Hang Gu

Differential Regulation of Glucose Transporters Mediated by CRH Receptor Type 1 and Type 2 in Human Placental Trophoblasts

Glucose transport across the placenta is mediated by glucose transporters (GLUT), which is critical for normal development and survival of the fetus. Regulatory mechanisms of GLUT in placenta have not been elucidated. Placental CRH has been implicated to play a key role in the control of fetal growth and development. We hypothesized that CRH, produced locally in placenta, could act to modulate GLUT in placenta. To investigate this, we obtained human placentas from uncomplicated term pregnancies and isolated and cultured trophoblast cells. GLUT1 and GLUT3 expressions in placenta were determined, and effects of CRH on GLUT1 and GLUT3 were examined. GLUT1 and GLUT3 were identified in placental villous syncytiotrophoblasts and the endothelium of vessels. Treatment of cultured placental trophoblasts with CRH resulted in an increase in GLUT1 expression while a decrease in GLUT3 expression in a dose-dependent manner. Cells treated with either CRH antibody or nonselective CRH receptor (CRH-R) antagonist astressin showed a decrease in GLUT1 and an increase in GLUT3 expression. CRH-R1 antagonist antalarmin decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 antagonist astressin2b increased the expression of both GLUT1 and GLUT3. Knockdown of CRH-R1 decreased GLUT1 expression while increased GLUT3 expression. CRH-R2 knockdown caused an increase in both GLUT1 and GLUT3 expression. Our data suggest that, in placenta, CRH produced locally regulates GLUT1 and GLUT3 expression, CRHR1 and CRHR2-mediated differential regulation of GLUT1 and GLUT3 expression. Placental CRH may regulate the growth of fetus and placenta by modulating the expression of GLUT in placenta during pregnancy.

Expression of Cystathionine β-synthase and Cystathionine γ-lyase in Human Pregnant Myometrium and Their Roles in the Control of Uterine Contractility

Background Human uterus undergoes distinct molecular and functional changes during pregnancy and parturition. Hydrogen sulfide (H2S) has recently been shown to play a key role in the control of smooth muscle tension. The role of endogenous H2S produced locally in the control of uterine contractility during labour is unknown. Methodology/Principal Findings Human myometrium biopsies were obtained from pregnant women undergoing cesarean section at term. Immunohistochemistry analysis showed that cystathionine-γ-lyase (CSE) and cystathionine-β-synthetase (CBS), the principle enzymes responsible for H2S generation, were mainly localized to smooth muscle cells of human pregnant myometrium. The mRNA and protein expression of CBS as well as H2S production rate were down-regulated in labouring tissues compared to nonlabouring tissues. Cumulative administration of L-cysteine (10−7–10−2 mol/L), a precursor of H2S, caused a dose-dependent decrease in the amplitude of spontaneous contractions in nonlabouring and labouring myometrium strips. L-cysteine at high concentration (10−3 mol/L) increased the frequency of spontaneous contractions and induced tonic contraction. These effects of L-cysteine were blocked by the inhibitors of CBS and CSE. Pre-treatment of myometrium strips with glibenclamide, an inhibitor of ATP-sensitive potassium (KATP) channels, abolished the inhibitory effect of L-cysteine on spontaneous contraction amplitude. The effects of L-cysteine on the amplitude of spontaneous contractions and baseline muscle tone were less potent in labouring tissues than that in nonlabouring strips. Conclusion/Significance H2S generated by CSE and CBS locally exerts dual effects on the contractility of pregnant myometrium. Expression of H2S synthetic enzymes is down-regulated during labour, suggesting that H2S is one of the factors involved in the transition of pregnant uterus from quiescence to contractile state after onset of parturition.

Reduced Expression of 11β-Hydroxysteroid Dehydrogenase Type 2 in Preeclamptic Placentas Is Associated With Decreased PPARγ but Increased PPARα Expression

Placental 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) is reduced in pregnancies complicated with preeclampsia (PE). Peroxisome proliferator-activated receptors β/δ (PPARβ/δ) have been shown to suppress 11β-HSD2 expression in human placental cells. Our objectives were to investigate whether the reduced 11β-HSD2 expression is associated with the changes in PPARs in PE placentas, and whether PPARα and PPARγ affect 11β-HSD2 expression in placental cells. PPARα and PPARβ/δ mRNA and protein expression was increased, whereas PPARγ mRNA and protein expression was decreased in PE placentas. 11β-HSD2 protein expression was inversely correlated with PPARβ/δ in normal placentas but correlated positively with PPARγ and inversely to PPARα in PE placentas. In cultured placental cells, PPARα agonist inhibited, whereas PPARγ agonist stimulated, 11β-HSD2 mRNA and protein expression and activity in a dose-dependent manner. Knockdown of retinoid X nuclear receptor α (RXRα) resulted in a loss of PPARγ effect but not PPARα effect on11β-HSD2. The PPARα effect remained, but the PPARγ effect was lost in the presence of the translational inhibitor cycloheximide. PPARγ agonist dose-dependently stimulated specificity protein 1 (Sp-1) protein expression. Inhibition or knockdown of Sp-1 resulted in a loss of the effects of PPARα and PPARγ. The Sp-1 protein level was not correlated with 11β-HSD2 and PPARs in normal placentas, whereas Sp-1 expression was correlated with 11β-HSD2, PPARγ, and PPARβ/δ in PE placentas. Our data indicate that 11β-HSD2 expression can be modulated by PPARα and PPARγ in placental trophoblasts through Sp-1. Decreased 11β-HSD2 expression in PE placenta might be associated with decreased PPARγ but increased PPARα expression.

Corticotropin-Releasing Hormone (CRH) Promotes Inflammation in Human Pregnant Myometrium: The Evidence of CRH Initiating Parturition?

CONTEXT An increasing body of evidence indicates that human labor, either term or preterm, is an inflammatory event. CRH has been implicated to be a trigger of human parturition. OBJECTIVE To investigate whether CRH induces the cascades of inflammation in human pregnant myometrium, thereby leading to activation of uterus. DESIGN The myometrial tissues were obtained from pregnant women who were in labor or not in labor at term. The output of cytokines and prostaglandins (PGs) was determined by Multiplex and ELISA. Western blot analysis was used to determine the levels of uterine activation proteins (UAPs). RESULTS The levels of chemokines and cytokines as well as activated nuclear factor-κB (NF-κB) were increased in the term labor group more than the not term labor group. CRH stimulated production of a number of chemokines and cytokines in cultured uterine smooth muscle cells (USMCs), which induced chemotaxis of monocytes. These effects were mediated by CRH receptor 1 (CRHR1) and dependent on adenylyl cyclase/protein kinase (PKA) and NF-κB signaling. Cocultures of CRH-treated USMCs with monocytes greatly enhanced the output of cytokines and chemokines as well as PGs in cultures and increased the expression of uterine activation proteins (UAPs) in USMCs. IL-1β, IL-6, and TNF-α stimulated the expression of UAPs and output of PGs in USMCs. CONCLUSIONS CRH induces the production of chemokines and cytokines in myometrium at term and subsequently results in the cascade of inflammation in uterus. The inflammation induced by CRH can lead to activation of uterus.

Hydrogen sulfide in the endocrine and reproductive systems

Hydrogen sulfide (H2S) is now considered a member of a group of signaling molecules termed ‘gasotransmitters’. H2S has been shown to be generated in the endocrine and reproductive organs and elicits various actions. H2S modulates insulin secretion in pancreatic islets. Adipose tissues have the ability to produce H2S, which regulates the local insulin sensitivity and vascular responsiveness. H2S also acts on the hypothalamic–pituitary–adrenal axis and is involved in stress responses. The effects of H2S on male and female reproductive function have also attracted great interest for their potential therapeutic implications in reproductive disorders. Alterations of H2S biosynthesis are associated with various endocrine disorders, and hormones can be important factors in the regulation of H2S production. Understanding the regulatory mechanisms for H2S synthesis pathways may help develop new therapeutic strategies.

CRH Acts on CRH-R1 and -R2 to Differentially Modulate the Expression of Large-Conductance Calcium-Activated Potassium Channels in Human Pregnant Myometrium

CRH has been implicated to play a key role in the control of human pregnancy and parturition. Large-conductance potassium channels (BKCa) play a pivotal role in the modulation of uterine contractility during pregnancy. The objectives of the present study were to investigate the effect of CRH on BKCa expression in human pregnant myometrial cells. Myometrial tissues were collected at cesarean section from pregnant women not-in-labor (TNL) or in-labor (TL) at term, and myocytes were isolated and cultured. CRH was identified in human pregnant myometrium and mainly expressed in myometrial myocytes. Cultured myometrial cells were able to secrete CRH. In TNL myometrial cells, CRH treatment increased the expression of BKCa α- and β-subunits. CRH receptor type 1 (CRH-R1) antagonist, antalarmin, decreased whereas CRH receptor type 2 (CRH-R2) antagonist, astressin2b, increased the expression of BKCa. CRH-R2 small interfering RNA (siRNA) caused an increase, but CRH-R1 siRNA resulted in a decrease, in BKCa expression. In contrast to TNL cells, CRH exhibited an opposite effect on BKCa expression in TL myometrial cells, i.e. decreased BKCa expression. Antalarmin enhanced but astressin2b reduced BKCa expression. CRH-R2 siRNA decreased whereas CRH-R1 siRNA increased BKCa expression. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one significantly inhibited the frequency of spontaneous contractions of myometrial strips, and this effect was significantly decreased in TL strips compared with TNL ones. Our data suggest that CRH-R1 and CRH-R2 show differential regulation of BKCa expression. These effects mediated by CRH-R1 and CRH-R2 are changed after the onset of labor. This leads us to suggest that CRH may fine-tune myometrial contractility by modulating the expression of BKCa during pregnancy and labor.

CRH Activation of Different Signaling Pathways Results in Differential Calcium Signaling in Human Pregnant Myometrium before and during Labor

CONTEXT Our previous study has demonstrated that CRH has differential effects on human uterine contractility before and after onset of labor. Intracellular Ca2+ concentration ([Ca2+]i) mobilization plays an important role in the control of uterine contraction. OBJECTIVE Our objective was to investigate the effects of CRH on [Ca2+]i homeostasis in laboring and nonlaboring myometrial cells and determine subsequent signaling involved in [Ca2+]i regulation by CRH. DESIGN The myometrial tissues were obtained from pregnant women who were undergoing or not undergoing labor at term. [Ca2+]i was determined by Ca2+ imaging system using the fluorescent dye fura-2-acetoxymethyl ester. Western blot analysis, ELISA, and RIA were used to determine the signaling pathways induced by CRH. RESULTS CRH induced Ca2+ transient in laboring cells, which was blocked by CRH receptor type 1 (CRHR1) antagonist antalarmin. CRHR1 knockdown impaired this effect of CRH. CRH activated Gi protein, decreased cAMP production, and induced phosphorylated phospholipase C-β3 and inositol-1,4,5-triphosphate production. Phospholipase C and inositol-1,4,5-triphosphate receptor inhibitors blocked the CRH-induced Ca2+ transient in laboring cells. CRH did not induce whereas antalarmin induced the Ca2+ transient in nonlaboring cells. Knockdown of CRHR1 impaired the effect of antalarmin. CRH acted on CRHR1 to activate Gs in nonlaboring cells. Forskolin blocked antalarmin-induced Ca2+ transient. CONCLUSIONS CRH acts on CRHR1 to activate different signaling pathways before and after onset of labor, thereby resulting in differential calcium signaling in response to CRH. The signaling pathways of CRHR1 might serve as a target for the development of new therapeutic strategies for preterm birth.

MiR 20a,-20b and -200c are involved in hydrogen sulfide stimulation of VEGF production in human placental trophoblasts.

UNLABELLED Hydrogen sulfide (H2S) has been implicated to angiogenesis in various tissues. We sought to investigate the role of hydrogen sulfide (H2S) in regulating production of vascular endothelial growth factor (VEGF) proteins, the key factors of angiogenesis and vasculogenesis, in placenta. METHODS Placental tissues were obtained from pregnant women with preeclampsia and healthy pregnant women who underwent elective cesarean section. Explants and trophoblasts were isolated from healthy placentas and treated with H2S donor and precursor. Western blotting was used to determine the levels of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). The levels of VEGF mRNA, miR miR-200c,-20a and -20b were determined by quantitative real time PCR. RESULTS NaHS and l-cysteine increased VEGF but not placenta growth factor (PlGF) production in cultured explants and trophoblasts. Transfection of CBS and CSE siRNA reversed the stimulatory effect of l-cysteine on VEGF production in placental cells. H2S prolonged the half-life of VEGF mRNA and decreased the expression of miR-200c,-20a and -20b in placental cells. MiR-200c mimic and inhibitor affected VEGF mRNA and protein level, whereas miR-20a or -20b mimic and inhibitor affect VEGF protein release but not mRNA expression. The expression level of miR-200c,-20a and -20b as well as the level of CBS, CSE and VEGF were downregulated in preeclamptic placentas. CONCLUSION H2S produced via CSE and CBS plays a critical role in VEGF production in human placenta. Reduced expression of CSE and CBS may contribute to the abnormal production of angiogenic factors in preeclamptic placenta.

MiR133b is involved in endogenous hydrogen sulfide suppression of sFlt-1 production in human placenta

Increased production of soluble fms-like tyrosine kinase-1 (sFlt-1) from placenta is one of the major contributors to the development of preeclampsia. Our previous study has shown that hydrogen sulfide (H2S) inhibits sFlt-1 release in placenta. In the present study, we sought to investigate whether endogenous H2S affects sFlt-1 production and elucidate which H2S-producing enzyme is responsible for its effect in placenta. It was found that, besides cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), 3-mercaptopyruvatesulfurtransferase (3-MST) was identified in human placenta and mainly localized in syncytiotrophoblasts. There was no significant difference in expression level of 3-MST among preeclamptic and normal placentas. Treatment of cultured syncytiotrophoblasts with NaHS and l-cysteine suppressed sFlt-1 mRNA expression and caused a decrease in sFlt-1 protein content in culture media of the cells. Transfection of syncytiotrophoblasts with CBS siRNA and CSE siRNA reversed the above effects of l-cysteine. Furthermore, NaHS and l-cysteine treatment decreased the half-life of sFlt-1 mRNA and increased the expression of miR-133b targeting sFlt-1. MiR-133b expression was downregulated in preeclamptic placentas and correlated with the level of CBS and CSE. These results indicate that H2S is an important regulatory factor in sFlt-1 production in placenta. Reduced H2S generation in placenta contributes to development of preeclampsia by enhancing sFlt-1 production.

PGF2α modulates the output of chemokines and pro-inflammatory cytokines in myometrial cells from term pregnant women through divergent signaling pathways

Prostaglandin F2α (PGF2α) plays a critical role in the initiation and process of parturition. Since human labor has been described as an inflammatory event, we investigated the role of PGF2α in the inflammatory process using cultured human uterine smooth muscle cells (HUSMCs) isolated from term pregnant women as a model. Using a multiplex assay, HUSMCs treated with PGF2α changed their output of a number of cytokines and chemokines, with a distinct response pattern that differed between HUSMCs isolated from the upper and lower segment region of the uterus. Confirmatory enzyme-linked immunosorbent assays (ELISAs) showed that PGF2α stimulated increased output of interleukin (IL) 1β, IL6, IL8 (CXCL8) and monocyte chemotactic protein-1 (MCP1, also known as chemokine (c-c motif) ligand 2, CCL2) by HUSMCs isolated from both upper and lower uterine segments. In contrast, PGF2α inhibited tumor necrosis factor α (TNFα) release by HUMSCs from the lower uterine segment while the output of TNFα was undetectable in the upper segment. Small interfering (si) RNA mediated knockdown of the PGF2α receptor prevented the changes in cytokine and chemokine output by the HUSMCs. Since the PGF2α receptor (PTGFR) couples via the Gq protein and subsequently activates the phospholipase C (PLC) and protein kinase C (PKC) signaling pathways, we examined the role of these pathways in PGF2α modulation of the cytokines. Inhibition of PLC and PKC reversed the effects of PGF2α. PGF2α activated multiple signaling pathways including extracellular signal-regulated kinases (ERK) 1/2, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K), P38, calcineurin/nuclear factor of activated T-cells (NFAT) and NF-κB signaling. Inhibition of ERK reversed PGF2α-induced IL1β, IL6 and CCL2 output, while inhibition of PI3K blocked the effect of PGF2α on IL6, CXCL8 and CCL2 output and inhibition of NF-κB reversed PGF2α-induced IL1β and CCL2 output. NFAT was involved in PGF2α modulation of CCL2 and TNFα output. In conclusion, our results support a role of PGF2α in creating an inflammatory environment during the late stage of human pregnancy.