Excessive expression of subunit 1 of GIRK1 in ER+ breast tumors is associated with reduced survival times and increased lymph node metastasis in patients. To investigate possible tumor-initiating properties, benign MCF10A and malign MCF7 mammary epithelial cells were engineered to overexpress GIRK1 neoplasia associated vital parameters and resting potentials were measured and compared to controls. The presence of GIRK1 resulted in resting potentials negative to the controls. Upon GIRK1 overexpression, several cellular pathways were regulated towards pro-tumorigenic action as revealed by comparison of transcriptomes of MCF10A(GIRK1) with the control (MCF10A(eGFP)). According to transcriptome analysis, cellular migration was promoted while wound healing and extracellular matrix interactions were impaired. Vital parameters in MCF7 cells were affected akin the benign MCF10A lines, but to a lesser extent. Thus, GIRK1 regulated cellular pathways in mammary epithelial cells are likely to contribute to the development and progression of breast cancer.

Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1–6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed “LPA” throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways.

BACKGROUND:Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS:In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS:Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION:We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS:The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.

Mechanical interaction between cells – specifically distortion of tensional homeostasis-emerged as an important aspect of breast cancer genesis and progression. We investigated the biophysical characteristics of mechanosensitive ion channels (MSCs) in the malignant MCF-7 breast cancer cell line. MSCs turned out to be the most abundant ion channel species and could be activated by negative pressure at the outer side of the cell membrane in a saturable manner. Assessing single channel conductance (G Λ ) for different monovalent cations revealed an increase in the succession: Li + < Na + < K + ≈Rb + ≈ Cs + . Divalent cations permeated also with the order: Ca 2+ < Ba 2+ . Comparison of biophysical properties enabled us to identify MSCs in MCF-7 as ion channels formed by the Piezo1 protein. Using patch clamp technique no functional MSCs were observed in the benign MCF-10A mammary epithelial cell line. Blocking of MSCs by GsMTx-4 resulted in decreased motility of MCF-7, but not of MCF-10A cells, underscoring a possible role of Piezo1 in invasion and metastatic propagation. The role of Piezo1 in biology and progression of breast cancer is further substantiated by markedly reduced overall survival in patients with increased Piezo1 mRNA levels in the primary tumor.

Mortality of breast cancer is very high when it metastases to distant organs. New early tumor markers and mechanistic insight into carcinogenesis and metastasisation are needed to treat the disease at curable stages. GIRK channels are activated by GPCRs and regulate resting potential and excitability of neurons, myocytes and other cells. Recently GIRK proteins have been associated with endocrine adenomas and also breast cancer (Stringer et al.2001, Brevet et a.l 2008 and Choi et al.2009). In this study we analyzed the vital parameters of cancerous and noncancerous breast cell lines when human GIRK1 and splice variants (hG1a, hG1c, hG1d) are overexpressed or silenced. GIRK1a, GIRK1c and GIRK1d were stably overexpressed and knocked out in breast cell lines MCF7, MDA-MB-231 and MCF 10A. Vital parameters including invasion, wound healing, adhesion, proliferation and motility were evaluated. Our findings revealed that overexpression of GIRK1a significantly increased the velocity 0.102±0.035 (μm/min) in overexpresssed vs. 0.05±0.02 in vector control cells (P˂0.001) and motility coefficient 0.2±0.3 (μm2/min) in overexpresssed vs. 0.05±0.06 in vector control cells (P˂0.001) of the MCF7 cell lines. Overexpression of hG1c in MCF7 significantly increased invasiveness of the cells (P˂0.001) as measured by matrigel assay. Overexpression of the GIRK1 and splice variants didn't have significant effect on proliferation, wound healing and adhesion of the MCF7 cells. Stably overexpression of GIRK1 and splice variants in MCF7 cause increased velocity, motility coefficient and invasion of the cells. Expression of GIRK might be a new diagnostic biomarker of breast cancer and possibly is causally involved in metastasisation. Acknowledgements: The research is funded by the Austrian Science Foundation (FWF; P22974-B19 to W. S and KLIF 182 to T. B).

Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo.The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling.RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53(wt) (U87MG, A172, and primary GBM2), and p53(mut) (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53(wt) and p53(mut) cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53(wt) and p53(mut) GBM cells. PRKD2 knockdown in p53(wt) cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53(mut) GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation.PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-JunS73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.

Lysophosphatidic acid (LPA), a naturally occurring bioactive phospholipid, mediates a multitude of (patho)physiological events including activation of mitogen-activated protein kinases (MAPKs). As LPA may induce cellular reponses in human osteosarcoma, the present study aimed at investigating expression of various LPA receptors, LPA-mediated activation of MAPK via G-protein coupling, and expression of early response genes in a cellular model for human osteosarcoma. We show that MG-63 cells express three members of the endothelial differentiation gene (Edg) family of G-protein coupled receptor transcripts (LPA(1-3)) but only two (LPA(4/5)) out of three members of the non-Edg family LPA receptor transcripts. Stimulation of MG-63 cells with LPA or synthetic LPA receptor agonists resulted in p42/44 MAPK phosphorylation via LPA(1)-LPA(3) receptors. Using pharmacological inhibitors, we show that LPA-mediated phosphorylation of p42/44 MAPK by LPA receptor engagement is transmitted by God-dependent pathways through the Src family of tyrosine kinases. As a consequence, a rapid and transient upregulation of the zinc finger transcription factor early growth response-1 (Egr-1) was observed. Egr-1 expression was strictly mediated via G(alpha i)/Src/p42/44 MAPK pathway; no involvement of the G(alpha q/11)/PLC/PKC or the PLD/PI3 kinase/Akt pathways was found. LPA-induced expression of functional Egr-1 in MG-63 cells could be confirmed by electrophoretic mobility shift assay. LPA-induced Egr-1 upregulation was accompanied by a time-dependent decrease of periostin (previously called osteoblast-specific factor 2), a cell adhesion protein for pre-osteoblasts. Silencing of LPA(1) and/or Egr-1 in MG-63 cells reversed LPA-mediated suppression of periostin. We here demonstrate a crosslink between Egr-1 and periostin in cancer cells, in particular in human osteosarcoma. (C) 2012 Elsevier Masson SAS. All rights reserved.

Objectives. To evaluate whether patients with abnormal Reflux Symptom Index (RSI) and Reflux Finding Score (RFS) benefit from proton pump inhibitor (PPI) therapy.Study Design. Open, multicenter, prospective longitudinal cohort study.Methods. Patients with suspected reflux-associated laryngologic symptoms were evaluated by 40 community practice otolaryngologists using RSI and RFS. Patients were treated with pantoprazole 40-80 mg/d for 8-12 weeks if RSI was greater than 9 and RFS greater than 7. Pre- and posttherapeutic RSI and RFS were compared using Wilcoxon signed rank test and additionally controlled with the symmetry test of Bowker.Results. A total of 1044 patients were included over a period of 20 months. Median total score of RSI before therapy was 12 and decreased to 3 (P << 0.001). Median total score of RFS before therapy was 16 and decreased to 6 (P << 0.001). Assessment of the treatment effect by otolaryngologists and patients was judged as being excellent in at least 50%. In 2% of the patients, gastrointestinal side effects were documented.Conclusion. RSI and RSF are easy to administer in the routine care of patients suspected of having laryngopharyngeal reflux. Patients identified by positive results of these tests have a high likelihood of excellent improvement after 8-12 weeks of PPI treatment. By implementation of RFS and RSI in daily use, most patients may not need time-consuming and cost-intensive examinations in the first-line assessment of LPR. These examinations can be reserved for nonresponders, and uncontrolled prescription of PPIs can be restricted.

PURPOSE:The aim of this study was the establishment of a minimally invasive technique of mesenchymal stem cell (MSC) harvesting and a predictable isolation and cultivation method on 2 different bone substitutes used as potential scaffolds. MATERIALS AND METHODS:Human MSCs isolated from the posterior maxilla were characterized by flow cytometric analysis. After in vitro expansion, cells were cultured and differentiated toward osteogenic, adipogenic, and chondrogenic lineages in 2-dimensional cultures and on natural bone mineral of bovine origin and β-tricalcium phosphate scaffolds. Three-dimensional growth was analyzed using live cell staining and confocal laser scanning microscopy. RESULTS:MSCs from all patients demonstrated the same immunophenotype, with expression of CD73, CD90, and CD105 but no expression of CD45, CD34, CD14, CD11, and HLA-DR. The potential of MSCs for multilineage differentiation along osteogenic, adipogenic, and chondrogenic lines was shown. Based on knowledge of the characteristics of the cells, a method was established to increase MSC expansion efficiency and seeding conditions on each scaffold. Results of the in vitro characterization and laser scanning microscopy visualized the 3-dimensional growth of MSCs on the 2 scaffold types. CONCLUSIONS:The present data showed that intraoral MSCs can be cultured predictably under 2- and 3-dimensional conditions, have proved multiple potencies, and thus seem to be potential candidates for tissue engineering approaches in maxillofacial reconstructions.

The aim of this study was to investigate the impact of increased mRNA levels encoding GIRK1 in breast tumours on GIRK protein expression. mRNA levels encoding hGIRK1 and hGIRK4 in the MCF7, MCF10A and MDA‐MB‐453 breast cancer cell lines were assessed and the corresponding proteins detected using Western blots. cDNAs encoding for four hGIRK1 splice variants (hGIRK1a, 1c, 1d and 1e) were cloned from the MCF7 cell line. Subcellular localisation of fluorescence labelled hGIRK1a–e and hGIRK4 and of endogenous GIRK1 and GIRK4 subunits was monitored in the MCF7 cell line. All hGIRK1 splice variants and hGIRK4 were predominantly located within the endoplasmic reticulum. Heterologous expression in Xenopus laevis oocytes and two electrode voltage clamp experiments together with confocal microscopy were performed. Only the hGIRK1a subunit was able to form functional GIRK channels in connection with hGIRK4. The other splice variants are expressed, but exert a dominant negative effect on heterooligomeric channel function. Hence, alternative splicing of the KCNJ3 gene transcript in the MCF7 cell line leads to a family of mRNA's, encoding truncated versions of the hGIRK1 protein. The very high abundance of mRNA's encoding GIRK1 together with the presence of GIRK1 protein suggests a pathophysiological role in breast cancer. J. Cell. Biochem. 110: 598–608, 2010. © 2010 Wiley‐Liss, Inc.

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA 1 , LPA 2 , and LPA 3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Background and purpose: Endothelins (ETs) and their G protein-coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal-derived cells.Experimental approach: The present study aimed at investigating ET receptor-mediated activation of the mitogen-activated protein kinase ( MAPK) pathway in human choriocarcinoma.Key results: Both JAR and Jeg-3 choriocarcinoma cell lines expressed ET receptor subtype B (ET(B)) but not ET(A) receptor transcripts. ET(B) receptor engagement by ET-1 and ET-3 resulted in a similar time-and concentration-dependent phosphorylation of p42/44 MAPK, also known as extracellular regulated kinase 1/2. Using specific pharmacological antagonists/inhibitors, we showed that ET-1/-3-mediated signal transduction by the ET(B) receptor is transmitted via G(i)- and G(q)-dependent pathways through activation of the Src (G(i)) and protein kinase C (G(q)) axis that converge at Ras/Raf, leading to downstream activation of p42/44. On a functional level, ET(B) engagement and subsequent phosphorylation of p42/44 resulted in enhanced transcription of the immediate early response genes c-fos and c-jun, a process commonly assumed to be mediated by the ET(A) receptor, and increased cell growth and relative cell area.Conclusions and implications: As human choriocarcinoma cells secrete ETs, pharmacological antagonism of ETs and/or ET(B) receptor-mediated signal transduction could represent a likely target therapy for choriocarcinoma.

Introduction: In many sports athletes with low body weight have a distinct advantage over their opponents. This can turn in it's extreme to a disadvantage owing to the associated health problems (Müller et al. 1996). A reduction in body mass entails fat mass and fat free mass. Because fatness is said to be more influenced by sport and, by inference, training than is the anatomical distribution of fat (Malina et al. 1982) we measured anthropometric parameters and subcutaneous fat patterning in world-class skijumpers. From a scientific point of view, we were interested to see i.) which kind of fat patterning is present in skijumpers and, ii.) whether differences exist in the fat patterning of skijumpers from different countries. Methods: 87 skijumpers (mean and SD, age: 21.8 ± 3.3 yrs.) from 15 countries were studied during the summer grand prix in Germany. Anthropometric characteristics, ie. body mass, body height, and waist circumference (waist) were measured. The thickness of subcutaneous adipose tissue-layers (SAT-layers) were measured by means of the optical device Lipometer at 15 different body sites from l-neck to 15-calf on the right side of the body (Möller et al. 2000). The datas according to skijumpers ancestry were collapsed. Skijumpers from Austria, France, Germany, Netherlands, Slovenija, Switzerland, and the Czech Republic served as the ‘mid-europe group’ (ME; N = 47). Skijumpers from Finland, Norway, and Sweden served as the ‘north-europe group’ (NE; N = 17). Skijumpers from Poland, Russia, and Kasachstan served as the ‘east-europe group’ (EE; N = 13). Skijumpers from Japan and Korea served as the ‘asia-group’ (AS; N = 10). Statistical evaluation of datas included ANOVA and factor analysis. Results: Factor analysis for measured SAT-layers extracted two factors which together explained ∼ 60% of the multivariate variance in SAT-layers. Factor 1 includes SAT-layers from the trunk and SAT-layer 3-biceps (trunk-fatness). Factor 2 includes SAT-layers from the lower extremities and SAT-layers 2-triceps (extremity-fatness) (Fig. 1). Trunk-fatness was greatest in skijumpers from NE (p = 0.02) but no differences were found in values of extremity fatness (p = 0.60). Skijumpers from AS had lower body mass, were of shorter stature and had also the smallest waist when compared with skijumpers from ME and NE. Anyway, body mass and body height were not different between skijumpers from AS and EE. No differences between skijumpers from ME and NE were found for the waist, body mass, and body height.Fig. 1: Mean (and SEM) standardized factor score values of subcutaneous fat patterning of world class skijumpers according to their ancestry. ME, skijumpers from mid-europe; NE, skijumpers from north-europe, EE, skijumpers from east-europe; AS, skijumpers from AsiaDiscussion: These findings suggest that even in elite skijumpers, the ‘typical’ kind of fat patterning (trunk- and extremity fatness) is present (2,4). Because of the small sample size of skijumpers from AS, less confidence can be placed in the trend of their reduced trunk- and extremity fatness (Fig. 1). This reduced fatness could also be the consequence of low body mass and short stature in ski-jumpers from AS. However, some differences in fat patterning can be obtained with respect to ancestry. Skijumpers from NE show an increased trunk-fatness whereas an increased extremity-fatness is observed in skijumpers from EE. Whether this is due to an influence of ethnic per se on fat patterning, or represents an environmental influence, ie. differences in training schedule and/or diet composition, remains to be elucidated. Acknowledgements: We gratefully acknowledge the support of the IOC, the FIS, and Parke Davis.