement C3.

The administration of estradiol to immature rats results in the increased synthesis and secretion of a 180-kDa protein, composed of 115- and 65-kDa subunits, by the uterine luminal epithelial cells. A monoclonal antibody against the 180-kDa protein was utilized to isolate the corresponding cDNA (LE-1) from a rat uterine luminal epithelial cell cDNA lambda gt11 expression library. This LE-1 cDNA was sequenced and shown to be homologous to complement component C3. The sequence was approximately 81 and 90% homologous to human and mouse C3, respectively. The LE-1 cDNA sequence was homologous with the 3' portion of the C3 mRNA containing the alpha subunit (115 kDa). Uterine mRNA isolated from immature rats treated with 1 microgram of estradiol for 24 h demonstrated a 25-fold increase in the concentration of a 6.0-kilobase mRNA by Northern hybridization with either LE-1 or authentic human C3 cDNA probes. To further examine the possibility that the estradiol-regulated secretory protein was C3, an aliquot of radiolabeled media protein from control and estradiol-stimulated rat uteri was incubated with goat anti-rat C3 antibody. The immunoprecipitated radiolabeled protein from estradiol-treated animals was increased significantly (p less than 0.01) compared to media from control animals. Analysis of the immunoprecipitated proteins on nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein of 180 kDa from estradiol-stimulated uterine media, whereas no detectable proteins were immunoprecipitated from media obtained from control uteri. Also, when the immunoprecipitated protein was reduced (20 mM dithiothreitol) it dissociated into two subunits of 115 and 65 kDa. Immunohistochemical studies demonstrated the presence of C3 only in the epithelial cells of estrogen-stimulated rat uteri. In addition, the estradiol-stimulated mRNA was only detectable in uterine epithelial cell RNA. Analysis of liver RNA demonstrated a 6.0-kilobase mRNA, as in the uterus, when hybridized with LE-1. However, unlike the uterus, its concentration was not influenced by estrogen administration with up to three daily injections of 100 micrograms of diethylstilbestrol. Based on biophysical, DNA sequence, and antibody data we conclude that rat uterine epithelial cells produce C3 in response to estradiol whereas the expression in the liver was not modulated by estrogens.

Animals↗