Progesterone receptor regulates decidual prolactin expression in differentiating human endometrial stromal cells.

Human endometrial stromal (ES) cells in culture express PRL, a marker of decidualization, in response to sustained activation of protein kinase A (PKA). Cotreatment with the progestin medroxyprogesterone acetate (MPA) enhanced decidual PRL gene activation in the presence of elevated intracellular cAMP levels. This synergy became apparent, at protein and promoter level, after a lag period of 2 days and increased in a time-dependent manner thereafter. Pretreatment with cAMP advanced the time at which synergy between cAMP and MPA was apparent, suggesting that PKA activation sensitized ES cells to the effects of progestins. Analysis of the progesterone receptor (PR) indicated that PR-A was the predominant form in differentiating ES cells, but its abundance decreased markedly during the course of the decidualization response. The decline in PR levels was of functional relevance, as expression of PR-B or PR-A, by transient transfection, dramatically inhibited the activity of a decidual PRL promoter-reporter construct in response to cAMP. Furthermore, the expression of endogenous PRL protein in response to cAMP or cAMP plus MPA was substantially decreased by constitutive expression of green fluorescence protein-tagged PR, which was localized in the nucleus even in the absence of added ligand. Ligand-independent PR inhibition of the decidual PRL promoter was receptor specific, independent of known PR phosphorylation sites, and required minimally a functional DNA-binding domain. Transient expression of steroid receptor coactivator-1e (SRC-1e), but not SRC-1a, allowed synergy between cAMP and MPA without the requirement of sensitization by pretreatment with cAMP. This raised the possibility that SRC-1e was a component of cAMP-dependent sensitization of ES cells, but there was no evidence of altered messenger RNA expression of either SRC-1 isoform during decidualization. In conclusion, cellular PR levels determine the onset of the decidualization response. Initiation of this process requires elevated intracellular cAMP levels that sensitize ES cells to the actions of progestins through down-regulation of cellular PR levels and possibly via modulation of function of an intermediate factor(s) such as SRC-1e.     

Integration of pro-inflammatory cytokines, 12-lipoxygenase and NOX-1 in pancreatic islet beta cell dysfunction

Elevated cellular reactive species, which can be produced by diabetic serum conditions such as elevated inflammatory cytokines, lipotoxicity or glucotoxicity contribute to islet beta cell dysfunction and cell death. Cellular pathways that result in beta cell oxidative stress are poorly resolved. In this study, stimulation of human donor islets, primary mouse islets or homogeneous beta cell lines with a cocktail of inflammatory cytokines (TNFα, IL-1β, and INFγ) significantly induced NADPH oxidase-1 (NOX-1) gene expression (p<0.05). This pro-inflammatory cytokine cocktail concomitantly induced loss of islet glucose stimulated insulin response (p<0.05), elevated expression of MCP-1 (p<0.01), increased cellular reactive oxygen species (ROS) and induced cell death. Inhibitors of NADPH oxidase, apocynin and diphenyleneiodonium, and a dual selective NOX1/4 inhibitor, blocked ROS generation (p<0.01) and induction of MCP-1 (p<0.05) by pro-inflammatory cytokines in beta cells. It has previously been reported that pro-inflammatory cytokine stimulation induces 12-lipoxygenase (12-LO) expression in human islets. 12-Hydroxyeicosatetraenoic acid (12-HETE), a product of 12-LO activity, stimulated NOX-1 expression in human islets (p<0.05). A novel selective inhibitor of 12-LO blocked induction of NOX-1, production of ROS and pro-caspase 3 cleavage by pro-inflammatory cytokines in INS-1 beta cells (p<0.01). Inhibition was not seen with a structurally related but inactive analog. Importantly, islets from human type 2 diabetic donors have an elevated expression of NOX-1 (p<0.05). This study describes an integrated pathway in beta cells that links beta cell dysfunction induced by pro-inflammatory cytokines with 12-lipoxygenase and NADPH oxidase (NOX-1) activation. Inhibitors of this pathway may provide a new therapeutic strategy to preserve beta cell mass in diabetes.     

FOXO1A differentially regulates genes of decidualization

The forkhead box O1A (FOXO1A) has been identified as one gene that is up-regulated early in the decidualization process. To further investigate the role of FOXO1A during this process, six genes, IGFBP1, PRL, TIMP3, LAMB1, CNR1, and DCN, shown to be up-regulated during decidualization, were chosen as potential targets of FOXO1A action. Treatment of human endometrial stromal cells with hormones (estradiol and medroxyprogesterone acetate) plus dibutyryl cAMP (H+dbcAMP) for 48 h increased expression of IGFBP1, PRL, TIMP3, CNR1, and DCN but not LAMB1, as measured by real-time PCR. Silencing of FOXO1A using small interfering RNA oligonucleotides decreased IGFBP1 and DCN levels and increased CNR1, TIMP3, and PRL levels. LAMB1 was not affected. When FOXO1A was overexpressed in human endometrial stromal cells, expression of IGFBP1, DCN, and PRL increased, whereas levels of TIMP3 and CNR1 decreased. Addition of H+dbcAMP caused an increased expression of IGFBP1, PRL, and DCN beyond that of FOXO1A alone. TIMP3 and CNR1 levels decreased even further in response to H+dbcAMP compared with FOXO1A alone. LAMB1, which was unresponsive to FOXO1A, decreased when H+dbcAMP was added. Overexpressing FOXO1A also caused a change in cell shape, in that the stromal fibroblasts acquired a rounded, epithelioid appearance. Finally, reporter studies showed that cotransfection of FOXO1A significantly increased PRL promoter activity but not TIMP3 promoter activity. Addition of H+dbcAMP resulted in a significant increase in PRL promoter activity and a significant decrease in TIMP3 promoter activity. In summary, this study demonstrates the versatile nature of FOXO1A in the regulation of a number of decidualization-specific genes.     

Modulation of p47PHOX activity by site-specific phosphorylation: Akt-dependent activation of the NADPH oxidase

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O(2)(-) at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47(PHOX) occurs during the activation of the enzyme in intact cells. p47(PHOX) carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47(PHOX) is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47(PHOX) whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47(PHOX) by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47(PHOX). These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase.     

Interleukin-2 inhibits the synthesis and release of prolactin from human decidual cells

PRL is synthesized and released by several extrapituitary tissues, including decidualized endometrial stromal cells. As interleukin-2 (IL-2) stimulates the synthesis and release of pituitary PRL, and decidual stromal cells have receptors for IL-2, we examined whether IL-2 also regulates the release of decidual PRL. Exposure of primary cultures of human decidual cells (10(6) cells/well) from term pregnancies to IL-2 (50 ng/mL) inhibited PRL release beginning 48 h after exposure. The inhibition by IL-2 was dose dependent, and the maximal inhibition of PRL release after 5 days of exposure to IL-2 was 71.0 +/- 0.9% (mean +/- SE). IL-2, however, had no effect on decidual cell viability. The inhibitory effect of IL-2 on PRL release was secondary to inhibition of PRL synthesis. Decidualized human endometrial stromal cells transfected with 3 kb of the extrapituitary PRL (exon 1a) promoter coupled to a luciferase expression vector responded to IL-2 (10 ng/mL) with a significant decrease in luciferase activity. These findings strongly suggest that IL-2 inhibits the synthesis and release of decidual PRL and provide further support for a critical role of cytokines in the regulation of decidual PRL gene expression.     

Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.

Endometrial stromal cells undergo decidual transformation, in response to epidermal growth factor (EGF) and progesterone. Since insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) are believed to be involved in endometrial differentiation, and insulin regulates IGFBP production in a variety of cells, we have investigated the modulatory roles of EGF, progesterone, and insulin on IGFBP secretion by long term cultures of human endometrial stromal cells. Without insulin, the principal IGFBP secreted into conditioned medium, detected by Western ligand blotting, was a 28-kilodalton (kDa) IGFBP, identified by immunoprecipitation as IGFBP-1. This was observed only when the stromal cells were decidualized. With increasing insulin, IGFBP-1 decreased to undetectable levels. Concomitantly, IGFBP-2 increased, as did a 24-kDa IGFBP (believed to be IGFBP-4) and a 28-kDa IGFBP, shown to be a glycoprotein by endoglycosidase sensitivity (and believed to be glycosylated IGFBP-4). In the nondecidualized state, insulin increased the secretion of IGFBP-3, IGFBP-2, and the 24-kDa IGFBP, which were slightly inhibited by EGF and relatively unaffected by progesterone alone. In the absence of insulin, progesterone weakly stimulated IGFBP-1 secretion, which increased markedly when the cells were decidualized by combined treatment with EGF and progesterone. These data show that IGFBP-3, IGFBP-2, IGFBP-1, and presumably IGFBP-4 and its glycosylated form are differentially regulated by peptide and steroid hormones in endometrial stromal cells and that their regulation is a function of stromal differentiation.     

Phosphodiesterase 4 inhibition synergizes with relaxin signaling to promote decidualization of human endometrial stromal cells

The decidualization of endometrial stromal cells in the secretory phase of the menstrual cycle is an essential prerequisite for the implantation of a blastocyst. This profound differentiation process is accompanied by sustained elevated intracellular cAMP concentrations in vivo. Primary cell cultures of endometrial stromal cells decidualize by treatment with cAMP-elevating agents in vitro. Our previous findings indicated that the cAMP-degrading activities of phosphodiesterases (PDE) and signaling of the peptide hormone relaxin are coupled in human endometrial stromal cells. In the present study we have chosen a pharmacological approach to test whether relaxin binding and PDE inhibition cooperate to induce decidualization. Measurement of PDE activity and relaxin-stimulated cAMP accumulation in the presence of diverse PDE inhibitors identified PDE4 and PDE8 as the principal PDE isoforms involved in human endometrial stromal cells. The PDE4 inhibitor rolipram was most effective in elevating intracellular cAMP concentrations and synergizing with relaxin to achieve maximal in vitro decidualization, as determined by measurement of the expression of the decidual marker genes for prolactin and IGF-binding protein-1 and measurement of prolactin secretion. Gene expression for PDE4D and PDE4C was significantly up-regulated during in vitro decidualization. Treatment of cell cultures with the protein kinase A inhibitor H89 revealed a minor role for protein kinase A-mediated positive feedback control of PDE4 activity in human endometrial stromal cells, consistent with sustained elevated cAMP essential for decidualization in vitro. These findings introduce the new idea of clinically applying the combination of a specific PDE4 inhibitor with an effector such as relaxin, thereby offering an alternative nonsteroidal luteal phase support for the endometrium to encourage endometrial development and implantation in subfertile women undergoing assisted reproductive technology procedures.     

Attenuation of NADPH oxidase activation and glomerular filtration barrier remodeling with statin treatment

Activation of reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase by angiotensin II is integral to the formation of oxidative stress in the vasculature and the kidney. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase inhibition is associated with reductions of oxidative stress in the vasculature and kidney and associated decreases in albuminuria. Effects of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibition on oxidative stress in the kidney and filtration barrier integrity are poorly understood. To investigate, we used transgenic TG(mRen2)27 (Ren2) rats, which harbor the mouse renin transgene and renin-angiotensin system activation, and an immortalized murine podocyte cell line. We treated young, male Ren2 and Sprague-Dawley rats with rosuvastatin (20 mg/kg IP) or placebo for 21 days. Compared with controls, we observed increases in systolic blood pressure, albuminuria, renal NADPH oxidase activity, and 3-nitrotryosine staining, with reductions in the rosuvastatin-treated Ren2. Structural changes on light and transmission electron microscopy, consistent with periarteriolar fibrosis and podocyte foot-process effacement, were attenuated with statin treatment. Nephrin expression was diminished in the Ren2 kidney and trended to normalize with statin treatment. Angiotensin II-dependent increases in podocyte NADPH oxidase activity and subunit expression (NOX2, NOX4, Rac, and p22(phox)) and reactive oxygen species generation were decreased after in vitro statin treatment. These data support a role for increased NADPH oxidase activity and subunit expression with resultant reactive oxygen species formation in the kidney and podocyte. Furthermore, statin attenuation of NADPH oxidase activation and reactive oxygen species formation in the kidney/podocyte seems to play roles in the abrogation of oxidative stress-induced filtration barrier injury and consequent albuminuria.     

Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells

Summary. Bone marrow stromal cells synthesize and secrete insulin-like growth factor (IGF)-I and IGF-binding proteins (IGFBP). IGFBPs may modulate the action of IGF-I or IGF-II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP-1 to -6 mRNA expression. [¹²⁵I]IGF-I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP-2 to -6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal-derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC-1 cells. Forskolin (10 ⁵ M) up-regulated IGFBP4 mRNA and protein secretion in a time-dependent manner. These findings suggest that IGFBP-4, -5 and -6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.     

Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells

Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22(phox) and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D(1)-like receptor agonist, fenoldopam (1 micromol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7+/-3.3%). In contrast, cholesterol depletion (2% methyl-beta-cyclodextrin [beta CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0+/-10.5% versus control, 103.1+/-3.4%), which was reversed by cholesterol repletion (118.9+/-9.9%). Moreover, NADPH oxidase activation by beta CD (145.5+/-9.0%; control: 98.6+/-1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4+/-3.2%) and diphenylene iodonium (9.5+/-3.3%). Furthermore, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta CD: 152.0+/-9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.     

Sex steroids and growth factors differentially regulate the growth and differentiation of cultured human endometrial stromal cells

We have studied the interaction between growth factors and sex steroids in regulating human endometrial stromal cell growth and differentiation using an in vitro serum-free cell culture model system. None of the growth factors [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin, insulin-like growth factor-I (IGF-I), IGF-II, or platelet-derived growth factor] stimulated the growth of human endometrial stromal cells grown in progestin-free medium. However, the growth of progestin-treated cultures was dramatically increased by EGF, bFGF, or platelet-derived growth factor, but not by insulin, IGF-I, or IGF-II. Estrogen could not substitute for progesterone in this protocol, and coadministration of estrogen with progestin did not enhance the response over that to progesterone alone. In contrast to their positive effects on growth, only EGF, not bFGF, stimulated stromal cell differentiation, as measured by an increase in PRL, laminin, and fibronectin production; moreover, stimulation of differentiation was dependent upon the presence of progestin in the culture medium. Thus, human endometrial stromal cell growth is 1) regulated by a discrete set of growth factors, only a subset of which regulates stromal cell differentiation; and 2) regulation of stromal cell growth and stromal cell differentiation by growth factors is progestin dependent. Our results provide direct evidence for interaction between growth factors and sex steroids in the regulation of stromal cell growth and differentiation in vitro and suggest that growth factors may be absolutely required in conjunction with progesterone for the decidual response in vivo.     

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.     

RNA silencing in vivo reveals role of p22phox in rat angiotensin slow pressor response

The angiotensin II (Ang II) slow-pressor response entails an increase in mean arterial pressure and reactive oxygen species. We used double-stranded interfering RNAs (siRNAs) in Sprague Dawley rats in vivo to test the hypothesis that an increase in the p22phox component of NADPH oxidase is required for this response. Reactive oxygen species were assessed from excretion of 8-isoprostane prostaglandin F2alpha and blood pressure by telemetry. Two siRNA sequences to p22phox (sip22phox) reduced mRNA >85% in cultured vascular smooth muscle cells. Rats received rapid (10 second) IV injections (50 to 100 microg) of 1 of 2 different sip22phox, control siRNA, or vehicle (TransIt in saline) during 14 day SC infusions of Ang II (200 ng.kg(-1).min(-1)) or sham infusions. In both groups, sip22phox, relative to control siRNA, led to significant (P<0.001; approximately 50%) reductions in expression of p22phox mRNA and protein and of NADPH oxidase activity in the kidney cortex. In Ang II-infused rats, sip22phox decreased protein expression for Nox-1, -2, and -4 but increased p47phox. Three days after sip22phox, conscious rats infused with Ang II had a reduced excretion of 8-isoprostane (10+/-1 versus 19+/-2 pg.24 h(-1); P<0.01) and a reduced mean arterial pressure (142+/-5 versus 168+/-4 mm Hg; P<0.005). An increase in p22phox is required for increased renal NADPH oxidase activity, expression of Nox proteins and oxidative stress, and contributes < or =50% to hypertension during an Ang II slow-pressor response.     

The Nox Family of NADPH Oxidases: Friend or Foe of the Vascular System?

NADPH (nicotinamide adenine dinucleotide phosphate) oxidases are important sources of reactive oxygen species (ROS). In the vascular system, ROS can have both beneficial and detrimental effects. Under physiologic conditions, ROS are involved in signaling pathways that regulate vascular tone as well as cellular processes like proliferation, migration and differentiation. However, high doses of ROS, which are produced after induction or activation of NADPH oxidases in response to cardiovascular risk factors and inflammation, contribute to the development of endothelial dysfunction and vascular disease. In vascular cells, the NADPH oxidase isoforms Nox1, Nox2, Nox4, and Nox5 are expressed, which differ in their activity, response to stimuli, and the type of ROS released. This review focuses on the specific role of different NADPH oxidase isoforms in vascular physiology and their potential contributions to vascular diseases.