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Biomedical subjects

Daria Mochly-Rosen

Publications and source records attributed to Daria Mochly-Rosen.

At least 37 records · Page 2Linked to original sources
PubMed2003

Preservation of base-line hemodynamic function and loss of inducible cardioprotection in adult mice lacking protein kinase C epsilon.

Signaling pathways involving protein kinase C isozymes are modulators of cardiovascular development and response to injury. Protein kinase C epsilon activation in cardiac myocytes reduces necrosis caused by coronary artery disease. However, it is unclear whether protein kinase C epsilon function is required for normal cardiac development or inducible protection against oxidative stress. Protein kinase C delta activation is also observed during cardiac preconditioning. However, its role as a promoter or inhibitor of injury is controversial. We examined hearts from protein kinase C epsilon knock-out mice under physiological conditions and during acute ischemia reperfusion. Null-mutant and wild-type mice displayed equivalent base-line morphology and hemodynamic function. Targeted disruption of the protein kinase C epsilon gene blocked cardioprotection caused by ischemic preconditioning and alpha(1)-adrenergic receptor stimulation. Protein kinase C delta activation increased in protein kinase C epsilon knock-out myocytes without altering resistance to injury. These observations support protein kinase C epsilon activation as an essential component of cardioprotective signaling. Our results favor protein kinase C delta activation as a mediator of normal growth. This study advances the understanding of cellular mechanisms responsible for preservation of myocardial integrity as potential targets for prevention and treatment of ischemic heart disease.

Animals↗
PubMed2003

Inhibition of delta-protein kinase C protects against reperfusion injury of the ischemic heart in vivo.

BACKGROUND: Current treatment for acute myocardial infarction (AMI) focuses on reestablishing blood flow (reperfusion). Paradoxically, reperfusion itself may cause additional injury to the heart. We previously found that delta-protein kinase C (deltaPKC) inhibition during simulated ischemia/reperfusion in isolated rat hearts is cardioprotective. We focus here on the role for deltaPKC during reperfusion only, using an in vivo porcine model of AMI. METHODS AND RESULTS: An intracoronary application of a selective deltaPKC inhibitor to the heart at the time of reperfusion reduced infarct size, improved cardiac function, inhibited troponin T release, and reduced apoptosis. Using 31P NMR in isolated perfused mouse hearts, we found a faster recovery of ATP levels in hearts treated with the deltaPKC inhibitor during reperfusion only. CONCLUSIONS: Reperfusion injury after cardiac ischemia is mediated, at least in part, by deltaPKC activation. This study suggests that including a deltaPKC inhibitor at reperfusion may improve the outcome for patients with AMI.

Animals↗
PubMed2003

Additive protection of the ischemic heart ex vivo by combined treatment with delta-protein kinase C inhibitor and epsilon-protein kinase C activator.

BACKGROUND: Protein kinase C (PKC) plays a major role in cardioprotection from ischemia/reperfusion injury. Using an HIV-1 Tat protein-derived peptide to mediate rapid and efficient transmembrane delivery of peptide regulators of PKC translocation and function, we examined the cardioprotective effect of selective delta-PKC inhibitor (deltaV1-1) and epsilon-PKC activator (psi(epsilon)RACK) peptides for ischemia/reperfusion damage in isolated perfused rat hearts. Furthermore, we examined the protective effects of these PKC isozymes in isolated perfused hearts subjected to ischemia/reperfusion damage using transgenic mice expressing these peptides specifically in their cardiomyocytes. METHODS AND RESULTS: In isolated perfused rat hearts, administration of deltaV1-1 but not psi(epsilon)RACK during reperfusion improved cardiac function and decreased creatine phosphokinase release. In contrast, pretreatment with psi(epsilon)RACK but not deltaV1-1, followed by a 10-minute washout before ischemia/reperfusion, also improved cardiac function and decreased creatine phosphokinase release. Furthermore, administration of psi(epsilon)RACK before ischemia followed by deltaV1-1 during reperfusion only conferred greater cardioprotective effects than that obtained by each peptide treatment alone. Both the delta-PKC inhibitor and epsilon-PKC activator conferred cardioprotection against ischemia/reperfusion injury in transgenic mice expressing these peptides in the heart, and coexpression of both peptides conferred greater cardioprotective effects than that obtained by the expression of each peptide alone. CONCLUSIONS: delta-PKC inhibitor prevents reperfusion injury, and epsilon-PKC activator mimics ischemic preconditioning. Furthermore, treatment with both peptides confers additive cardioprotective effects. Therefore, these peptides mediate cardioprotection by regulating ischemia/reperfusion damage at distinct time points.

Amino Acid Sequence↗
PubMed2003

PKC isozyme selective regulation of cloned human cardiac delayed slow rectifier K current.

Delayed rectifying K(+) channel, I(Ks), plays a vital role in normal and arrhythmogenic heart. I(Ks) is modulated by PKC but the identity of which PKC isozymes is involved in this modulation is not known. To dissect the role of individual PKC isozymes in the regulation of I(Ks), human cardiac I(Ks) channel (minK+KvLQT1) was expressed in Xenopus oocytes. Peptide PKC isozyme-specific activator and inhibitors, in addition to the general PKC activator, PMA, were used. Whole-cell I(Ks) was recorded using two-electrode voltage clamp technique. PMA and epsilon PKC specific activator peptide, but not the inactive analog, 4alphaPDD, significantly increased I(Ks). Peptide specific inhibitors for beta(II)PKC, and a general PKC inhibitor, calphostin C antagonized PMA-induced activation of I(Ks). However, control peptide, pentalysine, and specific inhibitor peptide for alphaPKC, beta(I)PKC, deltaPKC, or etaPKC did not alter PMA effect on I(Ks). The present study demonstrates that beta(II)PKC, epsilon PKC but not beta(I)PKC, alphaPKC, deltaPKC, and etaPKC, are involved in PMA-induced activation of the cloned human I(Ks) expressed in Xenopus oocyte. Furthermore, this is the first report to dissect the fine functional role of beta(II)PKC and beta(I)PKC in the regulation of I(Ks). Identification of the particular isozyme(s) that mediates the regulation of I(Ks) channels is of importance for the understanding of the mechanism of ion channel regulation and the development of new therapeutic agents.

Animals↗
PubMed2003

Epsilon PKC is required for the induction of tolerance by ischemic and NMDA-mediated preconditioning in the organotypic hippocampal slice.

Glutamate receptors and calcium have been implicated as triggering factors in the induction of tolerance by ischemic preconditioning (IPC) in the brain. However, little is known about the signal transduction pathway that ensues after the IPC induction pathway. The main goals of the present study were to determine whether NMDA induces preconditioning via a calcium pathway and promotes translocation of the protein kinase C epsilon (epsilonPKC) isozyme and whether this PKC isozyme is key in the IPC signal transduction pathway. We corroborate here that IPC and a sublethal dose of NMDA were neuroprotective, whereas blockade of NMDA receptors during IPC diminished IPC-induced neuroprotection. Calcium chelation blocked the protection afforded by both NMDA and ischemic preconditioning significantly, suggesting a significant role of calcium. Pharmacological preconditioning with the nonselective PKC isozyme activator phorbol myristate acetate could not emulate IPC, but blockade of PKC activation with chelerythrine during IPC blocked its neuroprotection. These results suggested that there might be a dual involvement of PKC isozymes during IPC. This was corroborated when neuroprotection was blocked when we inhibited epsilonPKC during IPC and NMDA preconditioning, and IPC neuroprotection was emulated with the activator of epsilonPKC. The possible correlation between NMDA, Ca2+, and epsilonPKC was found when we emulated IPC with the diacylglycerol analog oleoylacetyl glycerol, suggesting an indirect pathway by which Ca2+ could activate the calcium-insensitive epsilonPKC isozyme. These results demonstrated that the epsilonPKC isozyme played a key role in both IPC- and NMDA-induced tolerance.

Animals↗
PubMed2003

Opposing effects of delta- and zeta-protein kinase C isozymes on cardiac fibroblast proliferation: use of isozyme-selective inhibitors.

Neonatal primary cardiac fibroblasts in defined medium continue to proliferate. Here, we show that phorbol ester inhibited and transforming growth factor-beta1 (TGFbeta1) stimulated this fibroblast proliferation. Cardiac fibroblasts contain six protein kinase C (PKC) isozymes: alpha-, delta-, epsilon-, betaI-, betaII-, and zeta-PKC. To evaluate the effect of different PKC isozymes on the proliferation of these cells, we used isozyme-selective PKC inhibitors. Inhibition of endogenous delta-PKC with deltaV1-1, an isozyme-selective translocation inhibitor, resulted in increased basal thymidine incorporation by 58 +/- 12% of control cells, but did not affect TGFbeta1-induced cell growth. Inhibition of endogenous zeta-PKC in neonatal rat cardiac fibroblasts with zeta-pseudosubstrate, a selective inhibitor for the atypical PKC isozymes, revealed an opposite effect; this inhibitor reduced basal growth to 45 +/- 11% and TGFbeta1-induced growth to 61 +/- 10%. Other isozyme-specific inhibitors used in this study did not alter basal or TGFbeta1-stimulated fibroblast growth. Taken together, our data provide evidence that delta-PKC inhibits and zeta-PKC stimulates proliferation of neonatal rat cardiac fibroblasts.

Animals↗
PubMed2003

Prevention of NMDA-induced death of cortical neurons by inhibition of protein kinase Czeta.

Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (alpha, beta, and gamma), novel PKCs delta and epsilon, and an atypical PKCzeta in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 micro m NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (alpha, beta, and gamma) or novel PKCs (delta or epsilon) had no effect, the PKCzeta inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCzeta translocation, which was blocked by the PKCzeta inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCzeta mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCzeta activation is a downstream signal in NMDA-induced death of cortical neurons.

Animals↗
PubMed2002

The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins.

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.

3T3 Cells↗
PubMed2002

Sequential activation of individual PKC isozymes in integrin-mediated muscle cell spreading: a role for MARCKS in an integrin signaling pathway.

To understand how muscle cell spreading and survival are mediated by integrins, we studied the signaling events initiated by the attachment of muscle cells to fibronectin (FN). We have previously demonstrated that muscle cell spreading on FN is mediated by alpha5beta1 integrin, is associated with rapid phosphorylation of focal adhesion kinase and is dependent on activation of protein kinase C (PKC). Here we investigated the role of individual PKC isozymes in these cellular processes. We show that alpha, delta and epsilonPKC are expressed in muscle cells and are activated upon integrin engagement with different kinetics - epsilonPKC was activated early, whereas alpha and deltaPKC were activated later. Using isozyme-specific inhibitors, we found that the activation of epsilonPKC was necessary for cell attachment to FN. However, using isozyme-specific activators, we found that activation of each of three isozymes was sufficient to promote the spreading of alpha5-integrin-deficient cells on FN. To investigate further the mechanism by which integrin signaling and PKC activation mediate cell spreading, we studied the effects of these processes on MARCKS, a substrate of PKC and a protein known to regulate actin dynamics. We found that MARCKS was localized to focal adhesion sites soon after cell adhesion and that MARCKS translocated from the membrane to the cytosol during the process of cell spreading. This translocation correlated with different phases of PKC activation and with reorganization of the actin cytoskeleton. Using MARCKS-antisense cDNA, we show that alpha5-expressing cells in which MARCKS expression is inhibited fail to spread on FN, providing evidence for the crucial role of MARCKS in muscle cell spreading. Together, the data suggest a model in which early activation of epsilonPKC is necessary for cell attachment; the later activation of alpha or deltaPKC may be necessary for the progression from attachment to spreading. The mechanism of PKC-mediated cell spreading may be via the phosphorylation of signaling proteins, such as MARCKS, that are involved in the reorganization of the actin cytoskeleton.

Animals↗
PubMed2002

Molecular dynamics characterization of the C2 domain of protein kinase Cbeta.

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation.

Amino Acid Sequence↗
PubMed2002

Tissue angiotensin II during progression or ventricular hypertrophy to heart failure in hypertensive rats; differential effects on PKC epsilon and PKC beta.

The protein kinase C (PKC) family has been implicated as second messengers in mechanosensitive modulation of cardiac hypertrophy. However, little information is available on the role of expression and activation of specific cardiac PKC isozymes during development of left ventricular hypertrophy (LVH) and failure (LVF). Dahl salt-sensitive rats fed an 8% salt diet developed systemic hypertension and concentric LVH at 11 weeks of age that is followed by left ventricle (LV) dilatation and global hypokinesis at 17 weeks. Among several PKC isozymes expressed in the LV myocardium, only PKC epsilon showed a 94% increase at the LVH stage. At the LVF stage, however, PKC epsilon returned to the control level, whereas PKC beta I and beta II increased by 158% and 155%, respectively. Hearts were studied at each stage using the Langendorff set-up, and a LV balloon was inflated to achieve an equivalent diastolic wall stress. Following mechanical stretch, PKC epsilon was significantly activated in LVH myocardium in which tissue angiotensin II levels were increased by 59%. Pre-treatment with valsartan, an AT(1)-receptor blocker, abolished the stretch-mediated PKC epsilon activation. Mechanical stretch no longer induced PKC epsilon activation in LVF. Chronic administration of valsartan blunted the progression of LVF and inhibited the increase in PKC beta. Mechanosensitive PKC epsilon activation is augmented and therefore may contribute to the development of compensatory hypertrophy. This effect was dependent on activation of tissue angiotensin II. However, this compensatory mechanism becomes inactive in LVF, where PKC beta may participate in the progression to cardiac dysfunction and LV remodeling.

Angiotensin II↗
PubMed2002

Isozyme-specific inhibitors and activators of protein kinase C.

We describe here the methods we have used to generate selective peptide inhibitors and activators of PKC-mediated signaling. These approaches should be applicable to any signaling event that is dependent on protein-protein interaction. Furthermore, targeting downstream enzymes in signal transduction has been notoriously difficult as there are often families of related enzymes in each cell. The approaches we have used overcame this difficulty and may prove useful not only in basic research, but also in drug discovery.

14-3-3 Proteins↗
PubMed2002

Dopamine-induced exocytosis of Na,K-ATPase is dependent on activation of protein kinase C-epsilon and -delta.

The purpose of this study was to define mechanisms by which dopamine (DA) regulates the Na,K-ATPase in alveolar epithelial type 2 (AT2) cells. The Na,K-ATPase activity increased by twofold in cells incubated with either 1 microM DA or a dopaminergic D(1) agonist, fenoldopam, but not with the dopaminergic D(2) agonist quinpirole. The increase in activity paralleled an increase in Na,K-ATPase alpha1 and beta1 protein abundance in the basolateral membrane (BLM) of AT2 cells. This increase in protein abundance was mediated by the exocytosis of Na,K-pumps from late endosomal compartments into the BLM. Down-regulation of diacylglycerol-sensitive types of protein kinase C (PKC) by pretreatment with phorbol 12-myristate 13-acetate or inhibition with bisindolylmaleimide prevented the DA-mediated increase in Na,K-ATPase activity and exocytosis of Na,K-pumps to the BLM. Preincubation of AT2 cells with either 2-[1-(3-dimethylaminopropyl)-5-methoxyindol-3-yl]-3-(1H-indol-3-yl)maleimide (Gö6983), a selective inhibitor of PKC-delta, or isozyme-specific inhibitor peptides for PKC-delta or PKC-epsilon inhibited the DA-mediated increase in Na,K-ATPase. PKC-delta and PKC-epsilon, but not PKC-alpha or -beta, translocated from the cytosol to the membrane fraction after exposure to DA. PKC-delta- and PKC-epsilon-specific peptide agonists increased Na,K-ATPase protein abundance in the BLM. Accordingly, dopamine increased Na,K-ATPase activity in alveolar epithelial cells through the exocytosis of Na,K-pumps from late endosomes into the basolateral membrane in a mechanism-dependent activation of the novel protein kinase C isozymes PKC-delta and PKC-epsilon.

Animals↗
PubMed2002

Intracellular transport mechanisms of signal transducers.

Recent discoveries have revolutionized our conceptions of enzyme-substrate specificity in signal transduction pathways. Protein kinases A and C are localized to discreet subcellular regions, and this localization changes in an isozyme-specific manner upon activation, a process referred to as translocation. The mechanisms for translocation involve interactions of soluble kinases with membrane-bound anchor proteins that recognize individual kinase isoenzymes and their state of activation. Recently, modulation of kinase-anchor protein interactions has been used to specifically regulate, positively or negatively, the activity of C kinase isozymes. Also described in this review is a role for the Rab family of small G proteins in regulating subcellular protein trafficking. The pathophysiological significance of disrupted subcellular protein transport in cell signaling and the potential therapeutic utility of targeted regulation of these events are in the process of being characterized.

Animals↗