scholarly journals Functional and Molecular Characterization of a Muscarinic Receptor Type and Evidence for Expression of Choline-Acetyltransferase and Vesicular Acetylcholine Transporter in Human Granulosa-Luteal Cells1

1999 ◽  
Vol 84 (5) ◽  
pp. 1744-1750 ◽  
Author(s):  
S. Fritz ◽  
K. J. Föhr ◽  
S. Boddien ◽  
U. Berg ◽  
C. Brucker ◽  
...  
Keyword(s):  
Western Blotting ◽  
Choline Acetyltransferase ◽  
Nerve Fibers ◽  
Intracellular Free Calcium ◽  
Vesicular Acetylcholine Transporter ◽  
Free Calcium ◽  
Rt Pcr ◽  
Natural Ligand ◽  
M1 Receptor

Previously, we provided evidence for the presence of a class of muscarinic receptors on human luteinized granulosa cells (human GC) that is linked to transient increases in intracellular free calcium levels, but not to steroid production. The precise nature of the receptor is not known, and neither its function nor the source of its natural ligand acetylcholine (ACh) is clear. To address these issues we used RT-PCR approaches and isolated complementary DNAs corresponding to the M1 receptor subtype from reverse transcribed human GC messenger ribonucleic acids. M1 receptors were further shown by immunocytochemistry, using a M1 receptor antiserum. Single cell calcium measurements showed that the M1 receptor was functionally active and linked to acute increases in intracellular free calcium, as the M1 receptor specific antagonist pirenzepine blocked the Ca2+-mobilizing effect of oxotremorine M (a muscarinic agonist). An unexpected consequence of M1 receptor activation was evidenced by the ability of muscarinic agonists to stimulate the proliferation of human GC within 24 h. In vivo, ACh, the natural ligand of these receptors is thought to be contained in cholinergic nerve fibers innervating the ovary. Surprisingly, the prerequisite for the synthesis of ACh, the enzyme choline-acetyltransferase (ChAT), is also expressed by human GC, as shown by Western blotting and immunocytochemistry. In addition, these cells express another marker for ACh synthesis, namely the gene for the vesicular acetylcholine transporter, as evidenced by RT-PCR cloning, Western blotting, and immunocytochemistry. In conclusion, our data identify the M1 receptor in human GC and point to a novel, trophic role of the neurotransmitter ACh. Furthermore, the presence of the prerequisites of ACh synthesis in human GC indicate that an autocrine/paracrine regulatory loop also exists in the in vivo counterparts of these cells in the ovary, i.e. in the cells of the preovulatory follicle and/or of the young corpus luteum.

Dysfunction of Ca2+/ CaM kinase IIA cascades in basolateral amygdala of posttraumatic stress disorder rats

2011 ◽  
Vol 26 (S2) ◽  
pp. 178-178
Author(s):  
Y. Shi ◽  
F. Han
Keyword(s):  
Normal Level ◽  
Calcium Level ◽  
Basolateral Amygdala ◽  
Intracellular Free Calcium ◽  
Free Calcium ◽  
Rt Pcr ◽  
Control Groups ◽  
Male Wistar Rats ◽  
Polymerase Chain ◽  
Set Up

ObjectiveTo explore changes of Ca2+-CaM-CaMKIIα in basolateral amygdala of PTSD rats may reveal part of the pathogensis.MethodsThe SPS-method was used to set up the rat PTSD models. A total of 90 male Wistar rats were randomly divided into1d, 4d, 7d, 14d groups of SPS and normal control groups. The intracellular free calcium level in basolateral amygdala was examined by fluorescence spectrophotometer. CaM and CaMKIIα expression in basolateral amygdala were examined by immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction (RT-PCR).ResultsThe intracellular free calcium level reached the peak 1 day after SPS stimulation, then gradually decreased to normal level. The expression of CaM 1day after SPS is also the most and then decreased to normal level. In contrast, CaMKIIα expression showed a significant down-regulation 1day after SPS throughout and then gradually increased to normal level. This findings suggest dysfunction of Ca2+-CaM-CaMKIIα in basolateral amygdala of PTSD rats.ConclusionThus, the trauma-induced enhanced anxiety appear to be associated with, and possibly caused by, changes of Ca2+-CaM-CaMKIIα in basolateral amygdala.

scholarly journals Taurocholate Induces Connective Tissue Growth Factor Expression in Hepatocytes Through ERK-YAP Signaling

2018 ◽  
Vol 50 (5) ◽  
pp. 1711-1725
Author(s):  
Bin Yu ◽  
Guan-nan Jin ◽  
Mei Ma ◽  
Hui-fang Liang ◽  
Bi-xiang Zhang ◽  
...  
Keyword(s):  
Growth Factor ◽  
Liver Fibrosis ◽  
Western Blotting ◽  
Tissue Growth ◽  
Hepatic Cell ◽  
Erk Signaling ◽  
Rt Pcr ◽  
Hepatic Cell Lines

Background/Aims: Cholestasis is characterized by intrahepatic accumulation of cytotoxic bile acids (BAs), ultimately leading to fibrosis and cirrhosis, but the precise role of BAs in cholestasis-induced liver fibrosis remains largely elusive. In this study, we investigated the role and the potential mechanisms of BAs during cholestasis in vivo and in vitro. Methods: The effect of BAs during cholestasis was studied in bile duct ligation (BDL) rat models in vivo. We performed immunohistochemistry, Western blotting, and quantitative RT-PCR to investigate the expression of connective tissue growth factor (CTGF/CCN2) in rat liver during cholestasis. The hepatic cell lines AML12 and BRL were stimulated with taurocholate (TC) and the level of CTGF/CCN2, and activation of ERK, Akt, p38 MAPK, JNK, YAP, and TGF-β/Smad signaling were examined using Western blotting. Next, to elucidate the mechanism underlying bile acid-induced CTGF/CCN2, we treated the cells with MEK1/2 inhibitor (U0126), YAP function inhibitor (verteporfin), p38 kinase inhibitor (SB203580), Akt inhibitor (MK2206), and small interfering RNA (siRNA) targeting mek1, erk, and yap in cooperation with TC. Besides, we confirmed the activation of these signaling pathways in BDL and sham rat livers by immunohistochemistry, Western blotting, and quantitative RT-PCR. Results: In this study, we confirmed that the expression of CTGF/CCN2 was increased in BDL-induced rodent cholestatic liver fibrosis. In addition, we showed that TC, the main component of BAs, enhanced the synthesis of CTGF/ CCN2 in AML12 and BRL hepatic cell lines. Moreover, we demonstrated that TC activated ERK, Akt, and YAP signaling in hepatocytes, but the precise roles of these signaling cascades in the expression of CTGF/CCN2 were different: TC-induced expression of CTGF/CCN2 was mediated by ERK-YAP signaling, whereas Akt signaling inhibited ERK signaling and YAP and subsequently the expression of CTGF/CCN2 in hepatocytes. Furthermore, YAP functioned as a downstream regulator of ERK signaling in TC-induced CTGF/CCN2 expression in hepatocytes. Conclusion: Our report provides evidence for the role of conjugated BAs in liver fibrosis and suggests that the production of CTGF/CCN2, induced by conjugated BAs via ERK-YAP axis activation, may be a therapeutic target in cholestasis-induced liver fibrosis.

216 HORMONAL REGULATION OF CLAUDIN-4 TIGHT JUNCTION MOLECULE IN MOUSE PLACENTA

2015 ◽  
Vol 27 (1) ◽  
pp. 198
Author(s):  
C. Ahn ◽  
D. Lee ◽  
E.-B. Jeung
Keyword(s):  
Real Time ◽  
Tight Junctions ◽  
Western Blotting ◽  
Hormonal Regulation ◽  
Paracellular Transport ◽  
Rt Pcr ◽  
Ionic Selectivity ◽  
Mouse Placenta ◽  
Transport Barriers

Tight junctions (TJ) are composed of a branching network of sealing strands. TJ regulate paracellular conductance and ionic selectivity. TJ components include the peripheral protein ZO-1, junctional adhesion molecules (JAM) and the integral proteins such as occludin and claudin. Claudins are a family of proteins that are the most important components in the tight junctions. The established paracellular transport barriers that control transportation of molecules within intercellular space. The present study focused on the expression of claudin, suggesting as major working molecules in the paracellular transport system. To study the regulation and roles of claudin family, we examined expression of mouse placental claudin family. Fifteen pregnant C57/BL6 mice were used in this study and TJ proteins including Claudin-1 to Claudin-24 expressions by real-time RT–PCR and Western blotting. The mice were divided into 3 groups depending on the gestational day (on Days 12, 16, and 20 of gestation).The localization of TJ proteins were examined by immunohistochemistry. After we identified the fluctuation of claudin expression during pregnancy, we assumed that the hormones are one regulator for claudin family. Therefore, we performed an in vivo study with hormone receptor antagonists (ICI 182, 780, and RU-486) for examining hormonal effect on claudin expression in the placenta. Forty-nine mice were divided into 7 groups. The changes of claudin expression were examined with real-time RT-PCR and Western blotting. In the transcription levels, Claudin-1, claudin-2, claudin-4, and Claudin-5 expression levels were relatively high compared to others in the claudin family in all periods of the pregnancy. The claudin-4 expression, which reduces permeability of ions, increased over a period of time. However, caludin-5 expression that is the responsive protein for a decrease in paracellular conductance, were decreased. Claudin-1 and -4 have been known as responsive genes for a decrease in paracellular conductance. On the other hand, claudin 2 and 5 have been known as increasing paracellular conductance. In addition, immunohistochemistry was performed to identify their localization for inferring permeability in placenta. In summary, we analysed the claudin expressions and presented possible important claudins among its family. Furthermore, their localization was also examined in the mouse placenta. In addition, the regulation of critically expressed claudins by pregnancy-associated hormones, E2 and P4, was examined. These results may provide functional and structural roles of claudins and their involvement in the maternal-fetal interaction and in the transportation of placental materials.

scholarly journals WTAP-mediated GPX4 m6A methylation triggers PASMCs ferroptosis and pulmonary vascular fibrosis in pulmonary artery hypertension

2021 ◽  
Author(s):  
Wei Xin ◽  
Siyu He ◽  
Yanling Du ◽  
Yang Yu ◽  
Xinyue Song ◽  
...  
Keyword(s):  
Cell Death ◽  
Western Blotting ◽  
Regulatory Mechanism ◽  
Therapeutic Potential ◽  
Pulmonary Artery Hypertension ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Pulmonary Vessel ◽  
Vessel Remodeling

Background and Purpose Ferroptosis is a new form of cell death discovered in recent years. PH is a pulmonary circulatory disease partially characterized by small pulmonary vessel remodeling and fibrosis. However, researchers have not clearly determined whether ferroptosis is involved in PH. Here, this study examined the role and regulatory mechanism of ferroptosis in PH and pulmonary fibrosis. Experimental Approach To evaluate the occurrence of ferroptosis in rat PH models and in hypoxic PASMCs, MDA, GSH and iron assay were performed. The therapeutic potential of ferroptosis inhibitor fer-1 was evaluated using echocardiography, hemodynamic analysis and ventricular weight measurement in rat PH models. Ferroptosis-related molecule was determined by western blotting and RT-PCR. Changes in autophagy and fibrosis were analyzed by western blotting analysis, RT-PCR and immunofluorescence. Key Results Ferroptosis was existence in vivo and vitro PH models. The fer-1 significantly improved the pathological symptoms of PH and inhibited the occurrence of pulmonary vascular fibrosis. GPX4 was significantly lower expression in PH models, and serves as a key driver of PH-related ferroptosis. A KEGG pathway analysis and RT-PCR detection revealed that GPX4 drives ferroptosis in an autophagy-dependent manner. The RIP experiment verified that WTAP bound to the GPX4 pre-mRNA, induced m6A methylation and promoted its pre-mRNA degradation, thereby reducing the expression of GPX4 in hypoxic PASMCs. Conclusion and Implications This study proposed ferroptosis as a novel form of cell death in PH, and revealed the regulatory mechanism of the ferroptosis in PH, which is based on GPX4 m6A methylation regulated by WTAP.

Up-regulation of p21WAF1/CIP1 by small activating RNA inhibits the in vitro and in vivo growth of pancreatic cancer cells

2012 ◽  
Vol 98 (6) ◽  
pp. 804-811 ◽  
Author(s):  
Zhiping Zhang ◽  
Zhou Wang ◽  
Xiangyan Liu ◽  
Jie Wang ◽  
Feng Li ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Cancer Cells ◽  
Western Blotting ◽  
Gene Promoter ◽  
Inhibitory Effect ◽  
Rt Pcr ◽  
Pancreatic Cancer Cells

Aims and background To study the inhibitory effect of p21WAF1/CIP1 activation by saRNA on the growth of human pancreatic cancer cells PANC-1 in vitro and in vivo. Methods and study design A dsRNA (dsP21) targeting the p21WAF1/CIP1 gene promoter at position-322 relative to the transcription start site was transfected into PANC-1 cells. Expression of mRNA and protein was evaluated by semiquantitative RT-PCR and Western blotting. Proliferation of PANC-1 cells was measured by the MTT method, and the apoptosis rate was detected by flow cytometry. PANC-1 cells were transplanted subcutaneously in nude mice, and the inhibitory effect of dsP21 on tumor growth was observed. Results The introduction of dsP21 was shown to efficiently up-regulate expression of the p21WAF1/CIP1 gene in PANC-1 cells according to the results of RT-PCR and Western blotting (P <0.01, compared with controls). The inhibitory effect on cell proliferation was confirmed by the MTT test (P <0.05, compared with controls). The apoptosis rate of PANC-1 cells treated with dsP21 was significantly higher than that of the control cells (P <0.01). Our experimental data showed that dsP21-mediated up-regulation of p21 expression exerted an apparent growth inhibitory effect on PANC-1 cells in vivo. Conclusions dsP21 targeting the p21WAF1/CIP1 gene promoter can specifically up-regulate expression of the p21WAF1/CIP1 gene in PANC-1 cells. It therefore has a substantially inhibitory effect on cell proliferation in vitro and in vivo and can be used as a new method and material for the gene therapy of pancreatic cancer.

Identified neurons in C. elegans coexpress vesicular transporters for acetylcholine and monoamines

2001 ◽  
Vol 280 (6) ◽  
pp. C1616-C1622 ◽  
Author(s):  
Janet S. Duerr ◽  
Jennifer Gaskin ◽  
James B. Rand
Keyword(s):  
Motor Neurons ◽  
Choline Acetyltransferase ◽  
Egg Laying ◽  
Vesicular Acetylcholine Transporter ◽  
Vesicular Monoamine Transporter ◽  
Monoamine Transporter ◽  
C Elegans ◽  
Vesicular Transporters ◽  
The Individual

We have identified four neurons (VC4, VC5, HSNL, HSNR) in Caenorhabditis elegans adult hermaphrodites that express both the vesicular acetylcholine transporter and the vesicular monoamine transporter. All four of these cells are motor neurons that innervate the egg-laying muscles of the vulva. In addition, they all express choline acetyltransferase, the synthetic enzyme for acetylcholine. The distributions of the vesicular acetylcholine transporter and the vesicular monoamine transporter are not identical within the individual cells. In mutants deficient for either of these transporters, there is no apparent compensatory change in the expression of the remaining transporter. This is the first report of neurons that express two different vesicular neurotransmitter transporters in vivo.

scholarly journals MORC2 Enhances Tumor Growth by Promoting Angiogenesis and Tumor-Associated Macrophage Recruitment via Wnt/β-Catenin in Lung Cancer

2018 ◽  
Vol 51 (4) ◽  
pp. 1679-1694 ◽  
Author(s):  
Meihan Liu ◽  
Xiaochun Sun ◽  
Shaomin  Shi
Keyword(s):  
Lung Cancer ◽  
Tumor Growth ◽  
Real Time ◽  
Western Blotting ◽  
Cancer Progression ◽  
Pharmacological Intervention ◽  
Migration And Invasion ◽  
Rt Pcr ◽  
Lung Cancers

Background/Aims: In this study, we aimed to investigate how MORC family CW-type zinc finger 2 (MORC2) affects tumor progression of lung cancer. Methods: The MORC2 level was analyzed by real-time RT-PCR and immunohistochemistry (IHC) in normal control tissues and lung cancers. LL/2 cells overexpressing MORC2 were used to study how MORC2 expression influences lung cancer progression. The effects of MORC2 on cell viability, migration and invasion were assessed by MTT assay, Western blotting, and transwell assays, respectively. Afterwards, the effects of MORC2 on the activation of the Wnt/β-catenin pathway were explored by Western blotting. The effects of MORC2 on tumor-associated macrophages (TAM) were determined by immunofluorescence (IF) staining, real-time RT-PCR and Western blotting. Results: Our results showed that MORC2 was upregulated in lung cancers relative to adjacent tissues. The results also demonstrated that MORC2 promoted lung cancer tumor growth in vivo. Additionally, MORC2 overexpression stimulated the upregulation of vascular endothelial growth factor (VEGF), driving angiogenesis. MORC2 overexpression in LL/2 also increased the amount of aldehyde dehydrogenase-1 (ALDH1) protein, indicating that MORC2 increased cancer stem cell features. We further determined that MORC2 activated Wnt/β-catenin signaling in lung cancer cells. Upregulation of macrophage-recruiting genes including VEGF and Macrophage-specific colony stimulating factor (CSF-1) recruits TAMs to the tumor site, which has the net effect of promoting additional tumor growth and metastasis. Conclusion: Our data suggest that MORC2 overexpression can drive lung cancer growth by stimulating the recruitment of TAMs in addition to angiogenesis and that activation of Wnt/β-signaling may be a key pathway underlying this phenotype that is amenable to pharmacological intervention.

Matrix Metalloproteinase 13 (MMP13) Upregulation Is Essential for Multiple Myeloma Related Bone Lytic Lesion

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4025-4025
Author(s):  
Jing Fu ◽  
Shirong Li ◽  
Rentian Feng ◽  
Huihui Ma ◽  
Farideh Sabeh ◽  
...  
Keyword(s):  
Tumor Growth ◽  
Western Blotting ◽  
Stromal Cells ◽  
Lytic Lesion ◽  
Rt Pcr ◽  
Matrix Metalloproteinase 13 ◽  
Lytic Lesions ◽  
Mmp13 Expression

Abstract Abstract 4025 Background: MM cells produce several osteoclast-activating factors, which result in highly activated osteoclasts and cause dysregulated bone remodeling with excessive bone resorption. Our previous data showed that matrix metalloproteinase 13 (MMP13) is highly expressed in MM cells, and co-culture with stromal cells further increased MMP13 levels. Most importantly, exogenous MMP13 increased OCL fusion and bone resorption activity induced by nuclear factor kappa-B ligand (RANKL). Here, we further addressed the mechanism of MMP13 upregulation in MM and its role in MM-related bone disease in vivo. Methods and Results: Based upon the previous results that IL-6 neutralizing antibody blocks MMP13 upregulation in MM cells co-cultured with stromal cells, we further investigated the role of IL-6 on MMP13 induction in MM cells. Human RPMI8266 MM cells were serum-starved for 24 hours, then treated with IL-6 for up to 96 hours. RT-PCR and western blotting showed that MMP13 expression and secretion by MM cells were upregulated after 24h and prolonged through 96h. AP-1 binding sites were identified in the MMP13 promoter, and we found that IL-6 induced upregulation of the AP-1 members c-Jun and c-fos in MM cells by RT-PCR and western blotting, which correlated with MMP13 induction. To address the role of heightened MMP13 expression/secretion of MM on OCL formation and development of lytic lesions, we silenced MMP13 expression in MM cells by lentiviral mediated shRNA transduction. 5TGM1 mouse MM cells were infected with the pKLO.1-puro-vector (EV) or pKLO.1-puro-sh-MMP13 (MMP13 Knock down [KD]) lentivirus particles and cells were selected by puromycin. MMP13 KD was confirmed by RT-PCR and western blotting. To investigate the effects of MMP13 KD on OCL formation, we co-cultured mouse bone marrow cells (BMC) with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells using a transwell system to permit only soluble molecule exchange. Co-culture with 5TGM1-EV MM cells significantly (p<0.01) induced OCL fusion, while this effect was largely impaired in the co-culture group with 5TGM1-MMP13 KD MM cells. Treatment with IL-6 further enhanced the OCL fusion activity in both groups. However, in 5TGM1-MMP13 KD group, IL-6 did not compensate for impaired OCL formation due to MMP13 silencing. This indicates that MMP13 secreted from MM cells is one of the key factors facilitating OCL formation/activity in MM. We further investigated the effects of MMP13 silencing on MM tumor progression and bone disease in vivo using the 5TGM1 intratibial tumor model. Recombination activating genes 2 (RAG2) knockout mice were intratibially injected with either 5TGM1-EV or 5TGM1-MMP13 KD MM cells, and 3 weeks later, mice were sacrificed and tumor growth and bone lytic lesion were monitored by serum IgG2b level (by Elisa) and micro-QCT respectively. Preliminary results show that MMP13 KD in MM cells inhibited tumor growth and lytic lesions in trabecular bone. Conclusion: Our results demonstrate that IL-6-induced high level of MMP13 in MM cells is essential for multiple myeloma tumor growth, OCL induction and development of lytic bone lesions. Disclosures: Lentzsch: Celgene: Consultancy, Research Funding.

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