Regulation von Kalium-Kanälen durch mTOR und PDK1 in Dendritischen und Mast-Zellen

Dendritic cells are antigen-presenting cells, central for the development of optimal T cell immunity, that are able to initiate primary immune responses and to establish immunological memory. Mast cells are tissue-based effector cells in allergic diseases, playing a central role in the propagation of IgE-dependent allergic reactions, such as allergic rhinitis, asthma, anaphylaxis and delayed hypersensitivity reactions. Upon stimulation of IgE receptors , mast cells release granules containing several mediators including histamine and cytokines, which regulate responses of other inflammatory cells. Dendritic and mast cell functions are regulated by phosphatidylinositol-3 (PI3) kinase signalling pathway.. The PI3 kinase is partially effective in dendritic and mast cells through alteration of their ion channel activity. Both PI3 kinase on the one hand and ion channels on the other hand are important for the regulation of mast and dendritic cell functions. However, little is known about downstream elements of the PI3 kinase that regulate ion channels in those cells. In the present project the question was addressed whether two PI3 kinase downstream targets, phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycine (mTOR), regulate ion channels and ion channel-dependent functions in dendritic and mast cell. We showed that treatment of dendritic cells with rapamycine, the mTOR inhibitor, led to inhibition of the currents through voltage-gated K+ channels, which in dendritic cells belong to Kv 1.3 and Kv 1.5 families. Analysis of the time constants of activation and inactivation demonstrated that rapamycin caused faster Kv channel inactivation. To test the hypothesis that Kv1.3 and/or Kv1.5 channels could be regulated by mTOR, cRNA encoding Kv1.3 or Kv1.5 was injected into Xenopus oocytes with or without additional injection of cRNA encoding mTOR. The Kv1.3 and Kv1.5 currents were significantly increased by additional coexpression of mTOR, an effect abolished by rapamycin. Analysis of activation and inactivation time constants of Kv1.3 and Kv1.5 revealed that mTOR affected tau activation and tau inactivation of Kv1.3, but not of Kv1.5. Coexpression with mTOR resulted in a decreased tau activation and an increased tau inactivation of Kv1.3, suggesting that mTOR causes faster Kv1.3 channel activation and slower Kv1.3 channel inactivation. We were interested to analyze whether rapamycin has also effects on non-voltage gated K+ channels. We performed experiments in mouse bone marrow-derived mast cells (BMMCs), which are known to express Ca2+-activated K+ channels KCa3.1. Our observations demonstrated that though rapamycine did not influence KCa3.1 channel activation directly, it impaired antigen-dependent increase of cytosolic Ca2+ in BMMCs and that secondary led to the blunted activation of the KCa3.1 channels. In BMMC we also studied the effect of another kinase of the PI3 kinase pathway, PDK1, on ion channels and cell functions. The present study demonstrated that the stimulation of Ca2+ entry, but not Ca2+ release from intracellular stores, following exposure to antigen was significantly less pronounced in mast cells from PDK1 hypomorphic mice (pdk1hm) than in mast cells from their wild type littermates (pdk1wt). PDK1 deficiency thus blunted Ca2+ entry and consequently indirectly also the currents through Ca2+-activated K+ channels KCa3.1. Partial PDK1 deficiency did not abrogate degranulation despite the reduced Ca2+ entry in pdk1hm BMMCs. Both ß-hexosaminidase release and histamine release were not significantly different between pdk1wt and pdk1hm BMMCs. We provided a possible mechanism explaining unaffected degranulation in pdk1hm BMMCs by showing that PDK1 led to the activation of two downstream kinases, SGK1 and PKCdelta;, positive and negative regulators of mast cell degranulation, respectively.Dendritische Zellen sind Antigen-präsentierende Zellen, die zentral in der Entwicklung einer optimalen T-Zell-Immunität stehen. So können sie primäre Immunantworten einleiten und ein immunologisches Gedächtnis aufbauen. Mastzellen sind im Gewebe beheimatete Effektorzellen, die eine zentrale Rolle bei der Ausweitung IgE-abhängiger allergischer Reaktionen einnehmen, wie zum Beispiel bei allergischer Rhinitis, Asthma, Anaphylaxie und verzögerten Hypersensitivitätsreaktionen. Nach der Stimulation von IgE-Rezeptoren setzen Mastzellen Granula mit verschiedenen Mediatoren frei, unter anderem Histamine und Zytokine, welche die Antworten anderer inflammatorischer Zellen regulieren. Die Funktionen von dendritischen und Mastzellen werden über den Phosphatidylinositol-3 (PI3) Kinase Signalweg reguliert. Der PI3 Kinase Signalweg erreicht seine Effektivität in dendritischen und Mastzellen zum Teil durch die Veränderung von Ionenkanal-Aktivitäten. Also sind beide, einerseits Pi3K und andererseits Ionenkanäle wichtig für die Regulation von Funktionen dendritischer und Mastzellen. Trotzdem ist wenig bekannt über die Elemente stromabwärts von PI3K, die die Ionenkanäle in diesen Zellen regulieren. In dem gegenwärtigen Projekt war die Frage, ob zwei der Kinasen die PI3K nachgeschalten sind, phosphoinositid-dependent kinase 1 (PDK1) und mammalian target of rapamycin (mTOR) Ionenkanäle und davon abhängige Funktionen in dendritischen und Mastzellen regulieren. Wir haben gezeigt, dass die Behandlung dendritischer Zellen mit dem mTOR-Inhibitor Rapamycin zur Inhibierung von Strömen durch spannungsgesteuerte K+ - Kanäle, die in dendritischen Zellen zu den Familien Kv 1.3 und Kv 1.5 gehören, führt. Die Analyse der Zeitkonstanten der Aktivierung und Inaktivierung zeigte, dass Rapamycin eine schnellere Kv-Inaktivierung bewirkt hat. Um die Hypothese zu testen, dass Kv 1.3 und/oder Kv 1.5 durch mTOR reguliert werden können, wurde cRNA für Kv 1.3 oder Kv 1.5 gleichzeitig mit oder ohne cRNA für mTOR in Xenopus Oocyten injiziert. Die Kv 1.3 – und Kv 1.5 – Ströme wurden signifikant gesteigert durch die zusätzliche Expression von mTOR, ein Effekt, der durch Rapamycin aufgehoben werden konnte. Die Analyse der Aktivierungs- und Inaktivierungszeitkonstanten tau von Kv 1.3 und Kv 1.5 ergab, dass mTOR die Tau -Aktivierung und –Inaktivierung von Kv 1.3 aber nicht die von Kv 1.5 beeinflusste. Die Koexpression mit mTOR führte zu einer abgeschwächten Tau -Aktivierung und einer gesteigerten Tau -Inaktivierung, also zu einer schnelleren Aktivierung und langsameren Inaktivierung von Kv 1.3. Wie waren weiterhin daran interessiert, zu untersuchen, ob Rapamycin auch einen Effekt auf spannungsunabhängige K+ - Kanäle hat. Die Experimente dazu führten wir in Mastzellen aus dem Knochenmark von Mäusen aus (BMMCs), die bekannter weise die Ca2+- abhängigen K+ - Kanäle KCa3.1 exprimieren. Unsere Beobachtungen zeigten, dass Rapamycin, obwohl es die KCa3.1 – Kanal – Aktivierung nicht direkt beeinflusste, den Antigen-induzierten Anstieg des cytosolischen Ca2+- Spiegels der BMMCs verminderte, was sekundär zur Dämpfung der KCa3.1 – Kanäle führte. In den BMMCs untersuchten wir außerdem den Effekt einer anderen Kinase im PI3K-Signalweg, PDK1, auf Ionenkanäle und Zellfunktionen. Diese Arbeit bewies, dass die Stimulation des Antigen induzierten Ca2+- Einstroms, aber nicht die Ca2+- Ausschüttung aus intrazellulären Speichern in Mastzellen aus PDK1 hypomorphen Mäusen (pdk1hm) weniger stark ausgeprägt war als in Mastzellen aus den Wildtyp-Geschwistertieren (pdk1wt). Demnach schwächte der PDK1-Mangel den Ca2+- Einstrom ab und folglich auch indirekt die Ströme durch die Ca2+- aktivierten K+ - Kanäle KCa3.1. Das partielle PDK1-Defizit verhinderte nicht die Degranulation trotz des geringeren Ca2+- Einstroms in pdk1hm BMMCs. Beides, die ß;-Hexosaminidase- und die Histamin – Ausschüttung waren nicht signifikant unterschiedlich zwischen pdk1wt und pdk1hm BMMCs. Wir erstellten einen möglichen Mechanismus, der die unbeeinträchtigte Degranulation in pdk1hm BMMCs erklärt, indem wir zeigten, dass PDK1 zur Aktivierung zweier nachgeordneter Kinasen, SGK1 und PKCdelta; führte, wobei erstere die Degranluation in Mastzellen positiv und letztere negativ reguliert

Membrane potential drives the exit from pluripotency and cell fate commitment via calcium and mTOR

Abstract Transitioning from pluripotency to differentiated cell fates is fundamental to both embryonic development and adult tissue homeostasis. Improving our understanding of this transition would facilitate our ability to manipulate pluripotent cells into tissues for therapeutic use. Here, we show that membrane voltage (Vm) regulates the exit from pluripotency and the onset of germ layer differentiation in the embryo, a process that affects both gastrulation and left-right patterning. By examining candidate genes of congenital heart disease and heterotaxy, we identify KCNH6, a member of the ether-a-go-go class of potassium channels that hyperpolarizes the Vm and thus limits the activation of voltage gated calcium channels, lowering intracellular calcium. In pluripotent embryonic cells, depletion of kcnh6 leads to membrane depolarization, elevation of intracellular calcium levels, and the maintenance of a pluripotent state at the expense of differentiation into ectodermal and myogenic lineages. Using high-resolution temporal transcriptome analysis, we identify the gene regulatory networks downstream of membrane depolarization and calcium signaling and discover that inhibition of the mTOR pathway transitions the pluripotent cell to a differentiated fate. By manipulating Vm using a suite of tools, we establish a bioelectric pathway that regulates pluripotency in vertebrates, including human embryonic stem cells

Electrophysiology and metabolism of caveolin-3-overexpressing mice.

Caveolin-3 (Cav-3) plays a critical role in organizing signaling molecules and ion channels involved in cardiac conduction and metabolism. Mutations in Cav-3 are implicated in cardiac conduction abnormalities and myopathies. Additionally, cardiac-specific overexpression of Cav-3 (Cav-3 OE) is protective against ischemic and hypertensive injury, suggesting a potential role for Cav-3 in basal cardiac electrophysiology and metabolism involved in stress adaptation. We hypothesized that overexpression of Cav-3 may alter baseline cardiac conduction and metabolism. We examined: (1) ECG telemetry recordings at baseline and during pharmacological interventions, (2) ion channels involved in cardiac conduction with immunoblotting and computational modeling, and (3) baseline metabolism in Cav-3 OE and transgene-negative littermate control mice. Cav-3 OE mice had decreased heart rates, prolonged PR intervals, and shortened QTc intervals with no difference in activity compared to control mice. Dobutamine or propranolol did not cause significant changes between experimental groups in maximal (dobutamine) or minimal (propranolol) heart rate. Cav-3 OE mice had an overall lower chronotropic response to atropine. The expression of Kv1.4 and Kv4.3 channels, Nav1.5 channels, and connexin 43 were increased in Cav-3 OE mice. A computational model integrating the immunoblotting results indicated shortened action potential duration in Cav-3 OE mice linking the change in channel expression to the observed electrophysiology phenotype. Metabolic profiling showed no gross differences in VO2, VCO2, respiratory exchange ratio, heat generation, and feeding or drinking. In conclusion, Cav-3 OE mice have changes in ECG intervals, heart rates, and cardiac ion channel expression. These findings give novel mechanistic insights into previously reported Cav-3 dependent cardioprotection

A calcium transport mechanism for atrial fibrillation in Tbx5-mutant mice

Risk for Atrial Fibrillation (AF), the most common human arrhythmia, has a major genetic component. The T-box transcription factor TBX5 influences human AF risk, and adultspecific Tbx5-mutant mice demonstrate spontaneous AF. We report that TBX5 is critical for cellular Ca2+ homeostasis, providing a molecular mechanism underlying the genetic implication of TBX5 in AF. We show that cardiomyocyte action potential (AP) abnormalities in Tbx5-deficient atrial cardiomyocytes are caused by a decreased sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2)- mediated SR calcium uptake which was balanced by enhanced trans-sarcolemmal calcium fluxes (calcium current and sodium/calcium exchanger), providing mechanisms for triggered activity. The AP defects, cardiomyocyte ectopy, and AF caused by TBX5 deficiency were rescued by phospholamban removal, which normalized SERCA function. These results directly link transcriptional control of SERCA2 activity, depressed SR Ca2+ sequestration, enhanced transsarcolemmal calcium fluxes, and AF, establishing a mechanism underlying the genetic basis for a Ca2+-dependent pathway for AF risk

Transcription-factor-dependent enhancer transcription defines a gene regulatory network for cardiac rhythm

The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor-(TF)-dependence of ncRNA expression to define enhancers and enhancer-associated ncRNAs that are involved in a TF-dependent regulatory network. TBX5, a cardiac TF, regulates a network of cardiac channel genes to maintain cardiac rhythm. We deep sequenced wildtype and Tbx5-mutant mouse atria, identifying ~2600 novel Tbx5-dependent ncRNAs. Tbx5-dependent ncRNAs were enriched for tissue-specific marks of active enhancers genome-wide. Tbx5-dependent ncRNAs emanated from regions that are enriched for TBX5-binding and that demonstrated Tbx5-dependent enhancer activity. Tbx5-dependent ncRNA transcription provided a quantitative metric of Tbx5-dependent enhancer activity, correlating with target gene expression. We identified RACER, a novel Tbx5-dependent long noncoding RNA (lncRNA) required for the expression of the calcium-handling gene Ryr2. We illustrate that TF-dependent enhancer transcription can illuminate components of TF-dependent gene regulatory networks

Transcription-factor-dependent enhancer transcription defines a gene regulatory network for cardiac rhythm

The noncoding genome is pervasively transcribed. Noncoding RNAs (ncRNAs) generated from enhancers have been proposed as a general facet of enhancer function and some have been shown to be required for enhancer activity. Here we examine the transcription-factor- (TF)-dependence of ncRNA expression to define enhancers and enhancer-associated ncRNAs that are involved in a TF-dependent regulatory network. TBX5, a cardiac TF, regulates a network of cardiac channel genes to maintain cardiac rhythm. We deep sequenced wildtype and Tbx5-mutant mouse atria, identifying ~2600 novel Tbx5-dependent ncRNAs. Tbx5-dependent ncRNAs were enriched for tissue-specific marks of active enhancers genome-wide. Tbx5-dependent ncRNAs emanated from regions that are enriched for TBX5-binding and that demonstrated Tbx5- dependent enhancer activity. Tbx5-dependent ncRNA transcription provided a quantitative metric of Tbx5-dependent enhancer activity, correlating with target gene expression. We identified RACER, a novel Tbx5-dependent long noncoding RNA (lncRNA) required for the expression of the calcium-handling gene Ryr2. We illustrate that TF-dependent enhancer transcription can illuminate components of TF-dependent gene regulatory networks