Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

Recent genome-wide association studies identified FGFR2 as one of breast cancer susceptibility genes. FGFR2 expression was down-regulated in breast carcinomas when compared with paired normal epithelium. Stable retroviral transduction of FGFR2-IIIb and its alternatively spliced FGFR2-IIIc variants was achieved in breast cancer MDA-MB-231, T47D and near normal MCF-10A cells. Our findings revealed a direct reduction of breast cancer cell growth and motility, a significant arrest of transformed morphogenetic changes including the Epithelial to Mesenchymal transition (EMT), anchorage independent growth, and the formation of growth-arrested 3D acinar architectures, and suppressive actions on orthotopically xenografted epithelial neoplasms and surrounding tumor stroma. These tumor protective effects were concordant with physical interactions between the two FGFR2 isoforms and IKKβ. Consistent with these interactions we noted FGFR2 to inhibit NF-κB signaling, including decreased nuclear RelA/p65 NF-κB localization, down-regulation of a transfected NF-κB luciferase reporter, reduced production of NF-κB-dependent transcripts, Interleukin-6 and p-STAT3.

No description available.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 101-109).

FGF21 is a stress-inducible hormone predominantly secreted from the liver. FGF21 acts as a downstream target gene of hepatic transcription factor PPARα that plays an obligatory role in mediating metabolic adaptation responses to prolonged fasting. However, the physiological roles of FGF21 in regulating glucose homeostasis during adaptive starvation responses and its underlying mechanisms remain unknown. Furthermore, FGF21 is induced by both acute and chronic exercise training in both rodents and human. The physiological significances and the underlying mechanisms for exercise-induced FGF21 production have not been explored so far. Therefore, this study aims to investigate: 1) the mechanisms whereby FGF21 mediates the role of PPARα activation in modulating hepatic gluconeogenesis in response to prolonged fasting; 2) the physiological roles and mechanisms whereby FGF21 regulates exercise capacity and insulin sensitivity via its actions in the muscle during exercise; 3) the roles of FGF receptor-1 (FGFR1) and co-receptor βKlotho in mediating the metabolic effects of FGF21 during fasting and exercise. Our results show thatFGF21 is induced by fasting in the liver and enters into the brain. Both FGF21knockout (KO) mice and PPARα KO mice exhibit severe hypoglycemia and defective hepatic gluconeogenesis during prolonged fasting, and these changes are accompanied by impaired activation of the hypothalamic-pituitary-adrenal (HPA)axis and blunted release of corticosterone from adrenalgland. Moreover, intracerebroventricular injection of recombinant FGF21 reverses fasting hypoglycemia and impairment in hepatic gluconeogenesis by restoring corticosterone production in both FGF21 KO and PPARα KO mice. These effects are abrogated by blockage of hypothalamic FGFR1or by pharmacological inhibition of ERK1/2in the hypothalamus. In addition,FGF21 acts directly on the hypothalamic neurons to activate FGFR1/βKlotho-ERK1/2-CREBsignaling pathway, thereby leading to the transcriptional activation of corticotropin-releasing hormone (CRH) and subsequent activation of the HPA axis. FGF21 is also induced in the liver by exercise and FGF21 KO mice have decreased exercise capacity associated with lower mitochondrial content and thus lower insulin sensitivity compared to wildtype (WT)mice after exercise training.AMPK-PGC-1α signaling pathway is impaired in the muscle of FGF21 KO mice during exercise.FGF21treatmentincreasesmitochondrial content in myotubes, while inhibition of AMPK and knockdown of FGFR1, βKlotho and PGC-1αblocksthis effect. Moreover, FGF21 regulates genes and proteins involved in different steps of the autophagy process and these alterations are mediated by FGFR1/βKlotho-PGC-1α signaling cascade. Furthermore, replenishment of FGF21 reverses the reduced mitochondrial content and insulin sensitivity by restoring PGC-1α expression and subsequent increase in mitochondrial biogenesis and suppression of autophagy in the muscle of FGF21 KO mice. In conclusion, our results identify FGF21 as a critical hormonal regulator of glucose homeostasis during prolonged fasting, by coupling hepatic PPARα activation to corticosterone release via stimulation of the HPA axis in the brain. Moreover, FGF21 acts in the muscle through two distinctive mechanism to maintain mitochondrial homeostasis during exercise training, involving both biogenesis of new mitochondria and decreased clearance of stressed mitochondria and thus to mediate the beneficial effects of exercise on lipid and glucose metabolism. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

No description available.

Myocardial fibrosis is a common pathological finding in patients with cardiovascular disease and is believed to be a major contributing factor in the development of end stage organ failure. Early events that promote the development of myocardial fibrosis are not well understood. Rapid cellular infiltration into the cardiac tissue is evident in fibrosis but the infiltrating populations and their functions have yet to be completely elucidated. The aim of this thesis was to characterize the phenotype and function of this cellular population in a model of hypertension mediated myocardial fibrosis. Furthermore, we intended to explore therapies that target this population and ameliorate fibrosis. We characterized a novel population of infiltrating cells as circulating fibroblast progenitor cells, termed fibrocytes. We determined that this population does not appear to specifically migrate in response to previously established chemotactic signals (CCL2 or CXCL12). We found that fibrocytes respond to fibrogenic stimuli (AngII and CTGF) by increasing the expression of collagen and CTGF, an early molecular mediator of fibrosis, while also promoting fibrocyte differentiation. Using an anti-hypertension treatment, we found that hypertension as a physiologic stimulus likely promotes cellular infiltration and corresponding fibrosis. We also established that treatment with activated protein C (aPC) conferred protection against the development of myocardial fibrosis, potentially by inhibiting fibrocyte recruitment and/or activation. Lastly, to assess fibrocyte involvement in the progression of human myocardial fibrosis we assessed fibrocytes in levels in the circulation of patients with ischemic heart disease compared to healthy controls. We found that patients with ischemic heart disease had an increase of circulating cells that have the potential to become fibrocytes compared to healthy controls and therefore likely contribute to myocardial fibrosis. From this data, we propose that fibrocytes are a key effector cell that directly promotes pathologic fibrosis within the injured myocardium. Understanding their migration and function is therefore essential to the development of future therapies targeting this cell type to inhibit their role in fibrosis.

No description available.

Herzinsuffizienz ist ein weltweites Gesundheitsproblem mit hoher Morbidität und Mortalität und immer noch schlechter Prognose. Ein charakteristisches Merkmal der molekularen und damit verbundenen strukturellen Veränderungen, die der terminalen Insuffizienz vorangehen ist die durch Desensitivierungsmechanismen vermittelte Abnahme des beta-adrenergen (β-AR) Signalmoleküls zyklisches Adenosinmonophosphat (cAMP) auf der einen Seite und der gleichzeitigen Zunahme des von natriuretischen Peptiden (NP) und Stickstoffmonoxid (NO) generierten zyklischen Guanosinmonophosphat (cGMP) auf der anderen Seite. Während hohe cAMP-Spiegel im Herzen als schädlich gelten, werden cGMP-abhängige Signalkaskaden vorwiegend als protektiv verstanden. Amplitude, Lokalisation und Halbwertszeit beider Signalmoleküle werden durch spezifische Enzyme, den Phosphodiesterasen (PDE) reguliert. Unter der PDE-Superfamilie wird die Isoform PDE2 als einzige von cGMP aktiviert, um dann verstärkt cAMP abzubauen und steht damit im Zentrum eines negativen Crosstalks dieser beiden Signalwege. PDE2 ist sowohl in der humanen als auch der experimentellen Herzinsuffizienz hochreguliert und scheint dort am β-AR Desensitivierungsprozess beteiligt zu sein. Im Rahmen dieser Arbeit wurde die pathophysiologische Rolle der PDE2 im Herzen näher charakterisiert. Es wird gezeigt, dass die PDE2 nicht nur in Kardiomyozyten, sondern auch in kardialen Fibroblasten exprimiert wird. In Fibroblasten inhibieren cAMP/cGMP-Signalwege die Transformation von kardialen Fibroblasten (CF) zu Myofibroblasten (MyoCF), einem zellulären Phänotyp, der unter anderem mit der persistenten Fibrotisierung des erkrankten Herzgewebes in Verbindung gebracht wird. In CF führte eine Überexpression von PDE2 zu eine starken Abnahme der basalen und β2-AR-vermittelten cAMP-Synthese und war ausreichend, um in Abwesenheit exogener, pro-fibrotischer Stimuli die Transformation zum MyoCF zu induzieren. In Übereinstimmung zeigten funktionale Analysen mit künstlich hergestelltem Bindegewebe aus PDE2-überexprimierenden CF eine deutliche Zunahme der Gewebssteifigkeit. PDE2 übte keinen Einfluss auf basale oder durch das atriale NP generiertes cGMP aus und reduzierte nur partiell die NO-induzierte cGMP-Akkumulation. Interessanterweise waren beide Stimuli in der Lage, trotz niedriger cAMP-Spiegel die PDE2-induzierte CF-Transformation zum MyoCF zu verhindern und lassen daher eine Redundanz dieser beiden sonst so gegensätzlichen Signalwege vermuten. Zur Untersuchung von PDE2 in Kardiomyozyten wurde ein transgenes (TG) Mausmodell mit spezifischer kardialer Überexpression herangezogen. Die Basalcharakterisierung zeigte eine erniedrigte Herzfrequenz (HR) mit kompensatorisch erhöhter, basaler Kontraktionskraft, sowie eine verminderte Maximalantwort bezüglich der HR nach akuter β-AR Stimulation. Auf molekularer Ebene war dieser Phänotyp mit einer verminderten Phosphorylierung verschiedener β-AR Zielstrukturen wie Troponin I, Phospholamban und Ryanodinrezeptor-2 assoziiert. Langzeitstudien belegten, dass eine Überexpression von PDE2 keine pathologischen Konsequenzen hat, sondern im Gegenteil die durchschnittliche Lebensspanne der Tiere eher verlängerte. Erste Studien im Herzinsuffizienzmodel der transversalen Aortenkonstriktion (TAC) zeigten bisher eine beständig erniedrigte HR und verminderte Wanddicken bei allerdings vergleichbarer Abnahme der kardialen Kontraktionskraft. Trotz der klaren Befunde und neuen Erkenntnisse über die vielfältige Rolle der PDE2 im Herzen lässt sich bisher noch nicht klar belegen, ob eine zusätzliche Aktivierung von myokardialen PDE2 tatsächlich im Sinne einer intrazellulären β-AR-Blockade die Progression zur Herzinsuffizienz verlangsamen oder verhindern könnte. Weitere darauf aufbauende Untersuchungen, wie z.B. eine akut induzierbare Aktivierung bzw. Deaktivierung in experimentellen Herzinsuffizienzmodellen könnten den Weg für die Entwicklung klinisch anwendbarer Ansätze zur therapeutischen Modulation dieser viel versprechenden Zielstruktur ebnen.

Cartilage development relies on the coordinated presentation of biological signals to direct chondrocyte morphology and function. This is largely controlled by perlecan, a heparan sulfate proteoglycan (HSPG). Understanding the role of perlecan and its pendant glycosaminoglycan chains (GAG) in cartilage development is essential for advances in tissue engineered cartilage replacement strategies. Perlecan was immunolocalised to the pericellular matrix of prehypertrophic and hypertrophic chondrocytes in human fetal feet. Human fetal chondrocytes were isolated and cultured in 3-dimensional (3D) scaffolds for a period of 4 weeks. Their chondrogenic phenotype, based on extracellular matrix (ECM) components, was assessed and compared to 2D cultures. Chondrocyte perlecan was immunopurified from human fetal chondrocytes grown in vitro and fetal cartilage tissue and characterised using a combination of antibody-based techniques (ELISA, Western blotting) and gel electrophoresis. The biological function of chondrocyte perlecan was determined by its ability to form ternary complexes with fibroblast growth factors (FGF) and their receptors (FGFR) using an antibody-based technique as well as a cell proliferation assay using cells expressing FGFR isotypes. Perelcan was restricted to the prehypertrophic and hypertrophic zones of cartilage. This zonal organisation of chondrocytes and chondrogenic properties, determined by their morphology and PG deposition, was recapitulated in the 3D constructs while 2D cultures displayed dedifferentiated chondrocytes. Exogenous FGF2 promoted chondrocyte proliferation, while FGF18 stimulated the synthesis of perlecan, reflecting chondrocyte hypertrophy. Chondrocyte perlecan (630kDa) contained HS, chondroitin sulfate (CS) and keratan sulfate (KS) chains. Chondrocyte perlecan formed HS dependent ternary complexes with FGF2-FGFR1c and FGF18-FGFR3c, while FGF18-FGFR3c binding to perlecan protein core was also observed. Binding of FGF18-FGFR3c to chondrocyte perlecan HS was more promiscuous than FGF2-FGFR1c. Furthermore, chondrocyte perlecan HS mediated biological activity with FGF18 via FGFR3c, which was modulated by mammalian heparanase, while no biological activity was elicited by FGF2-FGFR1c. The findings underline how perlecan and its GAGs interact with FGF and FGFR in a spatio-temporal manner to promote signalling, effecting chondrocyte behaviour and morphology in cartilage development. This insight can be utilised in tissue engineering to improve the development of biologically functional cartilage replacements.