Retinoic acid enhances progesterone production via the cAMP/PKA signaling pathway in immature rat granulosa cells

Retinoic acid (RA) is a metabolite of vitamin A and has important roles in development, differentiation, and reproduction. Activin has been shown to regulate the RA pathway and affect granulosa cell (GC) proliferation, suggesting that RA is important for early follicle development. However, little is known about the effects of RA on GC functions, particularly steroidogenesis, during the early follicle stage. The aim of this study was to investigate the effects of all-trans-RA (atRA) on progesterone production in immature rat GCs cultured without gonadotropin. Our results demonstrated that atRA enhanced progesterone production by upregulating the levels of steroidogenic acute regulatory protein (StAR) and cytochrome P450scc (Cyp11a1) mRNAs, but not 3β-hydroxysteroid dehydrogenase mRNA in immature rat GCs. Additionally, analysis of the mechanisms through which atRA upregulated StAR and Cyp11a1 mRNAs revealed that atRA enhanced intracellular cAMP accumulation and phosphorylation of cAMP response-element binding protein (CREB). In addition, H-89, an inhibitor of protein kinase A (PKA), abolished the stimulatory effects of atRA, indicating that atRA enhanced progesterone synthesis through cAMP/PKA signaling. In conclusion, our data demonstrated that atRA has a crucial role in progesterone synthesis in rat GCs during the early follicle stage.     

Cooperation of Epac1/Rap1/Akt and PKA in prostaglandin E(2) -induced proliferation of human umbilical cord blood derived mesenchymal stem cells: Involvement of c-Myc and VEGF expression

Prostaglandin E₂ (PGE₂) is well known to regulate cell functions through cAMP; however, the role of exchange protein directly activated by cAMP (Epac1) and protein kinase A (PKA) in modulating such functions is unknown in human umbilical cord blood‐derived mesenchymal stem cells (hUCB‐MSCs). Therefore, we investigated the relationship between Epac1 and PKA during PGE₂‐induced hUCB‐MSC proliferation and its related signaling pathways. PGE₂ increased cell proliferation, and E‐type prostaglandin (EP) 2 receptor mRNA expression level and activated cAMP generation, which were blocked by EP2 receptor selective antagonist AH 6809. PGE₂ increased Epac1 expression, Ras‐related protein 1 (Rap1) activation level, and Akt phosphorylation, which were inhibited by AH 6809, adenylyl cyclase inhibitor SQ 22536, and Epac1/Rap1‐specific siRNA. Also, PGE₂ increased PKA activity, which was inhibited by AH 6809, SQ 22536, and PKA inhibitor PKI. HUCB‐MSCs were incubated with the Epac agonist 8‐pCPT‐cAMP or the PKA agonist 6‐phe‐cAMP to examine whether Epac1/Rap1/Akt activation was independent of PKA activation. 8‐pCPT‐cAMP increased Akt phosphorylation but not PKA activity. 6‐Phe‐cAMP increased PKA activity, but not Akt phosphorylation. Additionally, an Akt inhibitor or PKA inhibitor (PKI) did not block the PGE₂‐induced increase in PKA activity or Akt phosphorylation, respectively. Moreover, PGE₂ increased glycogen synthase kinase (GSK)‐3β phosphorylation and nuclear translocation of active‐β‐catenin, which were inhibited by Akt inhibitor or/and PKI. PGE₂ increased c‐Myc and vascular endothelial growth factor (VEGF) expression levels, which were blocked by β‐catenin siRNA. In conclusion, PGE₂ stimulated hUCB‐MSC proliferation through β‐catenin‐mediated c‐Myc and VEGF expression via Epac/Rap1/Akt and PKA cooperation. J. Cell. Physiol. 227: 3756–3767, 2012. © 2012 Wiley Periodicals, Inc.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Arachidonic acid alleviates the detrimental effects of acetylsalicylic acid on human granulosa cells performance in vitro

Here, we investigated the biological effects of arachidonic acid (AA) in human cumulus granulosa cells (CGCs) after exposure to ASA. Cells were isolated from the follicular fluid and incubated with 0.5 mM acetylsalicylic acid (ASA) and 50 µM AA. Cell viability was analyzed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assay. E₂ and P₄ levels were measured by chemiluminescence assay. Expression of genes including CYP19A1, FACN, and SCD1 was measured by real‐time polymerase chain reaction assay. Oxidative status was analyzed by monitoring glutathione peroxidase activity. The fatty acid profile was analyzed by the gas chromatography technique. Enzyme‐linked immunosorbent assay was used to measure prostaglandin E₂ (PGE₂) in CGCs after exposure to ASA and AA. Protein levels of the estrogen receptor were studied by immunofluorescence staining. Ultrastructural changes were evaluated by transmission electron microscopy imaging. ASA treatment reduced E₂ production, Cyp19a1 expression, glutathione peroxidase (GPx) activity, and estradiol receptor expression in CGCs. The addition of AA prevented the ASA‐induced E₂ reduction (p < .05) and expression of Cyp19a1. Moreover, AA increased the antioxidant capacity of CGCs exposed to ASA by promoting GPx activity (p < .05). AA increased monounsaturated fatty acid/saturated fatty acid ratio compared with the ASA group (p < .05). AA supplementation triggered the synthesis and secretion of PGE₂ in ASA‐treated CGCS (p < .05). Cytoplasmic vacuolation observed in the ASA group and treatment with AA intensified vacuolation rate. The expression of the estrogen receptor was increased after AA supplementation. Data demonstrated that AA decreased the detrimental effects of ASA on human CGCs after 72 hr.     

Effects of lipoic acid on lipolysis in 3T3-L1 adipocytes

Lipoic acid (LA) is a naturally occurring compound with beneficial effects on obesity. The aim of this study was to evaluate its effects on lipolysis in 3T3-L1 adipocytes and the mechanisms involved. Our results revealed that LA induced a dose- and time-dependent lipolytic action, which was reversed by pretreatment with the c-Jun N-terminal kinase inhibitor SP600125, the PKA inhibitor H89, and the AMP-activated protein kinase activator AICAR. In contrast, the PI3K/Akt inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and the PDE3B antagonist cilostamide enhanced LA-induced lipolysis. LA treatment for 1 h did not modify total protein content of hormone-sensitive lipase (HSL) but significantly increased the phosphorylation of HSL at Ser⁵⁶³ and at Ser⁶⁶⁰, which was reversed by H89. LA treatment also induced a marked increase in PKA-mediated perilipin phosphorylation. LA did not significantly modify the protein levels of adipose triglyceride lipase or its activator comparative gene identification 58 (CGI-58) and inhibitor G(0)/G(1) switch gene 2 (G0S2). Furthermore, LA caused a significant inhibition of adipose-specific phospholipase A2 (AdPLA) protein and mRNA levels in parallel with a decrease in the amount of prostaglandin E₂ released and an increase in cAMP content. Together, these data suggest that the lipolytic actions of LA are mainly mediated by phosphorylation of HSL through cAMP-mediated activation of protein kinase A probably through the inhibition of AdPLA and prostaglandin E₂.     

Arsenic induced progesterone production in a caspase-3-dependent manner and changed redox status in preovulatory granulosa cells

Arsenic contamination is a principal environmental health threat throughout the world. However, little is known about the effect of arsenic on steroidogenesis in granulosa cells (GCs). We found that the treatment of preovulatory GCs with arsenite stimulated progesterone production. A significant increase in serum level of progesterone was observed in female Sprague-Dawley rats following arsenite treatment at a dose of 10 mg/L/rat/day for 7 days. Further experiments demonstrated that arsenite treatment did not change the level of intracellular cyclic AMP (cAMP) or phosphorylated ERK1/2 in preovulatory GCs; however, progesterone production was significantly decreased when cAMP-dependent protein kinase (PKA) or ERK1/2 pathway was inhibited. This implied that the effect of arsenite on progesterone production may require cAMP/PKA and ERK1/2 signaling but not depend on them. Furthermore, we found that arsenite decreased intracellular reactive oxygen species (ROS) but increased the antioxidant glutathione (GSH) levels and mitochondrial membrane potential (ΔΨm) in parallel to the changes in progesterone production. Progesterone antagonist blocked the arsenic-stimulated increase of GSH levels. Arsenite treatment induced caspase-3 activation, although no apoptosis was observed. Inhibition of caspase-3 activity significantly decreased progesterone production stimulated by arsenite or follicle-stimulating hormone (FSH). GSH depletion with buthionine sulfoximine led to cell apoptosis in response to arsenite treatment. Collectively, this study demonstrated for the first time that arsenite stimulates progesterone production through cleaved/active caspase-3-dependent pathway, and the increase of GSH level promoted by progesterone production may protect GCs against apoptosis and maintain the steroidogenesis of GCs in response to arsenite treatment.     

Effect of insulin-like growth factor-binding protein 7 on steroidogenesis in granulosa cells derived from equine chorionic gonadotropin-primed immature rat ovaries

Insulin-like growth factor (IGF)-binding protein (IGFBP) 7 is a secreted protein that regulates cellular proliferation, adhesion, and angiogenesis, and has low affinity for IGF compared with that of IGFBP1-IGFBP6. We sought to determine whether IGFBP7 is present in follicular fluid and to elucidate whether IGFBP7 participates in the steroidogenesis of rat mature follicles. Follicular fluid and granulosa cells (GCs) were collected from immature rats 2 days after their treatment with equine chorionic gonadotropin (eCG). IGFBP7 protein was detected in the follicular fluid and the conditioned medium of cultured ovarian GCs by immunoblot analysis. When subconfluent GCs were cultured and treated with FSH and activin, coincubation with FSH and activin markedly increased GC expression of Cyp19a1 (aromatase) mRNA and 17beta-estradiol (E(2)) secretion. The addition of recombinant murine IGFBP7 to these cultures decreased in the activin-enhanced, FSH-stimulated Cyp19a1 mRNA levels in the cells and suppressed the 17beta-E(2) levels in the culture medium. Treatment of GCs with Igfbp7-specific small interfering RNA (siRNA), which knocked down Igfbp7 expression, increased the FSH-stimulated levels of Cyp19a1 but not Cyp11a1 expression. Basal and FSH-stimulated 17beta-E(2) secretion into the culture medium was also enhanced by Igfbp7 siRNA. These results suggest that IGFBP7 suppresses estrogen production in GCs. These observations support the notion that this protein, which is secreted into the follicular fluid, may serve as an intraovarian factor that negatively regulates GC differentiation.     

Nrf2‐Mediated Resistance to Oxidant‐Induced Redox Disruption in Embryos

Events that control developmental changes occur during specific windows of gestation and if disrupted, can lead to dysmorphogenesis or embryolethality. One largely understudied aspect of developmental control is redox regulation, where the untimely disruption of intracellular redox potentials (E_h) may alter development, suggesting that tight control of developmental‐stage–specific redox states is necessary to support normal development. In this study, mouse gestational day 8.5 embryos in whole embryo culture were treated with 10 μM dithiole‐3‐thione (D3T), an inducer of nuclear factor (erythroid‐derived 2)‐like 2 (Nrf2). After 14 hr, D3T‐treated and ‐untreated conceptuses were challenged with 200 μM hydrogen peroxide (H₂O₂) to induce oxidant‐induced change to intracellular E_hs. Redox potentials of glutathione (GSH), thioredoxin‐1 (Trx1), and mitochondrial thioredoxin‐2 (Trx2) were then measured over a 2‐hr rebounding period following H₂O₂ treatment. D3T treatment increased embryonic expression of known Nrf2‐regulated genes, including those responsible for redox regulation of major intracellular redox couples. Exposure to H₂O₂ without prior D3T treatment produced significant oxidation of GSH, Trx1, and Trx2, based on E_h values, where GSH and Trx2 E_h recovered, reaching to pre‐H₂O₂ E_h ranges, but Trx1 E_h remained oxidized. Following H₂O₂ addition in culture to embryos that received D3T pretreatments, GSH, Trx1, and Trx2 were insulated from significant oxidation. These data show that Nrf2 activation may serve as a means to protect the embryo from chemically induced oxidative stress through the preservation of intracellular redox states during development, allowing normal morphogenesis to ensue.     

Ginsenosides promote proliferation of granulosa cells from chicken prehierarchical follicles through PKC activation and up‐regulated cyclin gene expression

The effect of GS (ginsenosides) on proliferation of chicken GCs (granulosa cells) from prehierarchical SYF (small yellow follicles) was evaluated, and involvement of the PKC (protein kinase C) signalling pathway as well as mRNA expression of cyclins and CDK (cyclin‐dependent kinase) were investigated. Whole SYF or GCs isolated from SYF were cultured in Medium 199 supplemented with 0.5% FCS (fetal calf serum). After 16 h, the cells were challenged with GS alone or in combination with PKC inhibitor H₇ or activator PMA (phorbol 12‐myristate 13‐acetate) for 24 h in serum‐free medium. Results showed that in both whole follicles and pure GCs monolayer culture system, GS (0.1–10 μg/ml) significantly increased the number of GCs in SYF in a dose‐dependent manner, and this stimulatory effect was inhibited by H₇, but enhanced by PMA. Meanwhile, the PCNA‐LI (proliferating cell nuclear antigen labelling index) of GCs displayed similar changes with the cell number. Mechanism of GS action was further evaluated in cultured GCs separated from SYF. Western blot analysis showed that 10 μg/ml GS increased PKC translocation from cytoplasm to the plasma membrane of the GCs to become the active state. This effect was blocked by H₇. Furthermore, GS up‐regulated the expression of cyclin D1/CDK6 and cyclin E/CDK2 mRNAs in GCs; however, inhibition of PKC with H₇ attenuated this stimulatory effect. These results indicated that GS could stimulate proliferation of chicken GCs through activated PKC‐involved up‐regulation of cyclin D1/CDK6 and cyclin E/CDK2 genes, subsequently promoting development of the chicken prehierarchical follicles.     

Antioxidant butylated hydroxyanisole inhibits estrogen‐induced breast carcinogenesis in female ACI rats

Exposure to estrogens is suggested to be a risk factor in human breast cancer development. The mechanisms underlying estrogen‐induced cancer have not been fully elucidated. Both estrogen receptor (ER)‐mediated proliferative processes and ER‐independent generation of oxidative stress are suggested to play important roles in estrogen‐induced breast carcinogenesis. In the current study, we investigated the role of oxidative stress in breast carcinogenesis using the ACI rat model of mammary tumorigenesis. Female ACI rats were treated with 17β‐estradiol (E₂), butylated hydroxyanisole (BHA), or a combination of E₂ + BHA for up to 240 days. Cotreatment of rats with E₂ + BHA reduced estrogen‐induced breast tumor development with tumor incidence of 24%, a significant decrease relative to E₂ where tumor incidence was 82%. Proliferative changes in the breast tissue of E₂ + BHA‐treated animals were similar to those observed in E₂‐treated animals. Tissue levels of 8‐isoprostane, a marker of oxidant stress, as well as the activities of antioxidant enzymes including glutathione peroxidase, superoxide dismutase, and catalase were quantified in the breast tissues of rats treated with E₂ + BHA and compared to activity levels found in E₂‐treated animals and respective age‐matched controls. Cotreatment with BHA inhibited E₂‐mediated increases in 8‐isoprostane levels as well as activities of antioxidant enzymes. In summary, these data suggest that estrogen‐mediated oxidant stress plays a critical role in the development of estrogen‐dependent breast cancers and BHA inhibits E₂‐dependent breast carcinogenesis by decreasing oxidant stress. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:202–211, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20281     

The role of glutathione, membrane sphingomyelin, and its metabolites in oxidative stress-induced calcium “dysregulation” in PC12 cells

Previous research showed that increasing membrane sphingomyelin (SPH) levels in rat pheochromocytoma (PC12) cells to the same extent as that seen in some brain regions with aging dramatically increases the vulnerability to oxidative stress (OS). These increases in vulnerability were determined by assessing deficits in the ability of these cells to extrude and/or sequester Ca²⁺ following 30 mM KCl-induced depolarization (recovery). The purpose of the present experiments was to discern whether increasing the levels of particular SPH metabolite(s), i.e., ceramide (Cer), sphingosine (Ssine), or sphingosine-1-phosphate (SPP), or indirectly increasing the concentrations of these metabolites with sphingomylinase (Sase), would interact with the cell’s sensitivity to OS induced by low (5 μM) or high (nonlethal, 300 μM) H₂O₂. In addition, the OS vulnerability was examined as above under decreased SPH levels by exposing the cells to L-cycloserine (Lcc), which prevents SPH synthesis. Both Sase and SPP significantly decreased Ca²⁺ recovery of PC12 cells after H₂O₂ exposure. Conversely, Lcc-treated cells showed no further OS-induced decrements in recovery below those seen in controls. SPP significantly decreased glutathione levels (GSH) in the absence of OS. Repletion of GSH with 20 mM N-acetylcysteine significantly attenuated the effect of 5 μM H₂O₂ on recovery in SPP-treated cells and decreased sensitivity of SPP-treated cells to low doses of OS. Overall, our results suggest a critical role for GSH and SPP in the regulation of OS vulnerability, especially as it relates to Ca²⁺ homeostasis.     

HT-29 human colon cancer cell proliferation is regulated by cytosolic phospholipase A2α dependent PGE2via both PKA and PKB pathways

Cytosolic phospholipase A₂α (cPLA₂α) up-regulation has been reported in human colorectal cancer cells, thus we aimed to elucidate its role in the proliferation of the human colorectal cancer cell line, HT-29. EGF caused a rapid activation of cPLA₂α which coincided with a significant increase in cell proliferation. The inhibition of cPLA₂α activity by pyrrophenone or by antisense oligonucleotide against cPLA₂α (AS) or inhibition of prostaglandin E₂ (PGE₂) production by indomethacin resulted with inhibition of cell proliferation, that was restored by addition of PGE₂. The secreted PGE₂ activated both protein kinase A (PKA) and PKB/Akt pathways via the EP2 and EP4 receptors. Either, the PKA inhibitor (H-89) or the PKB/Akt inhibitor (Ly294002) caused a partial inhibition of cell proliferation which was restored by PGE₂. But, inhibited proliferation in the presence of both inhibitors could not be restored by addition of PGE₂. AS or H-89, but not Ly294002, inhibited CREB activation, suggesting that CREB activation is mediated by PKA. AS or Ly294002, but not H-89, decreased PKB/Akt activation as well as the nuclear localization of β-catenin and cyclin D1 and increased the plasma membrane localization of β-catenin with E-cadherin, suggesting that these processes are regulated by the PKB pathway. Similarly, Caco-2 cells exhibited cPLA₂α dependent proliferation via activation of both PKA and PKB/Akt pathways. In conclusion, our findings suggest that the regulation of HT-29 proliferation is mediated by cPLA₂α-dependent PGE₂ production. PGE₂via EP induces CREB phosphorylation by the PKA pathway and regulates β-catenin and cyclin D1 cellular localization by PKB/Akt pathway.     

Involvement of Ca<Superscript>2+</Superscript>/calmodulin kinase II (CaMK II) in genistein-induced potentiation of leucine/glutamine-stimulated insulin secretion

Genistein has been reported to potentiate glucose-stimulated insulin secretion (GSIS). Inhibitory activity on tyrosine kinase or activation of protein kinase A (PKA) was shown to play a role in the genistein-induced potentiation effect on GSIS. The aim of the present study was to elucidate the mechanism of genistein-induced potentiation of insulin secretion. Genistein augmented insulin secretion in INS-1 cells stimulated by various energy-generating nutrients such as glucose, pyruvate, or leucine/glutamine (Leu/Gln), but not the secretion stimulated by depolarizing agents such as KCl and tolbutamide, or Ca²⁺ channel opener Bay K8644. Genistein at a concentration of 50 μM showed a maximum potentiation effect on Leu/Gln-stimulated insulin secretion, but this was not sufficient to inhibit the activity of tyrosine kinase. Inhibitor studies as well as immunoblotting analysis demonstrated that activation of PKA was little involved in genistein-induced potentiation of Leu/Gln-stimulated insulin secretion. On the other hand, all the inhibitors of Ca²⁺/calmodulin kinase II tested, significantly diminished genistein-induced potentiation. Genistein also elevated the levels of [Ca²⁺]_i and phospho-CaMK II. Furthermore, genistein augmented Leu/Gln-stimulated insulin secretion in CaMK II-overexpressing INS-1 cells. These data suggest that the activation of CaMK II played a role in genistein-induced potentiation of insulin secretion.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.