Forskolin Treatment Research Articles

Pre-transplantational Control of the Post-transplantational Fate of Human Pluripotent Stem Cell-Derived Cartilage.

SummaryCartilage pellets generated from ectomesenchymal progeny of human pluripotent stem cells (hPSCs) in vitro eventually show signs of commitment of chondrocytes to hypertrophic differentiation. When transplanted subcutaneously, most of the surviving pellets were fully mineralized by 8 weeks. In contrast, treatment with the adenylyl cyclase activator, forskolin, in vitro resulted in slightly enlarged cartilage pellets containing an increased proportion of proliferating immature chondrocytes that expressed very low levels of hypertrophic/terminally matured chondrocyte-specific genes. Forskolin treatment also enhanced hyaline cartilage formation by reducing type I collagen gene expression and increasing sulfated glycosaminoglycan accumulation in the developed cartilage. Chondrogenic mesoderm from hPSCs and dedifferentiated nasal chondrocytes responded similarly to forskolin. Furthermore, forskolin treatment in vitro increased the frequency at which the cartilage pellets maintained unmineralized chondrocytes after subcutaneous transplantation. Thus, the post-transplantational fate of chondrocytes originating from hPSC-derived chondroprogenitors can be controlled during their genesis in vitro.

Amyloid Precursor Protein Overexpression in Down Syndrome Trophoblast Reduces Cell Invasiveness and Interferes with Syncytialization

The placentas of Down syndrome (DS) pregnancies exhibit morphologic and functional abnormalities. Although the increase in dosage of certain genes on chromosome 21 has been associated with the DS phenotype, the effects on placenta have seldom been studied. Herein, we examine the expression of four dosage-sensitive genes (APP, ETS2, SOD1, and HMGN1) in normal and DS placentas. We demonstrated significant overexpression of amyloid precursor protein (APP) in DS placentas at RNA and protein levels by real-time quantitative PCR, Western blot analysis, and immunohistochemistry. Inducible APP overexpression trophoblast cell line models were established using a Tet-On system. APP induction in HTR-8/SVneo dose-dependently decelerated cell growth, enhanced apoptosis, and reduced cell migration and invasion when compared with the uninduced controls. Concomitantly, decreased β-human chorionic gonadotropin in the culture medium was also detected on induction. Moreover, although forskolin treatment induced α/β-human chorionic gonadotropin and syncytin expression in BeWo cells, such induction of syncytialization was inhibited by APP overexpression. E-cadherin immunofluorescence also demonstrated a decrease in syncytia formation in forskolin-treated BeWo-overexpressing APP. By liquid chromatography-tandem mass spectrometry, proteins related to cell-cell adhesion, protein translation, processing, and folding were found to be up-regulated in APP-induced HTR-8/SVneo clones. Our data demonstrated, for the first time, the effects of increased APP expression in DS placenta.

Single Local Application of TGF-β Promotes a Proregenerative State Throughout a Chronically Injured Nerve

The lack of nerve regeneration and functional recovery occurs frequently when injuries involve large nerve trunks because insufficient mature axons reach their targets in the distal stump and because of the loss of neurotrophic support, primarily from Schwann cells (SCs). To investigate whether a single application of transforming growth factor-beta (TGF-β) plus forskolin or forskolin alone can promote and support axonal regeneration through the distal nerve stump. Using a delayed repair rat model of nerve injury, we transected the tibial nerve. After 8 wk, end-to-end repair was done and the repair site was treated with saline, forskolin, or TGF- β plus forskolin. After 6 wk, nerve sections consisting of the proximal stump, distal to the site of repair, and the most distal part of the nerve stump were removed for nerve histology, axon counts, and immunohistochemistry for activated SCs (S100), macrophages (CD68), cell proliferation (Ki67), p75NGFR, and apoptosis (activated caspase-3). TGF-β plus forskolin significantly increased the numbers of axons regenerated distal to the repair site and the most distal nerve sections. Both treatments significantly increased the numbers of axons regenerated in the most distal nerve sections compared to saline treated. Both treatments exhibited extended expression of regeneration-associated marker proteins. TGF-β plus forskolin treatment of chronically injured nerve improved axonal regeneration and increased expression of regeneration-associated proteins beyond the repair site. This suggests that a single application at the site of repair has mitogenic effects that extended distally and may potentially overcome the decrease in regenerated axon over long distance.

Characterization of transgenic mice expressing EGFP under the control of the monkey 20α-hydroxysteroid dehydrogenase promoter.

To directly assess the molecular function of the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) promoter, we generated transgenic mice (tg) expressing enhanced green fluorescent protein (EGFP) under control of this promoter. We demonstrated that prostaglandin F2α induced 20α-HSD promoter activity in CHO cells in a dose-dependent manner. Furthermore, forskolin treatment markedly reduced 20α-HSD promoter activity, and prolactin exhibited weak inhibitory activity. The transgenic mouse obtained one positive founder male. The transgene was propagated in 10 successive generations without any notable defects to the progeny. EGFP and 20α-HSD in the tg mice were colocalized in the luteal cells of the ovary during late pregnancy. Strong EGFP and 20α-HSD protein signals were also detected in the adult testis. Immunohistochemical analysis revealed high EGFP levels in the seminiferous epithelium, whereas 20α-HSD was expressed in the seminiferous tubules. Our data suggest that the ovaries in monkey and mouse exhibit similar expression patterns of 20α-HSD during pregnancy. However, the expression pattern of EGFP in tg mice testis slightly differed from that of the endogenous 20α-HSD. Further investigation is required to elucidate the functional mechanisms underlying regulation of the monkey 20α-HSD promoter in the tg mice.

Microparticle Release from Cell Lines and Its Anti-Influenza Activity.

Microparticles (MPs) are vesicles that are released by budding from plasma membrane of living cells. Recently, the role of MPs in antiviral activity has been proposed. We investigated quantity and anti-influenza activity of MPs from human alveolar epithelial cells A549, human bronchial epithelial cells BEAS-2B, human colon adenocarcinoma cells HT-29, and the human lung fibroblast cells MRC-5. MPs were found from all four cell lines. However, anti-influenza activity against an H1N1 influenza virus was found only from MPs of A549 and BEAS-2B. BEAS-2B cell differentiation did not increase MP release. Methyl-β-cyclodextrin (MβCD) increased MP release and anti-influenza activity in HT-29 and A549. MP release increased after calcium ionophore A23187 treatment in three cell lines but only in HT-29 after forskolin treatment. These findings provide in vitro data supporting the role of MPs as an innate defense against influenza virus and as an approach to enhance the defense.

Cystic Fibrosis Transmembrane Conductance Regulator Regulates Pannexin 1 Channel Opening for Acidosis‐induced ATP Release from Cardiomyocytes

ATP is released in the heart under a variety of conditions, including hypoxia, ischaemia and catecholamine stimulation. Under physiological conditions, extracellular ATP, and its breakdown product, adenosine, contribute to local vasodilation and regulation of coronary blood flow. We investigated the mechanism of ATP release in cardiomyocytes isolated from adult rat heart.Treatment with lactic acid produced pH depression and enhanced ATP release from cardiomyocytes. On the other hand, simulated ischaemia (metabolic inhibition plus anoxia) lowered ATP release. The acidosis‐induced ATP release was abolished by the specific CFTR inhibitor, CFTRinh‐172, CFTR pore blocker, GlyH‐101, or CFTR siRNA, suggesting that CFTR was involved. Forskolin and IBMX, agents that activate CFTR by elevating the intracellular cAMP, also increased ATP release, confirming the role of CFTR. However, blockade of Pannexin1 channels by Fast Green FCF, Brilliant Blue FCF, or pannexin1 siRNA, attenuated the ATP release during acidosis or forskolin treatment, and further lowered the ATP release during simulated ischaemia, suggesting that Pannexin1 functions as the ATP release channel. Immunofluorescence imaging suggested that CFTR and Pannexin1 were not co‐localized. Inhibition of either the Na+/H+ exchanger (NHE) with amiloride or the Na+/Ca2+ exchanger (NCX) with SN6 or KB‐R7943 abolished acidosis‐induced ATP release. FAK inhibitor 14, FAK autophosphorylation inhibitor, can attenuate the forskolin or lactic‐acid‐stimulated ATP release. Furthermore, forskolin or lactic‐acid‐stimulated ATP release was abolished by bicarbonate free medium or caspase inhibitor. Thus, we have demonstrated that the acidosis‐induced ATP release was involved in calcium induced CFTR open. The cyclic AMP, which activates mitochondrial PKA, is generated within mitochondria by the bicarbonate‐regulated soluble adenylyl cyclase (sAC). Hence, we propose that the CFTR Cl channel opens during acidosis, allowing the movement of bicarbonate into the cell, which activate the HCO3‐sAC‐cAMP‐PKA (mito‐sAC) signaling cascade and indirectly modulates Pannexin1 gating by activation of caspase.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced Placental Expression of Regulator of G‐Protein Signaling‐2 (RGS2) and Preeclampsia

Although late‐gestational mechanisms of preeclampsia (PreE) have been described, the genetic risk factors and mechanisms driving early‐gestational pathogenesis remain largely unknown. Various activators of Gαq‐mediated signaling pathways have been identified as possible early mediators of PreE, including vasopressin, endothelin, and angiotensin. Regulator of G‐protein Signaling 2 (RGS2) acts as an endogenous terminator of Gαq signaling and previous studies have identified a single nucleotide polymorphism (rs4606) in the RGS2 gene that is associated with increased risk for PreE, and which may reduce RGS2 mRNA. We therefore hypothesize that reduced placental expression of RGS2 increases the risk of PreE due to the subsequent disinhibition of Gαq‐mediated signaling. In silico reanalysis of a publicly‐available dataset (GSE75010) describing the transcriptomes of human placentas uncovered a significant reduction in RGS2 mRNA in placentas from pre‐term PreE pregnancies compared to control pre‐term pregnancies (Con n=35, PreE n=49, p<0.05). This reduction in RGS2 mRNA was confirmed by qPCR using human placental tissue samples from the University of Iowa Maternal‐Fetal Tissue Bank (PreE 19% of Con, n=11 vs 9, p<0.05), though no significant correlation was observed between the rs4606 SNP and PreE in this cohort. In silico reanalysis of a separate dataset (GSE93839) revealed RGS2 mRNA is among the highest‐expressed RGS family members in normal human placenta, and that it may be selectively reduced in both syncytiotrophoblast (73% of Con) and invasive cytotrophoblast (68% of Con) layers of the placenta during PreE. To examine the sufficiency of feto‐placental‐specific reduction in RGS2 on the development of PreE phenotypes, wildtype C57BL/6J female mice were mated with RGS2‐deficient (RGS2‐KO) sires, which results in a wildtype dam carrying all heterozygous feto‐placental units, or wildtype littermates of the RGS2‐KO males. Dams mated with RGS2‐KO sires developed diastolic hypertension (Con 92 ± 2 vs RGS2‐KO 98.2 ± 2 [24 hr avg mmHg]; p<0.05 vs pre‐pregnancy RGS2‐KO, and pregnant control) and increased proteinuria compared to dams mated with littermate males (18.2 ± 2.2, n=7 vs 28.4 ± 2.8, n=10 mg/day, p<0.05), although no intrauterine growth restriction, changes in placental PlGF (Con n=6, 9.8 ± 1.1 vs KO n=12, 11.8 ± 0.8 μg/g) or FLT1 protein (103 ± 30.2 vs 114.6 ± 42.7 ng/g), or markers of hypoxia were observed. Previous studies have indicated that placentas from PreE patients exhibit reduced cAMP content compared to normal placentas, and in vascular smooth muscle, transcription of RGS2 is promoted by CREB occupancy at a cAMP Response Element (CRE) within its promoter. Thus we hypothesized a potential role for cAMP/CREB control of RGS2 in trophoblasts. Forskolin treatment increased CREB binding to the RGS2 promoter (by chromatin immunoprecipitation) and RGS2 mRNA expression (by qPCR) in cultured human HTR8/SVneo trophoblasts. Preliminary data support reduced CREB occupancy at the RGS2 promoter in PreE placenta. These data support a role for reduced placental RGS2 expression in the pathogenesis of PreE, and demonstrate that reduced placental cAMP levels may contribute to the loss of RGS2 expression in placental trophoblasts regardless of rs4606 SNP genotype.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Entrainment of the Circadian Clock in Neural Stem Cells by Epidermal Growth Factor is Closely Associated with ERK1/2-mediated Induction of Multiple Clock-related Genes

The mitotic activity of certain tissues in the body is closely associated with circadian clock function. However, the effects of growth factors on the molecular clockwork are not fully understood. Stimulation of neural stem cells (NSCs) with epidermal growth factor (EGF), a well-known mitogen, is known to cause synchronized cell cycle progression with a period of approximately 24 h, closely associated with the Per2 gene expression rhythm. Here, we examined the effects of EGF on the molecular clockwork of NSCs. Treatment of cultured NSCs derived from embryonic mouse forebrain with EGF (20 ng/mL) caused a phase shift in the PER2::LUCIFERASE bioluminescence rhythm in a stimulation time-dependent manner. The EGF phase-response curve differed from that of forskolin (FK)—a well-known chemical resetting stimulus—both in the advance/delay ratio and stimulation time-dependency. PCR array analysis followed by quantitative PCR validation demonstrated that EGF treatment transiently induced multiple clock-related genes including Per1, Per2, Dec1, e4bp4, and Noct, whereas FK treatment induced a limited number of genes (Per1 and Dec1), suggesting that the mode of entrainment of NSC molecular clock was different for EGF and FK. EGF led to gene induction in the presence of cycloheximide, suggesting that de novo protein synthesis is unnecessary. Pretreatment with the MEK1/2 inhibitor U0126 significantly suppressed the acute induction of Per2, Dec1, and Noct by EGF and also abolished the EGF-induced phase shift of the PER2::LUCIFERASE rhythm in NSCs. These results suggest a unique effect of EGF on the molecular clockwork of NSCs.

Forskolin-mediated BeWo cell fusion involves down-regulation of miR-92a-1-5p that targets dysferlin and protein kinase cAMP-activated catalytic subunit alpha.

To study the role of miRNA(s) during trophoblastic BeWo cell fusion. Changes in miRNA(s) profile of BeWo cells treated with forskolin were analyzed using Affymetrix miRNA microarray platform. Down-regulated miRNA, miR-92a-1-5p, was overexpressed in BeWo cells followed by forskolin treatment to understand its relevance in the process of BeWo cell fusion by desmoplakin I+II staining and hCG secretion by ELISA. Predicted targets of miR-92a-1-5p were also confirmed by qRT-PCR/Western blotting. The miRNA profiling of BeWo cells after forskolin (25μmol/L) treatment identified miR-92a-1-5p as the most significantly down-regulated miRNA both at 24 and 48hours time points. Overexpression of miR-92a-1-5p in these cells led to a significant decrease in forskolin-mediated cell fusion and hCG secretion. miRNA target prediction software, TargetScan, revealed dysferlin (DYSF) and protein kinase cAMP-activated catalytic subunit alpha (PRKACA), as target genes of miR-92a-1-5p. Overexpression of miR-92a-1-5p in BeWo cells showed reduction in forskolin-induced transcripts for DYSF and PRKACA. Further, reduction in DYSF (~2.6-fold) at protein level and PRKACA-encoded protein kinase A catalytic subunit alpha (PKAC-α; ~1.6-fold) were also observed. These observations suggest that miR-92a-1-5p regulates forskolin-mediated BeWo cell fusion and hCG secretion by regulating PKA signaling pathway and dysferlin expression.

Increase in Ca2 + current by sustained cAMP levels enhances proliferation rate in GH3 cells

AimsCa2+ and cAMP are important intracellular modulators. In order to generate intracellular signals with various amplitudes, as well as different temporal and spatial properties, a tightly and precise control of these modulators in intracellular compartments is necessary. The aim of this study was to evaluate the effects of elevated and sustained cAMP levels on voltage-dependent Ca2+ currents and proliferation in pituitary tumor GH3 cells. Main methodsEffect of long-term exposure to forskolin and dibutyryl-cyclic AMP (dbcAMP) on Ca2+ current density and cell proliferation rate were determined by using the whole-cell patch-clamp technique and real time cell monitoring system. The cAMP levels were assayed, after exposing transfected GH3 cells with the EPAC-1 cAMP sensor to forskolin and dbcAMP, by FRET analysis. Key findingsSustained forskolin treatment (24 and 48h) induced a significant increase in total Ca2+ current density in GH3 cells. Accordingly, dibutyryl-cAMP incubation (dbcAMP) also elicited increase in Ca2+ current density. However, the maximum effect of dbcAMP occurred only after 72h incubation, whereas forskolin showed maximal effect at 48h. FRET-experiments confirmed that the time-course to elevate intracellular cAMP was distinct between forskolin and dbcAMP. Mibefradil inhibited the fast inactivating current component selectively, indicating the recruitment of T-type Ca2+ channels. A significant increase on cell proliferation rate, which could be related to the elevated and sustained intracellular levels of cAMP was observed. SignificanceWe conclude that maintaining high levels of intracellular cAMP will cause an increase in Ca2+ current density and this phenomenon impacts proliferation rate in GH3 cells.

Forskolin attenuates retinal inflammation in diabetic mice.

The present study aimed to investigate the effect of forskolin on retinal inflammation under diabetic conditions. C57BL/6 mice were randomly divided into normal control, diabetic control and forskolin treatment groups. The diabetic model was established by intraperitoneal injection of streptozotocin. The forskolin treatment group received intragastrical administration of forskolin for 12 weeks, the other two groups received an equal amount of PBS. At 21 weeks following diabetic induction, an immunoblotting test was conducted to investigate the expression of two inflammatory factors: Intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-α (TNF-α). Glucose concentration was additionally calculated. A leukostasis assay was utilized to compare microvasculature pathological alterations. It was demonstrated that retinal glucose concentration of diabetic control and forskolin treatment were both increased compared with normal control, however the forskolin treatment group was only ~68.06% of the diabetic control due to downregulated glucose transporter 1 expression. The expression of ICAM-1 and TNF-α were upregulated in the forskolin treatment and diabetic control groups compared with the normal control, however these two inflammatory factor expression levels in the forskolin treatment group were ~68.75 and 75.37% of diabetic control. It was additionally observed that there were less adherent leukocytes in retinal microvasculature in the forskolin treatment group compared with diabetic control. All the differences observed were significant. Overall, by means of limiting glucose transport into the retina via forskolin, the retinal environment with lower glucose concentration alleviates the inflammatory response under diabetic conditions.

Relevance of the NR4A sub-family of nuclear orphan receptors in trophoblastic BeWo cell differentiation

Nur-77, a member of the NR4A sub-family of nuclear orphan receptors, is downregulated in the placentae of pre-eclamptic women. Here, we investigate the relevance of Nor-1, Nurr-1 and Nur-77 in trophoblastic cell differentiation. Their transcript levels were found to be significantly upregulated in BeWo cells treated with forskolin. The maximum increase was observed after 2 h, with a second peak in the expression levels after 48 h. The expression of NR4A sub-family members was also found to be upregulated in BeWo cells after treatment with hCG and GnRH. A similar significant increase was observed at the respective protein levels after 2 and 48 h of treatment with forskolin, hCG or GnRH. Silencing Nor-1, Nurr-1 or Nur-77 individually did not show any effect on forskolin-, hCG- and/or GnRH-mediated BeWo cell fusion and/or hCG secretion. After silencing any one member of the NR4A sub-family, an increase in the transcript levels of the other sub-family members was observed, indicating a compensatory effect due to their functional redundancy. Simultaneously silencing all three NR4A sub-family members significantly downregulated forskolin- and hCG-mediated BeWo cell fusion and/or hCG secretion. However, a considerable amount of cell death occurred after forskolin or hCG treatment as compared to the control siRNA-transfected cells. These results suggest that the NR4A sub-family of nuclear orphan receptors has a role in trophoblastic cell differentiation.

Claudin5a is required for proper inflation of Kupffer's vesicle lumen and organ laterality.

Left-right asymmetric organ development is critical to establish a proper body plan of vertebrates. In zebrafish, the Kupffer’s vesicle (KV) is a fluid-filled sac which controls asymmetric organ development, and a properly inflated KV lumen by means of fluid influx is a prerequisite for the asymmetric signal transmission. However, little is known about the components that support the paracellular tightness between the KV luminal epithelial cells to sustain hydrostatic pressure during KV lumen expansion. Here, we identified that the claudin5a (cldn5a) is highly expressed at the apical surface of KV epithelial cells and tightly seals the KV lumen. Downregulation of cldn5a in zebrafish showed a failure in organ laterality that resulted from malformed KV. In addition, accelerated fluid influx into KV by combined treatment of forskolin and 3-isobutyl-1-methylxanthine failed to expand the partially-formed KV lumen in cldn5a morphants. However, malformed KV lumen and defective heart laterality in cldn5a morphants were significantly rescued by exogenous cldn5a mRNA, suggesting that the tightness between the luminal epithelial cells is important for KV lumen formation. Taken together, these findings suggest that cldn5a is required for KV lumen inflation and left-right asymmetric organ development.

Chronic hypertension increases aortic endothelial hydraulic conductivity by upregulating endothelial aquaporin-1 expression.

Numerous studies have examined the role of aquaporins in osmotic water transport in various systems, but virtually none have focused on the role of aquaporin in hydrostatically driven water transport involving mammalian cells save for our laboratory's recent study of aortic endothelial cells. Here, we investigated aquaporin-1 expression and function in the aortic endothelium in two high-renin rat models of hypertension, the spontaneously hypertensive genetically altered Wistar-Kyoto rat variant and Sprague-Dawley rats made hypertensive by two-kidney, one-clip Goldblatt surgery. We measured aquaporin-1 expression in aortic endothelial cells from whole rat aortas by quantitative immunohistochemistry and function by measuring the pressure-driven hydraulic conductivities of excised rat aortas with both intact and denuded endothelia on the same vessel. We used them to calculate the effective intimal hydraulic conductivity, which is a combination of endothelial and subendothelial components. We observed well-correlated enhancements in aquaporin-1 expression and function in both hypertensive rat models as well as in aortas from normotensive rats whose expression was upregulated by 2 h of forskolin treatment. Upregulated aquaporin-1 expression and function may be a response to hypertension that critically determines conduit artery vessel wall viability and long-term susceptibility to atherosclerosis.NEW & NOTEWORTHY The aortic endothelia of two high-renin hypertensive rat models express greater than two times the aquaporin-1 and, at low pressures, have greater than two times the endothelial hydraulic conductivity of normotensive rats. Data are consistent with theory predicting that higher endothelial aquaporin-1 expression raises the critical pressure for subendothelial intima compression and for artery wall hydraulic conductivity to drop.

Differential proteome expression analysis of androgen-dependent and -independent pathways in LNCaP prostate cancer cells

Prostate cancer (PC) is one of the leading causes of cancer death in men. It commonly develops in older males, but the number of younger men diagnosed with the disease has increased in recent years. Hormone therapies, such as chemical and surgical methods that inhibit androgen synthesis or androgen receptor (AR) activation, have been used for advanced disease. However, castration-resistant PC (CRPC), which exhibits androgen-independent mechanisms for activating AR, develops after a few years of such treatment and no therapy is available. In this study, we examined differences in the proteomes associated with the androgen-dependent (DHT treatment) and -independent (FSK, forskolin treatment) AR signaling conditions in LNCaP prostate cancer cells. Moreover, we used EPI-001, which inhibits AR-mediated transcriptional activity, to examine whether the observed differences in protein expression were directly affected by AR-mediated mechanisms. A total of 213 protein spots were matched in our 2-dimensional gel electrophoresis (2DE) analysis and 8 proteins with significant expression changes in our 5 different treatment groups were identified by mass spectrometry. Among these proteins, expression levels of PEPCK-M, catalase, tubulin alpha chain, alpha-enolase, and endoplasmic reticulum resident protein 29 were significantly altered by DHT and the levels of HSP 90 and EF-Tu were changed by FSK. These changes were blocked by EPI-001 in DHT-regulated proteins, PEPCK-M, catalase, and tubulin alpha chain and FSK-regulated EF-Tu protein. The results from our immunohistochemical analysis using in vivo xenograft models were consistent with the 2DE data. We therefore report the first identification of differences in proteins that are significantly regulated under androgen-dependent and -independent AR signaling conditions. Our findings could suggest a possible molecular mechanism through which AR is activated in the absence and/or presence of androgen, and might help explain the inhibitory action of EPI-001 on CRPC.

Establishment and characterization of cell lines derived from complete hydatidiform mole.

Gestational trophoblastic diseases (GTDs) are a group of diseases characterized by abnormal cellular proliferation of atypical trophoblasts. A hydatidiform mole is an abnormal pregnancy caused by genetic fertilization disorders, and it can be classified as a complete hydatidiform mole (CHM) or a partial hydatidiform mole. The aim of this study was to establish cell lines from CHMs and to characterize the cells for future studies concerning GTD. HMol1-2C, HMol1-3B, HMol1-8 and HMol3-1B were established from primary cultures of CHM explants following the introduction of different combinations of genes including human telomerase reverse transcriptase (hTERT), a mutant form of CDK (CDK4R24C), cyclin D1, p53C234, MYC and HRAS. HMol1-2C, HMol1-3B, and HMol3-1B were confirmed to originate from trophoblasts of androgenic, homozygous CHMs. These three cell lines exhibited low human chorionic gonadotropin secretion, low migration and invasion abilities, and the potential to differentiate into syncytiotrophoblastic cells via forskolin treatment. These results suggest that these cells exhibit characteristics of trophoblastic cells, especially cytotrophoblastic cells. HMol1-8 was found to consist of diploid cells and originated from maternal cells, suggesting that they were derived from decidual cells. In conclusion, we successfully established three cell lines from CHMs by introduction of hTERT and other genes. Analysis revealed that the genetic origin of each cell line was identical with that of the original molar tissue, and the cell lines exhibited characteristics of trophoblastic cells, which are similar to undifferentiated cytotrophoblasts.

Anatomical and functional implications of corticotrophin-releasing hormone neurones in a septal nucleus of the avian brain: an emphasis on glial-neuronal interaction via V1a receptors in vitro.

Previously, we showed that corticotrophin-releasing hormone immunoreactive (CRH-IR) neurones in a septal structure are associated with stress and the hypothalamic-pituitary-adrenal axis in birds. In the present study, we focused upon CRH-IR neurones located within the septal structure called the nucleus of the hippocampal commissure (NHpC). Immunocytochemical and gene expression analyses were used to identify the anatomical and functional characteristics of cells within the NHpC. A comparative morphometry analysis showed that CRH-IR neurones in the NHpC were significantly larger than CRH-IR parvocellular neurones in the paraventricular nucleus of the hypothalamus (PVN) and lateral bed nucleus of the stria terminalis. Furthermore, these large neurones in the NHpC usually have more than two processes, showing characteristics of multipolar neurones. Utilisation of an organotypic slice culture method enabled testing of how CRH-IR neurones could be regulated within the NHpC. Similar to the PVN, CRH mRNA levels in the NHpC were increased following forskolin treatment. However, dexamethasone decreased forskolin-induced CRH gene expression only in the PVN and not in the NHpC, indicating differential inhibitory mechanisms in the PVN and the NHpC of the avian brain. Moreover, immunocytochemical evidence also showed that CRH-IR neurones reside in the NHpC along with the vasotocinergic system, comprising arginine vasotocin (AVT) nerve terminals and immunoreactive vasotocin V1a receptors (V1aR) in glia. Hence, we hypothesised that AVT acts as a neuromodulator within the NHpC to modulate activity of CRH neurones via glial V1aR. Gene expression analysis of cultured slices revealed that AVT treatment increased CRH mRNA levels, whereas a combination of AVT and a V1aR antagonist treatment decreased CRH mRNA expression. Furthermore, an attempt to identify an intercellular mechanism in glial-neuronal communication in the NHpC revealed that brain-derived neurotrophic factor (BDNF) and its receptor (TrkB) could be involved in the signalling mechanism. Immunocytochemical results further showed that both BDNF and TrkB receptors were found in glia of the NHpC. Interestingly, in cultured brain slices containing the NHpC, the use of a selective TrkB antagonist decreased the AVT-induced increase in CRH gene expression levels. The results from the present study collectively suggest that CRH neuronal activity is modulated by AVT via V1aR involving BDNF and TrkB glia in the NHpC.

Abstract 858: The circadian clock of glioma cells undergoing epithelial-mesenchymal transition

Abstract Highly invasive gliomas contain many cells that have changed to an enhanced migratory state through an epithelial-mesenchymal transition (EMT). These cells have the phenotype of glioma stem cells (GSCs). Similar to GSCs, glioma cells undergoing EMT show phenotypic heterogeneity, altered gene expression, and resistance to anticancer drugs along with increased invasiveness. Circadian rhythms in tumor cells influence the progression and severity of cancer and appear to regulate cell division cycles. Increased cancer incidence and progression have often been linked to disruption or deregulation of the molecular mechanism of the circadian clock. One of the major clock proteins, PER2, has been shown to play a regulatory role as a tumor and EMT suppressor in metastatic breast cancer cell lines. GSCs in tumorsphere cultures contain circadian clocks that may regulate their cancer properties. The possibility of a regulatory role for circadian rhythms in EMT was examined here. We used a standard method to induce EMT in the C6 rat glioma cell line that has known circadian rhythms in gene expression generated by the circadian clocks within its cells. EMT was induced by exchanging the cell culture medium with a serum-free stem cell medium (SCM) containing growth factors (EGF, FGF2, PDGF-alpha-beta). Cell cultures were imaged continuously with a microscope and digital camera in a cell incubator to monitor cell shape, cell death, migration, and apoptosis. During EMT, cells changed from an extended flat state to rounded and spindle shapes, ceased proliferating, and expressed EMT markers ZEB1 and vimentin. At the end of two days in SCM, 33.4% of the cells were ZEB1-positive and only 1.26% were GFAP-positive (n=3 cultures). Cell diameters after EMT were within the size range of C6 GSCs described as Hoechst-negative cells positive for stem cell markers nestin and CD133. Following EMT, small tumorspheres began to form. After initiating EMT, the rounded cells were counted at hourly intervals for up to four days after the medium exchange. As the number of post-EMT cells increased, the population size oscillated, and when examined by Lomb-Scargle periodogram analysis, four cultures had a significant period within the circadian range, 19-29 hours, (average 22.20 ±2.45 SD, p<0.05). One had a 16-hour period and one lacked a significant rhythm. Rhythms were present even when circadian clocks in the cultures were not synchronized with an initial forskolin treatment. We conclude that EMT may be timed by endogenous circadian oscillators in gliomas that favor larger numbers of post-EMT cells at a particular time of day. These results suggest that pharmacological treatments that suppress EMT or target GSCs would be more effective when delivered at particular times during the circadian cycle of the tumor. Citation Format: Arpan De, Dilshan Harshajith Beligala, Vishal Premdev Sharma, Benjamin Ryan Fry, Michael Eric Geusz. The circadian clock of glioma cells undergoing epithelial-mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 858. doi:10.1158/1538-7445.AM2017-858

Pharmacological activation of protein kinase A improves memory loss and neuropathological changes in a mouse model of dementia of Alzheimer's type.

The study investigates the therapeutic potential of the protein kinase A (PKA) activator forskolin in cognitive deficits of mice. Streptozotocin (STZ) [3 mg/kg, intracerebroventricularly (i.c.v.)] was used to induce memory deficits in mice, whereas aged mice served as natural model of dementia. Forskolin (2.5, 5, and 10 mg/kg/day, oral) treatment was administered to i.c.v. STZ-treated and aged mice for 14 days. The Morris Water Maze test was used to evaluate learning and memory. Estimation of brain acetylcholinesterase (AChE) activity, brain glutathione, thiobarbituric acid-reactive species, brain myeloperoxidase levels, and histopathological studies were also performed. Both STZ i.c.v. and aging resulted in a marked decline in Morris Water Maze performance, reflecting impairment of learning and memory. STZ i.c.v.-treated mice and aged mice showed a marked accentuation of AChE activity, thiobarbituric acid-reactive species and myeloperoxidase levels along with a decrease in the glutathione level. Further, the stained micrographs of STZ-treated mice and aged mice indicated pathological changes, severe neutrophilic infiltration, and amyloid deposition. Forskolin treatment significantly attenuated STZ-induced and age-related memory deficits, and biochemical and histopathological alterations. The findings indicate that the PKA activator forskolin probably alleviated memory deficits by virtue of its anticholinesterase, antiamyloid, antioxidative, and anti-inflammatory effects. It is concluded that PKA could be explored as a potential therapeutic target in dementia.

Gs\u03b1 Controls Cortical Bone Quality by Regulating Osteoclast Differentiation via cAMP/PKA and \u03b2-Catenin Pathways

Skeletal bone formation and maintenance requires coordinate functions of several cell types, including bone forming osteoblasts and bone resorbing osteoclasts. Gsα, the stimulatory subunit of heterotrimeric G proteins, activates downstream signaling through cAMP and plays important roles in skeletal development by regulating osteoblast differentiation. Here, we demonstrate that Gsα signaling also regulates osteoclast differentiation during bone modeling and remodeling. Gnas, the gene encoding Gsα, is imprinted. Mice with paternal allele deletion of Gnas (Gnas+/p−) have defects in cortical bone quality and strength during early development (bone modeling) that persist during adult bone remodeling. Reduced bone quality in Gnas+/p− mice was associated with increased endosteal osteoclast numbers, with no significant effects on osteoblast number and function. Osteoclast differentiation and resorption activity was enhanced in Gnas+/p− cells. During differentiation, Gnas+/p− cells showed diminished pCREB, β-catenin and cyclin D1, and enhanced Nfatc1 levels, conditions favoring osteoclastogenesis. Forskolin treatment increased pCREB and rescued osteoclast differentiation in Gnas+/p− by reducing Nfatc1 levels. Cortical bone of Gnas+/p− mice showed elevated expression of Wnt inhibitors sclerostin and Sfrp4 consistent with reduced Wnt/β-catenin signaling. Our data identify a new role for Gsα signaling in maintaining bone quality by regulating osteoclast differentiation and function through cAMP/PKA and Wnt/β-catenin pathways.