Biomedical subjects

Robert Feil

Publications and source records attributed to Robert Feil.

At least 55 records · Page 3Linked to original sources

PubMed2003

A proatherogenic role for cGMP-dependent protein kinase in vascular smooth muscle cells.

Nitric oxide (NO) exerts both antiatherogenic and proatherogenic effects, but the cellular and molecular mechanisms that contribute to modulation of atherosclerosis by NO are not understood completely. The cGMP-dependent protein kinase I (cGKI) is a potential mediator of NO signaling in vascular smooth muscle cells (SMCs). Postnatal ablation of cGKI selectively in the SMCs of mice reduced atherosclerotic lesion area, demonstrating that smooth muscle cGKI promotes atherogenesis. Cell-fate mapping indicated that cGKI is involved in the development of SMC-derived plaque cells. Activation of endogenous cGKI in primary aortic SMCs resulted in cells with increased levels of proliferation; increased levels of vascular cell adhesion molecule-1, peroxisome proliferator-activated receptor gamma, and phosphatidylinositol 3-kinase/Akt signaling; and decreased plasminogen activator inhibitor 1 mRNA, which all are potentially proatherogenic properties. Taken together, these results highlight the pathophysiologic significance of vascular SMCs in atherogenesis and identify a key role for cGKI in the development of atherogenic SMCs in vitro and in vivo. We suggest that activation of smooth muscle cGKI contributes to the proatherogenic effect of NO and that inhibition of cGKI might be a therapeutic option for treating atherosclerosis in humans.

Alleles↗

PubMed2003

Impairment of LTD and cerebellar learning by Purkinje cell-specific ablation of cGMP-dependent protein kinase I.

The molecular basis for cerebellar plasticity and motor learning remains controversial. Cerebellar Purkinje cells (PCs) contain a high concentration of cGMP-dependent protein kinase type I (cGKI). To investigate the function of cGKI in long-term depression (LTD) and cerebellar learning, we have generated conditional knockout mice lacking cGKI selectively in PCs. These cGKI mutants had a normal cerebellar morphology and intact synaptic calcium signaling, but strongly reduced LTD. Interestingly, no defects in general behavior and motor performance could be detected in the LTD-deficient mice, but the mutants exhibited an impaired adaptation of the vestibulo-ocular reflex (VOR). These results indicate that cGKI in PCs is dispensable for general motor coordination, but that it is required for cerebellar LTD and specific forms of motor learning, namely the adaptation of the VOR.

Adaptation, Physiological↗

PubMed2003

Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice.

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.

Animals↗

PubMed2003

Individual cerebellar Purkinje cells express different cGMP phosphodiesterases (PDEs): in vivo phosphorylation of cGMP-specific PDE (PDE5) as an indicator of cGMP-dependent protein kinase (PKG) activation.

The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed. Although expression of guanylate cyclase and/or cGMP-dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cells in vivo has been demonstrated. To determine if changes in PKG activation and PDE5 regulation occur in vivo we have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylation in vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as an in vivo indicator for PKG activation.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2003

Hippocampal cGMP-dependent protein kinase I supports an age- and protein synthesis-dependent component of long-term potentiation but is not essential for spatial reference and contextual memory.

cGMP-dependent protein kinase I (cGKI) is expressed in the hippocampus, but its role in hippocampal long-term potentiation (LTP) is controversial. In addition, whether cGKI is involved in spatial learning has not been investigated. To address these issues, we generated mice with a hippocampus-specific deletion of cGKI (cGKIhko mice). Unlike conventional cGKI knock-out mice, cGKIhko mice lack gastrointestinal and cardiovascular phenotypes and have a normal life expectancy, which enables us to analyze hippocampal synaptic plasticity and learning in young and adult animals. Hippocampal LTP after repetitive episodes of theta burst stimulation was impaired in adult (12-14 weeks of age) but not in juvenile (3-4 weeks of age) cGKIhko mice. The difference in LTP between adult control and cGKIhko mice was abolished by the protein synthesis inhibitor anisomycin, suggesting that the impairment of LTP in adult cGKIhko mice reflects a defect in late-phase LTP. Despite their deficit in LTP, adult cGKIhko mutants showed normal performance in a discriminatory water maze and had intact contextual fear conditioning. These results suggest that hippocampal cGKI supports an age- and protein synthesis-dependent form of hippocampal LTP, whereas it is dispensable for hippocampus-dependent spatial reference and contextual memory.

Age Factors↗

PubMed2003

cGMP-dependent protein kinase II modulates mPer1 and mPer2 gene induction and influences phase shifts of the circadian clock.

BACKGROUND: In mammals, the master circadian clock that drives many biochemical, physiological, and behavioral rhythms is located in the suprachiasmatic nuclei (SCN) of the hypothalamus. Generation and maintenance of circadian rhythmicity rely on complex interlocked transcriptional/translational feedback loops involving a set of clock genes. Among the molecular components driving the mammalian circadian clock are the Period 1 and 2 (mPer1 and mPer2) genes. Because the periodicity of the clock is not exactly 24 hr, it has to be adjusted periodically. The major stimulus for adjustment (resetting) of the clock is nocturnal light. It evokes activation of signaling pathways in the SCN that ultimately lead to expression of mPer1 and mPer2 genes conveying adjustment of the clock. RESULTS: We show that mice deficient in cGMP-dependent protein kinase II (cGKII, also known as PKGII), despite regular retinal function, are defective in resetting the circadian clock, as assessed by changes in the onset of wheel running activity after a light pulse. At the molecular level, light induction of mPer2 in the SCN is strongly reduced in the early period of the night, whereas mPer1 induction is elevated in cGKII-deficient mice. Additionally, we show that light induction of cfos and light-dependent phosphorylation of CREB at serine 133 are not affected in these animals. CONCLUSIONS: cGKII plays a role in the clock-resetting mechanism. In particular, the ability to delay clock phase is affected in cGKII-deficient mice. It seems that the signaling pathway involving cGKII influences in an opposite manner the light-induced induction of mPer1 and mPer2 genes and thereby influences the direction of a phase shift of the circadian clock.

Animals↗

PubMed2003

Significance and therapeutic potential of the natriuretic peptides/cGMP/cGMP-dependent protein kinase pathway in vascular regeneration.

Natriuretic peptides (NPs), which consist of atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP, respectively), are characterized as cardiac or vascular hormones that elicit their biological effects by activation of the cGMPcGMP-dependent protein kinase (cGK) pathway. We recently reported that adenoviral gene transfer of CNP into rabbit blood vessels not only suppressed neointimal formation but also accelerated reendothelialization, a required step for endothelium-dependent vasorelaxation and antithrombogenicity. Accordingly, we investigated the therapeutic potential of the NPscGMPcGK pathway for vascular regeneration. In transgenic (Tg) mice that overexpress BNP in response to hindlimb ischemia, neovascularization with appropriate mural cell coating was accelerated without edema or bleeding, and impaired angiogenesis by the suppression of nitric oxide production was effectively rescued. Furthermore, in BNP-Tg mice, inflammatory cell infiltration in ischemic tissue and vascular superoxide production were suppressed compared with control mice. Ischemia-induced angiogenesis was also significantly potentiated in cGK type I Tg mice, but attenuated in cGK type I knockout mice. NPs significantly stimulated capillary network formation of cultured endothelial cells by cGK stimulation and subsequent Erk12 activation. Furthermore, gene transfer of CNP into ischemic muscles effectively accelerated angiogenesis. These findings reveal an action of the NPscGMPcGK pathway to exert multiple vasculoprotective and regenerative actions in the absence of apparent adverse effects, and therefore suggest that NPs as the endogenous cardiovascular hormone can be used as a strategy of therapeutic angiogenesis in patients with tissue ischemia.

Animals↗

PubMed2003

Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling.

In platelets, the nitric oxide (NO)-induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed2003

Absence epilepsy and sinus dysrhythmia in mice lacking the pacemaker channel HCN2.

Hyperpolarization-activated cation (HCN) channels are believed to be involved in the generation of cardiac pacemaker depolarizations as well as in the control of neuronal excitability and plasticity. The contributions of the four individual HCN channel isoforms (HCN1-4) to these diverse functions are not known. Here we show that HCN2-deficient mice exhibit spontaneous absence seizures. The thalamocortical relay neurons of these mice displayed a near complete loss of the HCN current, resulting in a pronounced hyperpolarizing shift of the resting membrane potential, an altered response to depolarizing inputs and an increased susceptibility for oscillations. HCN2-null mice also displayed cardiac sinus dysrhythmia, a reduction of the sinoatrial HCN current and a shift of the maximum diastolic potential to hyperpolarized values. Mice with cardiomyocyte- specific deletion of HCN2 displayed the same dysrhythmia as mice lacking HCN2 globally, indicating that the dysrhythmia is indeed caused by sinoatrial dysfunction. Our results define the physiological role of the HCN2 subunit as a major determinant of membrane resting potential that is required for regular cardiac and neuronal rhythmicity.

Animals↗

PubMed2003

A stimulatory role for cGMP-dependent protein kinase in platelet activation.

It is currently accepted that cGMP-dependent protein kinase (PKG) inhibits platelet activation. Here, we show that PKG plays an important stimulatory role in platelet activation. Expression of recombinant PKG in a reconstituted cell model enhanced von Willebrand factor (vWF)-induced activation of the platelet integrin alpha(IIb)beta(3). PKG knockout mice showed impaired platelet responses to vWF or low doses of thrombin and prolonged bleeding time. Human platelet aggregation induced by vWF or low-dose thrombin was inhibited by PKG inhibitors but enhanced by cGMP. Furthermore, a cGMP-enhancing agent, sildenafil, promoted vWF- or thrombin-induced platelet aggregation. The cGMP-stimulated platelet responses are biphasic, consisting of an initial transient stimulatory response that promotes platelet aggregation and a subsequent inhibitory response that limits the size of thrombi.

Animals↗

PubMed2003

Conservation of IGF2-H19 and IGF2R imprinting in sheep: effects of somatic cell nuclear transfer.

In different mammalian species, in vitro culture and manipulation can lead to aberrant fetal and peri-natal development. It has been postulated that these diverse abnormalities are caused by epigenetic alterations and that these could affect genes that are regulated by genomic imprinting. To explore this hypothesis relative to somatic cell nuclear transfer in sheep, we investigated whether the ovine H19-IGF2 and IGF2R loci are imprinted and analysed their DNA methylation status in cloned lambs. A comparison between parthenogenetic and control concepti established that imprinting at these two growth-related loci is evolutionarily conserved in sheep. As in humans and mice, IGF2R and H19 comprise differentially methylated regions (DMRs) that are methylated on one of the two parental alleles predominantly. In tongue tissue from 12 out of 13 cloned lambs analysed, the DMR in the second intron of IGF2R had strongly reduced levels of DNA methylation. The DMR located upstream of the ovine H19 gene was found to be similarly organised as in humans and mice, with multiple CTCF binding sites. At this DMR, however, aberrant methylation was observed in only one of the cloned lambs. Although the underlying mechanisms remain to be determined, our data indicate that somatic cell nuclear transfer procedures can lead to epigenetic deregulation at imprinted loci.

Alleles↗

PubMed2003

Signaling through NO and cGMP-dependent protein kinases.

The gaseous molecule nitric oxide (NO) modulates a large variety of physiological functions including vascular tone, intestinal motility, platelet aggregation, proliferation, apoptosis, and neurotransmission. NO initiates diverse cellular signaling cascades which comprise nitrosylation of proteins, adenosine 5'-diphosphate (ADP)-ribosylation, or stimulation of soluble guanylyl cyclases which catalyze intracellular guanosine 3',5'-cyclic monophosphate (cGMP) synthesis. cGMP activates cGMP-dependent protein kinases (cGK) which mediate localized and global signaling. Furthermore, cGMP regulates the activity of phosphodiesterases (PDE) which modulate the duration and amplitude of cyclic nucleotide signaling. Two different types of cGK are expressed in mammals, cGKI and cGKII. Activation of the NO/cGMP/cGKI pathway induces relaxation of smooth muscle by lowering the cytosolic calcium level and/or by calcium desensitization of the contractile elements.

Calcium Signaling↗

PubMed2003

Epigenetic properties and identification of an imprint mark in the Nesp-Gnasxl domain of the mouse Gnas imprinted locus.

The Gnas locus in the mouse is imprinted with a complex arrangement of alternative transcripts defined by promoters with different patterns of monoallelic expression. The Gnas transcript is subject to tissue-specific imprinted expression, Nesp is expressed only from the maternal allele, and Gnasxl is expressed only from the paternal allele. The mechanisms controlling these expression patterns are not known. To identify potential imprinting regulatory regions, particularly for the reciprocally expressed Nesp and Gnasxl promoters, we examined epigenetic properties of the locus in gametes, embryonic stem cells, and fetal and adult tissues. The Nesp and Gnasxl promoter regions are contained in extensive CpG islands with methylation of the paternal allele at Nesp and the maternal allele at Gnasxl. Parental allele-specific DNase I-hypersensitive sites were found at these regions, which correlate with hypomethylation rather than actual expression status. A germ line methylation mark was identified covering the promoters for Gnasxl and the antisense transcript Nespas. Prominent DNase I-hypersensitive sites present on paternal alleles in embryonic stem cells are contained within this mark. This is the second gametic mark identified at Gnas and suggests that the Nesp and Gnasxl promoters are under separate control from the Gnas promoter. We propose models to account for the regulation of imprinting at the locus.

Alleles↗

PubMed2002

Allele-specific histone lysine methylation marks regulatory regions at imprinted mouse genes.

In different eukaryotic model systems, chromatin and gene expression are modulated by post-translational modification of histone tails. In this in vivo study, histone methylation and acetylation are investigated along the imprinted mouse genes Snrpn, Igf2r and U2af1-rs1. These imprinted genes all have a CpG-rich regulatory element at which methylation is present on the maternal allele, and originates from the female germ line. At these 'differentially methylated regions' (DMRs), histone H3 on the paternal allele has lysine-4 methylation and is acetylated. On the maternally inherited allele, in contrast, chromatin is marked by hypermethylation on lysine-9 of H3. Allele-specific patterns of lysine-4 and lysine-9 methylation are also detected at other regions of the imprinted loci. For the DMR at the U2af1-rs1 gene, we establish that the methyl-CpG-binding-domain (MBD) proteins MeCP2, MBD1 and MBD3 are associated with the maternal allele. These data support the hypothesis that MBD protein-associated histone deacetylase/chromatin-remodelling complexes are recruited to the parental allele that has methylated DNA and H3-K9 methylation, and are prevented from binding to the opposite allele by H3 lysine-4 methylation.

Animals↗

PubMed2002

Allelic inactivation of the pseudoautosomal gene SYBL1 is controlled by epigenetic mechanisms common to the X and Y chromosomes.

On the human long-arm pseudoautosomal region (XqPAR), genes that are subject to inactivation are closely linked with those that escape. Genes subject to inactivation are not only silenced on the inactive X in females, but they are also inactivated on the Y chromosome in males. One of the genes subject to this unusual inactivation pattern is the synaptobrevin-like 1 gene (SYBL1). Previously we showed that its silencing on the inactive X and the Y allele involves DNA methylation. This study explores the molecular events associated with SYBL1 silencing and investigates their relationship. Promoter DNA methylation profiles were determined by bisulfite sequencing and immunoprecipitation experiments demonstrate that chromatin on the repressed Xi and the Y alleles has underacetylated histones H3 and H4 and H3-lysine 9 methylation. In addition, the inactive X and the Y allele were found to have a condensed chromatin conformation. In contrast, the expressed allele shows H3 and H4 acetylation, H3-lysine 4 methylation and a less compacted chromatin conformation. In ICF syndrome, a human disease affecting DNA methylation, SYBL1 escapes from silencing and this correlates with altered patterns of histone methylation and acetylation. Combined, our data suggest that specific combinations of histone methylation and acetylation are involved in the somatic maintenance of permissive and repressed chromatin states at SYBL1. Although it is unclear at present how this allele-specific silencing comes about, the data also indicate that the epigenetic features of the 'Y inactivation' of SYBL1 are mechanistically similar to those associated with X-chromosome inactivation.

Alleles↗

PubMed2002

cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons.

Previous in vitro studies using cGMP or cAMP revealed a cross-talk between signaling mechanisms activated by axonal guidance receptors. However, the molecular elements modulated by cyclic nucleotides in growth cones are not well understood. cGMP is a second messenger with several distinct targets including cGMP-dependent protein kinase I (cGKI). Our studies indicated that the alpha isoform of cGKI is predominantly expressed by sensory axons during developmental stages, whereas most spinal cord neurons are negative for cGKI. Analysis of the trajectories of axons within the spinal cord showed a longitudinal guidance defect of sensory axons within the developing dorsal root entry zone in the absence of cGKI. Consequently, in cGKI-deficient mice, fewer axons grow within the dorsal funiculus of the spinal cord, and lamina-specific innervation, especially by nociceptive sensory neurons, is strongly reduced as deduced from anti-trkA staining. These axon guidance defects in cGKI-deficient mice lead to a substantial impairment in nociceptive flexion reflexes, shown using electrophysiology. In vitro studies revealed that activation of cGKI in embryonic dorsal root ganglia counteracts semaphorin 3A-induced growth cone collapse. Our studies therefore reveal that cGMP signaling is important for axonal growth in vivo and in vitro.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2002

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.

Animals↗

PubMed2002

p8-deficient fibroblasts grow more rapidly and are more resistant to adriamycin-induced apoptosis.

p8 is a stress-induced DNA-binding protein, biochemically related to the architectural chromatin binding HMG protein family and whose function is presently unknown. We obtained fibroblast from mice lacking p8 and found that p8 is involved in cell growth regulation and in apoptosis. p8(-/-) mouse embryonic fibroblasts (MEFs) grow more rapidly than p8(+/+) MEFs. This might be explained by the higher intracellular level and activity of the Cdk2 and Cdk4 observed in p8(-/-) MEFs, which in turn may result, at least in part, from the concomitant decrease observed in the amount of cyclin-dependent kinase inhibitor p27. We also report that p8 mRNA expression is strongly activated in fibroblasts after cell growth arrest induced by serum deprivation or confluence. As expected, MEFs expressing p8 arrest their growth more rapidly after serum deprivation than MEFs lacking p8, which strongly suggests that p8 over-expression is implicated in cell growth arrest. On the other hand, p8(+/+) MEFs are more sensitive than p8(-/-) MEFs to the apoptosis induced by adriamycin treatment. p53 might be involved, as p8 expression increases its intracellular amount and trans-activation capacity. Finally, demonstration that p53 is a negative trans-activator of p8 suggests the presence of a complex autoregulatory loop. In conclusion, p8 is a cell growth inhibitor that facilitates apoptosis induced in fibroblasts by DNA damage.

Animals↗