Biomedical subjects

Paul F Terranova

Publications and source records attributed to Paul F Terranova.

14 recordsLinked to original sources

PubMed2006

Tumor necrosis factor (TNF) receptor type 2 is an important mediator of TNF alpha function in the mouse ovary.

It is believed that a finite pool of primordial follicles is established during embryonic and neonatal life. At birth, the mouse ovary consists of clusters of interconnected oocytes surrounded by pregranulosa cells. Shortly after birth these structures, termed germ cell cysts or nests (GCN), break down to facilitate primordial follicle formation. Tumor necrosis factor alpha (TNF) is a widely expressed protein with myriad functions. TNF is expressed in the ovary and may regulate GCN breakdown in rats. We investigated whether it participates in GCN breakdown and follicle formation in mice by using an in vitro ovary culture system as well as mutant animal models. We found that TNF and both receptors (TNFRSF1A and TNFRSF1B) are expressed in neonatal mouse ovaries and that TNF promotes oocyte death in neonatal ovaries in vitro. However, deletion of either receptor did not affect follicle endowment, suggesting that TNF does not regulate GCN breakdown in vivo. Tnfrsf1b deletion led to an apparent acceleration of follicular growth and a concomitant expansion of the primordial follicle population. This expansion of the primordial follicle population does not appear to be due to decreased primordial follicle atresia, although this cannot be ruled out completely. This study demonstrates that mouse oocytes express both TNF receptors and are sensitive to TNF-induced death. Additionally, TNFRSF1B is demonstrated to be an important mediator of TNF function in the mouse ovary and an important regulator of folliculogenesis.

Animals↗

PubMed2006

Conditional Akt activation promotes androgen-independent progression of prostate cancer.

Aggressive androgen-independent (also termed as hormone-refractory) prostate cancer is a major clinical obstacle because there is no means to cure. Previous studies have shown that Akt activation is associated with prostate cancer progression from androgen-dependent to androgen-independent stage. However, its causative role in this process has not been established. One of the major limitations is the lack of a well-controlled inducible system to study Akt involvement. Recently, we developed a novel inducible Akt (iAKT) system based on a chemically induced dimerization (CID) approach. This system allows for conditional activation of Akt in a physiological setting. Utilizing this iAKT system, we found that Akt activation prevented cell death after serum withdrawal and promoted cell proliferation in the absence of androgen in vitro in human prostate cancer LNCaP cells, which should stop growing after androgen withdrawal or even die after serum starvation. The iAKT-induced death protection and growth promotion were further demonstrated in vivo using a transgenic mouse model that expresses the iAKT system conditionally in the prostate epithelium. Most importantly, in a mouse xenograft model derived from LNCaP cells, iAKT activation promoted tumor growth in castrated animals by enhancing cell proliferation and inhibiting apoptosis. Taken together, our data suggest that Akt activation is playing a causative role in androgen-independent progression of prostate cancer. This study provides a significant relevance of Akt-targeted therapy for hormone-refractory prostate cancers.

Androgens↗

PubMed2005

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) alter body weight by decreasing insulin-like growth factor I (IGF-I) signaling.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) affects glycemia due to reduced gluconeogenesis; when combined with a reduction in feed intake, this culminates in decreased body weight. We investigated the effects of steady-state levels of TCDD (loading dose rates of 0.0125, 0.05, 0.2, 0.8, and 3.2 microg/kg) or approximately isoeffective dose rates of 1,2,3,4,7,8-hexachlorodibenzo-p-dioxin (HxCDD) (loading dose rates of 0.3125, 1.25, 5, 20, and 80 microg/kg) on body weight, phosphoenolpyruvate carboxykinase (PEPCK) mRNA expression and activity, and circulating concentrations of insulin, glucose, and insulin-like growth factor-I (IGF-I), and expression of hepatic phosphorylated AMP kinase-alpha (p-AMPK) protein in female Sprague-Dawley rats (approximately 250 gm) at 2, 4, 8, 16, 32, 64, and 128 days after commencement of treatment. At the 0.05 and 1.25 microg/kg loading dose rates of TCDD and HxCDD, respectively, there was a slight increase in body weight as compared to controls, whereas at the 3.2 and 80 microg/kg loading dose rates of TCDD and HxCDD, respectively, body weight of the rats was significantly decreased. TCDD and HxCDD also inhibited PEPCK activity in a dose-dependent fashion, as demonstrated by reductions in PEPCK mRNA and protein. Serum IGF-I levels of rats treated initially with 3.2 microg/kg TCDD or 80 microg/kg HxCDD started to decline at day 4 and decreased to about 40% of levels seen in controls after day 16, remaining low for the duration of the study. Eight days after initial dosing, hepatic p-AMPK protein was increased in a dose-dependent manner with higher doses of TCDD and HxCDD. There was no effect with any dose of TCDD or HxCDD on circulating insulin or glucose levels. In conclusion, doses of TCDD or HxCDD that began to inhibit body weight in female rats also started to inhibit PEPCK, inhibited IGF-I, while at the same time inducing p-AMPK.

Adenylate Kinase↗

PubMed2005

Alterations in reproductive function in SRC tyrosine kinase knockout mice.

The role of Src tyrosine kinase in regulating reproductive processes in female mice was investigated using Src wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice. Ovarian Src kinase activity transiently increased in Src +/+ mice following eCG administration. Src knockout mice were infertile. Estrous cycles and vaginal opening in Src knockouts were variable and altered compared with Src +/+ and +/- mice. Follicle development was compromised in Src knockout mice as evidenced by reduced numbers of large pre-antral and antral follicles compared to Src +/+ mice. Corpora lutea were not observed in the ovaries of Src knockout mice; however, administration of eCG and hCG did result in ovulation. Serum LH and FSH on d 40 and 52 of age did not differ between Src wild-type and knockout females. Results from these studies reveal that female Src knockout mice are infertile due to reduced follicle development and anovulation.

Animals↗

PubMed2005

Tumor necrosis factor alpha (TNF) suppresses cAMP response element (CRE) activity and nuclear CRE binding protein in MA-10 mouse Leydig tumor cells.

TNF is known to suppress gonadotropin-induced steroid secretion by Leydig cells. However, the mechanisms by which this occurs are largely unknown. Because expression of many steroidogenic proteins is regulated by the PKA pathway, effects of TNF on CRE activity were examined using MA-10 mouse Leydig tumor cells. The cells were transfected with a CRE-luciferase construct, and stimulated with either LH or 8Br-cAMP in the presence or absence of TNF. TNF suppressed, LH-stimulated and 8Br-cAMP stimulated CRE activity. TNF also suppressed CRE activity stimulated with a PKA expression vector. Further experiments suggested that the effect of TNF on CRE activity was not mediated by the NF-kappaB pathway. TNF did not affect levels of either CREB or phospho-CREB in whole cell lysates; however, TNF decreased both CREB and phospho-CREB in nuclear extracts in a time-dependent manner. The decrease in nuclear CREB is likely to be a major mechanism of the suppressive effects of TNF on steroidogenesis in MA-10 Leydig cells.

Animals↗

PubMed2004

Tumor necrosis factor-alpha induces serum amyloid A3 in mouse granulosa cells.

TNF-alpha has significant inhibitory effects on steroidogenesis and folliculogenesis and is associated with several inflammatory responses. Because ovulation is an inflammatory reaction, the effects of TNF on the family of acute-phase proteins in granulosa cells were investigated. Granulosa cells from immature mice at 28 d of age were cultured in the presence of 10 ng TNF/ml for 24 h. Serum amyloid A3 (SAA3), a main acute-phase protein, was induced by TNF in granulosa cells. The other isoforms of serum amyloid proteins SAA1, SAA2, and SAA4 were neither expressed in granulosa cells nor induced by TNF. TNF did not induce SAA3 mRNA in granulosa cells from TNF receptor type 1 (TNFR1) knockout mice, although SAA3 mRNA was induced within 3 h after TNF treatment in wild-type cells. Two SAA3 promoters, -617/+73 and -198/+73, were responsive to TNF and to p65, a component of the TNF signaling molecule nuclear factor (NF)-kappaB. The -106/+73 promoter of SAA3 lacking a NF-kappaB-like site was not responsive to TNF or p65. In granulosa cells from TNFR1 knockout mice, the SAA3 promoter (-198/+73) was responsive to transfection with the p65 component of NF-kappaB, but neither TNF treatment nor overexpression of the p50 component of NF-kappaB increased promoter activity. Similar results were observed in the murine ovarian granulosa tumor cell line (OV3121-1). Overexpression of the inhibitor of NF-kappaB (called IkappaB) blocked SAA3 promoter activity induced by TNF and by p65 in OV3121-1 cells. Closer analysis of deletion mutants of the SAA3 promoter revealed the necessity of a NF-kappaB like site for responsiveness to TNF in the OV3121-1 cells. TNF rapidly increased p65 in OV3121-1 nuclei when compared with controls not treated with TNF. TNF also increased phospho-IkB and SAA3 in whole-cell homogenates as determined by Western blots. Thus, TNF likely increased SAA3 promoter activity and protein by activating NF-kappaB signaling via TNFR1 in mouse granulosa cells. SAA3 is a novel gene in granulosa cells with yet unknown functions in the ovary.

Animals↗

PubMed2004

Protein kinase C delta is activated in mouse ovarian surface epithelial cancer cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

Interactions between the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and protein kinase C (PKC) signaling pathways are governed in cell and tissue-specific manners, albeit the physiological significance of which is unclear. This research sought to define the effects of TCDD on the PKC pathway using a mouse ovarian surface epithelial cancer cell line (ID8). Phorbol-12-myristate-13-acetate (PMA) potentiated (1 nM) TCDD-induced 7-ethoxyresorufin-O-deethylase (EROD) activity after 24h of treatment, and pre-treatment with (1 microM) of either a general PKC inhibitor (BisI) or PKCdelta-specific inhibitor (Rotterlin) abolished the potentiation indicating that activation of PKC enhances TCDD signal transduction. Western blot analysis revealed that unstimulated ID8 cells express PKCalpha, beta, epsilon, tau, lambda and RACK1. PKCgamma, eta, theta and DGKtheta were not detected. TCDD (1 nM) increased PKCdelta protein approximately eight-fold after 24h of treatment and this effect was dose-dependent (0.1-100 nM); other PKC isoforms and related signaling proteins tested were unaffected by TCDD treatment. Immunofluorescent microscopy revealed that TCDD (1 nM) promoted the subcellular redistribution of PKCdelta, from the cytoplasm and the nucleus to the perinuclear area after 2h of treatment, however, after 24h of treatment PKCdelta was observed in nuclear structures that resembled nucleoli. TCDD (1 nM) also increased total PKC and PKCdelta-specific kinase activities in biphasic time-responsive manners. Total PKC and PKCdelta-specific activities increased after 1-2h of treatment. Then TCDD increased the total PKC activity again after 12h of treatment, whereas, PKCdelta-specific activity resurged at 24h and remained elevated at 48 h after treatment. The results indicate that TCDD preferentially induces PKCdelta protein expression and phosphotransferase activity, and its membrane translocation, indicating a potential intracellular role for PKCdelta as an effector molecule for TCDD-mediated biological events in this ovarian cancer cell line.

Animals↗

PubMed2004

Src tyrosine kinase regulates CYP17 expression and androstenedione secretion in theca-enriched mouse ovarian cells.

Src tyrosine kinase belongs to a non-receptor tyrosine kinase family and has been shown to be involved in G protein-coupled receptor desensitization and internalization. Stimulation of ovarian thecal cells with lutein-izing hormone (LH) activates adenylyl cyclase via a G protein-coupled LH receptor leading to an increase in cAMP. Subsequently, cAMP activates protein kinase A (PKA) that increases steroidogenesis. In order to evaluate the role of Src in thecal cell steroidogenesis, a pharmacological approach was utilized by treating a population of mouse ovarian theca-enriched cells (TEC) in vitro with two Src inhibitors, geldanamycin (GA) and herbimycin A (HA). Treatment of TEC with either GA or HA increased basal androstenedione secretion without alteration of cAMP. In the presence of forskolin, GA and HA treatment further increased androstenedione secretion. RT-PCR analysis of RNA from cells treated with GA for 8, 24, and 48 h revealed that GA increased cytochrome P450 17alpha-hydroxylase/lyase (CYP17) mRNA at 48 h. CYP17 promoter activity also increased after treatment of cells with GA and after co-transfection with a Src dominant negative plasmid. Inhibition of PKA using H89 blocked the effect GA and HA on androstenedione secretion. These results indicate that the pharmacological inhibitors of Src, GA and HA, tested in vitro increased thecal CYP17 promoter activity, CYP17 mRNA, and androstenedione secretion. In addition, GA and HA induced thecal androstenedione secretion may be cAMP independent but possibly requires PKA.

3-Hydroxysteroid Dehydrogenases↗

PubMed2003

Tumor necrosis factor alpha (TNF) increases granulosa cell proliferation: dependence on c-Jun and TNF receptor type 1.

TNF alpha has significant in vitro effects on steroidogenesis and folliculogenesis and reproductive alterations occur in TNF receptor type 1 (TNFR1) knockout mice. The present study investigated the effect of in vitro TNF on granulosa cell proliferation from immature mice at 28 d of age, with emphasis on intracellular signaling that regulates granulosa cell proliferation. TNF dose dependently increased granulosa cell proliferation and the proto-oncogene c-Jun protein. However, other Jun family members such as JunD was expressed constitutively and JunB was not expressed. In vitro TNF did not increase c-Jun and proliferation in granulosa cells from TNFR1 knockout mice. The time course of TNF-induced c-Jun revealed biphasic patterns of short-term (3 h) and long-term (24 h) induction. The time courses of Ser63- and Ser73-phospho c-Jun coincided with changes in total c-Jun. Among MAPK cascades, stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling was increased transiently in TNF-treated cells, whereas p38MAPK and ERK1 and 2 were not changed. In addition, overexpression of nuclear factor-kappa B and addition of ceramide and 8-bromo-cAMP did not increase c-Jun or proliferation. Antisense oligonucleotides for c-Jun blocked cell proliferation induced by TNF. In conclusion, the above results demonstrate that TNF increased c-Jun by activating stress-activated protein kinase/c-Jun-NH(2)-teminal kinase signaling via TNFR1 in mouse granulosa cells, and the induced c-Jun resulted in increased cell proliferation.

Age Factors↗

PubMed2003

Src tyrosine kinase promotes survival and resistance to chemotherapeutics in a mouse ovarian cancer cell line.

The vast majority of ovarian cancers originate in the ovarian surface epithelium. Unfortunately, there is a lack of appropriate animal models for ovarian cancer research. Spontaneously transformed mouse ovarian surface epithelial cells may provide a faithful animal model for human ovarian cancer. One such cell line (ID8) has been partially characterized. ID8 cells demonstrate constitutive Src tyrosine kinase activation with resulting phosphatidylinositol-3-kinase activation and Akt and forkhead phosphorylation. In addition, focal adhesion kinase is constitutively phosphorylated at tyrosine 925, a Src phosphorylation site, resulting in increased Ras activation. These features are common to human ovarian cancer cell lines. Inhibition of Src enhances the cell killing effects of both paclitaxel and cisplatinum. Finally, Src inhibition restores sensitivity of a drug resistant ID8 cell line. The ID8 mouse ovarian cancer cell line presents new opportunities to study ovarian cancer progression and pre-therapeutic trials in an immune competent background.

Animals↗

PubMed2002

Endocrine disruption by indole-3-carbinol and tamoxifen: blockage of ovulation.

Immature Sprague-Dawley rats received daily doses of indole-3-carbinol (I3C, 0-1.5 g/kg/day), 3,3'-diindolymethane (DIM, 0-400 mg/kg/day), tamoxifen (TAM, 0-0.5 mg/kg/day), or vehicle to determine if their antiestrogenic effects occur by the same mechanism and whether I3C's action is mediated by DIM. Follicular development was induced on day 24 of age by equine chorionic gonadotropin (eCG, 5 IU) 1 day after the initial dose. In a hormone replacement study, human chorionic gonadotropin (hCG, 10 IU sc, 48 h post-eCG) was used to mimic a normal preovulatoy luteinizing hormone (LH) surge following treatment with either I3C or TAM. Blood and ovaries were collected throughout follicular development and the number of ova shed was measured on the morning following expected ovulation (72 h post-eCG). I3C but not TAM reduced body weight gain at higher doses after 4 days of dosing. Ovarian weight gain and ovulation were inhibited by both I3C and TAM in a dose-dependent fashion. During the preovulatory period, both I3C and TAM blocked normal LH and follicle-stimulating hormone (FSH) surges and suppressed serum progesterone (P(4)) profoundly without changing circulating levels of estrogen (E(2)). At the time of expected ovulation, serum E(2) was increased in rats receiving I3C or tamoxifen, whereas serum P(4) was dose-dependently decreased. DIM exerted no significant effects on any of the endpoints studied, even at the highest dose, indicating that the antiestrogenic effects of I3C are not mediated by this metabolite of I3C. hCG successfully restored ovarian weight gain and ovulation in TAM-treated rats. However, hCG only partially reversed the blockage of ovulation by I3C, although ovarian weight gain was restored to normal. In summary, both I3C and TAM block ovulation by altering preovulatory concentrations of LH and FSH, but I3C appears to exert its effect(s) by (a) different mechanism(s) of action. I3C seems to act at both the ovarian and hypothalamic levels by mechanisms similar to those seen in TCDD-treated rats, whereas TAM appears to act only on the hypothalamic-pituitary axis as an anti-estrogen.

Animals↗

PubMed2002

Estradiol enhances and estriol inhibits the expression of CYP1A1 induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in a mouse ovarian cancer cell line.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a ubiquitous pollutant and promoter of carcinogenesis. This study investigated the interaction between TCDD and different estrogens in a cancer cell line (ID8) derived from mouse ovarian epithelium. TCDD-induced ethoxyresorufin-O-deethylase (EROD) activity and cytochrome P4501A1 (CYP1A1) expression in a dose- and time-dependent manner. Estrogen receptor (ER) alpha mRNAs were constitutively expressed, but ER beta and progesterone receptor (PR) mRNAs were not expressed. Induction of EROD by TCDD was completely inhibited by a alpha-naphthoflavone and phenanthroline, two aryl hydrocarbon receptor (AhR) antagonists. Progesterone and gonadotropins (FSH and LH) had no effect on the induction of EROD by TCDD. Congeners of 17beta-estradiol (E2) increased the induction of EROD activity by TCDD dose-dependently in the relative potency order: estrone (El)>E2> or = 4-hydroxyestradiol (4OHE2)> or = 2-hydroxyestradiol (2OHE2). In contrast, estriol (E3) decreased EROD activity induced by TCDD. E2 increased TCDD-induced CYP1A1 protein and mRNA whereas E3 decreased both the protein and mRNA. E2 did not alter luciferase activity induced by TCDD in cells transfected with a luciferase reporter containing dioxin response elements (DRE) or a CYP1A1 promoter. In contrast, E3 dose-dependently decreased the luciferase activity. A pure anti-estrogen (ICI 182780) inhibited the interaction between E2 and TCDD but did not block E3's effect on EROD activity. These results indicate that E2 may affect TCDD-induced CYP1A1 expression by a mechanism different from E3 in ID8 cells. It appears that the potentiation of E2 in the induction of CYP1A1 by TCDD occurs by a mechanism involving ER alpha since a specific ER antagonist blocked the potentiation. The inhibitory effect of E3 may be due to a rapid direct effect on EROD and a later suppression of CYP1A1 expression.

Animals↗

PubMedDate not supplied

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on serum inhibin concentrations and inhibin immunostaining during follicular development in female Sprague-Dawley rats.

Intact and hypophysectomized immature rats were pretreated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0 or 32 microg/kg p.o.) and sacrificed throughout synchronized follicular development (0, 12, 24, 48, and 72 h after equine chorionic gonadotropin, eCG). TCDD administration to intact rats resulted in a premature elevation of serum FSH and LH by 12 h post-eCG. In intact rats pretreated with TCDD, the intensity of ovarian immunoreactivity for inhibin and the number of ovarian follicles staining for inhibin in midsaggital ovarian sections were decreased at the time of eCG administration (24 h post-TCDD) in comparison to controls. However, this decreased ovarian staining for inhibin was not associated with alterations in serum inhibin concentrations. Serum inhibin was suppressed in TCDD-treated rats when compared to intact controls only at 24 h post-eCG. Hypophysectomized animals exhibited no effect of TCDD on serum inhibin at any timepoint but did have decreased estradiol concentrations during follicular development. In summary, TCDD reduced serum concentrations of inhibin after the premature increases in FSH and LH suggesting that inhibin is not important in the initial elevation of FSH following exposure to TCDD.

Animals↗

PubMedDate not supplied

Alteration in ovarian gene expression in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin: reduction of cyclooxygenase-2 in the blockage of ovulation.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a reproductive toxicant and endocrine disrupter that is known to block ovulation. This study was designed to investigate alterations in relevant ovarian genes that may be involved in the blockage of ovulation by TCDD in immature intact rats primed with equine chorionic gonadotropin (eCG). In this ovulation model, rats were given either 32 microg/kg TCDD or corn oil by gavage on 25 days of age. The next day, eCG (5 IU) was injected subcutaneously (s.c.) to stimulate follicular development. Ovulation occurs 72 h after administration of eCG in controls of this model. TCDD blocked ovulation at the expected time and also reduced both ovarian and body weights. At 72 h after eCG (the morning after expected ovulation), TCDD did not alter significantly serum concentrations of progesterone (P4) and androstenedione (A4). However, estradiol (E2) was significantly higher at 72 h after eCG in TCDD-treated rats when compared with controls. Western blots revealed that ovarian CYP1A1 was induced by TCDD. In addition, the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (ARNT) were down- and up-regulated by TCDD, respectively, indicating that AhR-mediated signal transduction was altered in the ovary. Ovarian estrogen receptor (ER)alpha, ER beta and progesterone receptor (PR) were not altered significantly by TCDD, but ovarian glucocorticoid receptor (GR) was increased at 24h after TCDD and decreased at 72 h after eCG when compared with controls. TCDD induced the early appearance of ovarian plasminogen activator inhibitor type-1 (PAI-1), plasminogen activator inhibitor type-2 (PAI-2), urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA) at 24h after dosing when compared with controls. On the morning after ovulation (72 h after eCG), no significant differences between control and TCDD-treated rats were observed except that TCDD had still increased tPA and decreased PAI-2 when compared with controls. Interestingly, ovarian COX-2 was induced on the morning after ovulation (72 h after eCG) in controls, but was greatly inhibited in TCDD-treated rats at that time. On the other hand, COX-1 was constitutively expressed throughout the ovulatory period and remained unaffected by TCDD. Immunolocalization of COX-2 in the ovary revealed that TCDD inhibited COX-2 expression in the granulosa cell layer when assessed in the morning of expected ovulation. In conclusion, AhR signaling is activated in the ovary by TCDD and inhibition of COX-2 appeared to be a critical step in the TCDD blockage of ovulation because blockage or reduction of COX-2 expression is well known to be associated with failure of ovulation.

Administration, Oral↗