Dean Zaragoza - Academia.edu (original) (raw)

Papers by Dean Zaragoza

Biochimica Et Biophysica Acta, Feb 1, 2004

The promoter region of the human prostaglandin F2a receptor (FP) gene was isolated, sequenced, an... more The promoter region of the human prostaglandin F2a receptor (FP) gene was isolated, sequenced, and characterized. The 5V-flanking region has minimal homology to the bovine, mouse, and rat FP promoters with the exception of a 100-bp region. The human promoter similarly lacks a canonical TATA-box and a CAAT-box. Potential binding sites for SP-1, GATA-1, STAT-1, and AP-1 are present in the 5V-flanking region. One major transcription start site was identified using 5V RLM-RACE analysis and mapped to an adenine residue 262 nucleotides upstream from the initiator codon in exon 2. Transfection of HeLa cells with FP promoter-GFP deletion constructs indicates that the 2437/1946 region contains repressor activity. DNase I footprinting analysis of this region identifies a footprint over the GATA-like site at 2400. This suggests repression of basal FP transcription may be mediated by a GATA binding site. D 2003 Elsevier B.V. All rights reserved.

Molecular and cellular biology, 1998

The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly... more The macrolide antibiotic rapamycin inhibits cellular proliferation by interfering with the highly conserved TOR (for target of rapamycin) signaling pathway. Growth arrest of budding yeast cells treated with rapamycin is followed by the program of molecular events that characterizes entry into G0 (stationary phase), including the induction of polymerase (Pol) II genes typically expressed only in G0. Normally, progression into G0 is characterized by transcriptional repression of the Pol I and III genes. Here, we show that rapamycin treatment also causes the transcriptional repression of Pol I and III genes. The down-regulation of Pol III transcription is TOR dependent. While it coincides with translational repression by rapamycin, transcriptional repression is due in part to a translation-independent effect that is evident in extracts from a conditional tor2 mutant. Biochemical experiments reveal that RNA Pol III and probably transcription initiation factor TFIIIB are targets of repre...

Placenta

To study how the decidua contributes to parturition, we examined prostaglandin F(2alpha) concentr... more To study how the decidua contributes to parturition, we examined prostaglandin F(2alpha) concentrations as well as prostaglandin 15-hydroxy dehydrogenase, prostaglandin F(2alpha) receptor, matrix metalloproteinases 2 and 9, oxytocin receptor, prostaglandin-H synthase-2, and the prostaglandin E(2) receptor expression in human decidua. Decidual samples were isolated from placentas collected from patients at preterm not in labor (PTNIL), preterm labor (PTL), term not in labor (TNIL), and term labor (TL). For immunohistochemistry, fresh membranes which included chorion, amnion and decidua from term patients were collected. Prostaglandin F(2alpha) receptor mRNA was low in all preterm patients and then significantly increased towards term (p=0.049). Prostaglandin F(2alpha) receptor protein was identified in the amnion epithelium and mesoderm, chorion trophoblasts and decidua by immunohistochemistry, and levels were highest at TNIL (p=0.007) as measured by western blot. Prostaglandin F(2al...

Seminars in Reproductive Medicine, 2007

Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua... more Prostaglandins produced by the intrauterine tissues of both mother and fetus (myometrium, decidua, placenta, chorion, and amnion) are involved with all of the physiologies of parturition (membrane rupture, cervical dilatation, myometrial contractility, placental separation, and uterine involution). For parturition to occur, however, the intrauterine tissues need to first be activated to prepare for the work of labor, then stimulated to initiate labor. Prostaglandins normally are considered to be stimulators of the physiologies of labor. This review presents evidence that one prostaglandin, PGF2alpha, and its receptor, FP, are also activators, especially of the decidua. Stimulated by cytokines, the decidual synthesis of PGF2alpha and the expression of FP lead to increased matrix metalloproteinase activity, further enhancement of cytokine activity, increased decidual oxytocin and oxytocin receptor expression, decreased progesterone responsiveness, and possibly, enhanced expression of vascular endothelial growth factor. These collective actions prepare the decidua for its role in parturition.

Placenta, 2003

An increase in the myometrial expression of the prostaglandin (PG) receptors, and especially the ... more An increase in the myometrial expression of the prostaglandin (PG) receptors, and especially the PGF 2 receptor (FP), may be an important component of the process initiating preterm labour. In this review of the literature and presentation of new possibilities, evidence will be discussed that demonstrates an increase in mouse uterine FP mRNA occurs at preterm birth whereas uterine PGF 2 concentrations do not increase, suggesting elevated uterine receptor expression and sensitivity is a mechanism for preterm labour initiation. The first examination of the complete human myometrial FP promoter will be described and evidence presented that demonstrates the pro-inflammatory cytokine, interleukin-1 , stimulates FP mRNA expression. Finally new data showing that administration of a specific FP antagonist delays preterm birth in sheep will be presented.

Pediatric Research, 2004

Renal prostaglandins (PG), renin, and cortisol are necessary for normal kidney development and fu... more Renal prostaglandins (PG), renin, and cortisol are necessary for normal kidney development and function during fetal life. We examined the effects of cortisol infusion before completion of nephrogenesis (d 109 -116 gestation; 2.0 -3.0 mg hydrocortisone succinate/24 h) on the renal mRNA expression of PGHS-2, the PGE 2 receptors, EP 2 and EP 4 , and renin in fetal sheep. Cortisol infusion raised plasma cortisol levels to 42.8 Ϯ 6.0 nmol/L compared with saline infusion levels of 1.5 Ϯ 0.5 nmol/L (p Ͻ 0.001), but had no effect on fetal body weight, proportional kidney mass, or blood gases. Cortisol decreased significantly the relative expression of renin mRNA (saline: 0.93 Ϯ 0.06 units; cortisol: 0.32 Ϯ 0.03 units, p Ͻ 0.05), however it had no effect upon the expression of PGHS-2, EP 2 , or EP 4 mRNA in fetal sheep kidney. Although there is substantial evidence that PGE 2 acting through either the EP 2 or EP 4 receptor stimulates renin synthesis in the adult kidney, our results have demonstrated that before the completion of nephrogenesis, cortisol down-regulation of renin mRNA expression is independent of any change in the expression of PGHS-2, EP 2 , or EP 4 mRNA expression. During nephrogenesis, the insensitivity of PGHS-2, EP 2 , and EP 4 expression to down-regulation by cortisol may permit continued PG regulation of renal development and urine formation. Abbreviations PG, prostaglandins RAS, renin-angiotensin system PGHS-1 -r -2, prostaglandin endoperoxide G/H synthase RT, reverse transcription

Pediatric Research, 2002

There is evidence that fetal growth restriction is associated with impaired nephrogenesis and red... more There is evidence that fetal growth restriction is associated with impaired nephrogenesis and reduced numbers of mature nephrons at birth. It has been proposed that such impairment of renal growth may contribute to increased blood pressure in later life. Although prostaglandins (PG) play a key role in kidney development, it is unknown whether a poor fetal substrate supply alters the synthesis or actions of PG within the fetal kidney. Using real-time reverse transcriptase PCR, we have measured the effect of chronic placental restriction (PR) on the renal expression of PG endoperoxide G/H synthase-2 (PGHS-2), PGE 2 receptors EP 2 and EP 4 , and renin mRNA in the sheep fetus in late gestation. Restriction of placental growth reduced fetal body weight (PR: 3.2 Ϯ 0.2 kg, control: 4.8 Ϯ 0.2 kg) and total kidney weight (PR: 19.7 Ϯ 1.8 g, control: 25.1 Ϯ 1.3 g). Mean fetal arterial PO 2 was reduced by PR (PR: 15.03 Ϯ 0.67 mm Hg, control: 21.3 Ϯ 0.87 mm Hg). Renal PGHS-2 mRNA was increased in the PR group (PR: 2.26 Ϯ 0.38, control: 1.20 Ϯ 0.31) and was inversely related to mean fetal arterial PO 2 in the PR and control groups [PGHS-2: Ϫ0.17 (PO 2 ) ϩ 4.69, r 2 ϭ 0.26]. PR also increased renal EP 2 (PR: 1.57 ϩ 0.24, control: 0.82 ϩ 0.13) but not EP 4 mRNA. Renin mRNA was directly related to renal EP 2 [renin ϭ 0.37 (EP 2 ) ϩ 0.97, r 2 ϭ 0.29] and EP 4 , [renin ϭ 0.75 (EP 4 ) ϩ 0.44, r 2 ϭ 0.38] mRNA expression. Thus, the restriction of placental growth and associated chronic hypoxemia appear to increase the renal capacity to synthesize and respond to PG, which may play an important role in maintaining renin mRNA expression in the growth-restricted fetus.

Biochimica Et Biophysica Acta-gene Structure and Expression - BBA-GENE STRUCT EXPRESS, 2004

The promoter region of the human prostaglandin F2α receptor (FP) gene was isolated, sequenced, an... more The promoter region of the human prostaglandin F2α receptor (FP) gene was isolated, sequenced, and characterized. The 5′-flanking region has minimal homology to the bovine, mouse, and rat FP promoters with the exception of a 100-bp region. The human promoter similarly lacks a canonical TATA-box and a CAAT-box. Potential binding sites for SP-1, GATA-1, STAT-1, and AP-1 are present in the 5′-flanking region. One major transcription start site was identified using 5′ RLM-RACE analysis and mapped to an adenine residue 262 nucleotides upstream from the initiator codon in exon 2. Transfection of HeLa cells with FP promoter-GFP deletion constructs indicates that the −2437/−1946 region contains repressor activity. DNase I footprinting analysis of this region identifies a footprint over the GATA-like site at −2400. This suggests repression of basal FP transcription may be mediated by a GATA binding site.

Journal of Reproductive Immunology, 2008

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis f... more A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Journal of Reproductive Immunology, 2006

Endocrinology, 2010

IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infectio... more IL-6 abundance in amniotic fluid and uterine tissues increases in late gestation or with infection-associated preterm labor. A role in regulation of labor onset is suggested by observations that IL-6 increases expression of genes controlling prostaglandin synthesis and signaling in isolated uterine cells, but whether IL-6 is essential for normal parturition is unknown. To evaluate the physiological role of IL-6 in parturition in mice, we investigated the effect of Il6 null mutation on the timing of parturition and expression of genes associated with uterine activation. Il6 null mutant mice delivered 24 h later than wild-type mice, although circulating progesterone fell similarly in both genotypes during the prepartal period. Il6 null mutant mice were also refractory to low doses of lipopolysaccharide sufficient to induce preterm delivery in wild-type mice. The characteristic late-gestation elevation in uterine expression of Oxtr mRNA encoding oxytocin receptor, and peripartal increases in Ptgfr and Ptgs2 mRNAs regulating prostaglandin synthesis and signaling were delayed by 24 h in Il6 null mutant mice. Conversely, Ptger4 mRNA encoding the prostaglandin E receptor-4 was abnormally elevated in late-gestation in Il6 null mutant mice. Administration of recombinant IL-6 from d 11.5 postcoitum until term restored the normal timing of delivery and normalized Ptger4 mRNA expression in late gestation. We conclude that IL-6 has a key role in controlling the progression of events culminating in parturition and that it acts downstream of luteolysis in the uterus to regulate genes involved in the prostaglandin-mediated uterine activation cascade.

Biology of Reproduction, 2006

The molecular mechanisms that regulate the expression of genes involved in parturition are poorly... more The molecular mechanisms that regulate the expression of genes involved in parturition are poorly understood. The mRNA expression of the prostaglandin F 2alpha receptor (PTGFR), a uterine activating gene, is increased at labor and is required for uterine contractile activity in numerous animal models, although the signaling pathways responsible for this increased expression have not been identified. Proinflammatory cytokines have been proposed to regulate the expression of the uterine activating genes via activation of the nuclear transcription factor, NFkappaB, and initiate labor. However, it is uncertain whether uterine PTGFR is regulated this way. In this report, we demonstrate for the first time that treatment of immortalized human myometrial-derived ULTR cells with the proinflammatory cytokine IL1beta causes an increase in PTGFR mRNA levels. Furthermore, IL1beta treatment increased the nuclear levels of the RELA subunit of NFkappaB and increased binding of RELA to the NFkappaB DNA-binding site. Inhibition of NFkappaB activation with either the proteasome inhibitor MG132 or phenethyl caffeiate reduced PTGFR mRNA levels, which indicates that this transcription factor is important for basal transcription. Furthermore, this inhibition prevented IL1beta induction of PTGFR mRNA, which confirms that NFkappaB is required for the IL1beta-induced increase in PTGFR. These results are consistent with the proposal that proinflammatory cytokines directly regulate uterine activation genes and that the transcription factor NFkappaB is involved in both basal and IL1beta-stimulated transcription of the PTGFR gene.

Biology of Reproduction, 2002

We explored a potential mechanism linking placental prostaglandins (PGs) with a fall in plasma pr... more We explored a potential mechanism linking placental prostaglandins (PGs) with a fall in plasma progesterone and increased expression of uterine activation proteins in the mouse. PG endoperoxide H synthase 2 (PGHS-2) mRNA expression increased in placenta in late gestation in association with an 8-fold increase in PGF(2alpha) concentration, reaching a peak on Gestational Day (GD) 18. This peak coincided with the final descent in plasma progesterone and birth on GD 19.3 +/- 0.2. Implantation of a progesterone-releasing pellet in intact pregnant dams on GD 16 delayed birth at term until GD 20.9 +/- 0.4 and inhibited the GD 18 increase in placental PGF(2alpha) levels in conjunction with a delayed fall in plasma progesterone that reached its lowest level 1 day after term birth. The mRNA levels of uterine activation proteins, connexin-43 (CX-43), oxytocin receptor, PGF(2alpha) receptor (FP), and PGHS-2, and the concentration of uterine PGF(2alpha) all increased at normal term birth. At progesterone-delayed term birth on GD 19.3, even though tissue PGF(2alpha) concentrations were at the same high levels observed at normal term birth, CX-43 and FP mRNA levels were lower than those at normal term birth, thereby possibly contributing to the delay of birth. These data are consistent with the hypotheses that fetal placental PGs affect the timing of birth by hastening luteolysis, that uterine activation initiates labor, and that birth may be delayed by blocking or decreasing the expression of two of the uterine activation proteins.

Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 2003

To elucidate the molecular basis of the expression of the membrane tethering protein p115, a geno... more To elucidate the molecular basis of the expression of the membrane tethering protein p115, a genomic DNA clone including the 5Vflanking region, first exon, and part of the first intron of the p115 gene was isolated from a bovine genomic DNA library in EFIX II. Nine transcriptional initiation sites (at À 189, À 183, À 178, À 177, À 164, À 151, À 149, À 133 and À 129 from the translation initiation site) were determined by the CapSiteR Hunting method. The 5V -flanking region (a 2.6-kb fragment) of the bovine p115 gene ligated to a luciferase reporter gene displayed promoter activity in primary cultured bovine mammary epithelial (BME) cells and in human MCF-7 breast carcinoma cells. The luciferase reporter gene assays of the promoter deletion constructs suggested the region required for promoter activity of the bovine p115 gene. The region that retained promoter activity contained a potential nuclear respiratory factor-1 (NRF-1) binding site. In both the BME and MCF-7 cells, mutagenesis to impair the NRF-1 consensus sequence in the p115 promoter gave substantially lowered levels of luciferase expression. An electrophoretic mobility shift assay (EMSA) showed that the NRF-1 consensus sequence and nuclear protein formed a complex that was abolished by oligonucleotides containing the authentic NRF-1 binding site, and was also supershifted with an antibody to NRF-1. In luciferase reporter gene assays of the p115 promoter constructs, treatment of the MCF-7 cells with estradiol-17h (E 2 ), insulin, or both stimulated the p115 promoter activity correlating with the cell proliferation rate. These results indicate that the NRF-1 response element in the p115 promoter is important for promoter function, and that it involves the binding of NRF-1. Furthermore, the results suggested that p115 gene transcription is activated by E 2 , by insulin, or by both in association with the stimulation of mammary epithelial cell proliferation. D

Biochemical and Biophysical Research Communications, 1999

American Journal of Obstetrics and Gynecology, 2001

We determined the relative abundance of prostaglandin endoperoxide H synthase type 1 and type 2 m... more We determined the relative abundance of prostaglandin endoperoxide H synthase type 1 and type 2 messenger ribonucleic acid levels in the human fetus and newborn infant. We used ribonuclease protection assays and normalized values to messenger ribonucleic acid of cyclophilin. Tissues were obtained from all 3 trimesters and in the first 9 days of the newborn period. Prostaglandin endoperoxide H synthase type 1 and type 2 messenger ribonucleic acid is present in every fetal tissue examined (lung, kidney, intestine, heart, brain, and stomach). Kidney and lung demonstrated no changes in the expression of prostaglandin endoperoxide H synthase type 1 messenger ribonucleic acid with gestational age, whereas postnatal levels in lung were one third those in the first trimester (P &amp;lt;.05). A large increase (5-fold to 30-fold; P &amp;lt;.05) occurred throughout gestation for the expression of prostaglandin endoperoxide H synthase type 2 messenger ribonucleic acid in intestine, lung, and kidney, which extended into the newborn period for lung and kidney. These data imply that the expression of prostaglandin endoperoxide H synthase type 2 messenger ribonucleic acid may be responsible for prostaglandin-related effects and is coordinated in several human fetal tissues in late gestation.

Alcoholism: Clinical and Experimental Research, 1999

Background Recently, an association between alcohol consumption during pregnancy and shortened ge... more Background Recently, an association between alcohol consumption during pregnancy and shortened gestational length has been reported, but the underlying mechanisms remain unknown. Progesterone (P,) and prostaglandins have been shown to play important roles in parturition in both human and animal models. Recently, it has been suggested that prostaglandin H synthase-2 (PGHS-2) is responsible for prostaglandin changes associated with term and preterm labor. It is possible that alcohol induces preterm birth by altering P, or PGHS-2 levels. These studies were designed to determine the role of P, and PGHS-2 in alcohol-induced preterm labor in mice.

Myometrial contractions of labor result from an increase in myometrial activation and stimulation... more Myometrial contractions of labor result from an increase in myometrial activation and stimulation. Activation develops through the expression of contraction associated proteins (CAPs), including oxytocin receptors (OTR), connexin-43 (Cx-43), and prostaglandin F 2␣ receptors (FP). Stimulation involves increases in contractile agonists including prostaglandin E 2 (PGE 2 ) and prostaglandin F 2␣ (PGF 2␣ ) that may result from increases in prostaglandin endoperoxide H synthase (PGHS)-2. A mouse model of preterm birth was used to study gene expression involved in myometrial activation and stimulation. To induce preterm birth, pregnant C57BL/6J mice were intubated with 6 g/kg ethanol on gestational day 16 and were killed every 6 h from treatment until birth. RIA was used to measure uterine PGE 2 and PGF 2␣ , while PGHS-2, OTR, Cx-43, and FP messenger RNA levels were measured by ribonuclease protection assay. Increases in CAP mRNA were associated with term and preterm birth. There were differences in stimulation effectors associated with preterm and term birth. Uterine PGF 2␣ values were increased only at the time of term birth, but PGE 2 was elevated during both preterm and term labor. These data suggest that existing levels of PGF 2␣ are sufficient for preterm birth when CAP expression is increased, but term labor requires increases in PGE 2 , PGF 2␣ , and CAPs. The PGHS-2 messenger RNA expression pattern suggests that it is a CAP.