Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study investigates the regulatory effects of growth factors upon angiotensin II type 2 (AT2) mRNA levels in neurons co-cultured from newborn rat hypothalamus and brainstem. Incubation of cultured neurons with nerve growth factor (NGF; 5-50 ng/ml) caused time-dependent changes in the steady-state levels of AT2 receptor mRNA. Short-term (0.5-1.0 h) incubations with NGF resulted in significant increases in AT2 receptor mRNA, whereas longer-term incubations (4-24 h) caused significant decreases. Activation of NGF receptors is known to stimulate phospholipase C-gamma and subsequently activate protein kinase C (PKC). Incubation of cultures with the PKC activator, phorbol-12-myristate-13-acetate (PMA; 100 nM), caused temporal changes in AT2 receptor mRNA levels similar to those observed with NGF. By contrast, insulin (0.1-10 microg/ml) elicited only significant decreases in AT2 receptor mRNA levels. The observed abilities of NGF and insulin to regulate the expression of AT2 receptor mRNA are consistent with the fact that the AT2 receptor gene promoter region contains several cis DNA regulatory elements that respond to growth factor-stimulated transcription factors. These novel observations which show that NGF and insulin can regulate AT2 receptor mRNA in neurons derived from neonatal rat CNS lend support to the idea that AT2 receptors have a role in development and differentiation.
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PMID:Modulation of angiotensin II type 2 receptor mRNA in rat hypothalamus and brainstem neuronal cultures by growth factors. 922 21

The induction of neurite outgrowth by NGF is a transcription-dependent process in PC12 cells, but the transcription factors that mediate this process are not known. Here we show that the bHLH transcriptional repressor HES-1 is a mediator of this process. Inactivation of endogenous HES-1 by forced expression of a dominant-negative protein induces neurite outgrowth in the absence of NGF and increases response to NGF. In contrast, expression of additional wild-type HES-1 protein represses and delays response to NGF. Endogenous HES-1 DNA-binding activity is post-translationally inhibited during NGF signaling in vivo, and phosphorylation of PKC consensus sites in the HES-1 DNA-binding domain inhibits DNA binding by purified HES-1 in vitro. Mutation of these sites generates a constitutively active protein that strongly and persistently blocks response to NGF. These results suggest that post-translational inhibition of HES-1 is both essential for and partially mediates the induction of neurite outgrowth by NGF signaling.
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PMID:Mediation of NGF signaling by post-translational inhibition of HES-1, a basic helix-loop-helix repressor of neuronal differentiation. 938 49

Oxytocin receptor (OTR) gene transcription has predominantly been thought to be regulated by estrogen. However, the continuous presence of receptors in certain brain regions after gonadectomy suggests the existence of alternate mechanisms of regulation. We have cloned and sequenced 4 kb of 5'-flanking DNA of the rat OTR gene and identified an internal segment which was absent in the initial publication of this promoter sequence. Sequence analysis of this segment, as well as of a novel upstream region, revealed the presence of a CRE as well as several other potential regulatory elements, including AP-1, AP-2, AP-3, AP-4 sites, an ERE, and a half-SRE (SRE/2). The effects of phorbol 12-myristate 13-acetate (PMA), forskolin, and NGF treatment on this promoter were tested in transfection experiments in MCF7 and SK-N-SH cells. Transcription of the full-length OTR promoter was induced by forskolin and by the phorbol ester PMA, and a synergistic (17-fold) effect was observed in MCF7 cells treated with both agents. Receptor binding studies using the OTR antagonist 125I-labeled ornithine vasotocin, and Western blot analyses of OTRs in MCF7 cells, showed that PMA and forskolin also increased the density of endogenous human oxytocin receptors. Mutational analyses of the CRE and half-SRE sites in this promoter indicated that these elements function as enhancers and support forskolin and NGF effects, respectively, on transcription. These studies have identified a novel region of the rat OTR promoter containing elements which impart cAMP and/or phorbol ester inducibility of OTR gene transcription. A potential role of the PKA and/or PKC pathways in OTR gene regulation is suggested.
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PMID:NGF, cyclic AMP, and phorbol esters regulate oxytocin receptor gene transcription in SK-N-SH and MCF7 cells. 947 29

The ectodomain of the neurotrophin receptor TrkA has been recovered as a soluble fragment from the culture media of cells by a process that involves endoproteolytic cleavage. This cleavage may be upregulated by several treatments, including NGF treatment or protein kinase C activation. In this report we have investigated the cellular site and proteolytic activities involved in TrkA cleavage, and the effects of ectodomain truncation on signalling. Cleavage occurs when the receptor is at, or near, the cell surface, and it can be prevented by agents that affect protein sorting. Cleavage generates several cell-bound fragments, and their generation can be differentially blocked by inhibitors, documenting the involvement of multiple plasma membrane metalloendoproteases. The major cell-bound receptor fragment (i) is tyrosine-phosphorylated in vivo; (ii) does autophosphorylate in vitro; and (iii) is able to associate with intracellular signalling substrates. Artificial deletion of the TrkA ectodomain results in an active receptor that induced neurite outgrowth in pheochromocytoma cells. Cleavage by this natural cellular mechanism appears thus to serve not only as an outlet of receptor binding fragments, but also to generate signalling-competent cell-bound receptor fragments. In the nervous system this ligand-independent receptor activation could play important roles in the development and survival of neurons.
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PMID:Cleavage of the TrkA neurotrophin receptor by multiple metalloproteases generates signalling-competent truncated forms. 1010 37

Removal of atypical PKC blocks NGF-induced differentiation of PC12 cells.1 We now examine the consequences that overexpression of atypical PKCs had upon NGF responses. PC12 cells were stably transfected with either PKC-iota or PKC-zeta. Overexpression of atypical PKCs markedly enhanced NGF- induced neurite outgrowth as well as enhanced NGF-stimulated JNK kinase. Cotransfection of HA-JNK1 along with increasing concentrations of PKC-iota, resulted in dose-dependent phosphorylation of GST c-Jun (1 - 79). NGF treatment of PC12 cells resulted in activation of NF-kappaB. In comparison, overexpression of atypical PKC-iota was by itself sufficient to activate NF-kappaB and shift the kinetics of NGF-induced kappaB activity. Furthermore, transfection of full-length antisense PKC-iota blocked basal and NGF-stimulated NF-kappaB. Differentiated and undifferentiated PC12 cells overexpressing atypical PKC-iota were protected from serum deprivation-induced cell death. Collectively, these findings demonstrate that atypical PKC-iota lies in a pathway that regulates NF-kappaB and contributes to both neurotrophin-mediated differentiation and survival signaling.
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PMID:Overexpression of atypical PKC in PC12 cells enhances NGF-responsiveness and survival through an NF-kappaB dependent pathway. 1046 49

Elevated vascular (VSMC) and bladder smooth muscle (BSMC) NGF are associated with altered visceral innervation in the spontaneously hypertensive rat (SHR: hypertensive, behaviorally hyperactive) compared with control Wistar-Kyotos (WKYs). Stretch stimulates increased NGF production in BSMCs. To elucidate whether stretch induces NGF synthesis in VSMCs, and to determine if disturbances in stretch-mediated NGF production contribute to the elevated tissue levels of NGF in SHRs, we subjected VSMCs and BSMCs cultured from four established inbred rat strains (WKY, WKHA: hyperactive; SHR and WKHT: hypertensive) to several stretch paradigms. For VSMCs, acute and cyclic stretch affected cells derived from hypertensive rats (80-100% increase over control) but not from normotensive strains. For BSMCs, cyclic and static stretch increased NGF secretion in all four strains, but had a two- to threefold greater effect in cells from SHRs and WKHTs (increase up to 600%) at early time points. At later time points of a 24-h experimental period, stretch increased NGF output up to 400% in SHR and WKHA cultures. Thus, defects that influence early induction of stretch-mediated SHR NGF secretion cosegregate with the hypertensive phenotype. Stretch-gated ion channel inhibitors, voltage-gated ion channel inhibitors, and protease inhibitors failed to affect stretch-induced BSMC NGF secretion. In contrast, gene transcription, intracellular calcium, protein kinase C (PKC), and autocrine release of an unknown factor may play a role in the elevated NGF secretion observed in smooth muscle from hypertensive animals. Altered stretch-induced smooth muscle NGF secretion may contribute to the augmented vascular and bladder NGF content associated with high blood pressure and hyperactive voiding in SHRs.
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PMID:Stretch-activated signaling of nerve growth factor secretion in bladder and vascular smooth muscle cells from hypertensive and hyperactive rats. 1079 3

The mechanisms by which excitable cells adapt and respond to changes in O2 levels remain largely unknown. We have investigated the effect of hypoxia on the cyclic AMP response element binding protein (CREB) transcription factor. PC12 cells were exposed to moderate levels of hypoxia (5% O2) for various times between 20 min and 6 hr. We found that hypoxia rapidly and persistently induced ser133 phosphorylation of CREB. This effect was more robust than that produced by exposing PC12 cells to either forskolin, KCl, or NGF. This effect was not due to activation of any of the previously known CREB kinases, including PKA, CaMK, PKC, p70s6k, or MAPKAP kinase-2. Thus, hypoxia may induce activation of a novel CREB kinase. To test whether phosphorylation of CREB was associated with an activation of CRE-dependent gene expression, cells were transfected with wild type and mutated regions of the 5'-flanking region of the tyrosine hydroxylase (TH) gene fused to a CAT reporter gene. Mutation of the CRE element in a TH reporter gene reduced, but did not abolish, the effects of hypoxia on TH gene expression. However, hypoxia did not induce transactivation of a GAL4-luciferase reporter by a GAL4-CREB fusion protein. Thus, the mechanism by which hypoxia regulates CREB is distinct, and more complex, than that induced by forskolin, depolarization, or nerve growth factor.
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PMID:Regulation of CREB by moderate hypoxia in PC12 cells. 1084 56

The dynamic properties of the cell cortex and its actin cytoskeleton determine important aspects of cell behavior and are a major target of cell regulation. GAP43, myristoylated alanine-rich C kinase substrate (MARCKS), and CAP23 (GMC) are locally abundant, plasmalemma-associated PKC substrates that affect actin cytoskeleton. Their expression correlates with morphogenic processes and cell motility, but their role in cortex regulation has been difficult to define mechanistically. We now show that the three proteins accumulate at rafts, where they codistribute with PI(4,5)P(2), and promote its retention and clustering. Binding and modulation of PI(4, 5)P(2) depended on the basic effector domain (ED) of these proteins, and constructs lacking the ED functioned as dominant inhibitors of plasmalemmal PI(4,5)P(2) modulation. In the neuron-like cell line, PC12, NGF- and substrate-induced peripheral actin structures, and neurite outgrowth were greatly augmented by any of the three proteins, and suppressed by DeltaED mutants. Agents that globally mask PI(4,5)P(2) mimicked the effects of GMC on peripheral actin recruitment and cell spreading, but interfered with polarization and process formation. Dominant negative GAP43(DeltaED) also interfered with peripheral nerve regeneration, stimulus-induced nerve sprouting and control of anatomical plasticity at the neuromuscular junction of transgenic mice. These results suggest that GMC are functionally and mechanistically related PI(4,5)P(2) modulating proteins, upstream of actin and cell cortex dynamics regulation.
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PMID:GAP43, MARCKS, and CAP23 modulate PI(4,5)P(2) at plasmalemmal rafts, and regulate cell cortex actin dynamics through a common mechanism. 1087 Dec 85

Zinc overload may be a key mechanism of neuronal death in acute brain injury. We have demonstrated previously that zinc overload neurotoxicity involves protein kinase C (PKC)-dependent rises in intracellular levels of reactive oxygen species (ROS). However, the cascade linking PKC activation to ROS generation in cultured cortical neurons has been unknown. A recent study has demonstrated that ROS-generating NADPH oxidase is present in sympathetic neurons and contributes to NGF deprivation-induced cell death. Because NADPH oxidase is activated by PKC, in the present study, we examined the possibility that NADPH oxidase is the effector for oxidative stress in zinc-overloaded cortical cells. Reverse transcription-PCR and Western blot analyses revealed that naive cultured cortical cells express subunits of NADPH oxidase at low levels. Exposure to zinc substantially increased levels of NADPH oxidase subunits in both neurons and astrocytes. In addition, zinc exposure induced translocation of the p47(PHOX) and p67(PHOX) subunits to the membrane, a signature event for NADPH oxidase activation. Addition of a selective PKC inhibitor, GF109203X, blocked both the induction and the membrane translocation of NADPH oxidase by zinc. Supporting the role for NADPH oxidase in zinc-triggered oxidative injury, NADPH oxidase inhibitors attenuated ROS production and cortical neuronal death induced by zinc. In addition, Cu/Zn-superoxide dismutase and catalase attenuated zinc-induced cortical neuronal death. Our results have demonstrated that zinc overload induces and activates NADPH oxidase in cortical neurons and astrocytes in a PKC-dependent manner. Thus, NADPH oxidase may be an enzyme contributing to ROS generation in zinc-overloaded cortical neurons and astrocytes.
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PMID:Induction and activation by zinc of NADPH oxidase in cultured cortical neurons and astrocytes. 1109 Jun 11

The human neuroblastoma cell line SH-SY5Y can differentiate into a functional sympathetic neuronal phenotype when treated with low concentrations of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of serum or defined growth factors. When TrkA is introduced into the cells, NGF also induces differentiation. In both cases, protein kinase C (PKC) is pivotal for induction and maintenance of the differentiated phenotype. We have recently shown that PKC activity is needed to enable the MAPK ERK to accumulate in the nucleus of SH-SY5Y cells and hence activate transcription. To find out whether this could be one reason for the PKC dependency in the differentiation process we have investigated the role of ERK during neuronal differentiation of these cells. The results show that ERK was needed for full upregulation of the neuronal marker genes NPY and GAP-43. However, ERK activity was not necessary for TPA-induced neurite formation. Neither was activation of ERK sufficient to promote neurite outgrowth. The results clearly show that there was no correlation between nuclear ERK activity, measured as SRE transactivation, and neurite formation in TPA-differentiated SH-SY5Y neuroblastoma cells.
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PMID:A functional role for ERK in gene induction, but not in neurite outgrowth in differentiating neuroblastoma cells. 1128 40


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