Epigenetic Modifications of White Blood Cell DNA Caused by Transient Fetal Infection with Bovine Viral Diarrhea Virus.

Bovine viral diarrhea virus (BVDV) infections cause USD 1.5-2 billion in losses annually. Maternal BVDV after 150 days of gestation causes transient fetal infection (TI) in which the fetal immune response clears the virus. The impact of fetal TI BVDV infections on postnatal growth and white blood cell (WBC) methylome as an index of epigenetic modifications was examined by inoculating pregnant heifers with noncytopathic type 2 BVDV or media (sham-inoculated controls) on Day 175 of gestation to generate TI (n = 11) and control heifer calves (n = 12). Fetal infection in TI calves was confirmed by virus-neutralizing antibody titers at birth and control calves were seronegative. Both control and TI calves were negative for BVDV RNA in WBCs by RT-PCR. The mean weight of the TI calves was less than that of the controls (p < 0.05). DNA methyl seq analysis of WBC DNA demonstrated 2349 differentially methylated cytosines (p ≤ 0.05) including 1277 hypomethylated cytosines, 1072 hypermethylated cytosines, 84 differentially methylated regions based on CpGs in promoters, and 89 DMRs in islands of TI WBC DNA compared to controls. Fetal BVDV infection during late gestation resulted in epigenomic modifications predicted to affect fetal development and immune pathways, suggesting potential consequences for postnatal growth and health of TI cattle.

Enrichment of ovine gonadotropes via adenovirus gene targeting enhances assessment of transcriptional changes in response to estradiol-17 beta†.

Gonadotropes represent approximately 5-15% of the total endocrine cell population in the mammalian anterior pituitary. Therefore, assessing the effects of experimental manipulation on virtually any parameter of gonadotrope biology is difficult to detect and parse from background noise. In non-rodent species, applying techniques such as high-throughput ribonucleic acid (RNA) sequencing is problematic due to difficulty in isolating and analyzing individual endocrine cell populations. Herein, we exploited cell-specific properties inherent to the proximal promoter of the human glycoprotein hormone alpha subunit gene (CGA) to genetically target the expression of a fluorescent reporter (green fluorescent protein [GFP]) selectively to ovine gonadotropes. Dissociated ovine pituitary cells were cultured and infected with an adenoviral reporter vector (Ad-hαCGA-eGFP). We established efficient gene targeting by successfully enriching dispersed GFP-positive cells with flow cytometry. Confirming enrichment of gonadotropes specifically, we detected elevated levels of luteinizing hormone (LH) but not thyrotropin-stimulating hormone (TSH) in GFP-positive cell populations compared to GFP-negative populations. Subsequently, we used next-generation sequencing to obtain the transcriptional profile of GFP-positive ovine gonadotropes in the presence or absence of estradiol 17-beta (E2), a key modulator of gonadotrope function. Compared to non-sorted cells, enriched GFP-positive cells revealed a distinct transcriptional profile consistent with established patterns of gonadotrope gene expression. Importantly, we also detected nearly 200 E2-responsive genes in enriched gonadotropes, which were not apparent in parallel experiments on non-enriched cell populations. From these data, we conclude that CGA-targeted adenoviral gene transfer is an effective means for selectively labeling and enriching ovine gonadotropes suitable for investigation by numerous experimental approaches.

Subplasmalemmal hydrogen peroxide triggers calcium influx in gonadotropes.

Gonadotropin-releasing hormone (GnRH) stimulation of its eponymous receptor on the surface of endocrine anterior pituitary gonadotrope cells (gonadotropes) initiates multiple signaling cascades that culminate in the secretion of luteinizing and follicle-stimulating hormones, which have critical roles in fertility and reproduction. Enhanced luteinizing hormone biosynthesis, a necessary event for ovulation, requires a signaling pathway characterized by calcium influx through L-type calcium channels and subsequent activation of the mitogen-activated protein kinase extracellular signal-regulated kinase (ERK). We previously reported that highly localized subplasmalemmal calcium microdomains produced by L-type calcium channels (calcium sparklets) play an essential part in GnRH-dependent ERK activation. Similar to calcium, reactive oxygen species (ROS) are ubiquitous intracellular signaling molecules whose subcellular localization determines their specificity. To investigate the potential influence of oxidant signaling in gonadotropes, here we examined the impact of ROS generation on L-type calcium channel function. Total internal reflection fluorescence (TIRF) microscopy revealed that GnRH induces spatially restricted sites of ROS generation in gonadotrope-derived αT3-1 cells. Furthermore, GnRH-dependent stimulation of L-type calcium channels required intracellular hydrogen peroxide signaling in these cells and in primary mouse gonadotropes. NADPH oxidase and mitochondrial ROS generation were each necessary for GnRH-mediated stimulation of L-type calcium channels. Congruently, GnRH increased oxidation within subplasmalemmal mitochondria, and L-type calcium channel activity correlated strongly with the presence of adjacent mitochondria. Collectively, our results provide compelling evidence that NADPH oxidase activity and mitochondria-derived hydrogen peroxide signaling play a fundamental role in GnRH-dependent stimulation of L-type calcium channels in anterior pituitary gonadotropes.

Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK.

We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3-1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3-1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca(2+) influx via the L-type Ca(2+) channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca(2+) influx through L-type Ca(2+) channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca(2+) influx via L-type channels to facilitate ERK phosphorylation.

GnRH evokes localized subplasmalemmal calcium signaling in gonadotropes.

The binding of GnRH to its receptor initiates signaling cascades in gonadotropes, which result in enhanced LH and FSH biosynthesis and secretion. This process is necessary for follicular maturation and ovulation. Calcium influx activates MAPKs, which lead to increased transcription of LH and FSH genes. Previous research suggests that two MAPK signaling pathways, ERK and jun-N-terminal kinase, are activated by either calcium influx through L-type calcium channels or by global calcium signals originating from intracellular stores, respectively. Here we continued this investigation to further elucidate molecular mechanisms transducing GnRH receptor stimulation to ERK activation. Although it is known that GnRH activation of ERK requires calcium influx through L-type calcium channels, direct evidence supporting an underlying local calcium signaling mechanism was lacking. Here we used a combination of electrophysiology and total internal reflection fluorescence microscopy to visualize discrete sites of calcium influx (calcium sparklets) in gonadotrope-derived αT3-1 cells in real time. GnRH increased localized calcium influx and promoted ERK activation. The L-type calcium channel agonist FPL 64176 enhanced calcium sparklets and ERK activation in a manner indistinguishable from GnRH. Conversely, the L-type calcium channel antagonist nicardipine inhibited not only localized calcium sparklets but also ERK activation in response to GnRH. GnRH-dependent stimulation of L-type calcium channels was found to require protein kinase C and a dynamic actin cytoskeleton. Taken together, we provide the first direct evidence for localized L-type calcium channel signaling in αT3-1 cells and demonstrate the utility of our approach for investigating signaling mechanisms and cellular organization in gonadotropes.

TRPC1, STIM1, and ORAI influence signal-regulated intracellular and endoplasmic reticulum calcium dynamics in human myometrial cells.

To explore the relationship between signal-stimulated increases in intracellular calcium ([Ca(2+)](i)) and depletion and refilling of the endoplasmic reticulum (ER) Ca(2+) stores ([Ca(2+)](L)) in human myometrial cells, we measured simultaneous changes in [Ca(2+)](i) and [Ca(2+)](L) using Fura-2 and Mag-fluo-4, respectively, in PHM1-41 immortalized and primary cells derived from pregnant myometrium and in primary cells derived from nonpregnant tissue. Signal- and extracellular Ca(2+)-dependent increases in [Ca(2+)](i) (SRCE) and ER refilling stimulated by oxytocin and cyclopiazonic acid were not inhibited by voltage-operated channel blocker nifedipine or mibefradil, inhibition of Na(+)/Ca(2+) exchange with KB-R7943, or zero extracellular Na(+) in PHM1-41 cells. Gadolinium-inhibited oxytocin- and cyclopiazonic acid-induced SRCE and slowed ER store refilling. TRPC1 mRNA knockdown specifically inhibited oxytocin-stimulated SRCE but had no statistically significant effect on ER store refilling and no effect on either parameter following cyclopiazonic acid treatment. Dominant negative STIMΔERM expression attenuated oxytocin- and thapsigargin-stimulated SRCE. Both STIM1 and ORAI1-ORAI3 mRNA knockdowns significantly attenuated oxytocin- and cyclopiazonic acid-stimulated SRCE. The data also suggest that reduction in STIM1 or ORAI1-ORAI3 mRNA can impede the rate of ER store refilling following removal of SERCA inhibition. These data provide evidence for both distinct and overlapping influences of TRPC1, STIM1, and ORAI1-ORAI3 on SRCE and ER store refilling in human myometrial cells that may contribute to the regulation of myometrial Ca(2+) dynamics. These findings have important implications for understanding the control of myometrial Ca(2+) dynamics in relation to myometrial contractile function.

Changes in rat myometrial plasma membrane protein kinase A are confined to parturition.

We have previously shown that pregnant rat myometrial plasma membrane-associated cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) decreases prior to delivery, coincident with a decline in the inhibitory effect of cAMP on contractant-stimulated parameters. We now find that rat myometrial membrane-associated PKA concentrations in early to mid-pregnancy are equivalent to those in cycling rats. Following the decline associated with parturition, membrane PKA recovers within 1 to 2 days postpartum. Treatment with the antiprogestin onapristone caused a decrease in myometrial membrane PKA catalytic and regulatory subunits compared to untreated controls by 12 hours. This coincided temporally with recently reported increases in electrical and contractile activity. In unilaterally pregnant rats, the decline in plasma membrane PKA was observed in both nonpregnant and pregnant horns but was more rapid in the pregnant horns. These data indicate that the myometrial plasma membrane PKA pattern before and during most of pregnancy is not consistent with progesterone exerting a primary influence on PKA membrane localization. Rather, the fall in membrane PKA associated with parturition may contribute to or be influenced by the increased contractile and electrical activity of labor that is a consequence of the loss of progesterone influence and is not absolutely dependent on the presence of fetuses.

Multiple signals regulate phospholipase CBeta3 in human myometrial cells.

Phospholipase CB3 (PLCB3) serine(1105) (S(1105)), a substrate for multiple protein kinases, represents a potential point of convergence of several signaling pathways in the myometrium. To explore this hypothesis, the regulation of PLCB3-S(1105) phosphorylation (P-S(1105)) was studied in immortalized and primary human myometrial cells. 8-[4-chlorophenylthio] (CPT)-cAMP and calcitonin gene-related peptide (CALCA) transiently increased P-S(1105). Relaxin also stimulated P-S(1105); this effect was partially blocked by the protein kinase A (PRKA) inhibitor, Rp-8-CPT-cAMPS. Oxytocin, which stimulates Galphaq-mediated pathways, also rapidly increased P-S(1105), as did prostaglandin F2alpha and ATP. Oxytocin-stimulated phosphorylation was blocked by protein kinase C (PRKC) inhibitor Go6976 and by pretreatment overnight with a phorbol ester. Cypermethrin, a PP2B phosphatase inhibitor, but not okadaic acid, a PP1/PP2A inhibitor, prolonged the effect of CALCA on P-S(1105), whereas the reverse was the case for the oxytocin-stimulated increase in P-S(1105). PLCB3 was the predominant PLC isoform expressed in the myometrial cells and PLCB3 short hairpin RNA constructs significantly attenuated oxytocin-stimulated increases in intracellular calcium. oxytocin-induced phosphatidylinositol (PI) turnover was inhibited by CPT-cAMP and okadaic acid, but was enhanced by pretreatment with Go6976. CPT-cAMP inhibited oxytocin-stimulated PI turnover in the presence of overexpressed PLCB3, but not overexpressed PLCB3-S(1105)A. These data demonstrate that both negative crosstalk from the cAMP/PRKA pathway and a negative feedback loop in the oxytocin/G protein/PLCB pathway involving PRKC operate in myometrial cells and suggest that different protein phosphatases predominate in mediating P-S(1105) dephosphorylation in these pathways. The integration of multiple signal components at the level of PLCB3 may be important to its function in the myometrium.

Amino acids in the COOH-terminal region of the oxytocin receptor third intracellular domain are important for receptor function.

Previously, residue K6.30 in the COOH-terminal region of the third intracellular domain (3iC) of the oxytocin (OT) receptor (OTR) was identified as important for receptor function leading to phospholipase C activation in both OTR and the vasopressin V(2) receptor (V(2)R) chimera V(2)ROTR3iC. Substitution of either A6.28K or V6.30K in wild-type V(2)R did not recapitulate the increase in phosphatidylinositide (PI) turnover observed in V(2)ROTR3iC. Hence, the role of K6.30 may be context-specific. Deletion of two NH(2)-terminal OTR3iC segments in the V(2)ROTR3iC chimera did not diminish vasopressin-stimulated PI turnover, whereas deletion of RVSSVKL (residues 6.19-6.25) reduced receptor expression. Deletion of this sequence in wild-type OTR reduced expression by 50% without affecting affinity for [(3)H]OT. This OTR mutant was unable to activate PI turnover or extracellular signal-regulated kinase 1/2 phosphorylation. The effects of alanine substitution for individual residues in RVSSVKL indicated differential importance for OTR function. The R6.19A substitution lost high-affinity sites for [(3)H]OT and the ability to stimulate PI turnover. Affinity for [(3)H]OT and membrane expression was not affected by any other substitutions. OTR-V6.20A and OTR-K6.24A mutants functioned as well as wild-type OTR, whereas OTR S6.21A, S6.22A, and V6.23A mutants exhibited impaired abilities to activate PI turnover (20-40% of OTR), and the OTR-L6.25A mutant exhibited constitutive activity. In conclusion, specific amino acids in the RVSSVKL segment in the COOH-terminal region of the third intracellular domain of OTR influence the ability of OTR to activate G protein-mediated actions.

Distinct binding of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol to cytochrome P450 27A1: evidence from modeling and site-directed mutagenesis studies.

Cytochrome P450 27A1 (P450 27A1 or CYP27A1) is an important enzyme that participates in different pathways of cholesterol degradation as well as in the activation of vitamin D(3). Several approaches were utilized to investigate how two physiological substrates, cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol, interact with CYP27A1. The enzyme active site was first probed spectrally by assessing binding of the two substrates and five substrate analogues followed by computer modeling and site-directed mutagenesis. The computer models suggest that the spatial positions and orientations of cholesterol and 5beta-cholestane-3alpha,7alpha,12alpha-triol are different in the enzyme active site. As a result, some of the active site residues interact with both substrates, although they are situated differently relative to each steroid, and some residues bind only one substrate. Mutation of the overlapping substrate-contact residues (W100, H103, T110, M301C, V367, I481, and V482) affected CYP27A1 binding and enzyme activity in a substrate-dependent manner and allowed identification of several important side chains. T110 is proposed to interact with the 12alpha-hydroxyl of 5beta-cholestane-3alpha,7alpha,12alpha-triol, whereas V367 seems to be crucial for correct positioning of the cholesterol C26 methyl group and for regioselective hydroxylation of this substrate. Distinct binding of the CYP27A1 substrates may provide insight into why phenotypic manifestations of cerebrotendinous xanthomatosis, a disease associated with CYP27A1 deficiency, are so diverse.

Phospholipids modify substrate binding and enzyme activity of human cytochrome P450 27A1.

Cytochrome P450 27A1 (P450 27A1) is an important metabolic enzyme involved in bile acid biosynthesis and the activation of vitamin D3 in mammals. Recombinant P450 27A1 heterologously expressed in Escherichia coli was found to be copurified with phospholipids (PLs). The PL content varied in different preparations and was dependent on the purification protocol. A link between the increased amounts of PLs and deterioration of the enzyme substrate binding properties was also observed. Tandem negative ionization mass spectrometry identified phosphatidylglycerol (PG) as the major PL copurified with P450 27A1. Subsequent reconstitution of P450 into exogenous PG vesicles assessed the effect of this contamination on substrate binding and enzyme activity. Two other PLs, phosphatidylethanolamine (PE) and phosphatidylserine (PS), were also tested. PG and PE increased the Kd for 5beta-cholestane-3alpha,7alpha,12alpha-triol and cholesterol binding, whereas PS had no effect on either substrate binding. PG and PE did not significantly alter 5beta-cholestane-3alpha,7alpha,12alpha-triol hydroxylase activity and even stimulated cholesterol hydroxylase activity. PS inhibited 5beta-cholestane-3alpha,7alpha,12alpha-triol hydrolyase activity and had no effect on cholesterol hydroxylase activity. Our study shows the potential for PLs to regulate the activity of P450 27A1 in vivo and alter the amount of cholesterol degraded through the "classical" and "alternative" bile acid biosynthetic pathways.

Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1.

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.