Cornelia Brunner

Modulation of Calcium-Activated Potassium Channels Induces Cardiogenesis of Pluripotent Stem Cells and Enrichment of Pacemaker-Like Cells

Background— Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca2+-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. Methods and Results— We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone–induced effects. Conclusions— SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

Cardiomyocyte-specific IκB kinase (IKK)/NF-κB activation induces reversible inflammatory cardiomyopathy and heart failure

Inflammation is a major factor in heart disease. IκB kinase (IKK) and its downstream target NF-κB are regulators of inflammation and are activated in cardiac disorders, but their precise contributions and targets are unclear. We analyzed IKK/NF-κB function in the heart by a gain-of-function approach, generating an inducible transgenic mouse model with cardiomyocyte-specific expression of constitutively active IKK2. In adult animals, IKK2 activation led to inflammatory dilated cardiomyopathy and heart failure. Transgenic hearts showed infiltration with CD11b+ cells, fibrosis, fetal reprogramming, and atrophy of myocytes with strong constitutively active IKK2 expression. Upon transgene inactivation, the disease was reversible even at an advanced stage. IKK-induced cardiomyopathy was dependent on NF-κB activation, as in vivo expression of IκBα superrepressor, an inhibitor of NF-κB, prevented the development of disease. Gene expression and proteomic analyses revealed enhanced expression of inflammatory cytokines, and an IFN type I signature with activation of the IFN-stimulated gene 15 (ISG15) pathway. In that respect, IKK-induced cardiomyopathy resembled Coxsackievirus-induced myocarditis, during which the NF-κB and ISG15 pathways were also activated. Vice versa, in cardiomyocytes lacking the regulatory subunit of IKK (IKKγ/NEMO), the induction of ISG15 was attenuated. We conclude that IKK/NF-κB activation in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure by inducing an excessive inflammatory response and myocyte atrophy.

NFATc1 affects mouse splenic B cell function by controlling the calcineurin--NFAT signaling network.

Mouse B cells lacking NFATc1 exhibit defective proliferation, survival, isotype class switching, cytokine production, and T cell help.

The Ca2+-dependent Phosphatase Calcineurin Controls the Formation of the Carma1-Bcl10-Malt1 Complex during T Cell Receptor-induced NF-κB Activation

T cell receptor (TCR) ligation induces increased diacylglycerol and Ca2+ levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity.

Neutrophil development and function critically depend on Bruton tyrosine kinase in a mouse model of X-linked agammaglobulinemia

Bruton tyrosine kinase (Btk) is essential for B cell development and function and also appears to be important for myeloid cells. The bone marrow of Btk-deficient mice shows enhanced granulopoiesis compared with that of wild-type mice. In purified granulocyte-monocyte-progenitors (GMP) from Btk-deficient mice, the development of granulocytes is favored at the expense of monocytes. However, Btk-deficient neutrophils are impaired in maturation and function. Using bone marrow chimeras, we show that this defect is cell-intrinsic to neutrophils. In GMP and neutrophils, Btk plays a role in GM-CSF- and Toll-like receptor-induced differentiation. Molecular analyses revealed that expression of the lineage-determining transcription factors C/EBPα, C/EBPβ, and PU.1, depends on Btk. In addition, expression of several granule proteins, including myeloperoxidase, neutrophilic granule protein, gelatinase and neutrophil elastase, is Btk-dependent. In the Arthus reaction, an acute inflammatory response, neutrophil migration into tissues, edema formation, and hemorrhage are significantly reduced in Btk-deficient animals. Together, our findings implicate Btk as an important regulator of neutrophilic granulocyte maturation and function in vivo.

B Cell–specific Transgenic Expression of Bcl2 Rescues Early B Lymphopoiesis but Not B Cell Responses in BOB.1/OBF.1-deficient Mice

Mice deficient for the transcriptional coactivator BOB.1/OBF.1 show several defects in B cell differentiation. Numbers of immature transitional B cells in the bone marrow are reduced and fewer B cells reach the periphery. Furthermore, germinal center B cells are absent and marginal zone (MZ) B lymphocytes are markedly reduced. Increased levels of B cell apoptosis in these mice prompted us to analyze expression and function of antiapoptotic proteins. Bcl2 expression is strongly reduced in BOB.1/OBF.1-deficient pre–B cells. When BOB.1/OBF.1-deficient mice were crossed with Bcl2-transgenic mice, B cell development in the bone marrow and numbers of B cells in peripheral lymphoid organs were normalized. However, neither germinal center B cells nor MZ B cells were rescued. Additionally, Bcl2 did not rescue the defects in signaling and affinity maturation found in BOB.1/OBF.1-deficient mice. Interestingly, Bcl2-transgenic mice by themselves show an MZ B cell defect. Virtually no functional MZ B cells were detected in these mice. In contrast, mice deficient for Bcl2 show a relative increase in MZ B cell numbers, indicating a previously undetected function of Bcl2 for this B cell compartment.

TBX3 Directs Cell-Fate Decision toward Mesendoderm

Summary Cell-fate decisions and pluripotency are dependent on networks of key transcriptional regulators. Recent reports demonstrated additional functions of pluripotency-associated factors during early lineage commitment. The T-box transcription factor TBX3 has been implicated in regulating embryonic stem cell self-renewal and cardiogenesis. Here, we show that TBX3 is dynamically expressed during specification of the mesendoderm lineages in differentiating embryonic stem cells (ESCs) in vitro and in developing mouse and Xenopus embryos in vivo. Forced TBX3 expression in ESCs promotes mesendoderm specification by directly activating key lineage specification factors and indirectly by enhancing paracrine Nodal/Smad2 signaling. TBX3 loss-of-function analyses in the Xenopus underline its requirement for mesendoderm lineage commitment. Moreover, we uncovered a functional redundancy between TBX3 and Tbx2 during Xenopus gastrulation. Taken together, we define further facets of TBX3 actions and map TBX3 as an upstream regulator of the mesendoderm transcriptional program during gastrulation.

BOB.1/OBF.1 controls the balance of TH1 and TH2 immune responses

BOB.1/OBF.1 is a transcriptional coactivator essential at several stages of B‐cell development. In T cells, BOB.1/OBF.1 expression is inducible by co‐stimulation. However, a defined role of BOB.1/OBF.1 for T‐cell function had not been discovered so far. Here, we show that BOB.1/OBF.1 is critical for T helper cell function. BOB.1/OBF.1−/− mice showed imbalanced immune responses, resulting in increased susceptibility to Leishmania major infection. Functional analyses revealed specific defects in TH1 and TH2 cells. Whereas expression levels of TH1 cytokines were reduced, the secretion of TH2 cytokines was increased. BOB.1/OBF.1 directly contributes to the IFNγ and IL2 promoter activities. In contrast, increased TH2 cytokine production is controlled indirectly, probably via the transcription factor PU.1, the expression of which is regulated by BOB.1/OBF.1. Thus, BOB.1/OBF.1 regulates the balance of TH1 versus TH2 mediated immunity.

Protein kinase D2 is a novel regulator of glioblastoma growth and tumor formation

Glioblastoma multiforme, a highly aggressive tumor of the central nervous system, has a dismal prognosis that is due in part to its resistance to radio- and chemotherapy. The protein kinase C (PKC) family of serine threonine kinases has been implicated in the formation and proliferation of glioblastoma multiforme. Members of the protein kinase D (PKD) family, which consists of PKD1, -2 and, -3, are prominent downstream targets of PKCs and could play a major role in glioblastoma growth. PKD2 was highly expressed in both low-grade and high-grade human gliomas. The number of PKD2-positive tumor cells increased with glioma grading (P < .001). PKD2 was also expressed in CD133-positive glioblastoma stem cells and various glioblastoma cell lines in which the kinase was found to be constitutively active. Inhibition of PKDs by pharmacological inhibitors resulted in substantial inhibition of glioblastoma proliferation. Furthermore, specific depletion of PKD2 by siRNA resulted in a marked inhibition of anchorage-dependent and -independent proliferation and an accumulation of glioblastoma cells in G0/G1, accompanied by a down-regulation of cyclin D1 expression. In addition, PKD2-depleted glioblastoma cells exhibited substantially reduced tumor formation in vivo on chicken chorioallantoic membranes. These findings identify PKD2 as a novel mediator of glioblastoma cell growth in vitro and in vivo and thereby as a potential therapeutic target for this devastating disease.

Ca2+ activated K channels-new tools to induce cardiac commitment from pluripotent stem cells in mice and men.

To date, the precise role of ion channels in developmental processes has remained elusive, although it is well accepted that cell differentiation and maturation affect the expression patterns of ion channels [1]. Calcium-activated potassium channels (SKCas) exhibit small (SK1-3, Kcnn1-3) or intermediate (SK4, IK, Kcnn4) unitary conductance for Stem Cell Rev and Rep (2012) 8:720–740 DOI 10.1007/s12015-011-9324-9