Chi Wing Chow

Hydrogen sulfide attenuates myocardial ischemia-reperfusion injury by preservation of mitochondrial function

The recent discovery that hydrogen sulfide (H2S) is an endogenously produced gaseous second messenger capable of modulating many physiological processes, much like nitric oxide, prompted us to investigate the potential of H2S as a cardioprotective agent. In the current study, we demonstrate that the delivery of H2S at the time of reperfusion limits infarct size and preserves left ventricular (LV) function in an in vivo model of myocardial ischemia-reperfusion (MI-R). This observed cytoprotection is associated with an inhibition of myocardial inflammation and a preservation of both mitochondrial structure and function after I-R injury. Additionally, we show that modulation of endogenously produced H2S by cardiac-specific overexpression of cystathionine γ-lyase (α-MHC-CGL-Tg mouse) significantly limits the extent of injury. These findings demonstrate that H2S may be of value in cytoprotection during the evolution of myocardial infarction and that either administration of H2S or the modulation of endogenous production may be of clinical benefit in ischemic disorders.

Microvascular Hyperpermeability in Caveolin-1 (−/−) Knock-out Mice TREATMENT WITH A SPECIFIC NITRIC-OXIDE SYNTHASE INHIBITOR,l-NAME, RESTORES NORMAL MICROVASCULAR PERMEABILITY IN Cav-1 NULL MICE

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (−/−) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis. Thus, a compensatory mechanism must be operating in these mice. One possible compensatory response would be an increase in the paracellular pathway, resulting in increased microvascular permeability. To test this hypothesis directly, we studied the microvascular permeability of Cav-1 null mice using a variety of complementary in vivo approaches. Radio-iodinated bovine serum albumin was injected into Cav-1-deficient mice, and its rate of clearance from the circulatory system was compared with that of wild type control mice. Our results indicate that iodinated bovine serum albumin is removed from the circulatory system of Cav-1-deficient mice at a substantially faster rate. To determine whether this defect is restricted to the paracellular movement of albumin, lungs from Cav-1-deficient mice were next perfused with the electron dense dye Ruthenium Red. Micrographs of lung endothelial cells from Cav-1-deficient mice demonstrate that the paracellular movement of Ruthenium Red is dramatically increased. In addition, electron micrographs of Cav-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane. This defect in cell-substrate attachment is consistent with the postulated role of caveolin-1 in positively regulating integrin signaling. Because loss of caveolin-1 expression results in constitutive activation of eNOS activity, we also examined whether these increases in microvascular permeability are NO-dependent. Interestingly, treatment with l-NAME (a well established nitric-oxide synthase inhibitor) successfully reversed the microvascular hyperpermeability phenotype of Cav-1 knock-out mice. Thus, caveolin-1 plays a dual regulatory role in controlling microvascular permeability: (i) as a structural protein that is required for caveolae formation and caveolar transcytosis and (ii) as a tonic inhibitor of eNOS activity to negatively regulate the paracellular pathway.

Phosphorylation of NFATc4 by p38 mitogen-activated protein kinases.

ABSTRACT Nuclear factor of activated T cells (NFAT) is implicated in multiple biological processes, including cytokine gene expression, cardiac hypertrophy, and adipocyte differentiation. A conserved NFAT homology domain is identified in all NFAT members. Dephosphorylation of the NFAT homology region is critical for NFAT nuclear translocation and transcriptional activation. Here we demonstrate that NFATc4 is phosphorylated by p38 mitogen-activated protein (MAP) kinase but not by JNK. The p38 MAP kinase phosphorylates multiple residues, including Ser168 and Ser170, in the NFAT homology domain of NFATc4. Replacement of Ser168,170 with Ala promotes nuclear localization of NFATc4 and increases NFAT-mediated transcription activity. Stable expression of Ala168,170 NFATc4, but not of wild-type NFATc4, in NIH 3T3 cells promotes adipocyte formation under differentiation conditions. Molecular analysis indicates that peroxisome proliferator-activated receptor γ2 (PPARγ2) is a target of NFAT. Two distinct NFAT binding elements are located in the PPARγ2 gene promoter. Stable expression of Ala168,170 NFATc4, but not of wild-type NFATc4, increases the expression of PPARγ, which contributes in part to increased adipocyte formation. Thus, NFAT regulates PPARγ gene expression and has a direct role in adipocyte differentiation.

Cardiomyocyte-Specific BMAL1 Plays Critical Roles in Metabolism, Signaling, and Maintenance of Contractile Function of the Heart

Circadian clocks are cell autonomous, transcriptionally based, molecular mechanisms that confer the selective advantage of anticipation, enabling cells/organs to respond to environmental factors in a temporally appropriate manner. Critical to circadian clock function are 2 transcription factors, CLOCK and BMAL1. The purpose of the present study was to reveal novel physiologic functions of BMAL1 in the heart, as well as to determine the pathologic consequences of chronic disruption of this circadian clock component. To address this goal, we generated cardiomyocyte-specific Bmal1 knockout (CBK) mice. Following validation of the CBK model, combined microarray and in silico analyses were performed, identifying 19 putative direct BMAL1 target genes, which included a number of metabolic (e.g., β-hydroxybutyrate dehydrogenase 1 [Bdh1]) and signaling (e.g., the p85α regulatory subunit of phosphatidylinositol 3-kinase [Pik3r1]) genes. Results from subsequent validation studies were consistent with regulation of Bdh1 and Pik3r1 by BMAL1, with predicted impairments in ketone body metabolism and signaling observed in CBK hearts. Furthermore, CBK hearts exhibited depressed glucose utilization, as well as a differential response to a physiologic metabolic stress (i.e., fasting). Consistent with BMAL1 influencing critical functions in the heart, echocardiographic, gravimetric, histologic, and molecular analyses revealed age-onset development of dilated cardiomyopathy in CBK mice, which was associated with a severe reduction in life span. Collectively, our studies reveal that BMAL1 influences metabolism, signaling, and contractile function of the heart.

Microtubule stabilization by bone morphogenetic protein receptor-mediated scaffolding of c-Jun N-terminal kinase promotes dendrite formation.

ABSTRACT Neuronal outgrowth occurs via coordinated remodeling of the cytoskeleton involving both actin and microtubules. Microtubule stabilization drives the extending neurite, yet little is known of the molecular mechanisms whereby extracellular cues regulate microtubule dynamics. Bone morphogenetic proteins (BMPs) play an important role in neuronal differentiation and morphogenesis, and BMP7 in particular induces the formation of dendrites. Here, we show that BMP7 induces stabilization of microtubules in both a MAP2-dependent neuronal cell culture model and in dendrites of primary cortical neurons. BMP7 rapidly activates c-Jun N-terminal kinases (JNKs), known regulators of microtubule dynamics, and we show that JNKs associate with the carboxy terminus of the BMP receptor, BMPRII. Activation and binding of JNKs to BMPRII is required for BMP7-induced microtubule stabilization and for BMP7-mediated dendrite formation in primary cortical neurons. These data indicate that BMPRII acts as a scaffold to localize and coordinate cytoskeletal remodeling and thereby provides an efficient means for extracellular cues, such as BMPs, to control neuronal dendritogenesis.

Recruitment of the Extracellular Signal-Regulated Kinase/Ribosomal S6 Kinase Signaling Pathway to the NFATc4 Transcription Activation Complex

ABSTRACT Integration of protein kinases into transcription activation complexes influences the magnitude of gene expression. The nuclear factor of activated T cells (NFAT) group of proteins are critical transcription factors that direct gene expression in immune and nonimmune cells. A balance of phosphotransferase activity is necessary for optimal NFAT activation. Activation of NFAT requires dephosphorylation by the calcium-mediated calcineurin phosphatase to promote NFAT nuclear accumulation, and the Ras-activated extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase, which targets NFAT partners, to potentiate transcription. Whether protein kinases operate on NFAT and contribute positively to transcription activation is not clear. Here, we coupled DNA affinity isolation with in-gel kinase assays to avidly pull down the activated NFAT and identify its associated protein kinases. We demonstrate that p90 ribosomal S6 kinase (RSK) is recruited to the NFAT-DNA transcription complex upon activation. The formation of RSK-NFATc4-DNA transcription complex is also apparent upon adipogenesis. Bound RSK phosphorylates Ser676 and potentiates NFATc4 DNA binding by escalating NFAT-DNA association. Ser676 is also targeted by the ERK MAP kinase, which interacts with NFAT at a distinct region than RSK. Thus, integration of the ERK/RSK signaling pathway provides a mechanism to modulate NFATc4 transcription activity.

Role of Transcription Factor NFAT in Glucose and Insulin Homeostasis

ABSTRACT Compromised immunoregulation contributes to obesity and complications in metabolic pathogenesis. Here, we demonstrate that the nuclear factor of activated T cell (NFAT) group of transcription factors contributes to glucose and insulin homeostasis. Expression of two members of the NFAT family (NFATc2 and NFATc4) is induced upon adipogenesis and in obese mice. Mice with the Nfatc2−/−Nfatc4−/− compound disruption exhibit defects in fat accumulation and are lean. Nfatc2−/−Nfatc4−/− mice are also protected from diet-induced obesity. Ablation of NFATc2 and NFATc4 increases insulin sensitivity, in part, by sustained activation of the insulin signaling pathway. Nfatc2−/−Nfatc4−/− mice also exhibit an altered adipokine profile, with reduced resistin and leptin levels. Mechanistically, NFAT is recruited to the transcription loci and regulates resistin gene expression upon insulin stimulation. Together, these results establish a role for NFAT in glucose/insulin homeostasis and expand the repertoire of NFAT function to metabolic pathogenesis and adipokine gene transcription.

Proteins kinases: chromatin-associated enzymes?

Protein kinases contribute to the regulation of gene expression by interacting with transcription factors that are recruited to the regulatory regions of genes. Previous studies investigated the role of protein kinases in transcription initiation. Here, we discuss new insights gleaned from recent work showing that kinases can also interact with chromatin throughout the entire transcribed region of target genes (Pokholok et al., 2006; Proft et al., 2006).

Integration of protein kinases mTOR and extracellular signal-regulated kinase 5 in regulating nucleocytoplasmic localization of NFATc4.

ABSTRACT The target of rapamycin (TOR) signaling regulates the nucleocytoplasmic shuttling of transcription factors in yeast. Whether the mammalian counterpart of TOR (mTOR) also regulates nucleocytoplasmic shuttling is not known. Using a phospho-specific monoclonal antibody, we demonstrate that mTOR phosphorylates Ser168,170 of endogenous NFATc4, which are conserved gate-keeping Ser residues that control NFAT subcellular distribution. The mTOR acts as a basal kinase during the resting state to maintain NFATc4 in the cytosol. Inactivation and nuclear export of NFATc4 are mediated by rephosphorylation of Ser168,170, which can be a nuclear event. Kinetic analyses demonstrate that rephosphorylation of Ser168,170 of endogenous NFATc4 is mediated by mTOR and, surprisingly, by extracellular signal-regulated kinase 5 (ERK5) mitogen-activated protein kinase as well. Ablation of ERK5 in the Erk5−/− cells ascertains defects in NFATc4 rephosphorylation and nucleocytoplasmic shuttling. In addition, phosphorylation of NFATc4 by ERK5 primes subsequent phosphorylation mediated by CK1α. These results demonstrate that distinct protein kinases are integrated to phosphorylate the gate-keeping residues Ser168,170 of NFATc4, to regulate subcellular distribution. These data also expand the repertoire of physiological substrates of mTOR and ERK5.

Mutation of SIMPLE in Charcot–Marie–Tooth 1C alters production of exosomes

Mutations in the protein SIMPLE account for the rare autosomal-dominant demyelination in type 1C CMT patients (CMT1C). SIMPLE plays a role in the production of exosomes. Dysregulated endosomal trafficking and changes in exosome-mediated intercellular communications might account for CMT1C molecular pathogenesis.